text:
Complement receptor C3aR1 controls neutrophil mobilization following spinal cord injury through physiological antagonism of CXCR2

1School of Biomedical Sciences, Faculty of Medicine, 2Blood and Bone Diseases Program, Mater Research Institute, 3School of Public Health, Faculty of Medicine, and 4 Clem Jones Centre for Ageing Dementia Research, Queensland Brain Institute, The University of Queensland, Brisbane, Australia.5Orthopaedic Department, Princess Alexandra Hospital, Brisbane, Australia.6Princess Alexandra Hospital – Southside Clinical School, Faculty of Medicine, The University of Queensland, Brisbane, Australia. 7Antigen Presentation and Immunoregulation Laboratory, QIMR Berghofer Medical Research Institute, Brisbane Australia. 8Trauma, Critical Care and Recovery, Brisbane Diamantina Health Partners, Brisbane, Australia.

Introduction
The bone marrow (BM) stores a large pool of mature neutrophils that is rapidly mobilized in response to injury and/or infection. Egress of these cells is critical for any successful immune response, but uncontrolled neutrophil accumulation and activity in inflamed tissues can also drive pathology (1,2). The mobilization of BM neutrophils is therefore tightly regulated by a complex interplay of cytokines, chemokines, pattern recognition receptors, and cell adhesion molecules (3). Our previous work indicated an unexpected negative regulatory role for the G protein-coupled receptor (GPCR) of complement activation product 3a, C3aR1, in this process (4). How C3aR1 controls the egress of BM neutrophils remained to be elucidated.
Activation of C3aR1 by its natural ligand, C3a, a 77-amino acid cleavage product of C3 proteolysis, was traditionally viewed as predominantly proinflammatory (5). Context-specific immunoregulatory roles for C3a/C3aR1 have, however, emerged over the last decade, and they include the homing and/or retention of hematopoietic stem/progenitor cells (6,7) as well as neutrophils (4) in the BM. Understanding how these C3a/C3aR1 effects are mediated is key to the development of novel therapeutic approaches to attenuate inflammation, particularly in neutrophil-driven pathologies.
Traumatic spinal cord injury (SCI) triggers an acute-phase response that leads to systemic inflammation and rapid mobilization of bone marrow (BM) neutrophils into the blood. These mobilized neutrophils then accumulate in visceral organs and the injured spinal cord where they cause inflammatory tissue damage. The receptor for complement activation product 3a, C3aR1, has been implicated in negatively regulating the BM neutrophil response to tissue injury. However, the mechanism via which C3aR1 controls BM neutrophil mobilization, and also its influence over SCI outcomes, are unknown. Here, we show that the C3a/C3aR1 axis exerts neuroprotection in SCI by acting as a physiological antagonist against neutrophil chemotactic signals. We show that C3aR1 engages phosphatase and tensin homolog (PTEN), a negative regulator of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, to restrain C-X-C chemokine receptor type 2-driven BM neutrophil mobilization following trauma. These findings are of direct clinical significance as lower circulating neutrophil numbers at presentation were identified as a marker for improved recovery in human SCI. Our work thus identifies C3aR1 and its downstream intermediary, PTEN, as therapeutic targets to broadly inhibit neutrophil mobilization/recruitment following tissue injury and reduce inflammatory pathology.
Neutrophil infiltration after insult is a prominent feature of both human and experimental traumatic spinal cord injury (SCI) (8)(9)(10), and presence of these cells at the lesion site is thought to contribute considerably to secondary inflammatory pathology and thus worse outcomes (11)(12)(13)(14). Relatively little is known, however, about the molecular mechanisms orchestrating neutrophil mobilization and recruitment to the injured spinal cord, and to the best of our knowledge no study to date has explored a role for C3aR1 in this pathology.
Here, we first delineated a role for C3a/C3aR1 in SCI by comparing the recovery of wild-type (WT) and C3ar1 -/-(i.e., knockout) mice from blunt spinal cord trauma, one of the most common types of SCI in humans (15). Because C3aR1 is expressed by cells of myeloid (16,17) and central nervous system (CNS) origin (18,19), we also used BM chimera approaches to disentangle peripheral from central C3a/C3aR1 roles in relation to SCI outcomes. We then employed a variety of genetic and pharmacological approaches, including in vitro and in vivo functional assays, antibody-mediated neutrophil depletion, and chemotaxis assays to demonstrate that C3aR1 engages phosphatase and tensin homolog (PTEN) to negatively regulate granulocyte egress from the BM into the circulation in response to inflammatory stimuli. These findings are significant from a therapeutic perspective, as a greater number of circulating neutrophils in the blood was associated with worse outcomes in both mouse and human SCI.

Results
SCI leads to C3a generation, leukocyte infiltration, and elevated C3aR1 expression. To begin exploring a role for C3a in SCI, we first assessed the time course of its generation. C3a levels in the mouse spinal cord rapidly increased following injury ( Figure 1A), and they were significantly elevated over sham-operated controls at 6, 12, and 24 hours after surgery (>5-fold increase; P < 0.001). Plasma C3a levels also rose sharply within 30 minutes of SCI, and remained elevated over sham-operated controls for at least 1 day after SCI ( Figure 1B). Select key comparisons of C3a levels in plasma and spinal cord samples of C3ar1 -/mice yielded similar results, suggesting a similar magnitude of complement system activation between genotypes (2 hours after surgery: C3ar1 -/plasma 7.93 ± 1.63 μg/ml vs. WT plasma 6.94 ± 0.90 μg/ml, n = 4-5 per genotype, P = 0.51; C3ar1 -/spinal cord 0.76 ± 0.10 pg/μg vs. WT spinal cord 0.68 ± 0.08 pg/μg, n = 4 per genotype, P = 0.58).
Widespread C3aR1 staining was observed at and around the site of SCI, and on a variety of cell types. In the acute phase, C3aR1-expressing Ly6B.2 + and CD11b + cells were abundant at and around the lesion site at 1 day after injury (Figure 1, C and D), a time point that coincides with peak neutrophil recruitment (20). The majority of infiltrating Ly6B.2 + cells were genuine neutrophils, as little overlap was observed between Ly6B.2 staining and GFP + cells of monocytic lineage in Cx3cr1 gfp/+ mice at this time point ( Figure  1E). Overall, these findings are consistent with C3aR1 being expressed by activated neutrophils (16,21). At 35 days after SCI, when the lesion site is dominated by macrophages and a glial scar (22), C3aR1 expression remained high and was observed on a variety of cell types; these included GFAP + astrocytes, Iba1 + microglia/macrophages, and at least a subset of residual Ly6B.2 + neutrophils ( Figure 1, F-H).
Genetic ablation of C3aR1 worsens SCI outcomes and exacerbates inflammation. To determine whether C3a/ C3aR1 affects the neurological outcome from SCI, we next assessed the recovery of WT and C3ar1 -/mice from SCI. Over-ground locomotion was normal in both genotypes prior to surgery (time point 0), and SCI induced near-complete paralysis of the hindlimbs at 1 day after injury ( Figure 2A). Strikingly, C3ar1 -/mice regained significantly less locomotor function during the postacute phase of injury compared with their WT counterparts, as evident from lower BMS scores at 14, 21, 28, and 35 days after SCI (P < 0.05; Figure 2, A and B). Consistent with worse functional outcomes, C3ar1 -/mice had larger lesion volumes on T 2 *-weighted MRI (Figure 2, C and D; P < 0.01). Myelin content at the lesion epicenter, which closely correlates with behavioral deficits/outcomes (24), was also significantly lower in the absence of C3aR1 (P < 0.01; Figure 2, E and F). Assessment of astrogliosis revealed no differences between genotypes in either the GFAP + area (Figure 2, E and G; P = 0.18) or the intensity of GFAP staining (P = 0.73) at 35 days after SCI. Nissl staining further revealed a significantly greater abnormal granular tissue presence (P < 0.01; Figure 2, H and I), and an increase in the inflammatory infiltrate at the lesion epicenter of C3ar1 -/mice at 35 days after SCI (P < 0.05; Supplemental Figure  1; supplemental material available online with this article; https://doi.org/10.1172/jci.insight.98254DS1).
Loss of C3aR1 leads to acute granulocytosis in response to trauma. Given the various indicators of worsened outcomes and increased post-SCI inflammation in C3ar1 -/mice, we next assessed whether a loss of C3aR1 changed the nature and/or magnitude of the inflammatory response at the outset. Ly6B.2 + neutrophils, i.e., the first white blood cells (WBCs) to enter into the lesioned spinal cord, were indeed present in significantly greater numbers in C3ar1 -/mice ( Figure 3A; P < 0.05); this difference from WT mice was apparent as early as 1 day after injury and did not normalize during the postacute phase. Thus, absence of C3aR1 augments the inflammatory response to SCI, beginning with an increased presence of Ly6B.2 + neutrophils.
We then explored if the greater presence of Ly6B.2 + cells at the site of SCI in C3ar1 -/mice resulted from increased BM neutrophil mobilization in response to trauma-induced tissue damage. WBC analysis indeed revealed that significantly more Gr-1 + SSC hi neutrophils were being mobilized into the blood of C3ar1 -/mice at 2 hours after SCI (C3ar1 -/-1.6 ± 0.17 × 10 6 cells/ml vs. WT 0.91 ± 0.18 × 10 6 cells/ml; P < 0.01; Figure 3B), with a concurrent reduction in the number of these cells at the level of the BM (P < 0.05; Figure 3C). Acute differences in SCI-induced BM neutrophil mobilization between genotypes could not be explained based on lesion severity (see Methods) and/or the degree of intraspinal hemorrhaging (Supplemental Figure 2A). By 1 day after injury, blood neutrophil numbers were again reduced in both genotypes, likely reflecting a transient exhaustion of the primed reservoir and/or recruitment of mobilized BM neutrophils to the injured spinal parenchyma (see below); Figure 1. C3a production after SCI and expression of its receptor, C3aR1, in the damaged neural parenchyma. (A) C3a levels in the spinal cord rapidly increased in response to injury, peaking at 1 day after SCI. (B) Circulating C3a levels were also significantly increased within 2 hours of SCI, and they remained well above those observed in sham-operated control mice for up to 1 day after injury. Data points are mean ± SEM (n = 4-5 per genotype per time point). **P < 0.01, ***P < 0.001; ****P < 0.0001 by 2-way ANOVA with Bonferroni's post hoc test (SCI vs. time-matched sham-operated control). (C-F) Representative images of C3aR1 (or Ly6B.2 in E; red) staining during the acute (1 day) and more chronic (35 days) phase of SCI. Merged images of costains (green and/or blue) with nuclear dye (cyan), and Imaris surface reconstructions for colocalization analysis are shown on the right (r 2 value = square of Pearson's correlation for colocalization). C3aR1 was expressed on both Ly6B.2 + (C) and CD11b + (D) cells. (E) Ly6B.2 + cells in the injured spinal cord of Cx3cr1 gfp/+ mice do not express GFP, indicating that they are infiltrating neutrophils. (F) Representative image showing C3aR1 staining in WT spinal cord at 35 days after injury. C3aR1 colocalized to amoeboid-shaped Iba1 + microglia/macrophages (blue) and fibrous GFAP + astrocytes (green); other C3aR1-expressing cells can also be seen (arrowheads). these possibilities are not mutually exclusive. Otherwise, the influence of C3aR1 over early leukocyte mobilization (2 hours) appeared mostly specific to neutrophils, as no differences were observed for other WBC populations, including lymphocytes (WT 3.9 ± 1.0 × 10 6 cells/ml vs. C3ar1 -/-4.2 ± 0.8 × 10 6 cells/ml; P = 0.88) and Gr-1 + SSC lo inflammatory monocytes (WT 2.1 ± 0.55 × 10 5 cells/ml vs. C3ar1 -/-2.5 ± 0.66 × 10 5 cells/ml; P = 0.67). Lastly, a splenic contribution to the acute SCI-induced neutrophil mobilization into the circulation can be ruled out, as granulocyte numbers for this organ in WT mice closely mirror blood data; that is, they are significantly increased over baseline (>7-fold) at the 2-hour time point (SCI 1.41 ± 0.18 × 10 6 vs. naive 0.18 ± 0.04 × 10 6 CD11b + Ly6G + SSC hi cells/spleen; n = 5 per group, P < 0.0001). Collectively, these findings suggest that C3aR1 negatively regulates the BM neutrophil mobilization response to (neuro-) trauma. C3aR1 regulates BM cytokine production/availability. We next measured select complement proteins, key chemokines/cytokines, their receptors, and proteases to begin addressing how C3aR1 regulates BM neutrophil mobilization. Intraspinal and/or plasma protein levels of complement component C5a, its main signaling receptor C5aR1, CXCL1 (KC/IL-8), G-CSF, and CXCL12 (SDF-1) were all regulated by SCI but not differentially expressed between genotypes (Supplemental Figure 2, B-I). In the BM, however, CXCL1 was significantly increased in C3ar1 -/extracellular fluid at 2 (P < 0.05) and 24 hours after SCI (P < 0.0001; Figure 4A). Surface expression of the main CXCL1 receptor, CXCR2, on BM neutrophils was not different between genotypes (P = 0.47; Supplemental Figure 3A). G-CSF, another key mobilizer, was similarly increased in BM extracellular fluid of C3ar1 -/mice, although only at 1 day after injury (1.5-fold, P < 0.01; Figure 4B). The retention factor CXCL12 was also acutely increased in C3ar1 -/-BM extracellular fluid at 2 hours after SCI (P < 0.05; Figure 4C), but then fell (1.6-fold) to below WT levels by 1 day after SCI (P < 0.05; Figure 4D). Like CXCR2, there was no difference in surface staining of CXCR4 (CXCL12 receptor) on BM neutrophils between WT and C3ar1 -/mice (P = 0.48; Supplemental Figure 3B). For complement, C3a levels in the BM extracellular fluid rapidly increased after SCI at 2 (P < 0.05) and 24 hours after injury (P < 0.001), but they were not different between genotypes ( Figure 4E). Levels of C5a -a known potent activator of neutrophils -were also not influenced here by C3aR1 deficiency (Supplemental Figure 3C).

