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Coordinated Assembly of the Bacillus anthracis Coat and Exosporium during Bacterial Spore Outer Layer Formation

This work dramatically improves our understanding of the assembly of the outermost layer of the B. anthracis spore, the exosporium, a layer that encases spores from many bacterial species and likely plays important roles in the spore’s interactions with the environment, including host tissues. Nonetheless, the mechanisms directing exosporium assembly into a shell surrounding the spore are still very poorly understood. In this study, we clarify these mechanisms by the identification of a novel protein interaction network that directs assembly to initiate at a specific subcellular location in the developing cell. Our results further suggest that the presence or absence of an exosporium has a major impact on the assembly of other more interior spore layers, thereby potentially explaining long-noted differences in spore assembly between B. anthracis and the model organism B. subtilis.

host, and pathogenesis (1)(2)(3). The spore is organized as a series of layers surrounding a DNA-containing core, each of which makes a critical contribution to resistance and/or dormancy. In the case of Bacillus anthracis, the causative agent of anthrax, these layers are the cortex, coat, interspace, and exosporium ( Fig. 1A) (3,4). The interspace and exosporium are present in a subset of spore-forming species, both pathogenic and nonpathogenic, while the coat and cortex are present in all species (5). Ongoing investigations in B. anthracis and the model organism Bacillus subtilis (which lacks the interspace and exosporium) seek to identify the functions of these layers and elucidate the mechanisms directing their assembly.  N). Arrows indicate the exosporium, while arrowheads indicate the coat. In panel A, the cortex (Cx) and interspace (IS) are also indicated. Images are representative of at least 80 spores visualized. The presence of the exosporium was also assessed by immunofluorescence microscopy and staining with anti-BclA antibodies (G to N). Images are representative of more than 100 spores visualized in three separate spore preparations. Scale bars ϭ 1 m.
Our understanding of interspace and exosporium function remains poor. Exosporium surface properties affect interactions with surfaces and cells in the environment, as well as early interactions with the host upon infection (4,(6)(7)(8). The exosporium also affects germination (the process by which spores return from dormancy to active metabolism) and may provide resistance to some stresses (9)(10)(11). However, B. anthracis spores lacking the exosporium are largely functional (12) and cause significant disease in animal models (13,14). These results argue that the exosporium is not required for infection per se but may play an important role in natural infection or in other environmental adaptations. Importantly, exosporia are present in many species throughout the Bacillales and Clostridiales that are not known to cause disease in mammals (15). The exosporium likely has additional unknown functions in B. anthracis, as well as in the large number of other phylogenetically diverse species possessing this structure.
Elucidating the mechanisms controlling interspace and exosporium assembly is an especially intriguing problem. These mechanisms will almost certainly be novel, as these two layers are morphologically distinct from the better-studied coat. In B. anthracis and the related species B. cereus and B. thuringiensis, the exosporium is composed of a protein shell, the basal layer, which is studded with hair-like projections ( Fig. 1A) (10,12,(16)(17)(18)(19)(20). Exosporium assembly initiates soon after the first hallmark event in sporulation, the appearance of the asymmetrically positioned sporulation septum (2 h into sporulation) that divides the cell into a smaller compartment (the forespore) and a larger compartment (the mother cell) (13,15). The mother cell then engulfs the forespore, resulting in a free protoplast that develops into the spore. Before engulfment is complete, a thin layer of material, the cap, appears at the mother cell side of the septum ( Fig. 2A) (13). At these early time points, the cap lacks the electron density and hair-like projections of the mature exosporium, and the gap between it and the forespore (the interspace) is narrower than the interspace seen in the mature spore (13).
The next stage in exosporium assembly is encirclement of the forespore and, ultimately, closure into a contiguous shell. Full encirclement does not occur until six hours into sporulation (12), concomitant with the appearance of the coat and widening of the interspace (13). Encirclement starts at the same time as the initial expression of exsY, encoding a major basal layer structural protein (10,12,21,22). The hair-like projections, composed of the proteins BclA, BclB, and possibly others (23)(24)(25)(26), are anchored to the basal layer proteins ExsFA/BxpB and ExsFB (19,27,28).
The current description of exosporium assembly leaves at least two fundamental mechanistic questions unanswered: (i) what directs the cap to form at the forespore? and (ii) what guides exosporium elongation to form a closed shell around the forespore, rather than disconnected sheets in the mother cell cytoplasm? Addressing these questions is essential to a robust explanatory model of the assembly of this distinctive and unique cell layer, both in B. anthracis and other species. Our incomplete understanding of exosporium assembly impedes the development of improved anthrax therapeutics and spore decontamination strategies that are effective, yet gentle enough for practical needs, because improving these technologies will likely rely on a deeper understanding of exosporium composition (29,30).
Despite the morphological differences between B. subtilis and B. anthracis spores, previous work shows that several proteins with important roles in spore assembly in these two species are conserved evolutionarily and, at least in some cases, functionally (6,12,13). Studies in B. subtilis led to the identification of so-called morphogenetic proteins, which guide assembly of the other coat proteins into their respective layers (31)(32)(33)(34). Orthologues of the B. subtilis morphogenetic proteins SpoIVA, CotE, and CotZ have major roles in B. anthracis coat and exosporium formation (10,12,13,21,31,(35)(36)(37).
In this study, we sought to further elucidate the mechanisms directing cap formation and exosporium elongation by characterizing the role of the B. anthracis orthologue of the B. subtilis coat protein gene cotO. Our results demonstrate that cotO is important for cap formation and assembly of the exosporium, similar to the role of cotE (13). We propose a model in which CotE and CotO form a transient network linking the exosporium to the forespore during development. Our results also provide mechanistic insight into a significant difference in coat assembly between B. subtilis and B. anthracis (15,20,38), suggesting that the cap alters the pattern of coat assembly by transiently interfering with the deposition of coat proteins. These results emphasize the importance of exploring differences in coat and exosporium assembly among species if we are to have an accurate understanding of the assembly mechanisms for these ubiquitous and environmentally important cell types.

