extfieldsofstudy: Medicine Biology

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Anti-mitochondrial autoantibodies in systemic lupus erythematosus and their association with disease manifestations

Mitochondria are organelles that govern energy supply and control cell death. Mitochondria also express bacterial features, such as the presence of inner membrane cardiolipin and a circular genome rich in hypomethylated CpG motifs. While mitochondrial extrusion by damaged organs or activated cells is thought to trigger innate immunity, it is unclear whether extracellular mitochondria also stimulate an adaptive immune response. We describe the development of novel assays to detect autoantibodies specific to two distinct components of the mitochondrion: the mitochondrial outer membrane and mitochondrial DNA. Antibodies to these two mitochondrial constituents were increased in both human and murine systemic lupus erythematosus (SLE), compared to controls, and were present at higher levels than in patients with antiphospholipid syndrome or primary biliary cirrhosis. In both bi- and multi-variate regression models, antibodies to mitochondrial DNA, but not whole mitochondria, were associated with increased anti-dsDNA antibodies and lupus nephritis. This study describes new and optimized methods for the assessment of anti-mitochondrial antibodies, and demonstrates their presence in both human and murine SLE. These findings suggest that different mitochondrial components are immunogenic in SLE, and support the concept that extracellular mitochondria may provide an important source of circulating autoantigens in SLE.

extracellular mitochondria. Mitochondria and their components (e.g. N-formylated peptides and mtDNA) are recognized as damage-associated molecular patterns (DAMPs), which activate the innate immune system and elicit an inflammatory response 6,[21][22][23] . Moreover, ATP and reactive oxygen species (ROS), produced by mitochondria are triggers of the nuclear oligomerization domain (NOD)-like receptors and contribute to inflammasome activation 21,22,24 . Extracellular mitochondria have been described in various clinical conditions, including trauma 25,26 , burn injury 27 , cancer 28 , rheumatoid arthritis 17,29 , systemic lupus erythematosus (SLE) 15,16 and transfusion adverse reactions 17,18,30,31 . Their pro-inflammatory potential is generally thought to occur through activation of Toll-like receptors (TLR), formyl peptide receptors, and cytosolic pathogen recognition receptors, all key components of the innate immune system 6,[21][22][23][24] . The adaptive immune system can also recognize mitochondria. This concept is important, as the immune response initiated by mitochondrial DAMPs may be different if the adaptive immune system is also implicated 32,33 . Different sets of anti-mitochondrial antibodies (AMA), namely AMA-M1 to -M9, have been characterized 34 (recapitulated in Table 1). The AMA-M2, -M4, -M8, and -M9 classes are well-described in primary biliary cirrhosis (PBC) [35][36][37] , an autoimmune disease characterized by a progressive destruction of the bile ducts due to the infiltration of autoreactive T-cells 38 . These antibodies target distinct mitochondrial proteins notably implicated in oxidative phosphorylation, and their differential induction depends on disease severity or stage. Conversely, AMA-M6 autoantibodies have been described in iatrogenic hepatitis induced by iproniazid 39 , while the AMA-M7 class of antibodies targets mitochondrial epitopes, identified as sarcosine dehydrogenase and enzymes associated with flavin adenine dinucleotide, in patients with cardiomyopathy and myocarditis 40 .
SLE is an autoimmune disease characterized by the presence of circulating antigen-autoantibody immune complexes and inflammation in multiple organs and tissues. In SLE, neutrophils were shown to release mtDNA through the generation of ROS, a process leading to activation of the DNA sensor stimulator of interferon genes (STING) and type-I IFN production 15,16 . Studies showed that anti-mtDNA antibodies (AmtDNA) are induced in a subset of SLE patients, suggesting that extruded mtDNA could be a relevant source of antigen for the anti-double stranded DNA (anti-dsDNA) antibodies that prevail in SLE 16,41,42 . Clinically, anti-DNA antibodies are screened initially by indirect immunofluorescence using human epithelial type 2 cells (HEp-2) and enzyme immunoassay using double-stranded DNA 43 . Presence of anti-DNA antibodies can also be assessed by indirect immunofluorescence using Crithidia luciliae, an hemoflagellate parasite of blow-flies that possesses a uniquely large mitochondrion that contains a high concentration of DNA, the kinetoplast [44][45][46] . Another method commonly used is Farr radioimmunoassay, which involves the precipitation of antibody-bound radiolabeled DNA and its detection with a scintillation counter 47 .
Antibodies targeting mitochondrial components other than mtDNA are also found in SLE. They include anti-cardiolipin [AMA-M1, also known as anti-cardiolipin antibodies (ACA)], anti-60kDa heat shock protein (anti-HSP60), as well as AMA-M3 and AMA-M5 antibodies. AMA-M1 antibodies recognize cardiolipin and were originally identified in syphilis-infected patients. Anti-cardiolipin antibodies are also found in patients with SLE and antiphospholipid syndrome (APS), resulting in false positive results in earlier syphilis detection assays 34,48 .
Interestingly, cardiolipin is a phospholipid that is found uniquely in the inner mitochondrial membrane in eukaryotic cells. However, damaged mitochondria may externalize their cardiolipin 49 , that may become accessible and induce the development of antibodies to cardiolipin. Clinically, the presence of ACA is associated with a greater risk of thrombotic events and thrombocytopenia 50,51 .
Another mitochondrial antigen, HSP60, is a mitochondrial chaperonin implicated in mitochondrial protein import 52 . Patients with SLE have antibodies against HSP60 53 , and their presence (when concomitant with anti-phospholid antibodies) is associated with vascular events 54 . AMA-M3 were described in patients using a drug called venocuran who developed a drug-induced syndrome ("pseudolupus") with clinical manifestations of arthralgia, fever, serositis, and lymphopenia. The immunological profile of pseudolupus is characterized by the absence of antinuclear antibody (ANA), but the presence of AMA-M3 55 . The antigenic target of AMA-M3 is different than that of the other AMA classes described in PBC 55 . It is resistant to trypsin and it is extracted by solvent, pointing to its lipid nature. AMA-M3 are no longer encountered clinically since the withdrawal of the drug venocuran.
AMA-M5 comprise another class of anti-mitochondrial antibodies identified in patients with SLE, as well in APS, Sjögren syndrome, recurrent fetal loss, and hemolytic anemia 56 . The precise antigenic target(s) of AMA-M5 is undefined, but lack of competition by cardiolipin-containing liposomes suggests that it is distinct from the target of AMA-M1 57 . Indirect immunofluorescence on human or rodent tissues is used to identify anti-M5 antibodies 58 . While immunofluorescence and complement fixation test revealed that only 2% of SLE patients were positive for AMA-M5, approximately 40% were positive by enzyme-linked immunosorbent assay (ELISA) 59 . However, in the latter approach, mitochondria were only partially purified and were sonicated, thus revealing antigenic epitopes that might not be recognized in tissues 60 .
Emerging evidence supports the liberation of mitochondria by activated cells and their potential contribution to inflammation in SLE 15,16 . Identification of mitochondrial antigens recognized by autoantibodies in SLE may provide information on the roles of extracellular mitochondria in autoimmunity and systemic inflammation. Herein, we developed new methods to detect the presence of two distinct types of circulating AMA: anti-whole mitochondria antibodies (AwMA) and AmtDNA. We then determined their usefulness in a murine model of SLE, as well as in a cohort of SLE patients. We evaluated these AMA in parallel with antibodies to the mitochondrial chaperonin HSP60 (a known mitochondrial target in SLE), and determined the associations of these autoantibodies with disease characteristics. To our knowledge, this is the first study that examines the association of these AMA with disease manifestations in SLE.