+ infiltrate in WT and
C3ar1 -/mice. Dashed red lines indicate the boundary between the dorsal columns and horns (1 day after SCI). Note that Ly6B.2 + cell numbers are significantly increased in the injured spinal cord of C3ar1 -/mice at 1, 10, and 35 days after SCI. Data points represent the mean ± SEM (n = 5-8). Scale bar: 50 μm. (B) Quantitative analysis of circulating blood granulocyte numbers acutely after SCI. Note the significant expansion of this population in C3ar1 -/mice at 2 hours after SCI. (C) BM granulocyte numbers were significantly lower in C3ar1 -/mice at 2 hours after SCI compared with their WT counterparts. Data points for B and C are mean ± SEM (n = 6 per genotype and time point). *P < 0.05; **P < 0.01; ***P < 0.001 by 2-way ANOVA with Bonferroni's post hoc test for matching time points.
We lastly measured several key proteases involved in neutrophil egress from the BM. Of these, we found that only neutrophil elastase was increased in C3ar1 -/-BM extracellular following SCI (24 hours after injury; Supplemental Figure 3D). There was no effect of genotype on cathepsin G levels in BM extracellular fluid (Supplemental Figure 3E); MMP-9 activity, although generally lower in C3ar1 -/mice, was also similar between genotypes in terms of its responsiveness to SCI (Supplemental Figure 3, F and G; see complete unedited blots in the supplemental material). Collectively, these findings indicate that C3a levels rapidly increase in BM extracellular fluid after SCI, and that C3aR1 normally negatively regulates the expression of select neutrophil-mobilizing factors (i.e., CXCL1, G-CSF, and neutrophil elastase) following CNS injury.
CXCL1/CXCR2 signaling drives BM neutrophil mobilization in response to SCI. Given that CXCL1 was the only differentially expressed chemokine between WT and C3ar1 -/-BM at 2 hours after SCI, we sought to determine its role in neutrophil mobilization and the blood granulocytosis phenotype of C3ar1 -/mice. Administration of a selective antagonist for the main CXCL1 receptor (hereafter referred to as CXCR2-A) blocked BM neutrophil egress in response to SCI and, in doing so, also prevented the acute blood granulocytosis phenotype of C3ar1 -/mice ( Figure 5, A-C). These findings indicate that C3aR1 acts as a negative regulator of CXCL1 expression and/or associated CXCR2 signaling, which in turn controls the magnitude of BM neutrophil mobilization following a major traumatic event like SCI.
Reinstatement of C3aR1 in the peripheral immune compartment, or neutrophil depletion, rescues the C3ar1 -/phenotype. We next hypothesized that if changes in BM neutrophil-mobilizing factors and the associated blood granulocytosis phenotype were directly influencing SCI outcomes, then reinstating the expression of C3aR1 within the peripheral immune compartment should counteract, at least in part, the impaired recovery of C3ar1 -/animals. Transplantation of WT BM into conditioned C3ar1 -/recipients indeed rescued their functional phenotype, with these mice now recovering to the same extent as WT counterparts ( Figure 6, A and B). We also found that WT mice receiving a C3ar1 -/-BM transplant phenocopied conventional C3ar1 -/mice, in that they displayed impaired recovery from SCI ( Figure 6, A and B). These experiments demonstrate that presence of C3aR1 on circulating leukocytes, but not CNS-resident cells, is a critical determinant in the functional outcome from SCI.
To directly link increased neutrophil mobilization under C3aR1-deficient conditions to worsened outcomes from SCI, we next selectively depleted these cells in vivo via anti-Ly6G (α-Ly6G) antibody treatment. We hypothesized that if the magnitude of neutrophil mobilization acutely after SCI was indeed driving the impaired recovery of C3ar1 -/mice, then ablating these cells should restore BMS locomotor scores to WT levels. Flow cytometry confirmed the efficacy of α-Ly6G-mediated neutrophil depletion at the level of the blood, BM, spleen, and injured spinal cord for up to at least 72 hours after SCI (Figure 7, A-F). Extending from earlier observations ( Figure 3B), significant granulocytosis was again observed at 72 hours after SCI in C3ar1 -/mice treated with a nonspecific IgG2α isotype (Iso) control antibody (C3ar1 -/plus Iso, 9.4 ± 0.79 × 10 5 cells/ml [n = 3] vs. WT plus Iso, 2.0 ± 1.0 × 10 5 cells/ml [n = 5]; P < 0.01). Regardless, α-Ly6G antibody treatment depleted circulating SSC hi Ly6C + cells with equal efficacy (WT plus α-Ly6G, 0.025 ± 0.014 × 10 5 cells/ml [n = 5] vs. C3ar1 -/plus α-Ly6G, 0.024 ± 0.012 × 10 5 cells/ml [n = 3]; P = 0.27). Consistent with our earlier observations, C3ar1 -/mice treated with isotype control antibody regained less hindlimb function than their WT counterparts (P < 0.05; Figure 7, G and H). Treatment with α-Ly6G, on the other hand, improved recovery of both WT and C3ar1 -/mice (P < 0.01), highlighting the injurious role of neutrophils in this condition. Most importantly, α-Ly6G treatment enhanced the neurological recovery of C3ar1 -/mice to a level that was indistinguishable from their WT counterparts (Figure 7, G and H). Postmortem analysis of histopathology independently confirmed improved SCI outcomes based on myelin content within spared white matter, and also a full rescue of the C3ar1 -/phenotype with α-Ly6G treatment ( Figure 7I). These findings thus provide direct evidence that the SCI-induced granulocytosis indeed drives the worsened neurological outcomes in C3ar1 -/mice, and also in more general terms that the extent of BM neutrophil mobilization has a direct negative impact on recovery from experimental SCI. This relationship was also found in human patients with traumatic SCI, as individuals with high circulating neutrophil numbers on admission were less likely to have a positive conversion in their neurological grading (American Spinal Injury Association Impairment Scale [AIS]) at discharge (P < 0.01; Figure 7J). This difference remained when taking into account patient age, sex, admission AIS grade, and new injury severity scores (NISS) as possible confounders through logistic regression analysis (odds ratio [OR] = 5.895; 95% confidence internal [CI] 1.421-24.453; P < 0.05). These findings thus support an injurious role for neutrophils in traumatic SCI that is conserved between mice and humans, and also highlight the importance of better understanding neutrophil mobilization (and function) for conditions that are hallmarked by systemic inflammation and granulocytosis.
We already explored here how early neutrophil depletion affects the recruitment of other immune cells during the acute, subacute, and intermediate phases of SCI. Of all the cell types examined, only the presence of a B cell subset (CD45 + B220 mid CD11ccells) and monocytes/macrophages (CD45 hi CD11b + C-D64 hi cells) was altered in α-Ly6G-treated mice at 1 and 14 days after SCI, respectively (P < 0.05; Supplemental Figure 4); no other differences were observed. These findings demonstrate that α-Ly6G treatment early after SCI has only a very modest impact on the (subsequent) recruitment of other immune cells. The observed benefits of α-Ly6G treatment therefore most likely result from an acute attenuation of secondary damage to the spinal cord that is caused directly by activated neutrophils themselves and/or the way in which they influence the activation states of other cells.
C3aR1 reduces cellular responsiveness to CXCL1/CXCR2 signaling. Having determined that eliminating C3aR1 changes the BM microenvironment to one that favors neutrophil mobilization under challenged conditions (Figure 4), and also that the resulting granulocytosis phenotype could be alleviated with CXCR2 antagonism ( Figure 5), we lastly investigated whether C3aR1 signaling may also directly regulate the responsiveness of neutrophils to mobilizing/chemotactic signals. In vitro chemotaxis assays revealed that the responsiveness of Ly6G-enriched mouse BM neutrophils toward CXCL1 was indeed suppressed (P < 0.01) by exposure to the selective C3aR1 peptide agonist E7 (4) for WT but not C3ar1 -/cells ( Figure 8A). As E7 does not have any chemotactic effect on BM neutrophils (Supplemental Figure 5), these findings indicate that signaling events downstream of C3aR1 negatively regulate intracellular pathways activated by CXCL1. The same phenomenon was observed when complement activation product C5a was used as the chemoattractant (Supplemental Figure 5), indicating that these C3aR1 effects apply more broadly and are not just restricted to the CXCL1/CXCR2 axis.
Activation of the PI3K/AKT pathway downstream of GPCRs like CXCR2 (and C5aR1) plays a key role in controlling cell motility towards chemotactic signals ( Figure 8B) (25), and stimulation of mouse BM neutrophils with CXCL1 indeed increased AKT phosphorylation as expected ( Figure 8C). Importantly, however, this CXCL1-induced increase in phosphorylated AKT was fully annulled by E7 exposure ( Figure  8, D-F), and we further corroborated these findings by demonstrating similar responses in human neutrophils stimulated with IL-8 (Supplemental Figure 6).
With a recent study linking ERK phosphorylation to cell membrane recruitment of PTEN (26), a well-established negative regulator of the PI3K/AKT pathway, we wondered whether this phosphatase could be the missing link as to how C3aR1 is able to control the responsiveness of neutrophils to mobilizing/chemotactic signals. We first confirmed that activation of C3aR1 leads to ERK1/2 phosphorylation in  Figure 6A), and were also able to show that this change was associated with an increase in the geometric mean fluorescence intensity (MFI) for PTEN staining following E7 but not CXCL1 exposure (Figure 8, J-L).
We next employed in vitro chemotaxis assays again to directly probe a role for PTEN in mediating the negative regulatory effect of the C3a/C3aR1 axis on mouse BM neutrophil migration. Addition of the PTEN inhibitor VO-OHpic (100 nM) (27) indeed completely reversed the C3aR1-mediated suppression of BM neutrophil chemotaxis, fully restoring the migration of these cells toward CXCL1; exposure to VO-OHpic alone (i.e., without E7-induced C3aR1 coactivation) did not change the migratory response ( Figure 8M). Although we did not investigate other mouse strains of different genetic backgrounds (28), involvement of PTEN in the negative modulatory effect of the C3a/C3aR1 axis over chemotactic signals appears to be conserved between mice and humans. Specifically, the IL-8-induced increase in phosphorylated AKT was retained in the presence of E7 when the PTEN inhibitor VO-OHpic was also added (Supplemental Figure 6B). We finally theorized that if C3aR1 signaling controls BM neutrophil migration/ mobilization in a PTEN-dependent manner, then blocking the actions of this phosphatase in vivo should induce granulocytosis in WT mice that is akin to their C3ar1 -/counterparts. Administration of VO-OHpic (2 mg/kg i.v.) to WT SCI mice indeed led to greater neutrophil presence in the blood ( Figure 8N), and their numbers were comparable to those seen in C3ar1 -/mice (see Figure 3B and Figure 5B). As VO-OHpic may also target SHP-1 (29), a separate cohort of mice was treated with the SHP-1/SHP-2 PTPase inhibitor NSC87877 (2.5 mg/kg i.p.). No change in neutrophil presence was seen at the level of the blood with this treatment (Supplemental Figure 7), strongly suggesting that the observed effects of VO-OHpic are indeed mediated via PTEN and not SHP-1.