RESULTS
cotO is required for exosporium formation. Previously, we demonstrated that inactivation of the B. anthracis cotO gene by an insertion-disruption mutation (in strain MGM76), resulted in spores lacking the exosporium (6). Analysis of spore morphology by transmission electron microscopy (TEM) and immunofluorescence microscopy (IFM), using antibodies against the exosporium protein BclA (11), revealed that the mutant spores had an intact coat, but 94% of the spores lacked the exosporium (Fig. 1A, B, and G to J). Exosporium fragments not attached to spores were readily detected. IFM analysis of unwashed spores revealed that 22% had exosporia. These results suggest and visualized by thin-section TEM. Sporangium preparations were assessed for the presence of a cap, which are indicated by arrows in panels A to C and E. Images are representative of more than 50 sporangia from each strain visualized at these time points. Scale bars ϭ 1 m.
that exosporium material is synthesized in this strain and may be loosely associated with the spores, but it does not form a closed shell around the spore. As a result, the exosporium is readily sloughed off during either sporulation or washing. We generated a new mutant strain missing the cotO open reading frame (TJB124). The resulting spores also had an intact coat, but ϳ96% lacked the exosporium (Fig. 1C). We also observed exosporium fragments disconnected from spores in this strain ( Fig. 1K and L); however, these fragments were smaller than those seen in MGM76 spores ( Fig. 1I and J), suggesting that exosporium elongation may be reduced in strain TJB124. Despite lacking the exosporium, we did not detect a difference in the patterns of extractible proteins between this strain and the wild type (see Fig. S1 in the supplemental material). There was also no difference in germination (Fig. S2).
The introduction of cotO into the TJB124 genome at an ectopic site (creating strain TJB130) restored the expression of cotO (Fig. 3F) and partially restored exosporium formation (Fig. 1D, M, and N). We found that 32% of spores were encircled by an exosporium (Fig. 1D), compared to 4% in TJB124, and an additional 50% of the released spores were partially encircled by an exosporium (Fig. 1M and N). The partially encircling exosporia were not readily sloughed off during washing, unlike the exosporia in strain MGM76 spores, suggesting that these exosporia are more tightly adhered to the underlying spore layer. From these data, we infer that CotO likely has a role in connecting the exosporium to the spore.
Interestingly, no complementation was detected when cotO was placed on a nonintegrating plasmid (data not shown). Possibly, this failure was due to increased cotO expression. To test the hypothesis that elevated CotO levels cause an exosporium assembly defect, we transformed the wild-type strain with a plasmid expressing cotO from its endogenous promoter. The resulting strain (TJB116) had increased CotO levels during sporulation (Fig. 3E), and 88% of the released spores (53 of 60) lacked a contiguous exosporium (Fig. 1E). Most likely, proper exosporium assembly requires CotO concentrations to be within a relatively narrow range.
cotO is required for cap formation. To identify the stage at which exosporium assembly requires CotO, we used TEM to analyze sporangia of MGM76 (cotO⍀pMGM3) and TJB124 (cotO), harvested between 1 and 8 h after the onset of sporulation (T 1 -T 8 ). Consistent with previous studies (13,20), in wild-type sporangia, we first detected the exosporium as a small patch (or cap) of material located at the mother cell-proximal forespore pole during engulfment ( Fig. 2A). We also visualized the cap in 54% (42 of 78) of the wild-type sporangia harvested between the completion of engulfment and the appearance of the coat (T 2 -T 5 ). At these times, the cap covered less than one-quarter of the two-dimensional circumference of the developing spore and was close (ϳ35nm) to the forespore membrane ( Fig. 2B and C). In contrast, the cap was absent in 93% (65 of 70) of TJB124 (ΔcotO mutant) and 80% (45 of 54) of MGM76 (cotO⍀pMGM3) sporangia at these times ( Fig. 2D and E). Cap-like structures were visible in the mother cell cytoplasm of 6% of TJB124 and 16% of MGM76 sporangia (Fig. 2E) at these times, but they were never observed in wild-type sporangia. These results argue that CotO plays a major role in cap formation and localizing the cap to the forespore. Consistent with a role in cap formation, we first detected CotO in wild-type cells by Western blotting at T 2 (Fig. 3A), and CotO-green fluorescent protein (CotO-GFP) localized to the mother cell-proximal pole of the forespore at this time (Fig. 4). Our results also suggest that the CotO N-terminus, which is produced in MGM76 (as an almost-full-length protein) (Fig. 3D), retains at least some ability to assemble a cap-like structure.
If CotO is required for cap formation, overproduction of CotO might result in the assembly of additional caps. To address this hypothesis, we used TEM to search for caps in strain TJB116 (Fig. 3E). Prior to T 5 , we did not detect caps (Fig. 2F). Strikingly, we observed multiple (between 6 and 9) short exosporium-like structures in the mother cell cytoplasm of all 21 sporangia visualized at T 6 ( Fig. 5K). We infer that the overexpression of cotO resulted in the initiation of multiple cap-like structures, which were too small to visualize by TEM until they began to elongate and gain significant electron density. Possibly, released TJB116 spores lack exosporia because the amount of the major basal layer protein (ExsY) is insufficient to complete multiple exosporia. These data suggest a role for CotO in initiating cap assembly at the mother cell pole.
Interaction between CotE, CotO, and ExsY analyzed by Western blotting. Proteins were extracted from wild-type and mutant (strain RG56, cotE; strain TJB124, cotO; strain MGM76, cotO⍀; strain TJB130, cotO complement; strain TJB116, cotO overexpression; strain TJB139, exsY) sporangia or spores. Proteins were extracted from wild-type and mutant released spores (B and I), by three rounds of boiling and vortexing in Laemmli buffer, and mother cell proteins were extracted from sporangia by bead beating in PBS (C). Total sporangial proteins (A, D to H, and J) were extracted by bead beating, followed by three rounds of boiling and vortexing in Laemmli buffer. Forty micrograms of the extracted proteins was separated by 15% SDS-PAGE gels and transferred to a nitrocellulose membrane. Membranes were blocked with 5% BSA and 0.1% Tween 20 in TBS and probed with anti-CotO (1:10,000 in blocking buffer) (A to H) or anti-CotE (1:50,000 in blocking buffer) (I and J) antibodies, followed by anti-rabbit IgG alkaline phosphatase conjugate antibodies (1:10,000 in blocking buffer). Membranes were developed using SigmaFast BCIP/NBT. CotO is first detected in sporangia at T 2 (A), consistent with the timing of cap formation, and is present throughout sporulation (A and H). When only soluble proteins are extracted from sporangia, CotO is not detectable after T 5 (C). A band, similar in size to CotO, is present early during MGM76 sporulation (D), suggesting that a fragment of CotO is produced in this strain but may not be stable. CotO is also present early during RG56 sporulation (G) but cannot be detected after T 5 . Less CotO was extracted from the RG56 and TJB139 spores (B), suggesting that its presence in the spore is at least partially dependent on the presence of an exosporium. In comparison, CotE levels were unaffected by the cotO and exsY mutations (I), consistent with its localization in the coat.
We expected that CotO would be restricted to the forespore. To test this, we measured CotO steady-state levels in total sporangia and in sporangial fractions containing only soluble mother cell contents (and possibly loosely bound forespore proteins). We detected CotO at all time points (starting at T 2 ) in total sporangia (Fig. 3A). However, in the mother cell fraction, CotO was not detectable after T 5 (Fig. 3C), suggesting that CotO is largely forespore associated after that time.
cotY is required for cap, but not exosporium, assembly. To better understand the role of the cap in exosporium assembly, we inactivated the cotY gene, encoding a major cap protein (10,12,21). As expected, the resulting strain (TJB239) lacked a readily detected cap (Fig. 2G). However, consistent with previous observations in B. cereus, virtually all released spores had an exosporium (Fig. 1F) (10).
To assess the effects of the cotY mutation on the timing of exosporium formation and the location of exosporium initiation, we compared the degree of exosporium assembly at intermediate times in wild-type and mutant sporangia. We also compared the stage of exosporium assembly to the stage of coat assembly as a separate marker of progression through sporulation. In wild-type sporangia, exosporium elongation occurs simultaneously with coat assembly (Fig. 5A), such that nearly all forespores visualized with a complete coat were also fully surrounded by an exosporium (Fig. 5B). In contrast, in cotY mutant sporangia, forespores with a complete coat but no exosporium, or only partially complete exosporium, were readily observed ( Fig. 5D and E and S3). In both wild-type and cotY mutant sporangia, forespores were fully surrounded by an exosporium by T 6 ( Fig. 5F and G). It was particularly challenging to identify cotY mutant sporangia at an intermediate stage of exosporium formation; however, in the 10 sporangia we were able to analyze at this stage, exosporium initiation does not appear limited solely to the mother cell pole of the spore (as in the wild type). Instead, we observed what appear to be small patches of exosporium at random locations around the spore (Fig. 5E and S3). This suggests that exosporium initiation at the mother cell-proximal pole of the wild-type forespore is a result of cap protein deposition prior to or during engulfment. A similar mechanism in B. subtilis directs coat assembly initiation to this same forespore location (15,38). When exosporium initiation is delayed by the deletion of cotY, exosporium assembly can initiate at any location on the forespore surface. Possible role for CotO in exosporium elongation around the spore. Our data suggest that while CotO is required for early cap formation, early cap formation is not essential for exosporium assembly. This suggests that CotO (possibly by interacting with ExsY) also plays a role in directing elongation of the exosporium around the forespore. To investigate the role of CotO in guiding exosporium elongation, we characterized exosporium assembly in the cotO mutant strains at intermediate and late times using TEM. In wild-type sporangia, while the interspace widens at the mother cell-proximal forespore pole (Fig. 5A), the leading edge of the encircling exosporium remains very close to the coat ( Fig. 5B and C). In contrast, this close association between the coat and exosporium is not seen in cotO mutant sporangia. In cotO mutant sporangia, the exosporia either partially encircle the forespore (Fig. 5H), are present as dispersed fragments (Fig. 5I), or appear as wide tubes (Fig. 5J). Our data suggest that the exosporium-leading edge is attached to the coat by a protein network, including CotO, and is likely similar or even the same as that connecting the cap to the forespore. Cap formation alters the pattern of coat assembly. In B. subtilis, coat assembly begins at the mother cell-proximal pole of the forespore (5,38). In contrast, coat material first appears on the long axis of the B. anthracis and B. cereus forespore (Fig. 6A to C) (20). This difference in assembly patterns is a strong indication of at least some important difference in coat assembly between B. cereus group and B. subtilis spores. We noticed specifically that in sporangia with partially formed coats, there was a gap in the densely staining coat layer at the mother cell-proximal forespore pole in 80% (41 of 51) of wild-type sporangia ( Fig. 6B and C). Ten sporangia lacked gaps at the mother cell pole, but these sporangia were almost completely surrounded by a coat, making it difficult to assess the location of coat initiation. We interpret these data as suggesting that in most sporangia, coat material first accumulates on the long side of the spore instead of the mother cell pole as in B. subtilis.
Our results raised the possibility that the cap, appearing at the mother cell pole prior to coat maturation, interferes with the deposition of some coat proteins at intermediate and late times. If the cap does interfere with coat formation, we hypothesized that sporangia unable to assemble the cap should deposit the electron densely staining coat material first on the mother cell-proximal spore pole. To test this, we used TEM to sporangia were harvested at T 5 and visualized using thin-section TEM. At this time, many forespores were only partially surrounded by densely staining coat material (arrows), indication that coat formation was actively occurring. A gap in the densely staining coat layer at the mother cell proximal pole of the forespore (arrowheads) was present in nearly all wild-type sporangia (A to C), while densely staining material was often observed at this location in the TJB124 and MGM76 sporangia (D and E). Scale bars ϭ 1 m.