Results
Different methodologies exist for the isolation of mitochondria, and several improvements have been introduced in recent years to enhance their purity and quality. We used a combination of previously published protocols to obtain highly purified mitochondria 61,62 . Mitochondria were isolated from mouse liver or a human hepatocyte cell line (Hep-G2) using a combination of previously published protocols 61,62 (Fig. 1a). The Percoll gradient included in the purification protocol eliminated contaminants from the endoplasmic reticulum and the proteasome ( Supplementary Fig. 1a) 62 . The purity of the mitochondrial preparations was high, based on the extremely low content of cytosolic and nuclear proteins, and the enrichment of three mitochondrial proteins [voltage-dependent anion channel (VDAC), cytochrome C, and translocase of the outer membrane 22 (TOMM22)] (Fig. 1b). The isolated mitochondria maintained their cytochrome C (Fig. 1b) and respiratory functions, suggesting that integrity was conserved during the isolation process (Fig. 1c).
Mitochondria were homogeneous in size, with a main peak detected at 820 nm by dynamic light scattering (Fig. 1d, left panel), and retained their canonical morphology when observed in electron microscopy (910 ± 210 nm) (Fig. 1d, right panel). High sensitivity flow cytometry, which permits the identification of submicron particles, was used as a quantitative approach. We estimated that 6.6 ± 1.9 × 10 6 mitochondria (Mitotracker + TOMM22 + ) (or 4.33 ± 1.17 µg mitochondrial proteins) could be isolated per mg of mouse liver (n = 6), which was sufficient to prepare approximately 400 wells in half-area 96-well microplates using one mouse liver (Fig. 1e). The yield obtained with Hep-G2 cells was lower, as 3.5 ± 0.18 µg of mitochondrial protein were isolated per 10 6 Hep-G2 cells (7.0 × ± 2.1 × 10 8 Hep-G2 cells were harvested for each 175 cm 2 flask at confluence), which can be used to prepare approximately two half-area 96-well microplates.
The quantity of purified mitochondrial protein (12.5 µg mitochondrial protein/well) required for coating half-area 96-well microplates ELISA plates was optimized using increasing concentrations of mitochondria ( Supplementary Fig. 1b). Wells were saturated with phosphate buffered saline (PBS) containing fetal calf serum (FCS) and gelatin, which proved optimal for blocking nonspecific binding of antibodies in comparison to non-fat dry milk or bovine serum albumin (data not shown). We used an inducible murine model of SLE to determine whether AwMA could be detected in serum 63 . This model is known to produce autoantibodies to nuclear and cellular antigens 63 , but the presence of AwMA has never been explored. We found high serum levels of AwMA in this induced murine lupus model, compared to healthy control mice (Fig. 2a).
Reactive oxygen species are generated under inflammatory conditions, and were reported during the release of mitochondria 15,16 . Thus, we assessed whether oxidation of mitochondria could impact mitochondrial recognition by AwMA. Isolated mitochondria were treated with increasing concentrations of the oxidant tert-butyl hydroperoxide (TBHP), and the oxidized protein and lipid contents were confirmed using commercial assays (Fig. 2b,c and Supplementary Fig. 2). We found that oxidation had no or very little impact on recognition of mitochondria by SLE antibodies (Fig. 2d) (Fold increase: 1.2 ± 0.2). The data suggest that mitochondria are immunogenic in SLE regardless of the oxidation status of their antigens.
We next used our quantitative AwMA ELISA to screen human sera. We included 175 SLE patients and 43 healthy controls (76% female, mean age 42 ± 12) ( Table 2). We also evaluated sera from APS patients (n = 12), given the high levels of anti-cardiolipin antibodies (AMA-M1) in APS, as well as sera from PBC patients (n = 12) confirmed positive for AMA by indirect immunofluorescence on mouse stomach/kidney slides (MSK).
Given the higher yield and purity of the mitochondria isolated from mouse liver, and the fact that mice are readily accessible in most research laboratories, we used intact murine mitochondria as coating antigens in our assay for the detection of autoantibodies targeting the outer mitochondrial membrane in humans. We found that AwMA were present in all healthy controls, but at much lower levels than those encountered in a large proportion of the SLE patients. SLE patients were more frequently positive and at higher levels for AwMA than healthy controls. APS and PBC patients also presented a significant increase in AwMA compared to healthy www.nature.com/scientificreports www.nature.com/scientificreports/ donors but signals detected for these patients were lower than those measured in SLE patients (Fig. 3a). As the ELISA is performed on intact mitochondria, these results suggest that AwMA are induced in SLE, and recognize autoantigens on the outer mitochondrial membrane that are distinct from the epitopes in APS (cardiolipin) and PBC (pyruvate dehydrogenase complex E2-component, PDC-E2), both located in the mitochondrial inner membrane. Consistent with this, we identified PBC patients positive for AMA when submitochondrial particles (i.e. sonicated mitochondria) were used as coating antigens, suggesting that certain antigens relevant to PBC may be exposed in these conditions ( Supplementary Fig. 3). Human and murine mitochondria were also compared in our assay using sera from a subset of SLE patients. Both sources of mitochondria (human Hep-G2 cells and mouse liver) were similarly recognized by human AwMA (Fig. 3b), suggesting that antigenic epitopes may be conserved across these species. Hence, mitochondria from murine, bovine and porcine tissues are routinely used to assess AwMA in humans, suggesting that interspecies differences play a negligible role, if any, in mitochondrial antigenicity.
Membrane-bound vesicles in the extracellular milieu, known as extracellular vesicles or microparticles, are proposed contributors to the antigenic load in SLE [64][65][66] . To determine whether AwMA could also recognize microparticles derived from membranes from cells or particles other than mitochondria, we utilized red blood cell microparticles ( RBC MP) as blood-borne microparticles devoid of mitochondria as a competitor in our AwMA-ELISA, and compared it to extracellular mitochondria in solution. Whereas increased concentrations of competing mitochondria decreased AwMA binding by up to 49.84 ± 15.01%, RBC MP showed no inhibition of binding of the SLE antibodies in our assay (Fig. 3c). While these results suggest that the antibodies detected in the AwMA-ELISA might have a preferred substrate originating in mitochondrial membrane, we cannot exclude the possibility that other membrane bound microparticles or even cells may also be recognized by AwMA, given the probable occurrence of numerous protein and non-protein antigens in mitochondria.
To determine whether mtDNA also represents an antigenic target of SLE autoantibodies, we isolated mtDNA from crude mitochondria preparations, using standard DNA extraction by alkaline lysis. Digestion of the mtDNA by Hae II, a restriction enzyme with a single restriction site on the murine mitochondrial genome ( Supplementary  Fig. 4a) yielded a single fragment of 16,569 base pairs, indicating the isolation of mtDNA with its circular conformation. As expected, digestion by Pst I generated two fragments of 12,751 and 3818 base pairs, further confirming the expected size of the isolated mtDNA. Moreover, we confirmed enrichment of mtDNA relative to genomic DNA ( Supplementary Fig. 4b). Up to 1.55 ± 0.35 µg mtDNA was obtained for each mg of mitochondrial protein used. Plate adhesion of different concentrations of mtDNA was enhanced by using plates pre-treated with protamine sulfate, and binding specificity was increased by blocking the plates with a PBS solution containing FCS and gelatin ( Supplementary Fig. 4c). Of interest, sera from mice with induced SLE were positive for AmtDNA, compared to control mice (Fig. 4a). Moreover, AmtDNA was significantly increased in SLE patients, but not in patients with APS or PBC, relative to healthy controls (Fig. 4b). www.nature.com/scientificreports www.nature.com/scientificreports/ Little is known about the association of AwMA and AmtDNA with the clinical characteristics of SLE. We assessed whether AwMA and AmtDNA were associated with disease manifestations in 175 SLE patients for whom detailed clinical information was available. AwMA levels correlated with AmtDNA levels in SLE patients (r s = 0.23, p = 0.003), but not in healthy controls (r s = 0.15, p = 0.33). In contrast, AwMA did not correlate with antibodies against other mitochondrial antigens (i.e. HSP60 and cardiolipin) and was not found to be associated with clinical outcomes (Tables 3 and 4). Interestingly, AmtDNA was associated with both increased anti-dsDNA antibodies (p = 0.02) and with a history of lupus nephritis (p = 0.007), but not with any of the other clinical outcomes (Table 4). When the duration of the disease, the age of the patients, their BMI, the use of prednisone and/ or antimalarial drugs, and circulating cholesterol LDL were taken into account, the associations of AmtDNA with increased anti-dsDNA antibodies and lupus nephritis remained significant in a multivariate logistic regression (p = 0.01 for both), indicating an association between AmtDNA, and these two clinical parameters. However, AmtDNA did not correlate with anti-dsDNA as measured by Farr assay in the cohort, suggesting that the results measured by our AmtDNA-ELISA and those obtained by Farr assay may not be redundant. Cut-off values were identified for AwMA and AmtDNA (Figs 3, 4 and Table 5). Our two ELISAs displayed high specificities (AwMA: 0.88; AmtDNA: 0.74) and allowed us to efficiently discriminate SLE patients from healthy donors (p < 0.001 for both AmtDNA and AwMA). Using these values, we determined that 110 patients were positive for AmtDNA (62.86%) and 101 patients (57.71%) were positive for AwMA.  Table 2. Demographics and clinical characteristics (ACR criteria) for SLE patients included in the study (n = 175). A >0.5 g per day of protein in urine or cellular cast or end-stage renal disease. B Hemolytic anemia (low red blood cell count) or leukopenia (White blood cells < 4000/µl), lymphopenia (<100 000/µl) in the absence of offending drug. C Positive anti-Smith, anti-dsDNA, antiphospholipid antibody and/or false positive serological test for syphilis. eGFR: estimated glomerular filtration test.
www.nature.com/scientificreports www.nature.com/scientificreports/ Thus, although more work is needed to explore the clinical associations of AmtDNA and AwMA in a larger lupus population and over time, different subsets of mitochondrial epitopes (i.e. mtDNA vs. outer membrane antigens) appear to measure different immune responses in SLE patients and may be associated with distinct disease characteristics.