Discussion
Excessive and/or deregulated complement system activation following trauma can increase host tissue damage, impair wound healing, and lead to unfavorable outcomes (30). Consistent with this, gene knockout or pharmacological approaches to inhibit complement at the level of C1q (31), factor B (32), C3 (33), C3b/C4b (34), and C5a (35) all improve recovery from a major neurotraumatic event like SCI. However, no study to our knowledge has directly explored a putative role for C3a/C3aR1 in this condition. The mechanism via which C3aR1 regulates BM neutrophil mobilization in response to tissue injury more generally has also remained elusive. Here, we identify the C3a/C3aR1 axis as a negative regulator that controls expression of select neutrophil-mobilizing factors, particularly CXCL1, in the BM microenvironment. We further show that activation of the C3a/C3aR1 axis controls the responsiveness of BM neutrophils to mobilizing factors like CXCL1. By acting as a physiological antagonist, C3a/C3aR1 limits neutrophil mobilization and associated inflammatory tissue pathology. Several other previous studies have positively implicated the C3a/C3aR1 axis in reparative and/or neuroprotective processes, including retinal regeneration (36), the expression of neurotropic factors by glia (37,38), and neurogenesis (39). Although the present findings do not exclude a role for C3aR1 within the CNS, our BM chimera experiments showed that a loss of this receptor on CNS-resident cells does not change the neurological outcome from SCI. Rather, its expression within the peripheral immune compartment was identified as the crucial determinant for SCI recovery. Consistent with this, targeted depletion of neutrophils fully rescued the C3ar1 -/phenotype and improved overall recovery. This outcome supports other studies that have negatively implicated neutrophils in secondary inflammatory pathology following experimental SCI (11)(12)(13)(14), and we further corroborated this by linking the extent of SCI-induced neutrophil mobilization to recovery prognosis in human patients. For previous studies showing either no or even adverse effects of neutrophil depletion (40)(41)(42), it is worth noting that these mostly used the anti-Gr-1 antibody (RB6-8C5), which depletes not only neutrophils but also Ly6G -Ly6C + monocytes/myeloid-derived suppressor cells (41), and can increase TNF responses (43).
Our animal experiments did not reveal any evidence for differential expression of neutrophil-mobilizing and/or -retention factors between genotypes under homeostatic conditions, or for altered maturation of BM neutrophils (44,45) that could otherwise explain the acute granulocytosis seen in C3ar1 -/mice after insult. Taken together with our previous observations that pharmacological activation of C3aR1 reduces neutrophil numbers in the blood in response to gut ischemia/reperfusion injury (4), the present findings therefore indicate a direct negative regulatory role for C3a/C3aR1 in BM neutrophil mobilization under inflammatory conditions, as also supported by our in vitro chemotaxis experiments. To understand how C3a/C3aR1 controls BM neutrophil egress, we screened a panel of proteins known to be associated with the retention, mobilization, and/or recruitment of these cells. These experiments revealed that the local BM microenvironment of injured C3ar1 -/mice was changed toward one that is more favorable for neutrophil mobilization. In particular, we found that BM CXCL1 levels were abnormally elevated at 2 and 24 hours after SCI, and that blocking the main CXCL1 receptor, CXCR2, prevented the blood granulocytosis phenotype in C3ar1 -/mice. The observed key role for the CXCL1/CXCR2 axis in BM neutrophil mobilization after SCI is consistent with previous reports that CXCR2 deficiency overall causes neutropenia and retains neutrophils in the BM (46) and, further, that inhibition of this pathway alleviates inflammation in SCI (47) and other neutrophil-mediated pathologies (48). G-CSF levels were also increased in C3ar1 -/mice, although not until 1 day after SCI. Consistent with the view that G-CSF can act cooperatively with CXCL1 to promote neutrophil mobilization by reducing CXCR4 signaling (44,(49)(50)(51)(52), CXCL12 levels in the BM microenvironment of C3ar1 -/mice were also lower at 1 day after SCI. Collectively, these findings suggest that C3a/C3aR1 acts to constrain the levels of neutrophil-mobilizing factors within the BM niche in response to tissue injury and associated inflammation.
Finally, we also uncovered downstream signaling events via which C3aR1 directly modulates the sensitivity of neutrophils to chemotactic cues like CXCL1. Activation of PI3Kγ downstream of CXCR2 normally converts the membrane lipid phosphatidylinositol-3,4-bisphosphate (PIP 2 ) into phosphatidylinositol-3,4,5-triphosphate (PIP 3 ) at the leading edge of the neutrophil. Membrane-bound PIP 3 then allows for the docking of Akt, triggering a series of signaling events that ultimately culminate in directional cell movement. Consistent with this, PI3Kγ-deficient mice have limited recruitment of neutrophils and do not produce PIP 3 or phosphorylated AKT following exposure to CXCL1, C5a, or fMLP in inflammatory pathologies such as septic peritonitis (53). Although we did not study the specific PI3K isoform involved, our experiments show that C3aR1 signaling can negatively regulate CXCL1-induced AKT phosphorylation and BM neutrophil chemotaxis. We identified the phosphatase PTEN, a known negative regulator of the PI3K/AKT pathway that normally localizes to the sides and back of the crawling cell where it converts PIP 3 back to PIP 2 in areas away from the leading edge (54), as the likely intermediary via which C3aR1 limits BM neutrophil chemotaxis/egress in vitro and in vivo. Overall, our studies uncover what we believe is a new negative regulatory mechanism that connects rapid injury-induced complement activation and associated C3aR1 signaling in the BM microenvironment with the magnitude of the ensuing cellular inflammatory response (Supplemental Figure 8).
The discovery of how C3aR1 controls acute inflammation and neutrophil responsiveness following injury has clear translational significance. Specifically, it identifies agonism of C3aR1, or its downstream intermediary PTEN, as a therapeutic option to broadly inhibit neutrophil mobilization/recruitment in response to multiple chemotactic signals (e.g., CXCL1 and C5a) for a range of inflammatory conditions where these cells have been associated with pathology (2). Our findings in relation to PTEN further warn against broad/global inhibition of this phosphatase as a means to promote CNS axon regeneration (55), as this would also increase the magnitude of the peripheral inflammatory response and thus likely be detrimental for recovery. Indeed, previous studies have shown that loss of PTEN activity enhances neutrophil recruitment to sites of inflammation in neutropenia-associated pneumonia (56), and similar observations were made here in the context of SCI. In summary, our findings highlight the importance of understanding the role of complement activation products like C3a across multiple tissue compartments. They also warrant further research into the therapeutic development and use of C3aR1 agonists to attenuate neutrophil-driven pathology in complex trauma cases like SCI and other inflammatory conditions. (G) Depletion of Ly6G + cells improves recovery from SCI and alleviates the functional phenotype of C3ar1 -/mice. Data points represent mean ± SEM (n = 5-9 per group). **P < 0.01; ***P < 0.001; ****P < 0.0001 by repeated-measures 2-way ANOVA with Bonferroni's post hoc test (asterisks indicate significance vs. the "C3ar1 -/-+ Iso" group). (H and I) Endpoint BMS scores (H) and epicenter myelin content (I) at 35 days after SCI. Bar graphs are mean ± SEM, with data points representing individual mice within each cohort (n = 5-9). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001 by 1-way ANOVA with Bonferroni's post hoc test. (J) Circulating neutrophil numbers at clinical presentation for SCI patients with and without a positive AIS grade conversion on discharge. **P < 0.01 by Mann-Whitney U test.

Mice
Adult female C57BL/6J (WT, n = 271), C3ar1 -/-(n = 123) (57), C5ar1 -/-(n = 14) (58), and Cx3cr1 gfp/+ mice (n = 3) (59) were used; genetically modified lines had been previously backcrossed for more than 10 generations onto a C57BL/6J genetic background. Mice were obtained from local breeding colonies at The University of Queensland's Biological Resources Facility, or the Animal Resources Centre (Perth, Western Australia), and housed in clean holding facilities on a 12-hour light/dark cycle with unlimited access to food and water. Mice were matched by age and weight within experiments. All experiments were conducted in accordance with the Australian Code for the Care and Use of Animals for Scientific Purposes, as well as the ARRIVE guidelines (60) with regards to randomization (order of surgery, treatment allocation) and blinding (surgery and analysis). . **P < 0.01; ***P < 0.001; ****P < 0.0001 by 1-way ANOVA with Dunnett's post hoc test versus baseline (i.e., no CXCL1; dashed line). (N) Administration of VO-OHpic to WT SCI mice leads to increased mobilization/circulating neutrophil numbers (2 hours after surgery; data are from 3 independent experiments). Error bars are SEM (n = 5 per group). *P < 0.05 by 2-tailed Student's t test.

SCI
Mice were anesthetized with tiletamine/zolazepam (50 mg/kg; Virbac) and xylazine (10 mg/kg; Troy Laboratories) and subjected to a 70-kilodyne contusive SCI using the infinite horizon impactor (61). Postoperatively, animals received a single injection of buprenorphine (0.5 mg/kg s.c.) in Hartmann's solution (Baxter) for analgesia, and were administered 1.0 mg/kg gentamicin s.c. for the first 5 days after injury. Bladders were manually voided twice daily for the duration of the experiments.