Bacillus anthracis Coat and Exosporium Assembly
® visualize coat formation in cotE, cotO, and cotY mutant sporangia. Similar to cotO and cotY mutant sporangia, most cotE mutant sporangia fail to form a cap (13). Consistent with our hypothesis, 51% (26 of 51) of cotO mutant sporangia, 42% (11 of 26) of cotE mutant sporangia, and 35% (9 of 26) of cotY mutant sporangia had electron-dense coat material covering the mother cell-proximal pole of the forespore at this stage ( Fig. 6D and E, and data not shown), suggesting that the cap may alter assembly of the coat. CotE interacts with CotO within a protein network linking the cap to the forespore. We hypothesized, based on the similarity of the observed cotO mutant phenotype with the previously published cotE mutant phenotype, that CotE and CotO are part of a protein network linking the exosporium to the developing spore (13). To address this hypothesis, Western blot analysis was used to assess the effect of a cotE mutation on CotO steady-state levels or a cotO mutation on CotE levels. While a cotO mutation had no detectable effect on the amount of CotE extracted from released spores (Fig. 3I), a cotE mutation resulted in reduced levels of spore-associated CotO (Fig. 3B). This suggests that CotE has a role in CotO stability, assembly into the spore, or extractability from the spore.
To address the possibility that CotE directs CotO assembly early in sporulation, we used epifluorescence microscopy to monitor the localization of a CotO-GFP fusion protein in wild-type and cotE mutant sporangia. We found that in otherwise wild-type sporangia harboring cotO-gfp on a plasmid (MUS8188), CotO-GFP localized to, and eventually surrounded, the forespore (Fig. 7). In a strain bearing a mutation in cotE (MUS8189), CotO-GFP accumulated in the mother cell cytoplasm early during sporulation but did not localize to the forespore. No fluorescence was visible in this strain at later time points (T 7 ). We conclude that CotE has a significant role in CotO localization and may impact CotO stability.
To address the hypothesis that cotE is required for CotO stability, we measured CotO steady-state levels throughout sporulation in wild-type and cotE mutant sporangia using Western blot analysis. In wild-type sporangia, we detected CotO at all time points after T 2 . In contrast, CotO was not detected after T 5 in cotE mutant sporangia (Fig. 3G). These data, taken together with the observation that CotO-GFP is not detectable at late time points in cotE mutant sporangia (Fig. 7), suggest that CotO is likely degraded in the cotE mutant spores. Importantly, CotO levels during sporulation were largely unaffected by a mutation in exsY (Fig. 3G and H). This suggests that the low level of extractable CotO is due to the loss of CotE specifically and not a result of a mislocalized exosporium. A simple mechanism by which CotE could recruit CotO to the forespore would be direct interaction between the two proteins. To test whether CotE and CotO can interact directly, we overproduced each protein as a His-tagged fusion protein in Escherichia coli, purified them, and tested their binding using an enzyme-linked immunosorbent assay (ELISA). We found that CotE bound to CotO immobilized to the wells of a 96-well plate in a concentration-dependent manner (Fig. 8). These data indicate that CotE and CotO interact directly in vitro and support the hypothesis that CotE and CotO interact during sporulation to direct exosporium assembly.