Discussion
Mitochondrial DAMPs are known to stimulate the innate immune response, but it is less clear whether mitochondrial antigens stimulate an antibody response and whether these antibodies impact the inflammatory reaction. Interestingly, in PBC, a paradigm of true organ-specific autoimmunity, evidence points to involvement of both innate and adaptive immunity with a specific antibody response to mitochondrial antigens 67 .
Although the prevalence of AMA in SLE was reported several decades ago 59 , this observation was not pursued. Our study confirms the presence of AwMA (directed against the mitochondrial surface and not redundant with that of PBC) and AmtDNA (directed against mtDNA) in patients with SLE. However, it remains to be established whether AwMA are pathogenic initiators of the auto-immune process or whether they are consequences of an a priori cell activation or injury with subsequent release of mitochondria in the extracellular milieu. These "free mitochondria" could subsequently become antigenic in predisposed individuals and be a marker of cell or tissue injury. They could also be a cause of further immune activation through the formation of circulating or in-situ immune complexes, constituting a secondary trigger of inflammation. The recognition of mitochondria by antibodies could also implicate Fc receptors and thus modulate a distinct immune response. For example, the intravenous injection of mtDNA into mice failed to induce proteinuria and kidney damage 68 , but the response may differ in a recipient with circulating antibodies to mitochondria.
Epitope modification, such as oxidation, can impact its antigenicity 69 . Notably, there are reports suggesting that the oxidation of mtDNA occurs during its extrusion from cells, and that the oxidized form is pathogenic 15,16 . Our findings using in vitro oxidation of the mitochondria suggest that mitochondrial epitopes, regardless of oxidation status, are targeted by autoantibodies in SLE. However, this does not exclude the possibility that oxidized mitochondria may be more antigenic in other pathogenesis, such as APS, or more efficient at promoting responses if recognized by the innate immune system. Moreover, it is not excluded that following oxidation, mitochondrial swelling and potential formation of pores within the mitochondrial membrane might have revealed new antigens detected in our anti-whole mitochondria ELISA using TBHP-treated mitochondria, but this treatment may also have caused a release of existing antigens that could have been lost in this modified assay, thus explaining the apparent absence of impact of oxidation of the antigenicity of the mitochondria 70 . Future investigations will be needed to determine the relative targeting of native versus oxidized forms of organelle components by immune cells. We could not verify the role of oxidation on the recognition of mtDNA by autoantibodies due to spontaneous oxidation of isolated mtDNA, an observation also made by other groups despite extreme preventive measures  Table 5). Kruskal-Wallis test with multiple comparisons to healthy donors; Dunn's correction. (b) No significant differences were detected by direct ELISA when either murine (Mm, gray symbols) or human mitochondria (Hs, black symbols) were used as coating antigens to detect AwMAs in control and SLE patient sera (1:100). Two-way ANOVA. (c) AwMA binding to coating mitochondria is inhibited in presence of mitochondria (filled circles) but not by red blood cells microparticles (filled squares). Two-way repeated measures ANOVA with multiple comparison (Dunnett's correction) to signals detected without competitors (i.e. 100%). Experiments presented in panels 3 a and 3 c were performed using mouse mitochondria, the experiment presented in panel 3 b was performed in parallel on murine and human mitochondria. Data are mean ± SD. Not significant (ns): p > 0.05. *p < 0.05. ***p < 0.001. ****p < 0.0001. Hs: Homo sapiens; Mm: Mus musculus.
www.nature.com/scientificreports www.nature.com/scientificreports/ taken to maintain the molecules under their reduced form 71 . Thus, it is likely that AmtDNA and anti-dsDNA antibodies routinely measured in clinical testing both evaluate antibodies to oxidized DNA.
We observed that AwMA levels were more elevated in SLE than in PBC. PBC patients, however, were positive for AMA when sonicated mitochondria were utilized in our ELISA. These observations are consistent with the fact that pyruvate dehydrogenase complex E2 (PDC-E2), the immunodominant epitope in PBC, is located in the mitochondrial inner membrane. Our AwMA-ELISA measures antibodies to epitopes on intact mitochondria, in which inner membrane epitopes remain unavailable 72 . This contrasts with the sonicated mitochondria used in previous studies on AMA in SLE 59 , which contained inverted membranous structures and thus revealed antigens located in the mitochondrial inner membrane 60 . Furthermore, we found that AwMA levels correlated with the levels of AmtDNA, but not with other antibodies directed at antigens located within the inner membrane of the organelle (e.g. cardiolipin or HSP60), pointing to the existence of mitochondrial antigens that remain to be identified 73 . Our study provides simple and quantitative assays for assessment of two types of AMA (i.e. AwMA  Table 5). Kruskal-Wallis test with multiple comparisons to controls/healthy donors; Dunn's correction). All experiment presented in the figure were performed using mouse mtDNA. Data are mean ± SD. Not significant (ns): p > 0.05. ***p < 0.001. PBC: primary biliary cirrhosis. www.nature.com/scientificreports www.nature.com/scientificreports/ and AmtDNA) likely specific to SLE. Further research is required to identify the mitochondrial antigen(s) and epitope(s) of our AMA assay in SLE.
Our analyses of AmtDNA antibodies in human sera showed an association with anti-dsDNA as well as with lupus nephritis, consistent with the documented associations between anti-dsDNA antibodies detected by Farr assay and lupus nephritis 74 . While dsDNA used in Farr assays is usually isolated from plasmids (i.e. E. coli) and thus may share similarities with mtDNA (e.g. hypomethylated CpG motives, circular tertiary structure) 75 , the Farr assay has been described for its specificity in the detection of high avidity antibodies of all classes (IgG and IgM, for instance). Our data suggest that the Farr assay probably detects AmtDNA. Consistent with this, nuclear DNA (nDNA) efficiently competed in our AmtDNA ELISA assay ( Supplementary Fig. 5a), further pointing to a certain degree of redundancy between anti-dsDNA and AmtDNA. When nuclear and mtDNA were treated with S1 nuclease, which digests potential single-stranded regions, both could compete in our AmtDNA ELISA assay ( Supplementary Fig. 5b), which suggests that most of the antigenicity of mtDNA is provided by its double-stranded conformation. There might exist subsets of antibodies that preferentially recognize mtDNA versus genomic DNA, but there is no evidence of their occurrence at this stage. As extracellular mtDNA is reportedly associated with various pathologies, including trauma 25,26 , burn injury 27 , cancer 28 and rheumatoid arthritis 17,29 , it is tempting to postulate that extruded mtDNA, beyond its role as a pro-inflammatory signal for the innate immune system, represents a significant antigenic load available for the formation of immune complexes.
In this study, we focused our attention on immunoglobulin (Ig) G (IgG), but the assays can be modified easily to quantify specific IgG subclasses (e.g. IgG1, IgG2a, IgG2b) or other isotypes (e.g. IgA and IgM), which may reveal distinct functions of these antibodies in disease. Both AwMA and AmtDNA were detected at low levels in healthy individuals, suggesting that these antibodies are further induced during SLE pathogenesis. These antibodies might be part of a pool of naturally occurring autoantibodies that are thought to contribute to the continual clearance of membrane vesicles in the circulation 76,77 . Protective natural autoantibodies have been described predominantly as IgM, but other isotypes such as IgG or IgA have also been reported [78][79][80] . It is thus tempting to postulate that antibodies targeting mitochondrial epitopes are present in healthy individuals and might be involved in clearance of extracellular mitochondria. A profound change in the balance of natural IgM and pathogenic IgG against mitochondrial epitopes may in part explain the pathogenesis in SLE.
While these observations require confirmation in an independent cohort of patients, they suggest that, in SLE, the adaptive immune system recognizes mitochondrial organelles. Furthermore, our findings suggest that clinical associations may differ according to antibody recognition of inner (e.g., ACA) versus outer mitochondrial (e.g., AwMA) membrane components. This leads to the postulation that pathogenicity of these antibodies may depend on whether extracellular mitochondria are intact or not. Evaluation of antibodies to mitochondrial components in SLE may provide novel information on patients, such as their risk for developing nephritis. If these findings are confirmed in a large prospective cohort of SLE patients, AwMA and AmtDNA may prove useful in predicting  www.nature.com/scientificreports www.nature.com/scientificreports/ disease activity and disease severity, and in stratifying SLE patients. The quantification of mitochondrial antibodies may thus open the way to novel directions in autoimmune disease research and may be useful for achieving a better understanding of disease mechanisms.