C3a ELISA
WT mice were subjected to either SCI or sham (i.e., laminectomy) surgery and then killed with sodium pentobarbitone (100 mg/kg i.p., Virbac) at 30 minutes, 2 hours, 6 hours, 12 hours, 1 day, 4 days, 7 days, or 35 days after injury (n = 4-5 per time point). Naive mice were used to assess baseline C3a (time point 0). Blood and spinal cord samples were collected and processed as described previously (35). C3a concentrations in blood plasma and spinal cord samples were measured as per the manufacturer's instructions (BD Biosciences; 558250, 558251, and 558618). For spinal cord, C3a levels were divided by the total protein content of the sample (Thermo Fisher Scientific, Pierce BCA protein assay, 23227).

MRI
Vertebral columns with the spinal cord in situ were washed in 0.1 M PBS, followed by immersion for 48 hours in PBS containing gadolinium contrast agent (0.2% Magnevist, Bayer HealthCare Pharmaceuticals). Samples were imaged on a 16.4-T small animal MRI (Bruker BioSpin) and lesion volumes analyzed using AVIZO 6.2 software (Visualisation Sciences Group) as detailed previously (35).

Histopathology
Nissl staining. Cresyl violet stain was performed to examine gross morphology of injured spinal cords. Areas densely populated by small Nissl + bodies were outlined in ImageJ (NIH). The volume of granular tissue was estimated by multiplying the granular area by the section thickness.
Myelin preservation and glial scarring. FluoroMyelin Red (1:150; Molecular Probes, 34652) was combined with rabbit anti-GFAP (1:1000; Dako, Z033401-2) and a goat anti-rabbit IgG-Alexa Fluor 488 secondary antibody (1:250; Invitrogen, A-11008). Stained spinal cord sections were photographed using an Olympus SZX12 Research Fluorescence Stereo Microscope (Spectra Services), and the proportional area of myelin and GFAP staining measured as described previously (35). The lesion epicenter was defined as the section with the least amount of FluoroMyelin Red staining relative to the section area.
Cellular inflammation. Immunohistochemical staining was performed as detailed previously (20) using rat anti-Ly6B.2, rabbit anti-Iba1, and rat anti-CD3 (1:400; BD Biosciences, clone 17A2). Slides were digitized using a ScanScope XT scanner (Aperio) with ImageScope software (Leica Biosystems). Ly6B.2 + and CD3 + cell numbers were counted using the manual tag function in ImagePro Plus Version 6.3 (Media Cybernetics) and expressed as the number of cells per mm 2 based on the section area. The Iba1 + infiltrate was quantified as the proportional area of stained tissue (8).

Flow cytometry
For quantification of circulating neutrophil numbers, 50 μl of blood was obtained via retro-orbital eye bleeds from WT (n = 6) and C3ar1 -/mice (n = 6) at 1 week prior to SCI, then again at 2 hours after SCI and 1 day after SCI. For each time point, equal numbers of mice from each genotype were always processed side by side. Mice were euthanized with sodium pentobarbitone immediately after their final eye bleed at 1 day after SCI. Blood was always collected and processed at the same time of day to minimize the impact of circadian variations (62). Samples were diluted 1:3 in anticoagulant buffer (DPBS with 4 mM EDTA, pH 7.2) on ice, then diluted 1:5 with red blood cell lysis buffer (0.85% NH 4 Cl, pH 7.2) and incubated for 5 minutes at room temperature. Cells were centrifuged (300 g for 10 minutes) and resuspended in PBS with Zombie Green viability dye (BioLegend) for dead cell exclusion. Cells were resuspended to a concentration of 1 × 10 7 cells/ml in cytometry blocking buffer (0.5% BSA, 2 mM EDTA, in DPBS, pH 7.2), then incubated with rat anti-CD16/32 (1:200; BD Biosciences, clone 2.4G2) for 10 minutes on ice. Biotinylated rat anti-Gr-1 (1:400; BD Biosciences, clone RB6-8C5) was added for 10 minutes, followed by streptavidin-Alexa Fluor 546 (1:400; Invitrogen, S11225). Cells were centrifuged (300 g for 10 minutes), washed, and resuspended in 395 μl DPBS with 2 mM EDTA. Flow-Count Fluorospheres (5 μl/sample, Beckman Coulter) were added as an internal standard to quantify absolute cell numbers. Samples were analyzed using an LSRII flow cytometer (BD Biosciences) and BD FACS Diva/ FlowJo software. Compensation was applied to remove spectral overlap. Doublets were excluded based on FSC-A/FSC-H linearity. Unstained and isotype controls were used to confirm staining specificity, and to determine gate placement. Similar procedures were used to determine C5aR1 expression using an Alexa Fluor 647-conjugated rat anti-mouse C5aR1 (1:20; BD Biosciences, clone 10/92). Isotype control antibodies (BD Biosciences), unstained samples, as well as blood from C5ar1 -/mice, were used to confirm antibody specificity.
For quantification of BM neutrophil numbers, WT and C3ar1 -/mice were sacrificed at 2 and 24 hours after SCI with sodium pentobarbitone. Naive mice from each genotype were used for assessing BM cellularity under steady-state conditions. BM was flushed from femurs with ice-cold anticoagulant buffer and then passed through a 40-μm cell strainer. Cells were centrifuged at 500 g for 5 minutes and resuspended in 5 ml of RBC lysis buffer. Further processing was similar to that described for blood samples, with the additional inclu-

Spinal cord and plasma protein analysis
C5a and cytokine levels. WT and C3ar1 -/mice were killed with sodium pentobarbitone at 2 hours after surgery. ELISAs were used to determine the levels of C5a (R&D Systems, DY2150), CXCL1/KC (R&D Systems, DY453), and G-CSF (R&D Systems, MCS00) in each sample. For spinal cord samples, measured C5a and cytokine concentrations were divided by the total protein content of the sample.
Hemoglobin. Spinal cord samples were diluted 1:30 in Drabkin's reagent (Sigma-Aldrich) and analyzed alongside a standard curve of bovine hemoglobin (Sigma-Aldrich). Samples were run in duplicate and absorbance measured at 540 nm using a Tecan Sunrise 96-well plate reader. Hemoglobin test results were divided by the total protein content of the sample.
Proteases. Neutrophil elastase, cathepsin G, and MMP-9 were also quantified in BM extracellular fluids of WT and C3ar1 -/mice (n = 6 per genotype and time point/condition). In brief, femurs were flushed with ice-cold PBS. Neutrophil elastase and cathepsin G concentrations were assessed spectrophotometrically using respective enzyme substrates, i.e., 0.8 mM elastase substrate 1 (Merck) or 0.8 mM cathepsin G substrate 1 (Calbiochem) alongside serial standards (0.0375-1 μg/ml neutrophil elastase; 0.015-2 μg/ml cathepsin G; Elastin Products Co.). Samples were run in duplicate and absorbance measured after 3 hours at 405 nm on a FLUOstar plate reader. Protease concentrations in each sample were calculated after correcting for the background color of BM fluid. For MMP-9 analysis, 10 μl of sample from each group was pooled and the average concentration between genotypes and conditions assessed via gel zymography (63).

In vivo CXCR2 antagonism
WT and C3ar1 -/mice were treated with SB225002 (Tocris; 4 mg/kg i.p.) 1 hour before SCI. At 2 hours after injury, blood and BM samples were collected and processed for flow cytometry as described above.

Mouse chemotaxis assays
BM neutrophils were isolated from the femurs and tibiae of WT and C3ar1 -/mice using the α-Ly6G microbead kit (Miltenyi Biotec, 130-092-332) and MiniMACS cell separation system. Migration assays were performed in duplicate using a modified Boyden chamber (Neuroprobe), which consisted of a 3-μm pore filter above a 96-well chamber. RPMI medium (31 μl) with or without recombinant mouse CXCL1 (R&D Systems, 453-KC; 30 nM) was added to wells below the filter. The filter was snapped in place and 5 × 10 4 neutrophils in 20 μl of RPMI were added to the hydrophobic rings above the filter, allowing contact with the media below. For C3aR1 activation, Ly6G-enriched cells from WT, C3ar1 -/-, and C5ar1 -/-BM were stimulated for 10-15 minutes (37°C, 5% CO 2 ) with the selective C3aR1 agonist WWGKKYRASKLGLAR (E7, 10 μM) prior to being added to the migration plate. Similar procedures were followed for inhibition of CXCR2 (Tocris Bioscience, SB225002; 10 μM), PTEN (Sigma-Aldrich, V8639; 100 nM), or pan-PI3K (Sigma-Aldrich, W3144; 100 nM). Plates were incubated for 1 hour (37°C, 5% CO 2 ), after which the filter was carefully removed and the number of transmigrated cells in the bottom compartment counted following dilution in trypan blue solution (1:1) using a Neubauer hemocytometer. This method was also used to evaluate in vitro chemotaxis of BM neutrophils toward E7 or C5a (BioVision Inc, 4895A; 50 nM).
In vivo PTEN and SHP-1 antagonism WT mice were administered VO-OHpic (Sigma-Aldrich, V8639; 2 mg/kg i.v.), NSC87877 (Tocris; 2.5 mg/ kg i.p.), or vehicle (DMSO in saline or PBS) at 12 hours and 30 minutes prior to SCI (n = 5 per group). At 2 hours after SCI, 100 μl of blood was collected via retro-orbital eye bleed and circulating neutrophil numbers quantified by flow cytometry as detailed above.

Human SCI data
Retrospective data were collected from a total of 97 SCI patients admitted to the Princess Alexandra Hospital in Brisbane between 2012 and 2017. For each patient, the extent of neurological impairment was assessed using the AIS grading criteria during the first week of admission to hospital (baseline) and then again at discharge. All patients had an AIS grade of A, B, or C upon admission; the neurological level of injury ranged between the cervical (62.8%), thoracic (34.0%), and lumbar (2.1%) region of the spinal cord. Circulating neutrophil numbers were obtained from WBC differentials in admission blood samples (Pathology Queensland). There were no significant differences between patients who showed either no improvement (n = 63) or an AIS grade conversion (n = 34) with regard to their age (37.5 years vs. 37.9 years, respectively; P = 0.9009) or sex (84.1% vs. 88.2% male, respectively; P = 0.7645).


extfieldsofstudy: Medicine Biology

text:
Microbial community structure analysis in Acer palmatum bark and isolation of novel bacteria IAD-21 of the candidate division FBP

Background The potential of unidentified microorganisms for academic and other applications is limitless. Plants have diverse microbial communities associated with their biomes. However, few studies have focused on the microbial community structure relevant to tree bark. Methods In this report, the microbial community structure of bark from the broad-leaved tree Acer palmatum was analyzed. Both a culture-independent approach using polymerase chain reaction (PCR) amplification and next generation sequencing, and bacterial isolation and sequence-based identification methods were used to explore the bark sample as a source of previously uncultured microorganisms. Molecular phylogenetic analyses based on PCR-amplified 16S rDNA sequences were performed. Results At the phylum level, Proteobacteria and Bacteroidetes were relatively abundant in the A. palmatum bark. In addition, microorganisms from the phyla Acidobacteria, Gemmatimonadetes, Verrucomicrobia, Armatimonadetes, and candidate division FBP, which contain many uncultured microbial species, existed in the A. palmatum bark. Of the 30 genera present at relatively high abundance in the bark, some genera belonging to the phyla mentioned were detected. A total of 70 isolates could be isolated and cultured using the low-nutrient agar media DR2A and PE03. Strains belonging to the phylum Actinobacteria were isolated most frequently. In addition, the newly identified bacterial strain IAP-33, presumed to belong to Acidobacteria, was isolated on PE03 medium. Of the isolated bacteria, 44 strains demonstrated less than 97% 16S rDNA sequence-similarity with type strains. Molecular phylogenetic analysis of IAD-21 showed the lowest similarity (79%), and analyses suggested it belongs to candidate division FBP. Culture of the strain IAD-21 was deposited in Japan Collection of Microorganisms (JCM) and Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ) as JCM 32665 and DSM 108248, respectively. Discussion Our results suggest that a variety of uncultured microorganisms exist in A. palmatum bark. Microorganisms acquirable from the bark may prove valuable for academic pursuits, such as studying microbial ecology, and the bark might be a promising source of uncultured bacterial isolates.