DISCUSSION
In this study, we address a fundamental question in spore formation: how is exosporium formation directed to initiate at one pole of the forespore surface? Our findings argue that, to a large degree, this is achieved by a protein network, consisting at a minimum of the proteins CotE, CotO, and CotY, which assemble at the forespore prior to engulfment (Fig. 9A, left image, and B). The resulting structure, the cap, ensures that continued exosporium assembly proceeds relatively rapidly from a single location (Fig. 9A, middle image). We infer, based on previous work, that the coat proteins SpoIVA and ExsA (the homologue of SafA in B. subtilis) likely reside between CotE and the forespore membrane and anchor the network to the forespore (13,(39)(40)(41)(42). This model of assembly resembles the coat protein network in B. subtilis, suggesting that there may be a core outer layer assembly program that is conserved among all spore-forming Bacillaceae. Our results also argue that the impact of the cap on coat assembly potentially explains a significant difference in spore morphogenesis between B. anthracis and B. subtilis, the location at which electron-dense coat material first appears. These findings emphasize the limitations of the current model of spore assembly, which fails to account for important features of B. anthracis coat assembly. While coat formation in B. subtilis is well characterized and is the basis for understanding coat formation in all spore-forming species, the observation that the exosporium alters the pattern of coat assembly in B. anthracis argues for a need to thoroughly investigate spore formation in other species with other morphologically diverse outer layers.
The protein network connecting the exosporium to the forespore cannot be static throughout sporulation, since the exosporium migrates away from the forespore during sporulation ( Fig. 2A and 5A). We observed that the distance between the cap and forespore first expands from 35 nm at T 4 to 71 nm with the appearance of densely Bacillus anthracis Coat and Exosporium Assembly ® staining coat material at T 5 . Despite this expansion, the exosporium appears to be tightly associated with the coat at the leading edge of elongation ( Fig. 5B and C). Possibly, transient protein-protein interactions maintain the connection between the coat and exosporium, even as new coat proteins are added. We speculate that the network connecting SpoIVA, ExsA, and CotE has a degree of flexibility that accommodates assembly of new proteins into the coat. Following this expansion, the protein network appears to be disrupted, as a significant portion of the interspace widens during exosporium encirclement. This widening implies that the protein network is further expanded, or disrupted completely, to allow the incorporation of interspace material. We prefer a model in which the network is disrupted, since the gap between the coat and exosporium is not uniform around the spore. We speculate that the disruption occurs between CotE and CotO, with CotE remaining in the coat (13), while CotO either becomes an exosporium protein or is lost from the spore (Fig. 9). A key open question is the contents of the interspace, a problem we are presently investigating.
Our second key finding is that, unlike B. subtilis, not every B. anthracis coat layer initiates at the mother cell-proximal spore pole. Instead, as previously demonstrated in B. cereus, at least some coat layers initiate on the long side of the spore (20). We found that mutations preventing cap assembly result in a pattern of coat assembly MC, mother cell. At an early time in sporulation and before completion of forespore engulfment, a thin layer of material forms on the mother cell side of the septum (green). This layer likely contains at least some coat (SpoIVA, ExsA, CotE, and CotO) and interspace material but is transparent when visualized by TEM. The cap assembles on top of this layer (yellow) (left-most cartoon), resulting in the gap observed by TEM between the cap and the forespore membrane. Soon after engulfment, (i) the thin layer of coat/interspace material assembles around the entire forespore (see Discussion), (ii) exosporium proteins (red) assemble at the cap's edge, on the interspace material, and proceed to encircle the forespore, while the interspace material underneath the cap region widens, and (iii) densely staining coat features (blue) first appear at a location immediately ahead of the leading edge of the exosporium (middle cartoon). As exosporium assembly continues, the interspace widens along the entire spore, and coat and exosporium assembly complete (right-most cartoon). (B) Roles of CotE (E) and CotO (O) in linking the cap and forespore. CotE (anchored to the surface of the coat, which contains SpoIVA and ExsA) and CotO (at the underside of the cap, yellow) are part of a network which resides in the same location as the interspace (green) and connects the cap and forespore (left-most cartoon). As exosporium assembly proceeds, the interspace widens, and the interactions between CotE and CotO are disrupted throughout most of the interspace. However, at the leading edge of the assembling exosporium, the interspace is thin, and we speculate that the CotE-CotO interaction remains intact until the exosporium elongates completely around the forespore.
B. subtilis. We speculate that assembly of the cap, after the deposition of early synthesized coat proteins (including SpoIVA, ExsA, CotE, and CotO) at the mother cell-proximal pole, occludes further deposition of coat proteins at this location. We argue that coat proteins are unable to diffuse through the cap and therefore must traffic between the exosporium and the forespore membrane surface, resulting in late coat formation proceeding from the edge of the cap. After interspace widening, however, the cap no longer interferes with coat assembly, and electron-dense layers form where the cap used to be. We suggest that the mechanism of coat assembly may be more flexible than previously thought, changing as a result of the evolution of additional outer structures. In this view, no single model of sporulation is likely to completely describe spore formation in all species. The present work reinforces the view that orthologues of morphogenetic coat protein genes can have very different functions in different species (12,13,31,37). The morphogenetic coat proteins, identified in B. subtilis as being important for proper coat assembly, are often conserved in other Bacillaceae (43). Our results add to the growing list of morphogenetic proteins that are important for spore assembly in both B. subtilis and B. anthracis, hinting at a core assembly program that is conserved among Bacillaceae (10,12,13,32,37,39). Our results also demonstrate that while the sequences of these proteins are conserved, their exact function may not be; like CotE, CotO is important for coat assembly in B. subtilis and exosporium assembly in B. anthracis (13,31,32). These results suggest that orthologues of known morphogenetic proteins may play roles in the assembly of other novel outer layers, such as the canoe-shaped parasporal body (CSPB) of Brevibacillus laterosporus or the spikes projecting from the coat of Bacillus clausii (15). We speculate that while unidentified, and species-specific, morphogenetic proteins likely exist (5), it would be very informative to understand how the functions of known morphogenetic proteins diverge in different species. This information would likely reveal currently undetected roles for these proteins and provide insight into the ecological forces driving the diversification of spore outer layers.