Material and Methods study approval. Patient population. The human sera tested in this study were obtained from a University
Health Network research ethics board (REB) approved study of SLE and APS patients in Toronto as well as from healthy controls and PBC patients recruited from CHU de Québec -Université Laval REB approved studies in Quebec City. SLE patients had to meet the 1982 ACR classification criteria for SLE revised in 1997 81,82 and APS patients met the 1999 Sapporo criteria for APS revised in 2006 83,84 . For SLE, consecutive female patients from the University of Toronto Lupus Clinic (UTLC) were approached and provided consent between August 2010 and October 2011 to be part of a study on the role of fatty acids and cardiovascular disease in lupus. They provided one blood specimen and had their anonymized clinical data linked to their biospecimen. Similarly, APS patients seen at the rheumatology clinic in Toronto were approached following a similar procedure. All remaining biospecimen could be used for future studies on biomarkers of lupus as per the original subjects' consent. Healthy control volunteers were recruited as part of study on markers of inflammation if they had no known illnesses and did not have infectious symptoms at the time of the blood draw. Age and sex were collected at that time.

Inducible mouse model of sLe.
Recommendations of the Canadian Council on Animal Care were followed in a protocol approved by the Animal Welfare Committee at Université Laval. C57BL/6 J were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and housed in an Elite-Plus specific-pathogen-free animal facility at CHU de Quebec. SLE autoantibodies were induced in these mice as previously described 63  ethics. Throughout the entire study, blood samples were obtained from patients under informed consent. All the methods presented in this study were performed in accordance with the relevant guidelines and regulations for both human and murine donors.
Mitochondria isolation. Mitochondria were isolated from the livers of C57BL/6 J mice following a combination of published protocols 61,62 . Mice were sacrificed by cervical dislocation, the liver was swiftly removed, the gallbladder excised, and the liver was rinsed in ice-cold PBS (137 mM NaCl, 3 mM KCl, 19 mM Na 2 HPO 4 , 2 mM KH 2 PO 4 ). The liver was then minced and transferred to a pre-cooled glass/Teflon tissue grinder containing 12 mL of ice-cold mitochondrial isolation buffer (10 mM Tris, 1 mM EGTA, 200 mM sucrose) per gram of liver then ground until an homogeneous suspension was obtained. Intact cells and nuclei were pelleted twice at 700 g, 4 °C, 10 min. Contaminating membranes, proteins and organelles were eliminated by two centrifugations at 7,000 g, 4 °C, 10 min followed by a centrifugation at 10,000 g, 4 °C, 10 min. The crude mitochondrial suspension was further purified by ultracentrifugation against a Percoll gradient (10 mM Tris, 1 mM EGTA, 30% v:v Percoll) at 95,000 g, 4 °C, 30 min. The band containing mitochondria was collected in PBS. A similar approach was used to isolate human mitochondria with the exception that up to 10 7 Hep-G2 cells were lysed by repeated passages through a narrow-gauge needle. The commercial kit QIAgen Qproteome (QIAgen, Hilden, Germany) was also used, following the manufacturer's instructions, to isolate mitochondria from Hep-G2 cells for quality comparison by western blotting with the aforementioned protocol. Isolated mitochondria were dosed by the bicinchoninic acid (BCA) method using BCA Protein Assay Kit (Thermo Fisher scientific, Waltham, MA, USA). Freshly isolated mitochondria were used in all experiments with the exception of the submitochondrial particles preparations for which pelleted mitochondria were kept at −80 °C until needed. submitochondrial particles (sMp) preparation. SMP were prepared as described elsewhere 60 . Briefly, frozen mitochondria were thawed at room temperature and diluted to 10 mg of mitochondrial proteins in 10 mM 4-Morpholinepropanesulfonic acid (MOPS). Samples were then sonicated using a Fisher Sonic Dismembrator Model 500 (Thermo Scientific) at 20% maximal output for 20 seconds then kept on a salt-ice water bath for 10 minutes. This cycle was repeated nine times. Samples were then centrifuged at 16,000 g, 4 °C, 10 minutes in order to discard unbroken mitochondria and other unwanted debris. Supernatants were collected in a fresh tube and their volumes adjusted to 2 mL in 10 mM MOPS. Samples were then ultracentrifuged at 150,000 g, 4 °C for 45 minutes. Pelleted SMP were resuspended in SMP buffer (225 mM mannitol, 75 mM sucrose, 10 mM HEPES, 0.1 mM EDTA, pH 7.4) and dosed. SMP preparations were kept at −80 °C until needed.
www.nature.com/scientificreports www.nature.com/scientificreports/ Red blood cells microparticles ( RBC Mp) preparation. Red blood cells (RBC) were isolated from the blood of healthy human volunteers by centrifugation at 282 g for 10 minutes at RT. Platelet-rich plasma and buffy coat were discarded, and the RBC fraction kept. RBC were counted (Cellometer Auto M10, Nexcelom Bioscience, Lawrence, MA, USA) and adjusted to a concentration of 5 × 10 8 cells/mL in Tyrode's buffer (12 mM NaHCO 3 , 127 mM NaCl, 5 mM KCl, 0.5 mM NaH 2 PO 4 , 1 mM MgCl 2 , 5 mM Glucose, 10 mM HEPES, pH 7.4). 10 9 cells were then diluted in 45 mL double-distilled water and incubated for 5 minutes. The tonicity of the buffer was then balanced by addition of 5 ml of PBS 10X (filtered through a 0.22-μm membrane). Remnant RBC were removed by centrifugation at 1,300 g for 5 minutes at RT and supernatant was centrifugated at 18,000 g for 90 minutes at 18 °C. RBC microparticle pellet was then suspended in 500 μl PBS. Protein concentration was measured with BCA assay. 4. An equivalent of 10 μg proteins was seeded on XF-96 plates (Agilent, Santa Clara, CA, USA). Plates were then centrifuged 2,000 g for 20 min at 4 °C. We visualized the distribution of mitochondria under a bright-field microscope to ensure adherence and homogeneous repartition. Plates were maintained at 37 °C without CO 2 for approximately 40 minutes prior to loading. Oxygen consumption rates (OCR) were measured in accordance with manufacturer instructions (Agilent/Seahorse Bioscience). Experiments were replicated in three wells and averaged for each experimental condition. A total of 3 measurements of oxygen consumption for each condition were made approximately every 6 minutes under basal conditions and after sequential addition of rotenone (2 μM), succinate (10 mM), antimycin A (40 μM) and TMPD (N,N,N′,N′-tetramethyl-p-phenylenediamine)/Ascorbate (100 μM/10 mM). Succinate was used as the electron donor for complex II, rotenone as a complex I inhibitor, antimycin A as a complex III inhibitor and respiration through complex IV was measured using TMPD/ascorbate. High sensitivity flow cytometry. Due to their small size, mitochondria were detected by high sensitivity flow cytometry, using a "small particle option" consisting of a forward scatter (FSC) coupled to a photomultiplier tube (PMT) mounted on a BD FACS Canto II Special Order Research Product (SORP, BD Biosciences). Mitochondria (0.5 µg) were stained with 1 µg of anti-TOMM22-Atto 488 (clone IC9-2, Sigma-Aldrich) and 1 µM of mitotracker deep red (Invitrogen, Carlsbad, CA, USA) for 30 minutes at 37 °C and diluted with PBS to a final volume of 500 µl before flow cytometry analysis. To quantitatively measure the number of mitochondria, we used 3 µm diameter polystyrene microsphere (Polysciences, PA, USA). 80,000 microspheres were added to each sample and 500 microspheres were acquired. Silica particles (Kisker Biotech, Steinfurt, Germany) were used to determine 100-1,000 nm size scale. ( www.nature.com/scientificreports www.nature.com/scientificreports/ agitation. mtDNA suspensions were neutralized with one volume (v:v) mtDNA isolation buffer C (1.32 M potassium acetate, pH 4.8 adjusted with ice-cold acetic acid) for 5 min. on ice, under gentle agitation. Mitochondrial debris were pelleted by centrifugation for 10 minutes at 14,000 g, RT. Supernatants were transferred to fresh tubes. mtDNA was precipitated overnight at −20 °C with 0.1 volume (v:v) potassium acetate (stock solution: 3 M sodium acetate, pH adjusted to 5.2 with ice-cold acetic acid) and 0.7 volume (v:v) absolute isopropanol. mtDNA was then pelleted at 14,000 g, washed thrice with 1.5 mL 70% ethanol and resuspended in DNA resuspension buffer (10 mM Tris-HCl, 10 mM EDTA, pH 8.0). Mitochondrial DNA concentration was determined by spectrophotometry (Nanodrop 1000, Thermo Fisher Scientific).
Nuclei isolation from mouse hepatocytes. Nuclei were isolated from mouse livers using published methods 86 . Briefly, during mitochondria isolation protocols, while supernatants were used for performing mitochondria isolations, pellets from the first 700 g centrifugation were resuspended in 10 mL of ice-cold nuclei isolation buffer A (5 mM MgCl 2 , 10 mM Tris-HCl, 250 mM Sucrose. pH 7.4) and ground in a pre-cooled glass/Teflon tissue grinder. The suspension was further disrupted by three passages through a 25 G 5 / 8 gauge needle followed by two passages through a 27 G ½ gauge needle. The suspension was then filtered against a 40 µM nylon cell strainer (Thermo Fisher Scientific) and centrifuged at 600 g, 4 °C, 10 min. Pellets were resuspended in 14 mL buffer A and centrifuged at 600 g, 4 °C, 10 min. Pellets were then resuspended in 9 volumes of ice-cold nuclei isolation buffer Mitochondrial DNA and nuclear DNA enrichments by qpCR. Thirty nanograms of mitochondrial DNA, nuclear DNA or the same amount of total DNA extracted from whole mouse liver using the aforementioned protocols, were amplified in a Rotor-Gene Q real time qPCR cycler (QIAgen) according to standard protocols with SsoAdvanced Universal SYBR ® Green Supermix (BioRad) in a 10 µL reaction volume. Two distinct primer couples were used: one specific to mitochondrial DNA (5′-GGAACAACCCTAGTCGAATGAA-3′/5′-GCTAGGGCCGCGATAATAAA-3′) and the other to nuclear DNA (5′-CCTGCTGCTTATCGTGGCTG-3′/5′-GCCAGGAGAATGAGGTGGTC-3′). The experimental conditions were: 50 °C -2 minutes in, 95 °C -10 min and 40 cycles (95 °C -15 seconds, 60 °C -1 minutes). Fold changes were calculated with the 2 −ΔCt method setting mean value for total liver DNA extracts as 1.