INTRODUCTION
The total number of microorganisms existing on the earth is speculated to range from 10 29 to 10 30 organisms (Whitman, Coleman & Wiebe, 1998;Kallmeyer et al., 2012). It is reported that the number of operational taxonomic units (OTUs) detected from one g of soil is up to 52,000 (Roesch et al., 2007). The number of bacterial species that currently have been isolated, investigated with regards to physiological properties, and assigned scientific names is about 15,000 (Parte, 2018). This is only 1% of the total number of bacterial species presumed to exist on earth, and the remaining 99% of uncultured microorganisms is called the "microbial dark matter" (Ledford, 2015). Until now, only cultivable microorganisms among the 1% have been used to comprehend the overall microbial ecosystem and identify novel useful genes, but the exploration of these cultivable microbes have reached a plateau in recent years (Puspita et al., 2012). Since the microbial dark matter is expected to potentially impact the current status of academic and industrial fields, comprehensive environmental genome analyses are being conducted around the world (Rinke et al., 2013;McCalley et al., 2014). However, unraveling microbial functions, which cannot be elucidated from the nucleotide sequence alone, or the practical utilization of uncultured microorganisms, requires pure culture isolation and cultivation (Stewart, 2012). Since cultivation of the remaining 99% of microorganisms holds great potential, exploration and isolation of microorganisms from various environments are desirable.
Numerous analyses on symbiotic microorganisms have been conducted for many terrestrial plants, and their microbial community structures are determined not only by plant species, but also by factors such as plant organs and environmental factors (Wieland, Neumann & Backhaus, 2001;Schlaeppi et al., 2010;Edwards et al., 2015;Zarraonaindia et al., 2015). Many of these microorganisms provide benefits to plants, such as the promotion of plant growth (Köberl et al., 2013), modification of plant-hormone production (Bodenhausen et al., 2014), and resistance to disease (Berendsen, Pieterse & Bakker, 2012). To understand the plant-microbial symbiotic relationship and its impact on the ecosystem, comprehensive analysis of the plant symbiotic microbial community structure and further isolation of symbiotic microorganisms, including uncultured microorganisms, are necessary. For example, in the case of agricultural crops and model plants, including Arabidopsis thaliana, exhaustive analyses of symbiotic microorganisms' function and community structure have been performed using both culture-independent and culture-dependent methods (Delmotte et al., 2009;Manter et al., 2010;Vorholt, 2012;Comby et al., 2016). However, for some plant types, the exploration of microbial resources has not been sufficiently conducted yet. Tanaka et al. (2012) focused on the rhizosphere of aquatic plants, which have not been thoroughly investigated for studying symbiotic microbial communities, and isolated Armatimonas rosea YO-36 T (Former Candidate division OP10) from the roots of Phragmites australis. Tanaka et al. (2017) also investigated the roots of the aquatic plants Iris pseudacorus and Scirpus juncoides, and isolated microorganisms belonging to Acidobacteria and Verrucomicrobia, which are relatively difficult to cultivate. Microorganisms isolated from such aquatic plants are relatively novel, even if they belong to taxa with high cultivation frequency. Since environmental samples that have not been explored thus far lack information on microbial communities and isolates in databases, it is suggested that the novelty of cultured microorganisms is necessarily high from such unexplored potential microbial resources.
Analyses of the structures of microbial communities present on trees have been previously conducted (Mocali et al., 2003;Moore et al., 2006;Taghavi et al., 2009;Redford et al., 2010;Filteau et al., 2010;Laforest-Lapointe, Messier & Kembel, 2016a, 2016b. However, very few studies have focused on the tree bark. The bark refers to the outer side of the cambium surrounding the xylem of the tree and is composed of an inner bark, which is the living tissue consisting of phloem, and an outer bark, which is the dead tissue of the cork. The bark is composed of polysaccharides (cellulose, hemicellulose), pectin substances, phenolic polymers such as lignin and high molecular weight tannins, and cross-linked polyesters such as suberin and cutin. The bark contains greater amounts of extracts (polyphenol and suberin), minerals, and lignin than the center of the tree (Feng et al., 2013). As a protective tissue, the bark consists of compounds that are resistant to microbial degradation, such as suberin (Baldrian, 2017). In addition, the bark is impregnated with resin that inhibits the growth of microorganisms (Baldrian, 2017). The bark protects the cambium from precipitation, heat, frost, and UV radiation and acts as a barrier against the attack of bacteria, fungi, parasitic plants, insects, and animals (Sakai, 2001). By adapting to tree bark, microorganisms may be able to acquire a stable habitat. In the case of bark (especially old bark), the tree canopy blocks precipitation and UV irradiation, and there is less disturbance than in other tissues such as leaves and branches, suggesting that microorganisms can stably inhabit areas for a long time (Leff et al., 2015). Further, microorganisms can colonize microsites such as cracks and lenticels, which represent a more favorable environment for microbial growth because they retain humidity and nutrients (Buck, Lachance & Traquair, 1998), and the symbiotic microorganisms can utilize plant biomass and photosynthetic products as carbon sources in such a stable habitat. Therefore, the bark presents a suitable habitat for slow-growing microbes and those susceptible to disturbance. However, compared with other tissues such as leaves and rhizosphere, microbial community structure analysis, and isolation of microorganisms (especially bacteria) including uncultured microorganisms from the bark have not been sufficiently performed. Shen & Fulthorpe (2015) revealed the differences among the microbial community structures within the tree branches of the species Acer negundo, Ulmus pumila, and U. parvifolia, using isolation of the microorganisms and various culture-independent analyses. Ulrich, Ulrich & Ewald (2007) demonstrated the impact of different hybrid poplar clones on the endophytic community structure in branches and leaves using terminal restriction fragment length polymorphism analysis and analyzed the microbial community structure within the branches and leaves of poplar trees using isolation of the microorganisms and clone analysis. However, these two studies used tree branches as the source material, where the environment is completely different from that of the bark.
In addition, Aschenbrenner et al. (2017) revealed the microbial community structure of Acer pseudoplatanus bark, symbiotic moss, and lichens using next generation sequencing. Interestingly, there are a few studies that suggested the bark microbial community is different from that in other organs. Martins et al. (2013) investigated the cultivation and isolation of microorganisms from grapevine and reported that bacterial genera obtained from the bark differed from those obtained from the fruits and leaves. Leff et al. (2015) conducted a culture-independent analysis of Ginkgo biloba bark, branches, young branches, and leaves using high throughput 454 pyrosequencing and showed that the diversity of microbial communities in the old bark was the highest. In addition, phyla containing bacterial species that are generally difficult to culture, or are uncultured, such as Acidobacteria, Armatimonadetes, and the candidate division WYO (Serkebaeva et al., 2013;Weiss et al., 2015), were detected in higher proportions in the bark than in other tree organs. However, since there are few reported cases of analysis of the microbial community structure of bark samples and the isolation (acquisition) of uncultured microorganisms at higher taxonomic levels such as the phylum or class level, there is insufficient evidence to deduce whether bark is an excellent source of uncultured microorganisms.
Nonetheless, previous observations suggested that tree bark may harbor special microbial communities and that isolation and analysis of microorganisms from bark may provide insights into unknown microbial ecosystems and tree-microbial symbiosis. In the current study, we targeted the bark of Acer palmatum, which is a deciduous broad-leaved tree widely growing in Japan, and analyzed the microbial community structure using MiSeq-based next generation sequencing. In addition, we attempted to isolate and cultivate microorganisms by standard methods using low-nutrient agar media.

Sample collection and pre-treatment
Bark sample was collected from mature Acer palmatum from the Ichimura Foundation for New Technology Botanical Research Gardens, Atami, Shizuoka, Japan (35.107336 N, 139.047729 E) using sterile tweezers and scissors. Acer palmatum trees used in this study were at least 80 years old or more and naturally occurring. The botanical research garden is 277-310 m above sea level, along a gentle slope facing south-south-east. Although the botanical garden is an artificially landscaped Japanese garden with artificially planted plants, it also contains several natural plants. A sample for culture-independent analysis was collected in November 2015, and a bark sample for microbial isolation was collected in June 2016. Bark samples for both analyses were collected from the same position (at a height of 1.0-1.5 m) on the same single tree. Further, in order to re-analyze the microbial community structure by culture-independent analysis, we collected three samples (at heights of 1.0, 1.5, and 2.0 m) from each of two individual trees (one being the previously analyzed tree) in February 2018. Approximately 4.7 g of bark fragments from the surface to a depth of about two mm were collected. Since the thickness of the bark (phloem and periderm) of another member of the same genus, Acer rubrum, is 0.8 ± 0.03 mm (Hammond et al., 2015), it was considered that the bark area should be covered by this sampling and that the collected sample contains both epiphyte and endophyte in this range. The collected bark samples were minced using sterilized tweezers and scissors, suspended in 40 ml of phosphate-buffered saline, thoroughly mixed by vortexing, and sonicated at 42 kHz for 3 min using a Bransonic Ultrasonic Cleaner 3510J-DTH (Branson Ultrasonic Corporation, Danbury, CT, USA) to detach the microorganisms adhering to the bark surface. The microbial suspensions from the bark samples were stored at −80 C in 10% (v/v) glycerol.