MATERIALS AND METHODS
General techniques and plasmid construction. All strains were cultured in Luria-Bertani (LB) medium. When needed, E. coli strains were grown in the presence of spectinomycin (100 g/ml), kanamycin (25 g/ml), or carbenicillin (80 g/ml). B. anthracis strains were grown in the presence of polymyxin B (60 units/ml), kanamycin (20 g/ml), erythromycin (10 g/ml), chloramphenicol (10 g/ml), or spectinomycin (250 g/ml). Recombinant DNA techniques were performed as previously described (44). PCR products were purified using the QIAquick PCR purification kit (Qiagen). Plasmids (Table S1) were purified using the Wizard plasmid purification kit (Promega). Enzymes used for cloning were purchased from Thermo Fisher Scientific and used according to the manufacturer's instructions.
Generation of mutant B. anthracis strains. The cotO gene was initially disrupted via Campbell-type single-reciprocal recombination, as previously described (13,45), resulting in strain MGM76. Markerless deletion mutations were generated in cotO, exsY, and cotY, resulting in strains TJB124, TJB139, and TJB239, respectively, and confirmed by PCR (46,47). Single-copy complementation of the cotO deletion was achieved by integrating a plasmid expressing cotO (pTJB126) under its native promoter, immediately downstream of the eag1 gene (18). To generate a strain of B. anthracis overproducing CotO, a plasmid expressing cotO from its native promoter was introduced into B. anthracis by electroporation, as previously described (13,48,49).
To generate strains of B. anthracis producing a CotO-GFP fusion protein, cotO was inserted into plasmid pDG4099 (pMK4 bearing the enhanced GFP [eGFP] reporter cassette). The resulting plasmid (pBT4653) was introduced into B. anthracis using electroporation, followed by selection with chloramphenicol.
Sporulation. B. anthracis spores were generated by growth to exhaustion in Difco sporulation medium (DSM) (13). Spores were then washed three times and resuspended in distilled water. After resuspension in DSM, growth was monitored by measuring the optical density at 600 nm (OD 600 ) to determine the initiation of sporulation (T 0 ), defined as the end of exponential-phase growth. Twomilliliter samples were taken every hour during sporulation (T 1 -T 12 ) and either immediately fixed for analysis by transmission electron microscopy (TEM) (13) or stored at Ϫ80°C until proteins could be extracted for Western blot analysis.
Purification of CotO and CotE and the generation of anti-CotO antibodies. The CotE and CotO proteins were overproduced and nickel-affinity purified, using the QIAexpressionist system, according to the manufacturer's instructions (Qiagen). Purified CotO was used to generate anti-CotO antibodies in New Zealand White rabbits (GenScript, Piscataway, NJ), which were protein A affinity purified.
Protein isolation from spores and sporangia. Coat and exosporium proteins were isolated from harvested spores by resuspending in Laemmli buffer, followed by three rounds of heating (100°C for 5 min) and then vortexing (1 min) (13). Soluble proteins were extracted from sporangia by resuspending the cells in phosphate-buffered saline (PBS) and bead beating (50). To characterize forespore-associated proteins in sporangia, cells were resuspended in Laemmli buffer and lysed by bead beating, and the forespore-associated proteins were extracted by three rounds of heating (100°C for 5 min) and vortexing (1 min). Samples were then centrifuged at 17,000 ϫ g for 5 min, and the supernatant was collected.
Immunofluorescence microscopy. Washed spores (10 l) were adhered to the wells of a multiwell microscope slide (MP Biomedicals) coated with poly-L-lysine (Sigma-Aldrich). Slides were washed with PBS, treated with blocking solution (2% BSA in PBS) for 30 min, washed 9ϫ with PBS, and incubated for 1 h with either polyclonal anti-BclA (diluted 1:1,000 in blocking buffer; BEI Resources) or anti-CotE (diluted 1:50,000 in blocking buffer) antibodies. Slides were washed 9ϫ with PBS and incubated for 1 h with goat anti-rabbit antibodies conjugated to Alexa Fluor 488 (1:500 in blocking buffer; Molecular Probes). Slides were washed 9ϫ with PBS, and coverslips were mounted using PermaFluor Mountant mounting medium (Thermo Scientific). Spores were then visualized using a Leica DM IRB fluorescence microscope. Images were collected with a MagnaFire cryo-cooled charge-coupled-device (CCD) camera and processed using the ImageJ software.
In vitro interaction between CotE and CotO measured by ELISA. The potential for CotE and CotO to interact in vitro was measured using an enzyme-linked immunosorbent assay (ELISA), as previously described (51). CotO was fixed to 96-well MaxiSorp plates (Thermo Scientific) at concentrations of 0, 0.125, 0.25, and 0.5mg/well and incubated for 1 h with blocking buffer (5% BSA, 0.1% Tween 20 in TBS), before incubating for 1 h with CotE (0 to 12 g/ml in blocking buffer). The amount of CotE retained in the wells was determined using anti-CotE antibodies (1:10,000 in blocking buffer), followed by antirabbit-IgG alkaline phosphatase conjugate antibodies (1:2,000 in blocking buffer). Finally, wells were developed for 30 min with 200 l of 4-nitrophenol phosphate before the reaction was stopped with 50 l of 3 M NaOH, and the absorbance at 405 nm (A 405 ) was measured.