Mitochondrial DNA digestion by restriction enzymes. Purified mouse mtDNA was digested by Hae II
or Pst I restriction endonucleases (NEB, Whitby, ON, Canada) according to the manufacturer's protocol. Digested products (1.5 μg) were then resolved on a 0.5% (w/v) agarose gel. Images were acquired on a Chemidoc MP gel documentation system (Bio-Rad). Full-length gel is presented in Supplementary Fig. 6d. S1 nuclease treatment of mitochondrial DNA and nuclear DNA. 30 µg of mtDNA and nDNA were diluted in 200 µL of 1X S1-buffer and treated with 50 U S1 nuclease (Thermo Fischer Scientific) for 5 minutes at 37 °C. Reaction was stopped by addition of 600 mM EDTA (final concentration) and incubation at 70 °C for 10 minutes. Samples were precipitated by addition of 300 mM Sodium Acetate (final concentration) and 2 volumes (v:v) absolute ethanol for 16 hours at −20 °C. Samples were pelleted at 14,000 g, RT, 20 minutes, and washed 1 mL 70% ethanol and resuspended in DNA resuspension buffer. For competition assays, untreated nDNA and mtDNA followed the same treatment without addition of the enzyme. Samples were dosed by qPCR.
Mitochondria oxidation in-vitro. Dosed mitochondria were pelleted and resuspended at a concentration of 1.5 mg of mitochondrial proteins/mL in PBS containing 500 µM of the oxidant tertbutyl hydroperoxide (TBHP) (Sigma-Aldrich). Mitochondria were oxidized for 1 h 30 min at 37 °C under gentle agitation then rinsed twice with ice-cold PBS. Oxidized mitochondria were subsequently quantified by BCA.

Mitochondrial lipid oxidation.
Thiobarbituric acid reactive substances (TBARS) formation was assessed, using the TBARS Parameter Kit (R&D systems, Minneapolis, MN, USA), following the manufacturer's instructions. Two hundred micrograms of mitochondria were lysed, the proteins were precipitated using trichloroacetic acid (TCA) and pelleted at 12,000 g, room temperature, 4 min. The supernatants were transferred to fresh tubes. Samples (75 µL) were incubated with 37.5 µL of thiobarbituric acid in a 96-well plate for 3 h at 50 °C. Absorbances were read at 532 nm on a SpectraMax 190 microplate reader (Molecular Devices, Sunnyvale, CA, USA) and TBARS quantification was performed using a standard curve provided in the kit. Oxidized samples were compared to control mitochondria that were incubated under the same conditions in PBS devoid of TBHP.
Mitochondrial protein oxidation. The formation of carbonyl moieties following in-vitro mitochondrial oxidation was measured using the Protein Carbonyl Content Assay Kit (Sigma Aldrich) following the manufacturer's instructions. Oxidized samples were compared to control mitochondria that were incubated under the same conditions in PBS devoid of TBHP.
Detection of antibodies targeting mitochondrial epitopes by eLIsA. For the detection of anti-whole mitochondrial antibodies (AwMA), murine mitochondria were diluted (500 µg/mL) in 50 mM carbonate/bicarbonate buffer, pH 9.6 and 25 µL per well were loaded onto 96-well half-area clear flat bottom polystyrene high-binding microplates (Corning, New York, USA). Plates were coated for 18 h at 4 °C then blocked for 4 h www.nature.com/scientificreports www.nature.com/scientificreports/ at 37 °C with PBS containing 10% FBS and 0.5% gelatin. After three washes with PBS, sera diluted 1:150 (unless otherwise specified) in PBS-10% FBS-0.3% gelatin were incubated overnight at 4 °C in duplicate. After three washes with PBS, plates were incubated for 1 h at room temperature with alkaline phosphatase-(AP) conjugated goat anti-mouse or anti-human IgG (Sigma-Aldrich) diluted 1:1,000 in PBS-0.4% bovine serum albumin (BSA). Plates were washed thrice with PBS and developed with p-nitrophenol phosphate (p-NPP) for ~30 min at 37 °C and optical densities (OD) were read at 405 nm on a microplate reader. The same protocol was used for the detection of autoantibodies targeting submitochondrial particles by using 25 µL per well of SMP diluted (50 µg/mL) in 50 mM carbonate/bicarbonate buffer, pH 9.6. A similar approach was used for human mitochondria with the following modification: plates were incubated with a horseradish peroxidase-conjugated anti-human IgG secondary antibody (1:3,000), peroxidase activity was revealed at room temperature for ~5 min with 3,3′,5,5′-tetramethylbenzidine (TMB). The reaction was stopped with 2 N H 2 SO 4 and the ODs read at 450 nm. For the anti-mtDNA ELISA, plates were pre-coated with 1% protamine sulfate in double-distilled water for 1 h at room temperature. Plates were washed three times with PBS and coated overnight at 4 °C with 400 ng mtDNA in PBS. All subsequent steps were identical to those used for AwMA-ELISAs. Blank values (mitochondrial antigens and no sera) were substracted from measured values for each patient. During the development of these assays, the proper coating of the wells was tested by using isotype-matched mouse monoclonal antibodies (mAb). A monoclonal antibody (Clone IV.3, 4 µg/mL) was used as a negative assay control in each instance while different monoclonal antibodies were used, depending on the coating antigens, as positive assay controls: an anti-TOMM22 mAb (Clone IC9-2, 4 µg/mL. Abcam) for intact mitochondria, an anti-DNA mAb (Clone 35I9 DNA, 10 µg/mL. Abcam) or an anti-Cytochrome C mAb (Clone 7H8.2C12, 5 µg/mL. BD Biosciences).
Competition assay. AwMA-ELISAs were performed as described in the previous sections with the following modifications: serum samples were pre-incubated in dilution buffer (1:150) spiked with various concentrations (0.25, 1 and 3 mg/mL) of competitors (i.e. mitochondria or red blood cells microparticles) for 3 hrs. at RT and incubated in duplicate overnight at 4 °C. Data are presented as the percentage of signal remaining after each competition, compared to the OD (405 nm) measured in absence of competitors.
A similar procedure was used for AmtDNA-ELISA, using increased concentrations (0, 3, 9 and 27 ng/µL) of competing DNA (extracted from nuclei or mitochondria, with or without S1 nuclease treatment).

statistics. Comparisons between groups were made using either Student's t-test, Wilcoxon test, Friedman or
Kruskal-Wallis tests, one-way ANOVA, two-way or repeated measures ANOVA depending on the outcome, as well as the number and type of groups. When multiple comparisons were assessed, appropriate post-hoc correction tests were used such as Dunn's, Dunnett's, Sidak's or Bonferroni's. Associations between AwMA, AmtDNA and anti-HSP60 were computed with Spearman correlations. Distribution of these antibodies according to ACA results were compared using Wilcoxon test. Associations between AwMA or AmtDNA and clinical outcomes were studied by bivariate and multivariate linear and logistic regressions. Clinical outcomes studied are average intima media thickness, percent change of the flow mediated dilatation (FMD) of the brachial artery, presence of FMD, presence of plaque in the carotid, thrombotic event ever, white blood cells count, platelet count, increased DNA binding by Farr assay above normal range for testing laboratory, presence of damage according to SLICC Damage Index (SDI > 0), high activity according to SLEDAI-2K activity score (SLEDAI ≥ 4), presence of lupus nephritis, biopsy class, as well as chronicity and activity index from the biopsy. The latter were adjusted for disease duration, age, body mass index (BMI), low-density lipoproteins (LDL) cholesterol, antimalarial medication and prednisone. Logistic regressions are presented with odd ratios and their 95% Wald confidence interval. Participants' results were considered positive for AwMA and AmtDNA when their value was above the cut-off value identified after maximizing Youden's Index. A 95% confidence interval was obtained for the cut-off using 10,000 bootstrap samples. Performance measures are presented with their 95% exact confidence interval.
software. Western blot images were acquired using Image Studio Digits 5.2 (LI-COR biotechnology). mtDNA migration through agarose gel was imaged using Image Lab 6.0.1 (Bio-Rad). DLS studies were carried using the built-in Zetasizer 7.10 software (Malvern Instruments). EM images were acquired with the Image Capture Software 601.384 (Advanced Microscopy Techniques Corp., Woburn, MA, USA). Flow cytometry was performed using the BD FACSDiva ™ 6.1.3 (BD Biosciences). Yields from DNA isolations were quantified using the ND-1000 3.8.1 software (Thermo Fisher Scientific) and qPCR were performed with RotorGene 6.1 (Corbett Research/QIAgen). Optical densities were measured using SoftMax Pro 5.