Culture-independent analysis
To assess the structure of the microbial community in the bark of Acer palmatum, 16S amplicon sequencing using MiSeq was performed. From the above bark suspension, five ml of the supernatant was collected by pipetting so as to minimize contamination of the bark fragments, and DNA was extracted using a Fast DNA Spin Kit (MP Biomedicals, LCC, Santa Ana, CA, USA) according to the manufacturer's instructions. The extracted DNA was adjusted with distilled water to a concentration of 30 ng/ml in a total volume of 50 ml. The DNA concentration was fluorometrically determined using Qubit Assay Kits (Thermo Fisher Scientific Inc., Waltham, MA, USA) and a Nanophotometer (Implen GmbH, Munich, Germany). Sequence analysis of the bark samples using a MiSeq system (Illumina, Inc., San Diego, CA, USA) was performed by Fasmac Co., Ltd (Atsugi, Japan). During the first round of polymerase chain reaction (PCR) amplification, template DNA was amplified using a primer set targeting the V4 region of 16S rDNA. The hot-start PCR reaction consisted of five ng of the starting template, 10 µM of the forward primer 1st_PCR_515F (5′-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT-[GTG CCA GCM GCC GCG GTA A]-3′) and the reverse primer 1st_PCR_806R (5′-GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T-[GGA CTA CHV GGG TWT CTA AT]-3′), 0.2 µl of ExTaq HS polymerase (Takara Bio Inc., Kusatsu, Japan), 1.6 µl of dNTPs, and two µl of 10× Ex Taq buffer in a total reaction volume of 20 ml. The first PCR primers included the adapter sequences for the second PCR and sequences homologous to the V4 region of 16S, as shown in parentheses. The thermal cycling profile included an initial denaturing cycle of 94 C for 30 s, followed by 20 sequential cycles of 94 C for 15 s, 50 C for 30 s, 72 C for 30 s, and a final extension period of 72 C for 5 min, ending with a hold cycle at 4 C. The PCR products were purified using an Agencourt AMPure XP Kit (Beckman Coulter, Inc., Brea, CA, USA) using the manufacturer's instructions. The second PCR reaction included two ml of the purified template DNA, 10 µM of the forward primer 2nd_F ( The thermal cycling profile for the second PCR was a single cycle of 94 C for 2 min, followed by eight cycles of 94 C for 10 s, 60 C for 30 s, 72 C for 30 s, with one final cycle of 72 C for 5 min, and a hold cycle of 4 C. The products from the second PCR were purified using an Agencourt AMPure XP Kit. The DNA concentrations were determined using Qubit Assay Kits, and the PCR amplicons were mixed and subjected to 2 × 250 bp paired-end sequencing using MiSeq System v2. Cluster formation was performed using MiSeq Reagent Kit v2 and PhiX Control Kit v3, and sequence analysis was performed using MiSeq Control Software ver 2.4.1.3, Real Time Analysis ver 1.18.54 and bcl2fastq ver 1.8.4. Analysis of the sequencing results included trimming of the primer region using Fastx toolkit, version 0.0.13.2 (Gordon & Hannon, 2010), joining of the forward and reverse reads using FLASH, version 1.2.10 (Magoč & Salzberg, 2011), and quality filtering with sickle tool, version 1.33 (Joshi & Fass, 2011). Filtering of the raw sequence reads was performed based on the following criteria: (1) the start region of both reads exactly matched the primer of the V4 region; (2) the minimum length was 40 bp, after the trimming of the primer region and the low-quality sequence; and (3) both reads could be joined, and the length after joining was 246-260 bp (amplicon sequence length was 285-299 bp). The 97% identity OTU clustering and chimera filtering were performed using UCHIME (USEARCH package v8.0.1623) (Edgar et al., 2011) in QIIME, version 1.9.0 (Caporaso et al., 2010). These data were then used to assign taxonomy against the Greengenes 13_8 database (DeSantis et al., 2006) with a 97% similarity threshold using the UCLUST v1.2.22q (Edgar, 2010) in the assign taxonomy script of QIIME. Details of commands and parameters are summarized in Table S1.

Isolation of bacteria
In order to examine whether previously uncultivated microorganisms could be acquired from the bark of Acer palmatum, cultivation was performed using a general low-nutrient agar plate medium. Bark-suspension supernatants (100 ml) were 10-fold serially diluted (10-10 3 fold) and were inoculated into Reasoner's 2A (R2A; Wako Pure Chemical Industries, Ltd., Osaka, Japan) culture medium that had been 10-fold diluted (DR2A) and PE03 medium (Tamaki et al., 2005), and incubated at 25 C for 2 weeks under dark conditions. The strains isolated from PE03 medium are represented as Strain No. IAP and the strains isolated from DR2A medium are represented as Strain No. IAD as shown in Tables 1 and 2, respectively. In order to selectively isolate slow-growing microbes, small colonies that were visible but less than one mm in diameter were targeted. For each medium, 48 colonies were isolated based on colony color and shape. Isolated colonies were suspended in 20 ml of Tris-EDTA (TE) buffer (Sigma-Aldrich Co. LCC, St. Louis, MO, USA) for DNA extraction, in addition to preparing one ml of glycerol stock (five mM Mops, 10% (v/v) Glycerol, pH 7.0) of each isolate. The DNA samples were stored at −20 C, and the glycerol stocks at −80 C.

Identification of isolates
The bacterial cells suspended in TE buffer for DNA extraction were thawed, added to 20 ml of phenol:chloroform:isoamyl alcohol (25:24:1; Wako, Monza, Lombardy), and mixed by vortexing for 30 s to lyse the bacterial cells. The lysed cells were clarified by centrifugation at 15,000 rpm for 5 min, and one ml of supernatant was used as template for PCR.
Template DNA was amplified with an iCycler (Bio-Rad Laboratories, Inc., Hercules, CA, USA) using 0.25 ml of TaKaRa Ex Taq (five U/ml), five ml of 10× Ex Taq Buffer, four ml of dNTP mix (Takara, Kusatsu, Japan), and 50 pmol of primers 8F (Weisburg et al., 1991; 5′-AGA GTT TGA TCM TGG CTC AG-3′) and 1492R (Lane, 1991; 5′-TAC GGY TAC CTT GTT ACG ACT T-3′) in a 50-ml reaction. The thermal cycling profile was one cycle at 94 C for 20 s, 30 sequential cycles of 94 C for 20 s, 55 C for 30 s and 72 C for 1 min, followed by a final extension at 72 C for 7 min and a hold at 4 C. The PCR products were purified using a QIAquick PCR Purification Kit (QIAGEN, Hilden, Germany). The concentration of the purified DNA was determined using a V-730BIO Spectrophotometer (JASCO Corporation, Tokyo, Japan). A 100-ng aliquot of PCR-amplified DNA and 7.5 pmol of 8F primer were mixed in a total volume of 15 ml and analyzed by Sanger sequencing by Takara Bio Inc. (Kusatsu, Japan). In general, the sequencing was performed with a BigDye Terminator v3.1 Cycle Sequencing Kit (Takara, Kusatsu, Japan) and an Applied Biosystems 3730xl DNA Analyzer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The sequence reads obtained were compared with those in the NCBI database of rRNA type strains/prokaryotic 16S ribosomal RNA (database of bacterial and archaeal type strains, except environmental clones, hereafter referred to as type strains) using the BLAST program. Taxonomic classification at the genus level was performed using RDP Classifier as previously described (Wang et al., 2007).

Nucleotide accession number
Culture-independent MiSeq sequence reads of the 16S rDNA have been deposited in the DDBJ sequence read archive (DRA) under accession numbers DRA006430 and DRA008228. Sequence reads of 16S rDNA from the bacterial isolates have been deposited in the DDBJ nucleotide sequences databank under accession numbers LC361357-LC361426.

RESULTS
By analyzing the microbial community structure of Acer palmatum bark using MiSeq, a total of 97,288 reads were detected. In total, 4,560 OTUs were defined with 97% sequence similarity. The phylogenetic distribution of the defined OTUs at the phylum level is shown in Fig. 1A. Sequence reads belonging to 27 bacterial phyla were detected from the Acer palmatum bark. Proteobacteria, at 34.9%, was the most abundant bacterial phylum, followed by 26.2% for Bacteroidetes, and 9.2% for Acidobacteria. In addition, Gemmatimonadetes (5.3%), Verrucomicrobia (4.0%), and Armatimonadetes (1.1%) were also detected, of which many bacteria were uncultured. Candidate division FBP (0.7%) was also detected at a relatively low abundance. In order to confirm whether the above phyla could be universally detected from Acer palmatum bark, three samples were collected from two individual trees (one of them being the previously analyzed tree), and re-analysis of the 16S amplicon sequencing was performed ( Fig. 2; Table S3). A total of 51,109-77,943 reads were detected, and 673-1,794 OTUs were defined with 97% sequence similarity. These phyla (Acidobacteria: 6.7-33.1%, Gemmatimonadetes: 0.1-1.3%, Verrucomicrobia: 1.9-9.1%, Armatimonadetes: 0.4-1.4%) and candidate division FBP (0.04-0.7%), although varying in abundance, were also detected in the re-analysis (Fig. 2). Consistently, Proteobacteria was the most abundant phylum in all samples, although the ranks in the lower abundances were quite variable. Thus, both candidate divisions and rarely cultivated groups were found in the Acer palmatum bark. The top 30 genera, found in the Acer palmatum bark based on MiSeq analysis, are shown in Table S3. Sphingomonas, Actinomycetospora, unidentified genus in Chitinophagaceae, unidentified genus in Sphingomonadaceae, unidentified genus in Methylocystaceae, and unidentified genus in Acetobacteraceae were commonly detected in the top 30 genera in all seven samples. It was suggested that these genera universally inhabit Acer palmatum bark. Furthermore, some genera belonging to phyla Acidobacteria, Verrucomicrobia, Gemmatimonadetes, and Armatimonadetes were among the top 30 at the genus-level in terms of relative abundance. These results revealed that many uncultured bacteria inhabited the Acer palmatum bark.
To determine whether these microorganisms could be cultured and isolated, we incubated Acer palmatum bark for 2 weeks in DR2A or PE03 agar medium. A large number of colonies were obtained on both media. Among the 96 isolated strains (48 strains isolated from each medium), there were nine strains from the PE03 medium and 17 strains from the DR2A medium that could not be sub-cultured. The remaining 39 strains from the PE03 medium and 31 strains from the DR2A medium could be sub-cultured and were subjected to sequence analysis. The phylogenetic distribution of the isolated strains at the phylum level is shown in Fig. 1B. Of the isolated strains, Actinobacteria was the most frequent with 29 strains (41.4%), followed by Proteobacteria with 27 strains (38.6%), and Bacteroidetes with 11 strains (15.7%). Compared with the results of MiSeq analysis, the results from the isolation analysis differed in the relative abundance at the phylum level. Compared with the top 30 genera detected by MiSeq analysis from the seven samples (Table S3), the genera that could be cultured and isolated had three genera in common (Sphingomonas, an unidentified genus in Sphingomonadaceae, and Actinomycetospora). Genus-level relative abundance was low for most of the cultured and isolated genera detected in the bark. For example, the relative abundances of Novosphingobium (0-0.03%) and Nocardioides (0.001-0.21%) in the Acer palmatum bark were very low. The results of culture-independent analysis do not always accurately reflect the actual microbial community structure in the bark due to variation among taxa in DNA extraction efficiency, 16S copy number variation and bias of universal primers. However, this suggested that whether microorganisms in the bark could be cultured or not was not predictable based on the relative abundance in the bark. The classification at the genus level of all isolated strains obtained on the two types of medium is shown in Table 3. Sphingomonas was the most frequently isolated genus in this study with nine strains, followed by Microbacterium with five strains, Spirosoma with four strains, an unclassified genus in Cellulomonadaceae with four strains, and an unclassified genus in Nocardioidaceae with four strains. In addition, a bacterial strain belonging to Acidobacteria, which is difficult to culture and has very few isolated strains (Eichorst, Kuske & Schmidt, 2011;Navarrete et al., 2013;Tanaka et al., 2017), was isolated on PE03 medium, and based on classifier and BLAST analysis was presumed to belong to the genus Terriglobus. According to Tamaki et al. (2009), the isolates were phylogenetically divided into two groups on the basis of their partial 16S rDNA sequence similarities to the reference sequences in the public databases: (i) ≤97% similarity to type strains: isolates with high phylogenetic novelty, and (ii) >97% similarity: isolates with low phylogenetic novelty. These criteria were used as objective indicators of the phylogenetic novelty of isolates, although they do not necessarily indicate taxonomic novelty at the genus or species level (Tamaki et al., 2009). The culture collection obtained from this study included 44 strains (62.8% of the total) that showed ≤97% 16S rDNA sequence similarity with type strains (Tables 1 and 2). In particular, a potentially novel microorganism, strain IAD-21, was isolated on DR2A medium ( Table 2). The full-length 16S rDNA sequence for IAD-21 was determined, and its sequence similarity with type strains was confirmed. Strain IAD-21 showed the highest similarity of 79% with Egibacter rhizosphaerae strain 80759 (Accession number KR605111) ( Table 2). We conducted a BLAST search including environmental clones and found high sequence similarity with clones belonging to candidate division FBP, including clone UMAB-cl-090 obtained from the Antarctic soil (sequence similarity 95.2%; accession number FR749715), clone ncd242h05c1 obtained from human volar forearm skin (sequence similarity 97.2%; accession number HM269099), and clone ncd1960f07c1 obtained from human antecubital fossa skin (sequence similarity 96.9%; accession number JF171142) (Lee et al., 2013). Since the full length of the 16S rDNA sequence from strain IAD-21 showed the highest sequence similarity with a bacterial strain belonging to Actinobacteria (E. rhizosphaerae strain 80759), we obtained 16S rDNA sequences from Actinobacteria, and from Armatimonadetes and Chloroflexi, which are Figure 2 Bacterial phyla detected from seven samples collected from two A. palmatum trees. Relative abundances of bacterial phyla detected from seven samples collected from two A. palmatum trees. Results from the analysis of 16S rDNA sequences detected by culture-independent evaluation of bark using next generation sequencing with a MiSeq system. A and B refer to the tree number, and 1-3 refer to biological replicates within a single tree. Sample A (Nov. 2015) is identical to that in Fig. 1A.
Full-size  DOI: 10.7717/peerj.7876/ fig-2 considered to be phylogenetically close to Actinobacteria. Sequences of the top 100 hits from the BLAST search of strain IAD-21, and sequences used for generating the phylogenetic tree of candidate division FBP and WS1 from Lee et al. (2013), were subjected to molecular phylogenetic analysis (Table S2). As a result, the cluster containing strain IAD-21 was determined to be phylogenetically separate from the known bacterial taxa, and it was suggested that strain IAD-21 belonged to the same cluster as sequences from the candidate division FBP (Fig. 3).