extfieldsofstudy: Chemistry Medicine

text:
Changes in the lipopolysaccharide of Proteus mirabilis 9B-m (O11a) clinical strain in response to planktonic or biofilm type of growth

The impact of planktonic and biofilm lifestyles of the clinical isolate Proteus mirabilis 9B-m on its lipopolysaccharide (O-polysaccharide, core region, and lipid A) was evaluated. Proteus mirabilis bacteria are able to form biofilm and lipopolysaccharide is one of the factors involved in the biofilm formation. Lipopolysaccharide was isolated from planktonic and biofilm cells of the investigated strain and analyzed by SDS–PAGE with silver staining, Western blotting and ELISA, as well as NMR and matrix-assisted laser desorption ionization time-of-flight mass spectrometry techniques. Chemical and NMR spectroscopic analyses revealed that the structure of the O-polysaccharide of P. mirabilis 9B-m strain did not depend on the form of cell growth, but the full-length chains of the O-antigen were reduced when bacteria grew in biofilm. The study also revealed structural modifications of the core region in the lipopolysaccharide of biofilm-associated cells—peaks assigned to compounds absent in cells from the planktonic culture and not previously detected in any of the known Proteus core oligosaccharides. No differences in the lipid A structure were observed. In summary, our study demonstrated for the first time that changes in the lifestyle of P. mirabilis bacteria leads to the modifications of their important virulence factor—lipopolysaccharide.

Introduction
Bacteria belonging to the genus Proteus are rod-shaped, Gram-negative, nonsporulating microorganisms which, due to their biochemical properties, are widely distributed in environment [1,2]. The systematics of these bacteria have been changed several times, since they were first described by Hauser in 1885, and currently, the genus is divided into the following species: P. mirabilis, P. vulgaris, P. penneri, P. hauseri, P. myxofaciens, P. terrae sp. nov., and P. cibarius sp. nov. as well as three unnamed Proteus genomospecies: 4, 5, and 6 [1,[3][4][5][6]. Except for P. myxofaciens, P. terrae sp. nov., and P. cibarius sp. nov., the strains from the mentioned species are classified as opportunistic pathogens. They are responsible for various human diseases such as meningitis, pneumonia, wounds and bone infections, brain abscesses, and bacteremia [7][8][9]. The most frequent among Proteus-mediated infections are recurrent urinary tract infections (UTIs), whose hallmark is formation of urinary stones, containing struvite and carbonate apatite. P. mirabilis strains are mentioned as the main etiological factor of these infections. The frequency of P. mirabilis infections rises during catheterization, especially in long-term catheterized patients [3,10,11]. The recurrent character and formation of urinary stones are the main problems in combating P. mirabilis infections, but bacterial biofilm, especially that formed on the catheter surface, may also complicate the treatment [12]. A unique feature of biofilms developed by Proteus rods is the fact that they are covered with struvite and apatite crystals, which precipitate in urine. Such biofilms are less sensitive to antimicrobial drugs, as well as more resistant to the host immune system [13,14].
Biofilm is a type of growth in which bacteria can attach to a solid surface and develop a well-organised structure. This form of bacterial population is immersed in self-produced, extracellular polymeric substances, called matrix. Many various factors are involved in the formation of a complex biofilm structure. One of these factors is lipopolysaccharide (LPS)-a component of the outer membrane of Gram-negative bacteria. LPS consists of three regions: the most external O-polysaccharide (OPS), the core region, and lipid A. LPS possessing all three regions is characteristic for smooth forms of bacteria and is not only responsible for the emergence of a wide spectrum of biological reactions in the cells of macroorganisms (especially endotoxic activity connected with the lipid A part), but also plays a role in the interactions between the bacterial cells and cells of an infected host [15][16][17]. Due to their acidic character, O-polysaccharide chains of Proteus LPS contribute to the urinary stones occurrence, and also participate in biofilm matrix formation [1,18]. This extracellular matrix enhances the adherence of Proteus rods to the solid surface and biofilm development.
It has been well demonstrated that sessile bacteria differ greatly from their planktonic counterparts, especially with regard to drug resistance. Many bacteria living in biofilm exhibit reduced (from 10 up to 1000 times) susceptibility to a broad range of antimicrobial agents in comparison with their planktonic forms [19,20]. Benamara and co-workers [21] showed that in Pseudomonas aeruginosa sessile cells, the inner membrane lipidome shows a decrease in the amount of the uneven-numbered chain phospholipids and accumulation of phosphatidylethanolamines with long-chain lipids. Little is known about changes in the structure of lipopolysaccharides (LPSs) and their properties depending on the form of bacterial growth; however, also in P. aeruginosa, it has been demonstrated that the chemical structure of this component, essential for the outer membrane stability, can be modified [22]. The lack of data concerning possible modifications of Proteus bacteria LPSs in different forms of growth encouraged us to investigate this problem. Therefore, the aim of this study was to check whether a form of growth (planktonic or biofilm) of the investigated P. mirabilis strain leads to structural modifications of its LPS, changes in the serological reactions profile or particular biological properties.