Data Availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Engineering

text:
Synechococcus elongatus as a model of photosynthetic bioreactor for expression of recombinant β-glucosidases

Background The production of glucose from cellulose requires cellulases, which are obtained from decomposing microorganisms such as fungi and bacteria. Among the cellulases, β-glucosidases convert cellobiose to glucose and have low concentration in commercial cocktails used for the production of second-generation (2G) ethanol. Genetic engineering can be used to produce recombinant β-glucosidases, and cyanobacteria may be interesting bioreactors. These photosynthetic microorganisms can be cultured using CO2 emitted from the first-generation ethanol (1G) industry as a carbon source. In addition, vinasse, an effluent of 1G ethanol production, can be used as a source of nitrogen for cyanobacteria growth. Thus, photosynthetic bioreactors cannot only produce cellulases at a lower cost, but also reduce the environmental impact caused by residues of 1G ethanol production. Results In the present work, we produced a strain of Synechococcus elongatus capable of expressing high levels of a heterologous β-glucosidase from a microorganism from the Amazonian soil. For this, the pET system was cloned into cyanobacteria genome. This system uses a dedicated T7 RNA polymerase for the expression of the gene of interest under the control of a nickel-inducible promoter. The results showed that the pET system functions efficiently in S. elongatus, once nickel induced T7 RNA polymerase expression which, in turn, induced expression of the gene of the microbial β-glucosidase at high levels when compared with non-induced double transgenic strain. β-glucosidase activity was more than sevenfold higher in the transformed cyanobacteria than in the wild-type strain. Conclusions The T7 system promotes high expression levels of the cloned gene in S. elongatus, demonstrating that the arrangement in which an exclusive RNA polymerase is used for transcription of heterologous genes may contribute to high-level gene expression in cyanobacteria. This work was the first to demonstrate the use of cyanobacteria for the production of recombinant β-glucosidases. This strategy could be an alternative to reduce the release of 1G ethanol by-products such as CO2 and vinasse, not only contributing to decrease the cost of β-glucosidase production, but also mitigating the environmental impacts of ethanol industrial plants.

Background
Concerns about world energy consumption and the need to reduce the emission of greenhouse gases have accelerated scientific research on new energy sources and/or more efficient use of available feedstocks. Agroindustrial by-products of vegetal origin have great potential for renewable and sustainable energy generation due to their abundance, biodegradability, hydrocarbon content and low-cost/benefit ratio [1]. One such example is sugarcane bagasse, a by-product of sugarcane-based bioethanol industry, with an annual estimated production of 540 Mt [2]. Its approximate composition is cellulose (50%), hemicellulose (25%) and lignin (25%) [3]. Although much of the bagasse produced is burned in the boilers for bioelectricity cogeneration for the industrial facility itself, studies show that the use of lignocellulosic biomass for the production of second-generation (2G) ethanol may present a lower risk and greater profitability for the investors [4]. Integration of 2G ethanol production into already existing 1G ethanol biorefineries may be an attractive business [5] because of the possibility of (1) increasing ethanol production by 30%, without the need to expand the sugarcane-planted area; (2) reducing CO 2 emissions during the production process when compared to 1G ethanol, (3) using the existing and consolidated logistics infrastructure for distribution and utilization (the same used for 1G ethanol production); (4) using by-products and wastewater from the production of 1G ethanol; and (5) production during the off-season of sugarcane [6,7].
Second-generation (2G) ethanol is produced in four stages: pre-treatment, hydrolysis, fermentation and distillation. In general, the process consists in the removal of lignin and reduction in the degree of cellulose crystallization (pre-treatment) to facilitate the enzymatic hydrolysis by cellulases and β-glucosidases, which act to convert cellulose into glucose. Subsequently, the glucose generated is fermented into ethanol by yeast [1]. Although 2G ethanol has a better energy balance than 1G ethanol and it is environmentally "friendlier", its production is still not economically viable due to the high cost of the pre-treatment and enzymatic hydrolysis steps [8,9].
Cellulose saccharification (hydrolysis) is catalyzed by three types of enzymes: endoglucanases (EC 3.2.1.4), exoglucanases (EC 3.2.1.91) and β-glucosidases (EC 3.2.1.21). Endoglucanases break the inner bonds of the cellulose, reducing its degree of polymerization and exposing the microfibrils to the action of the exoglucanases at the ends of the chains, thus releasing glucose and cellobiose. The produced cellobiose acts as inhibitor of the endo-and exoglucanases, negatively affecting enzymatic hydrolysis. Therefore, β-glucosidases have a crucial role in the process. As they convert cellobiose to glucose, they not only make glucose available for yeast fermentation, but also they prevent cellulase inhibition by cellobiose. Although the action of the β-glucosidases represents the critical point of the enzymatic hydrolysis of the cellulose, its concentration in the currently commercialized enzymatic cocktails is low, increasing the cost of 2G ethanol production [10]. Finding organisms able to produce large amounts of cellulose deconstruction enzymes has been an obstacle to the generation of 2G ethanol [11]. Through genetic engineering, it is possible to develop biofactories for the production of recombinant proteins with desirable characteristics. Some genetically modified organisms have been shown to be more efficient in the production of β-glucosidases than non-transgenic organisms traditionally used in the production of enzymes [10]. For example, a Pichia pastoris strain was genetically engineered for the expression of a novel β-glucosidase (NpaBGS) isolated from the fungus Neocallimastix patriciarum, which showed better performance than commercial Novozym − 188 β-glucosidase (Novozymes) [11].
Cyanobacteria are considered attractive hosts for the production of recombinant proteins due to their photosynthetic efficiency (superior to terrestrial plants) [12], cosmopolitan distribution, minimum growth requirements (basically solar energy, water and CO 2 ), short life cycle, easy genetic manipulation and high biomass productivity compared to agricultural crops [13,14]. Genetic engineering has been widely employed in cyanobacteria for the development of biofactories to produce molecules of commercial interest at a reduced cost. Functional enzymes such as ketoacid decarboxylase from Lactococcus lactis, used to produce isobutyraldehyde and isobutanol [15], isoprene synthase from Pueraria montana, for isoprene generation [16] and larvicidal proteins from Bacillus thuringiensis against the mosquito Aedes aegypti [17] are some examples of such enzymes successfully produced in cyanobacteria.
To improve the yield of biomolecules in cyanobacteria, several vectors for chromosome integration, called SyneBricks, have been described for expression of heterologous genes in Synechococcus elongatus PCC 7942 [18]. One of the proposed expression systems was based on the pET expression system developed for expression of heterologous genes in Escherichia coli [19]. This system consists of an insertion of the bacteriophage T7 RNA polymerase into the cyanobacteria genome under the control of an inducible promoter. T7 RNA polymerase binds with high specificity to the T7 promoter (P T7 ), thus triggering targeted transcription of the heterologous gene cloned downstream of this promoter. With such an expression platform, it is not only possible to increase the production of recombinant proteins, but also to control expression of the gene of interest, yielding the protein only when desired.
It has been estimated that recombinant protein production in cyanobacteria enables conversion of more than 50% of atmospheric CO 2 into protein biomass [20]. In addition to CO 2 , other residues of the sugarcanederived 1G ethanol production can be used as a source of nutrients to further contribute to a sustainable and low-cost production of enzymes used in the 2G ethanol industry. Vinasse, a nitrogen-rich effluent derived from alcoholic fermentation, can be used in the cultivation of cyanobacteria for biofuel (e.g., biodiesel) production [21]. In this context, this study aimed to transform the cyanobacteria Synechococcus elongatus PCC 7942 into a photosynthetic bioreactor for the production of recombinant β-glucosidases. Due to the fact that CO 2 and vinasse can be used for Synechococcus elongatus biomass production, it is possible to integrate the 1G and 2G ethanol processes as previously suggested by Brasil et al. [5].