DISCUSSION
Although many studies are being carried out on the microbial community structure associated with the tree phyllosphere or rhizosphere, there are few reports that focus on the microbial community structure existing in the bark. In this study, we performed culture-independent analysis using MiSeq and isolation experiments to investigate the microbial community structure existing in Acer palmatum bark. As a result of cultivation and isolation experiments, Microbacterium, an unclassified genus in Cellulomonadaceae, an unclassified genus in Microbacteriaceae, an unclassified genus in Nocardioidaceae (all belonging to Actinobacteria), and Sphingomonas were frequently isolated in this study. Microorganisms from numerous closely related genera have also been detected in culture-dependent and -independent analyses of other barks and branches including elm, poplar, grapevine, Acer negundo, Acer pseudoplatanus, and G. biloba (Mocali et al., 2003;Ulrich, Ulrich & Ewald, 2007;Martins et al., 2013;Shen & Fulthorpe, 2015;Leff et al., 2015;Aschenbrenner et al., 2017), and are considered to be the natural inhabitants of bark. To the best of our knowledge, there are no prior reports of Spirosoma being cultured and isolated from bark. Since the chemical constituents of the bark differ depending on the tree species (Feng et al., 2013), it is considered that the bark of different tree species would have different microbial communities. In the future, more detailed analysis of microbial community structure in the bark will be required with respect to changes in the community depending on the tree species and localization of microorganisms in bark organs, using both culture-dependent and -independent analyses. Through the current culture-dependent analysis, a wide range of microbial species was identified.
By culture-independent analysis of Acer palmatum bark, members of rarely cultivated phyla such as Acidobacteria, Armatimonadetes, Verrucomicrobia, and Gemmatimonadetes were detected. This is consistent with other culture-independent analyses of the microbial community structures of bark samples. For instance, Acidobacteria and Verrucomicrobia were detected in samples from Acer pseudoplatanus bark at frequencies of 10.7% and 4.0%, respectively (Aschenbrenner et al., 2017), and Acidobacteria and Armatimonadetes were detected from G. biloba bark samples at 13.1% and 1.0%, respectively (Leff et al., 2015). Since these exist universally in soil environments (Bergmann et al., 2011;DeBruyn et al., 2011;Lee, Dunfield & Stott, 2014;Kielak et al., 2016), it is believed that they are spread by means such as the wind or insects and colonize the bark. Although Acidobacteria are believed to be as environmentally widespread as Proteobacteria (Barns, Takala & Kuske, 1999), many of them are slow growing and oligotrophic bacteria that are largely comprised of uncultured taxa (Da Rocha, Van Overbeek & Van Elsas, 2009;Ward et al., 2009). Leff et al. (2015) suggested that old bark environments provide more suitable locales for stable inhabitation over long periods of time for slow-growing and oligotrophic bacteria such as Acidobacteria than do the leaf or branch environments. In addition, there is little disturbance from UV radiation or precipitation in the old bark environment. They cited this limited disturbance in the old bark as a factor for the richness of the microbial community in G. biloba bark and for the detection of rarely cultivated phyla. In a few reports from analyses of the microbial community structure of bark, it was stated that comparison of results from previous work was difficult due to the scarcity of available data (Leff et al., 2015). In reviewing the results from this study, we agree with this impression. While comparisons may be difficult, it is still possible to speculate. In addition, in the outer bark consisting of dead cells, light irradiation and symbiosis with lichens and cyanobacteria may occur, and in the inner bark consisting of living cells, flow of photosynthates may affect the symbiotic microbial community (Baldrian, 2017). It is possible that the long-term existence of these factors in a stable environment may promote the growth of a microorganism on the bark. Martins et al. (2013) also reported the high diversity of acquired microorganisms in the bark as compared with that in organs such as leaves and fruits of grapevine. Recently in Figure 3 Phylogenetic tree of strain IAD-21. Phylogenetic tree of strain IAD-21 and related sequences of candidate division FBP based on 16S rDNA. In part, the full-length reads of 16S rDNA sequences were compared to sequences in international nucleotide sequence databases including the DNA Data Bank of Japan, the European Nucleotide Archive, and GenBank (DDBJ/ENA/GenBank). The phylogenetic tree was constructed using the neighbor-joining method and the Kimura 2-parameter model for estimating nucleotide substitutions. Bootstrap values were determined from 1,000 re-samplings. The newly identified and unique strain IAD-21 is located within the candidate division FBP cluster. The scale is given below the phylogenetic tree.
Full-size  DOI: 10.7717/peerj.7876/ fig-3 the bark tissue, microorganisms belonging to Acidobacteria (Yamada et al., 2014) and Armatimonadetes (Li, Kudo & Tonouchi, 2018) that are difficult-to-cultivate and slow-growing taxa were isolated. It can be inferred that the bark environment is a convenient residence for such bacterial taxa. Moreover, the fact that the bark harbors diverse microbial communities may have some meaning for trees. According to Khorsandy et al. (2016), the frequency of fungal endophytes in the bark of Platanus orientalis L. was significantly greater in older trees (60.04%) than in younger ones (39.96%). Existence of such fungal endophytes was positively correlated with the iron and potassium concentrations of the leaves, tree height, circumference, and improved visual appearance. These results suggested that fungal endophytes enhanced nutrient assimilation in trees, at least partly contributing to increased survival of the older trees (Khorsandy et al., 2016). Thus, there is no denying that old bark may benefit by harboring diverse microbial communities. However, since Khorsandy et al. (2016) reported fungal endophytes, while Leff et al. (2015) and Martins et al. (2013) referred to bacterial epiphytes, it is necessary to gain an understanding of the microbial community structure (both fungal and bacterial) of each organ of the bark area. It has been reported that members of Acidobacteria contribute to increases in biomass, rhizosphere morphology changes, production of indole-3-acetic acid, and iron absorption in Arabidopsis thaliana (Kielak, Cipriano & Kuramae, 2016). Further isolation and cultivation of microbes from phyla like Acidobacteria, which contain a considerable number of uncultured microbes, will lead to a better understanding of the tree-microbiota symbiotic system. In this study, we successfully isolated a novel microorganism, strain IAD-21. Based on molecular phylogenetic analyses, it was suggested that IAD-21 belongs to candidate division FBP. In addition to bark, candidate division FBP has been detected by culture-independent analysis of Antarctic soil (Tytgat et al., 2016), with two strains belonging to this division isolated from Antarctic soil (Tahon & Willems, 2017). These strains were successfully isolated by mimicking the Antarctic environment, using a low-nutrient medium for phototrophic bacteria, and adjusting the photoperiod over 10 weeks. However, in the current study, strain IAD-21 was relatively easy to culture, as we succeeded in its isolation by simply using a general low-nutrient medium during a 2-week cultivation period. In addition, isolated strains from Acer palmatum bark were relatively novel, even if they belonged to taxa with high cultivation frequencies.
The results of subsequent experiments exhibited the high phylogenetic novelty of isolates from Acer palmatum bark (Table S4). Furthermore, it is necessary to identify the reasons for obtaining high phylogenetic novelty of isolated strains and why strain IAD-21 could be cultivated with ease. One probable cause for the easy cultivation of strain IAD-21 could be its ability to grow in the relatively stable, less disturbed and unexplored environment of the bark. It is desirable that the culture efficiency be evaluated by the performance of comprehensive cultivation and isolation of microorganisms from the bark, and that the relationship between the poorly cultivated microorganisms and the tree bark be clarified.

CONCLUSIONS
Based on our study, we propose that Acer palmatum bark might prove to be a promising source of novel microorganisms. Since the culture conditions used in this study were relatively non-exceptional and only involved using low-nutrient media, it is possible that additional microorganisms could be acquired utilizing special culture methods and conditions (e.g., modification of the culture substrate, gelling agents, and medium composition). Analysis of the microbial community structure of various tree species and the isolation of uncultured microorganisms may lead to a more comprehensive understanding of the yet uncharacterized tree-microbiota symbiotic system. Microorganisms from bark samples may also be important from an academic point of view to understand microbial ecology, and further research is expected to clarify the unknown sectors of the microbial phylogenetic tree.


Determination of Substrate Specificity of the Purified Novel Plant Cysteine Protease Solanain From the Latex ofVallaris solanacea

Peptide synthesis refers to the production of peptides. Proteases are the enzymes that degrade proteins. They hold first place in the world market of enzymes, estimated at US$3 billion. Proteases are distributed widely in different parts of the biological sources. In occurrence of proteases, plant kingdom occupies the highest rank. The increasing demand for proteases and the need for economical production of commercially useful industrial proteases from novel sources are taken into consideration. A novel plant latex cysteine protease namely Vallaris solanacea was identified in Biodiversity Park, Visakhapatnam which showed maximum protease activity. It has been shown that the latex of Vallaris solanacea contains a high protease activity. The cysteine protease solanain was purified by fractionation with ammonium sulphate followed by DEAE-cellulose ion exchange and gel chromatography. Specificity studies towards synthetic peptide and ester substrates by the protease purified from the latex of Vallaris solanacea were performed. The purified solanain exhibited broad specificity similar to other cysteine proteases. However considerable differences were also noticed in the rate of hydrolysis and specificity towards simple peptide substrates.

Peptide synthesis
Peptide synthesis refers to the production of peptides. Peptides and proteins are linear polymers of amino acids linked by amide peptide bonds. Amino acids are primary amines that contain an anomeric carbon that is connected to a hydrogen atom (H), an amino (NH 3 ) group, a carboxyl group (COOH) and a variable side group (R) (Figure 1).
Peptides are chemically synthesized by the condensation reaction of the carboxyl group of one amino acid to the amino group of another. The formation of peptide bonds by linking amino acids together dates back to 100 years. The first peptides to be synthesized, including oxytocin and insulin took about 50-60 years which clearly indicates chemical synthesis of peptides is a difficult task [1]. But advances in protein synthesis today made peptide synthesis easier today which has varied applications in high-throughput biological research and drug development [2] (Figure 2).