Bacterial strain, antiserum, culture conditions, extraction of lipopolysaccharides
The Proteus mirabilis 9B-m (O11a) strain was isolated from urine of a patient of Barlicki Hospital in Łódź (Poland), and was kindly provided by D. Drzewiecka, Ph.D., the Laboratory of General Microbiology, University of Łódź. The strain was selected for the study on the basis of the results of preliminary investigation (data not shown) which demonstrated its great ability to form biofilm, in flat-bottomed polystyrene plates, among 30 tested clinical Proteus spp. isolates. Bacteria were stored in Luria broth with 25% glycerol, at − 80 °C. The polyclonal rabbit serum against the investigated P. mirabilis 9B-m strain (obtained as described by Sidorczyk and co-workers [23]) came from the Laboratory of General Microbiology, University of Łódź, Poland.
Planktonic as well as biofilm-associated bacteria were cultivated in nutrient broth (BTL, Poland). Biomass of planktonic cells was obtained by aeration of the bacteria in liquid medium at 37 °C for 18 h. Next, the bacteria were killed by the addition of phenol at 1% concentration, washed twice with distilled water, centrifuged, and finally lyophilized.
To obtain biomass of sessile bacteria, the microorganisms were cultured at 37 °C for 72 h in a sterile glass bioreactor with a constant medium flow (1 ml per minute), fitted with vertical glass plates for biofilm formation. Obtained biofilm was collected manually, washed twice with water and centrifuged. Next, the bacteria were inactivated by moist heat sterilization (115 °C, 20 min) and lyophilized.
To demonstrate that after changing the lifestyle from biofilm to the planktonic form the investigated strain regain its ability to synthesize full-length-LPS, sessile bacteria were transferred to liquid medium and cultured in planktonic conditions described above.
LPSs from dry masses of planktonic and biofilmforming bacteria were extracted using the phenol-water method by Westphal and Jann [24], modified as described by Palusiak [25]. They were subsequently used for silver staining, stimulation of THP-1 cells, and as antigens in serological assays, as well as for the chemical analysis of O-polysaccharides, core regions, and lipids A.

Silver staining of lipopolysaccharides and serological assays
The LPSs (3 µg) from both types of cultures were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with further Alcian blue prestaining [26] and subsequent silver staining of separated samples according to Tsai and Frasch [27].
Serological investigations were performed using the ELISA and the Western-blotting technique described in detail by Sidorczyk et al. [23]. For the Western blotting, 2 µg (short path) and 5 µg (extended path of separation) of each LPS in the loading buffer were added per single lane. In ELISA, native as well as adsorbed polyclonal O-antisera against P. mirabilis 9B-m were used. Adsorption of the antibodies from the serum was carried out by a threefold addition of whole cells of the planktonic or sessile bacteria to the diluted serum according to the method given by Drzewiecka et al. [28].

THP-1 cell culture, differentiation, and lipopolysaccharide stimulation
THP-1 cells (ATCC TIB-2) were cultured in RPMI 1640 medium, supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 U penicillin ml −1 , and 100 µg ml −1 streptomycin (all components obtained from Cytogen). The culture was performed at 37 °C, in a moist atmosphere containing 5% CO 2 . THP-1 cells in RPMI medium (at a density of 10 5 ml −1 ) were differentiated into a macrophage-like phenotype by the treatment with PMA (Phorbol Myristate Acetate); (50 nM, Sigma) for 48 h. Next, the cells were stimulated with LPSs suspended in a fresh medium, obtained from biofilm and planktonic cultures of the P. mirabilis 9B-m strain (62.5-1000 ng ml −1 ) at 37 °C for 48 h. LPS of E. coli O55:B5 (Sigma, kindly provided by A. Torzewska, Ph.D., the Department of Biology of Bacteria, University of Łódź) was applied as a positive control of the THP-1 activity. THP-1 macrophages, not stimulated with LPS, were used as a negative control. Next, THP-1 cells were centrifuged, and culture supernatants were collected and frozen at − 20 °C, until analyses. The studies were performed in three independent replicates.

Assessment of the tumour necrosis factor alpha (TNF-α) level in response to lipopolysaccharide stimulation, statistical analysis
TNF-α concentrations in obtained culture supernatants were determined using the Ready-Set-Go ELISA kit (eBioscience) according to the manufacturer's instructions. Statistical analyses were performed using CCS StatSoft Statistica 12.5 PL. Differences between TNF-α levels in response to LPS stimulation were evaluated using the nonparametric Kruskal-Wallis test (p values < 0.05 were considered significant).

Degradation of the lipopolysaccharide, isolation of lipid A, O-polysaccharide, and core oligosaccharide
Acid degradation of LPS was performed with 1% aqueous acetic acid at 100 °C until precipitation of lipid A (2 h). The precipitate was separated by centrifugation (13,000×g, 20 min), and the supernatant was fractionated by gel-exclusion chromatography on a column (56 × 2.6 cm) of Sephadex G-50 (Amersham Biosciences, Sweden) in 0.05 M pyridinium acetate buffer (pH 4.5) monitored using a differential refractometer (Knauer, Germany) to give a high-molecular O-polysaccharide fraction (I) and core oligosaccharide fractions (II): one from planktonic cells (IIa) and two from biofilm-associated cells (IIa and IIb).
NMR spectra of the polysaccharides were recorded with a Bruker DRX-500 spectrometer for solutions in 2 H 2 O at 30 °C using internal acetone (δ H 2.225, δ C 31.45) as reference. Standard Bruker software (XWINNMR 2.6) was used to acquire and maintain the NMR data. A mixing time of 200 and 100 ms was applied in two-dimensional TOCSY (total correlation spectroscopy) and ROESY (rotating-frame Overhauser spectroscopy) experiments, respectively.