Construction of pET expression system and insertion into S. elongatus PCC 7942
Studier and Moffatt developed a gene expression system based on the use of an exclusive RNA polymerase for the expression of heterologous genes [19]. Known as pET expression system, this protein expression platform was developed by inserting the T7 bacteriophage RNA polymerase gene into the Escherichia coli genome. T7 RNA polymerase is characterized by having highly selective and efficient transcription due to high specific binding to the T7 promoter, which does not occur naturally in the host cell genome. Thus, this platform is widely employed for heterologous protein expression in E. coli. In the present work, we used the pET system for expression of a cellulolytic enzyme in S. elongatus PCC 7942.
Bergmann et al. [22] constructed a metagenomic library to isolate new β-glucosidases from the microbial community of Amazonian soil. Two prokaryotic genes belonging to the GH3 and GH1 families (AMBGL17 and AMBGL18, respectively) were isolated. Although there is no evidence that AMBGL17 gene codes for a true cellobiase, it has been functionally considered as one because its ORF region codes for a β-glucosidase belonging to GH3 family. As the enzymatic activity of AMBGL17 has already been biochemically characterized and revealed that this enzyme not only possesses a high enzymatic activity and affinity with the substrate (V max : 16 mM s −1 , K m : 0.30 ± 0.017 mM, K cat : 38.57 s −1 ) but also a higher catalytic efficiency (K cat /K m ) than many β-glucosidases described in the literature (128.56 s −1 mM −1 ), it was chosen for the present work [22].
For the construction of the pET expression system, two neutral sites (NSI and NSIII) were used for the insertion of genes into the cyanobacterial genome through homologous recombination [23]. The gene encoding T7 RNA polymerase (T7RNAP) was inserted at neutral site 1 (NSI) under control of the Ni 2+ -inducible P nrsB promoter (Fig. 1a). Insertion of the T7RNAP expression cassette into the cyanobacterial genome was confirmed by PCR with the amplification of a 1.4-kb fragment, which corresponds to the distance between the genomic locus Synpcc7942_2499 (flotillin membrane protein; flanking the NSI site) and the spectinomycin resistance gene (aadA gene: aminoglycoside resistance protein AadA) present in the genetic construct (Fig. 1a, c). After confirmation of T7RNAP gene insertion into the cyanobacterial genome, a second transformation was performed with the pHN1_AMBGL17 plasmid ( Fig. 1b) for integration of the β-glucosidase gene (AMBGL17) at neutral site 3 (NSIII). Integration was confirmed by the amplification of a 1.8-kb fragment corresponding to the distance between the genomic locus Synpcc7942_0741 (Phage tail protein I gene) and the chloramphenicol resistance gene (cmlR gene: chloramphenicol resistance protein) present in the second genetic construct (Fig. 1b, c).
The double-transformed colonies called S. elongatus P T7 AMBGL17 were confirmed by the amplification of a 1.8-kb DNA fragment consisting of part of the genomic locus Synpcc7942_0741 (Phage tail protein I) and part of the chloramphenicol resistance gene present in the genetic construct (Fig. 1c). Vector choice (integrative or replicative) for genetic manipulation is essential to ensure successful transformation. In this work, we chose to use integrative vectors to guarantee gene stability through recombination, with long-term maintenance of the transgenic lineage, as suggested by Heidorn et al. [24].

Effects of transgenesis and nickel addition on cell growth rates
As Synechococcus elongatus PCC 7942 may harbor from 1 to 10 genome copies, we cannot guarantee that heterologous genes (T7RNAP and AMBGL17) integrated in all genome copies, being virtually impossible to get a homogeneous expression among different strains. Besides that, those genes can be lost during cell division, even in the presence of a selective agent. Due this fact, we performed a qPCR screening 10 strains of double transgenic cyanobacterium (P T7 AMBGL17) to evaluate the difference of gene expression among them and select the strain that presented the best level of expression to be used in the further experiments (data not shown). No significant statistical differences in growth were observed when wildtype and double transgenic (P T7 AMBGL17) strains were compared, indicating that foreign DNA integration did not affect cyanobacteria growth (Fig. 2a). However, addition of 5 μM Ni 2+ in the latency phase (Lag) had a toxic effect to both transformed and wild-type cells, leading to mortality of both strains (Fig. 2a). Lag phase is a period when cyanobacteria adapt to the environmental conditions and is characterized by little or no cell division, but intense metabolic activity [25]. The visible toxic effects that resulted from nickel addition at this stage of cultivation suggest that nickel efflux pumps were not activated, probably causing nickel to accumulate inside the cell, leading to the observed toxic effect. At the stationary phase, when metabolic activity decreases and cell division increases, nickel did not affect cellular viability, demonstrating that the activation of the pET system is not toxic for the wild-type or the double transgenic strains (Fig. 2c). Based on these results, induction at the stationary phase was used in all subsequent experiments.

Determination of expression levels from T7 system
Induction of expression of the target gene in the pET system is based on the use of the P nrsB promoter, which is part of the nrsBACD operon, responsible for cyanobacterium resistance to nickel exposure [26]. When present in the cell, nickel "sequesters" the regulatory protein that blocks interaction between the endogenous RNA polymerase and P nrsB by modifying its conformation, preventing its blocking activity and thus releasing the promoter region, so that transcription of the T7 RNA polymerase gene can start (Fig. 3a, b). The synthesized T7 RNA polymerase binds to its specific promoter (P T7 ) initiating the exclusive transcription of recombinant β-glucosidase (AMBGL17) (Fig. 3b). Although P nrsB is considered a promoter with low non-specific activity, our gene expression data show that in S. elongatus the pET system was activated at basal, even without inducer addition to the culture (0 h) (Fig. 3c). This was probably due to the presence of divalent metal ions such as Co 2+ , Zn 2+ and Cu 2+ in the BG-11 medium, which are capable of inducing the expression of some   Expression of the T7 system in Synechococcus elongatus PCC 7942. a Double-transgenic Synechococcus elongatus (P T7 AMBGL17) prior to Ni 2+ addition. Repressive protein is bound to the DNA and prevents endogenous RNA polymerase interaction with P nrsB that results in T7 RNA polymerase expression. b Mechanism of activation of the T7 system in S. elongatus P T7 AMBGL17. After Ni 2+ addition, this cation binds to the repressive protein, resulting in a change of conformation that blocks its interaction with DNA at the P nrsB region and results in T7RNA polymerase transcription by endogenous RNA polymerase. High levels of expression of T7 RNA polymerase, which action P T7 promoter and produces high levels of expression of the AMBGL gene. The thickness of the arrows indicates the intensity of transcription. c Relative expression of the T7 RNA polymerase (T7RNAP) and β-glucosidase (AMBGL17) genes in S. elongatus P T7 AMBGL17. The level of gene expression was determined by qPCR, and results were normalized using the constitutive genes rnpB (R-subunit of ribonuclease P) and rpoD (Delta subunit of RNA polymerase). Different letters (uppercase: T7RNAP; lowercase: AMBGL17) show significant statistical differences (p < 0.05) promoters, as previously reported to Synechocystis sp. PCC 6803, another cyanobacterium species [27]. After 24 h of induction, T7RNAP expression increased approximately 5.5-fold over the uninduced (0 h) timepoint, which was sufficient to raise AMBGL17 gene expression by 22-fold (Fig. 3c).
T7 RNA polymerase is known for its high level of transcription and a low error rate, and thus, relatively small amounts of this enzyme activate the system [19]. The use of the pET system allowed strong gene expression of AMBGL17 in S. elongatus PCC 7942, demonstrating that the system is active and efficient. Therefore, in our work, high levels of T7RNAP expression are not required for high expression of the gene of interest, as previously observed [18].
By analyzing the data over the course of days, we observed a decrease in T7RNAP gene expression and, as a consequence, AMBGL17 reaching approximately zero on the eighth day of induction (Fig. 3c). Probably, this occurred because when nickel was added to activate the pET system, the nrsBACD operon was also activated. This operon contains genes encoding proteins that form complex whose function is to pump nickel out of the cell. Therefore, over time, the system was inactivated and AMBGL17 induction stopped.
Englund and Lindberg suggested that one way to prevent the pumping of nickel out of the cell would be to utilize the genes constituting the nrsBACD operon as sites for homologous recombination of the transgene [27]. In this case, the knockout of the nickel efflux system could prolong the expression of the system by potentiating protein production using a lower concentration of the inducer. However, the functionality of the nrsBACD operon remains to be evaluated.