Peptide synthesis
They are two methods of peptide synthesis, namely • Liquid-phase peptide synthesis • Solid-phase peptide synthesis Liquid-phase peptide synthesis is the classical method that is slow and labour intensive in which separate peptides are synthesized and then coupled together to create larger peptides. In this method chemical group is used to protect the Cterminus of the first amino acid.
Solid-phase peptide synthesis is the most common method where the Cterminus of the first amino acid is coupled to an activated solid support, such as polystyrene or polyacrylamide. This method is advantageous as the resin acts as the  C-terminal protecting group and it is a rapid method to separate the growing peptide product from the different reaction mixtures during synthesis.
These two ways are combined in an exceedingly method known as native chemical tying. LifeTein's customary amide synthesis method uses the solid part. The liquid-phase approach is employed for the synthesis of short peptides, like diand tripeptides, and C-terminally changed peptides, like accelerator substrates ( Figure 3).

Solid phase synthesis
The important method for the synthesis of peptides in the lab is known as solidphase peptide synthesis (SPPS) [3]. Merrifield [4] developed this method of SPPS which allows the rapid assembly of a peptide chain through successive reactions of amino acid derivatives on an insoluble porous support. Unlike ribosome protein synthesis, artificial synthesis builds peptides in the C to N direction. During solidphase peptide synthesis, each peptide is anchored to an insoluble polymer at the Cterminus.

Solid support
Solid support in SPPS is Polystyrene, a styrene cross-linked with 1-2% divinylbenzene which is a popular carrier resin in SPPS. Other common gel-type supports include polyacrylamide and polyethylene glycol (PEG). Polystyrene is chemically inert under SPSS conditions. The solid support helps in anchoring the amino acids and formation of peptide.

Protecting groups
Amino acids contain side chains with different functional groups with different reactivity. Thus different protecting groups are required. A major problem in peptide synthesis is the side reactions due to multiple reactive groups in amino acids. In order to perform peptide formation with minimal side reactions or to protect the functional groups from non-specific reactions reactive groups in the amino acids need to be blocked or protected. For this many chemical groups have been developed that bind to the amino acids and protect it. They are two types of protecting groups, namely • Temporary protecting groups • Permanent protecting groups

Temporary protecting groups
N-terminal ends of amino acids are protected by groups called 'temporary' protecting groups as they are easily removed to allow peptide bond formation.

Permanent protecting groups
But Side chain protecting groups are known as permanent protecting groups as they remain at all the multiple cycles of chemical treatment during the synthesis phase and they can be removed only on treatment with strong acids after synthesis is complete (Figure 4).
Peptide synthesis involves coupling of carboxyl group and the incoming amino acid to the N-terminus of the growing peptide chain. It is a step wise method and also a cyclic process. It involves the following steps.
1. Blocking of amino group of amino acid by N-terminal protecting groups Prior to protein synthesis, individual amino acids are reacted with these protecting groups. Two common N-terminal protecting groups are tertbutoxycarbonyl (Boc) and 9-fluorenylmethoxycarbonyl (Fmoc), and each group has distinct characteristics that determine their use (Table 1; Figure 5).

Blocking of carbonyl group of amino acid by C-terminal protecting groups
Similarly the carbonyl group of amino acid is protected by C-terminal protecting group. The C-terminal protecting group depends on the type of peptide synthesis used. For liquid-phase peptide synthesis C-terminus of the first amino acid is protected by chemical reagent while in solid-phase peptide synthesis solid support (resin) acts as the protecting group for the C-terminal amino acid ( Figure 6).

Coupling of the protected amino acids to form a peptide
Once peptide synthesis is completed, all the protecting groups are removed from the nascent peptides. First C-terminal carboxylic acid is activated using carbodiimides like dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC). These coupling reagents respond with the carboxyl gathering to shape an exceptionally receptive O-acylisourea moderate that is immediately dislodged by nucleophilic assault from the deprotected essential amino gathering on the   N-end of the developing peptide chain to frame the beginning peptide security.
To affirm total coupling a test is performed called Kaiser Test (Figure 7).

Deblocking the amino group of amino acid
Removal of specific protecting groups from the newly added amino acid immediately after coupling to allow the next incoming amino acid to bind to the growing peptide chain is called deprotection. Boc is removed using moderately strong acid such as trifluoroacetic acid (TFA) while Fmoc is a base-labile protecting group that is removed with a mild base such as piperidine.

Deblocking the carbonyl group of amino acid
The synthetic peptide purification is by compounds like water, anisol or thiol derivatives are added in excess during the deprotection step to react with any of  these free reactive species. This cycle of deprotection and coupling is repeated. The remaining protecting groups are cleaved by acidolysis, using; strong acids such as hydrogen fluoride (HF), hydrogen bromide (HBr) or trifluoromethane sulphonic acid (TFMSA) (Figure 8).

Peptide purification
Peptides are purified by the following techniques namely Ion exchange chromatography (IEC), Partition chromatography, Size-exclusion chromatography, Reverse-phase chromatography (RPC) and High-performance liquid chromatography (HPLC).

Applications of synthetic peptides
The Peptide synthesis ( Figure 9) holds varied applications including • Synthetic peptides are used to study enzyme-substrate interactions within important enzyme classes such as kinases and proteases, which play a crucial role in cell signalling.
• The development of epitope-specific antibodies against pathogenic proteins.
• The study of protein functions and the identification and characterization of proteins.

Proteases
For about a century, the enzymes that play the central role in the degradation of proteins by hydrolysing peptide bonds have been known as 'proteases' and the term protease is therefore equivalent to 'peptide hydrolase'. They hold first place in the world market of enzymes, estimated at $US$3 billion [5]. Proteases are distributed widely in different parts of the biological sources. In occurrence of proteases, plant kingdom occupies the highest rank (43.85%) followed by bacteria (18.09%), fungi (15.08%), animals (11.15%), algae (7.42%) and viruses (     Barrett and McDonald [6], plant proteases are classified into serine, cysteine, aspartic and metalloproteases. Cysteine proteases (EC 3.4.2.2) are found in bacteria [7], eukaryotic microorganisms [8], plants [9] and animals. Cysteine proteases are represented by 70 families belonging to 12 different classes [10] ( Table 2; Figures 10-13).

Screening of plant lattices for novel plant latex cysteine protease
Taking into consideration, the increasing demand for proteases and the need for economical production of commercially useful industrial proteases from novel sources. A number of plant lattices belonging to different plant families have been collected in and around Visakhapatnam and screened for protease activity. Their activities were assayed immediately and were stored in ice for further investigations. A novel plant latex cysteine protease namely Vallaris solanacea was identified in Biodiversity Park, Visakhapatnam which showed maximum protease activity. Preliminary studies on protease activity from the latex of Vallaris solanacea were carried out. The protease was purified and characterized. Specificity studies towards synthetic peptide and ester substrates by the protease purified solanain.

Substrate specificity of the purified solanain from the latex of Vallaris solanacea
Plant lattices are rich source of proteases. Latex of Vallaris solanacea also showed high protease activity. The cysteine protease solanain was purified by ammonium sulphate precipitation followed by DEAE-cellulose ion exchange and gel chromatography. The purpose of the present study is determination of specificity of purified solanain towards synthetic peptide and ester substrates. Extensive and more systematic studies [13][14][15][16] have been made on papain, ficin [17] and bromelain [18].

Preparation of peptide solutions
First step is preparation of 10 mM peptide solution having tryptophan, tyrosine and phenylalanine. Peptides were solubilized using dilute acid, the assay mixture contained higher buffer concentration viz. 0.15 M.

Solanain action on peptides
Peptide was incubated by solanain in 0.05 M Phosphate buffer (pH 7.0), 10 mm β-mercaptoethanol, 2 mM EDTA, 2.5 mm amide and 0.5 mg to 1.0 mg/ml catalyst protein. Incubation was administered for 48 h at 30°C. The reaction was detected by sampling the digestible mixture on aid. For every digestion mixture, a bearing while not substrate or without catalyst was analysed. Skinny layer activity was performed as delineated by Renderath (1963).

Preparation of colloid silica gel G plates
About 25 weight unit of colloid Silica Gel G was mixed with 50 cm 3 of H 2 O and stirred smartly during a closed round shape flask till completely distributed. The suspension was transferred into Stahl's mechanical spreader adjusted to 250 μ thickness, victimization the spreader; layers were ready on 20 Â 20 cm glass plates. The gel was allowed to dry for a couple of minutes and so activated by drying in associate degree kitchen appliance at 1100°C for half-hour.

Developing the chromatographic plate
About 5-10 μl of the sample was loaded. Solvent used was 4:1:1 butanol: carboxylic acid: water (v/v/v). After development the plates were removed, dried and detected by spraying with 0.2% ninhydrin in butanol: ethanoic acid (95, 5 v/v) mixture. Rf values of the spots were calculated. Amino acids were identified.

Results and discussion
Studies on substrate specificity were done and results were tabulated. Solanain was capable of hydrolysing peptide bonds involving the amino groups of hydrophilic amino acid residues (peptides 1 to 5) and incapable of peptide bonds involving the groups of deliquescent organic compound residues (peptides one to 5) and incapable of hydrolysing amide bonds wherever amino group was given by a
Reaction of di-and tripeptides by refined Solanain.
hydrophobic residues (peptides half dozen to 9) with a large aspect chain e.g., leucine, essential amino acid, essential amino acid and essential amino acid. However, if the organic compound contributory the group of the bond was aromatic the bond was not hydrolysed although the C-terminal amino acid was deliquescent. Neither L-Trp-Gly nor L-Tyr-Gly was hydrolysed. It seems that the presence of a deliquescent organic compound at the N-terminal finish is not needed for the protein.
In Table 3, the results of the speed measurements given for 10 parts as V o /E o wherever V o is initial rate and E o is concentration of the protein in moles. The concentration of substrate used was one.56 Â 10-4 M. If the metric linear unit values of the p-nitrophenyl esters of CBZ-amino acids were of constant magnitude as for different thiol proteases like papain [9], ficin [17], bromelain [18] and ananain [12] then V o /E o values obtained for Glycine and amino acid were love the Kcat values of the corresponding esters of different proteases.
The refined solanain of Vallaris family Solanaceae showed close to identical specificity towards all the substrates.
Solanain, differs from the amino alkanoic acid proteases in having low amidase activity with BAPNA and conjointly did not show any esterase activity with BAEE. This finding suggests considerably low specificity for essential amino acid residues. It differs from enzyme, ficin and bromelain during this facet. By showing broad specificity, Solanain resembled enzyme [11], ficin [14,17] and bromelain [18] that change a spread of amide bonds. All these, however, showed a preference for basic amino acids. The solanain of Vallaris potato family hydrolysed leucyl bonds with efficiency and during this respect resembled ficin [17] and differed from bromelain [18] (Table 4).

Conclusion
Substrate specificity studies showed solanain exhibited broad specificity. It showed peptidase activity, amidase activity. The enzyme was capable of catalysing the hydrolysis of p-nitrophenyl esters of amino acids. It exhibited difference in specificity towards simple peptide substrates.  Table 4.
Hydrolysis of various synthetic ester substrates with purified solanain.