β-Glucosidase production
Although most enzyme assays involve cell lysis, tests previously performed in our laboratory (data not shown) have shown that the presence of large amounts of phycocyanin pigment present in cyanobacteria lysate interferes with the reading of the colorimetric compound (para-nitrophenol-pNP). As the substrate (pNPβG) and enzyme product (pNP) are permeable, being able to cross the cell membrane, it was chosen to carry out the enzymatic activity in the culture medium, without lysing the cells.
By measuring β-glucosidase activity over 24 h, we observed a significant difference between the S. elongatus P T7 AMBGL17 and wild-type strains (Fig. 4). Three genes coding for potential endogenous β-glucosidases were identified in the genome of S. elongatus PCC 7942; these genes are located at loci Synpcc7942_0354, Syn-pcc7942_0854 and Synpcc7942_1574. It is likely that at least one of these potential β-glucosidases is functional, which would explain the activity observed in the wildtype strain.
In the first 6 h of incubation, β-glucosidase activity was 1.70-fold higher in the transgenic strain compared to wild type, with this difference increasing to 7.4 times after 18 h of incubation, indicating that β-glucosidase activity in transgenic strain would mostly be performed by the heterologous AMBGL17. However, within 24 h this difference decreased to 3.5-fold, probably because most of the substrates (pNPβG) added to the culture were rapidly consumed by the greater amount of β-glucosidases produced by the transgenic strain. This did not occur with the wild strain, which has a lower amount of enzymes, thus taking more time to consume the substrate. If more pNPβG were added to the culture medium, an increase in activity would probably be observed. Several recombinant β-glucosidases have been expressed in bacteria and yeasts [28], but there are still no reports of the use of cyanobacteria as a production platform for this enzyme. The present work demonstrated, for the first time, that it is possible to use cyanobacteria as biofactories to produce recombinant β-glucosidases.

Conclusions
This is the first work to report an engineered strain of Synechococcus elongatus PCC 7942 able to produce β-glucosidase from the pET expression system. Our data show that the pET system is active in the cyanobacterium and promotes high expression of the AMBGL17 gene. It has been shown that the use of an exclusive RNA polymerase for the transcription of heterologous genes can contribute to high levels of transgene expression in cyanobacteria. This strategy can be further applied to the production of other proteins of commercial value. The use of cyanobacteria as biofactories for the production of β-glucosidases could enable the use of CO 2 and vinasse, effluents of the 1G ethanol plant, as a source of carbon and nitrogen. This could contribute not only to lower production costs, making ethanol 2G a competitive product in the market, but also to integrate 2G production with 1G already existing biorefineries, mitigating the environmental impacts of bioethanol production from sugarcane.

Strains and culture conditions
Escherichia coli One Shot TOP10 Electrocomp (Invitrogen) was used for cloning and replication of expression vectors. Bacteria were cultured in Luria-Bertani medium [29] supplemented with 100 μg mL −1 spectinomycin, at 37 °C, with constant shaking at 250 rpm. Synechococcus elongatus PCC 7942 (Thermo Fisher Scientific) was cultured in BG-11 medium [30], at 34 °C, with constant illumination of 8.000 lx. Transgenic cyanobacterial cultures were supplemented with spectinomycin (10 μg mL −1 ), and expression of the gene of interest was induced with 5 μM NiSO 4 for 24 h when the absorbance (750 nm) of the cultures reached the value of 1 [31]. Optical density (OD) was monitored in a spectrophotometer (BioMate 3, Thermo Scientific).

DNA cloning and vector construction
Most important elements of all of the expression vectors used in this work are presented in Table 1. For the production of pSyn1_T7RNAP, the T7 RNA polymerase (T7RNAP) gene was isolated from the E. coli BL21 (DE3) genome with the primers T7RNAP-FOR/ REV and cloned into pSyn_1 plasmid. The pHN1_ T7AMBGL17 vector was constructed by insertion of the β-glucosidase gene (AMBGL17; GenBank: KF433952.1) into the pHN1_lacUV5 vector, amplified by PCR using primers AMBGL17-FOR/REV and PNH1-FOR/REV, respectively. PCR products and plasmids were digested with specific restriction enzymes to enable cloning (pSyn_1 and T7RNAP: BamHI and KpnI; pHN1_lacUV5 and AMBGL17: EcoRI and MluI). All enzymes were obtained from New England Biolabs. All DNA fragments were purified (Illustra GFX PCR DNA and gel band purification kit, GE HealthCare) and quantified (Qubit Fluorimeter, Life Technologies), and vectors/inserts were ligated (T7 DNA ligase, New England Biolabs). All procedures were performed following the manufacturer's protocol, and all genetic constructs were sequenced for confirmation of correct cloning.  shows the sequences of primers used, which were designed with Primer Express 3.0 software (Applied Biosystems).

Transformation of S. elongatus
Transformation of S. elongatus PCC 7942 followed the protocol described in the Gene Art Synechococcus Engineering kit (Invitrogen). Cyanobacterial strain was first transformed with the pSyn_1T7RNAP vector. After the transgenic confirmation, the strain was again transformed with the second plasmid (pHN1_T7AMBGL17). As a control, S. elongatus was also incubated without the expressions vectors. After the transformation process, samples were plated on 1.5% BG-11 agar supplemented with 10 μg mL −1 of spectinomycin or 12.5 μg mL −1 chloramphenicol.  Table 2.

Growth measurement
Inoculum (1.6%) of wild-type and double transgenic (P T7 AMBGL17) S. elongatus cultures were added to erlenmeyers (125 mL) containing 50 mL of BG-11 medium. All cultures were carried out following the conditions described in item 4.1. The cultures used to evaluate the effect of the transgene on growth and addition of nickel to Lag phase cultures started from the same optical density (OD 750 : 0.156 ± 0.003). To evaluate the effect of nickel (Ni 2+ ) at the start of the stationary phase, the cultures started from an OD 750 of 1.047. Growth was monitored in the spectrophotometer (BioMate 3, Thermo Scientific) for 10 days, and 5 μM NiSO 4 was added at different stages of cultures (OD 750 : 0.156 ± 0.003 and OD 750 : 1.047 ± 0.019) to induce the pET system and evaluate its effect on growth.

Gene expression analysis
To evaluate expression of the heterologous genes, six samples were collected on days 0 (non-induced), 1, 4 and 8 after induction with 5 μM NiSO 4 . Total RNA was extracted with Trizol (Invitrogen) and treated with DNAse I (Invitrogen). The cDNA synthesis was performed with the High Capacity cDNA Reverse Transcription kit (Applied Biosystems), and cDNA was quantified using the Qubit fluorimeter (Invitrogen). Gene expression was analyzed by quantitative real-time PCR (qPCR) using Platinum SYBR Green qPCR Super-Mix-UDG (Invitrogen). Serial dilutions of cDNA were performed to test the efficiency of all primers. The conditions of qPCRs were 50 °C for 120 s, 95 °C for 120 s, followed by 40 cycles of 95 °C for 15 s and 60 °C for 30 s. The expression of the target genes was normalized by the constitutive genes rnpB and rpoD [33]. All procedures were performed following the manufacturer's protocol, and the primers used are described on Table 2. Data from qPCR were analyzed by the delta C T method [34].

β-Glucosidase activity
β-Glucosidase activity was determined by using p-nitrophenyl-β-d-glucopyranoside substrate (pNPβG) as described by Strahsburger et al. [35], who performed in vivo assays of β-glucosidase activity on strains of Bifidobacterium and Lactobacillus by incubating microorganisms with pNPβG. After 24 h of induction with 5 μM NiSO 4 , 1.600 μg mL −1 of pNPβG was added to wild-type and transgenic S. elongatus cultures. Culture samples were collected for the determination of total protein concentration (Qubit Fluorimeter, Life Technologies) and β-glucosidase activity after 6, 18 and 24 h of the addition of pNPβG. Cells were precipitated by centrifugation at 10,000g for 5 min. The supernatant (150 μL) was transferred to a clear 96-well plate containing 50 μL of 0.1 M Na 2 CO 3 . Absorbance was measured at the wavelength of 405 nm (BioTek ELx800 Microplate Reader). A unit (U) of enzymatic activity is defined as the amount of β-glucosidase required to release 1 μmol of pNPβG per minute per gram of protein under the assay conditions.

Statistical analysis
For gene expression analysis, it was used six samples for each treatment. Growth measurement and β-glucosidase activity were performed in triplicate. One-way ANOVA was used to evaluate expression of T7RNAP and AMBGL17 genes over time. The effect of transgenesis and nickel addition on the growth of S. elongatus PCC 7942 was evaluated by two-way ANOVA with repeated measures (factors: transgenesis and time/nickel and time). The effect of transgenesis on enzymatic activity was analyzed by two-way ANOVA with repeated measures (factors: transgenesis and time). All data were tested for their normality and homoscedasticity using the Shapiro-Wilk and Bartlett tests, respectively. A p < 0.05 was considered, and all the statistical analyses were performed with R Software [36].