Analyses of core oligosaccharides and lipids A
Core oligosaccharides were analyzed by ESI HR MS (electrospray ionization high-resolution mass spectrometry) in the negative mode using a micrOTOF instrument (Bruker Daltonics). Capillary entrance voltage was set to 3200 V, and the drying nitrogen temperature was 180 °C. The samples were dissolved in a 1:1 (v/v) H 2 O/MeCN mixture (∼ 50 ng µl −1 ) and sprayed at a flow rate of 3 µl min −1 .

Results
The present study was designed to investigate changes in the LPS of the P. mirabilis 9B-m strain in response to a switch between the planktonic and biofilm form of growth. The LPSs from both types of cultures were obtained (the yields amounted to 6.26 and 5.3% for planktonic and biofilm forms, respectively) and first characterized by SDS-PAGE and silver staining. Figure 1 shows an electropherogram of the LPSs from both (planktonic and biofilm) types of P. mirabilis 9B-m cultures. LPS from the planktonic culture displays a pattern with a fast-migrating fraction corresponding with low-molecular mass (O-polysaccharide-lacking) molecules, as well as a slower migrating, high-molecular mass fraction referring to the molecules containing a longchain O-antigen region. In contrast, LPS derived from sessile cells differs in its electrophoretic pattern by a decrease in the slow-migrating fraction. LPS containing high-molecularweight OPS was observed again in the cells reverted to the planktonic form.
We have also checked whether the changes in the lifestyle of P. mirabilis 9B-m can affect the profile of serological reactions of LPSs from both types of cultures. To answer this question, serological investigations using the ELISA and Western-blotting techniques were performed. In ELISA, the reactivity of the investigated LPSs was examined with polyclonal O-antiserum against P. mirabilis 9B-m (obtained for planktonic forms). To compare serological specificities of the used LPSs, two variants of the serum were applied: non-adsorbed and adsorbed with P. mirabilis 9B-m biofilm or planktonic cells. The results of this part of work are presented in Table 1. Antigens from biofilm or planktonic cultures reacted similarly, but non-identically with native O-antiserum (a slightly weaker reaction was observed for LPS from biofilm cells). In addition, the use of the adsorption procedure allowed noting some differences in the reactivity profiles. There was no reactivity observed for both LPSs when the antiserum was adsorbed with planktonic cells (as was mentioned above, O-antiserum had been obtained for planktonic cells). However, when sessile bacteria were added to the native antiserum to remove cross-reacting antibodies, no complete abolition of reactivity with the LPS from planktonic cells was noted. The serum still contained antibodies which reacted with this LPS to the titre 1:4000 (Table 1). These differences in reactivity of both types of LPSs prompted us to use the Western-blotting technique. The results of this stage of investigations are presented in Fig. 2. Qualitative differences in the LPS separation profiles are shown both on the short and on the extended path of electrophoretic separation. The investigated O-antiserum recognized and reacted much stronger with the epitopes in the LPS from planktonic cells. The reaction with the LPS from biofilm cells was much weaker in the fraction of molecules containing the highest molecular-weight O-antigen and, which is particularly noticeable, there was no visible reaction with the fast-migrating LPS fraction (lipid A-core oligosaccharide).
The obtained results suggested the form of growth of the investigated P. mirabilis clinical strain might influence the  changes in the length of polysaccharide chains of the LPS molecules on the bacterial cells, or even the modification of the chemical structure of this component. Therefore, in the next stage of our investigation, chemical analyses were performed.

Determination of the O-polysaccharide structure
Recently, the structure of the OPS of LPS from planktonic P. mirabilis 9B-m cells has been elucidated [30]. It has been found that the 9B-m OPS is closely related to that of P. mirabilis 24/57 (serogroup O11), which differs by the presence of an additional side-chain Glc residue only [31] (Fig. 3). Therefore, it has been proposed to divide Proteus serogroup O11 into two subgroups, including subgroup O11a for the strain 9B-m [30]. Our studies by GLC and NMR spectroscopy showed that the OPS of LPS from biofilm-associated P. mirabilis 9B-m cells had the same structure as that from planktonic cells. The 1 H and 13 C NMR spectra obtained for the samples from both types of cultures were essentially identical to each other and to those reported earlier [30]. This finding indicates that the outer part of the P. mirabilis 9B-m LPS having the structure shown in Fig. 3 did not change under the applied conditions.

Analyses of the core oligosaccharide
Fraction IIa core oligosaccharides were studied by negative ion mode high-resolution electrospray ionization mass spectrometry. The charge deconvoluted mass spectra of the compounds from both kinds of cells (Fig. 4a, b) showed the same major [M−H] − ion peak at m/z 2311.74, which could be assigned to HexNAc 1 HexN 1 HexA 2 Hex 1 Hep 5 anhKdo 1 Ara4N 1 PEtN 1 compound, where anhKdo, Ara4N, and PEtN indicate an anhydro form of 3-deoxy-d-manno-oct-2-ulosonic acid, 4-amino-4-deoxyarabinose, and phosphoethanolamine, respectively. This and all other peaks in the mass spectra were accompanied by less intense peaks of higher masses by 18 a.m.u. for the corresponding compounds containing a regular Kdo residue. The 9B-m fraction IIa core may represent a minor core species of P. mirabilis O34 LPS (Fig. 4, inset), which differs from the major core species by the lack of one of the PEtN groups and a terminal Hex residue [32]. In addition, both spectra contained a minor ion peak at m/z 2381.82 for an unidentified core oligosaccharide, and the spectrum of the sample from planktonic cells also contained a peak at m/z 2258.81 for the same oligosaccharide lacking a PEtN group. The spectrum of the sample from the sessile cells showed a minor peak for the compound lacking an Ara4N residue.
The most important difference between core oligosaccharides of LPS from sessile and planktonic cells is presented in Fig. 4c. The mass spectrum of the fraction IIb core oligosaccharide, which was present in the products from biofilm-associated cells only, showed [M−H] − ion peaks at m/z 1978.54 and 1847.48 for compounds differing by the presence or absence of an Ara4N group, as well as for the corresponding monoacetylated compounds. It needs to be highlighted that these peaks could not be assigned to any known Proteus core oligosaccharide [32,33].

Analyses of lipid A
To compare lipid A samples isolated from LPS of planktonic and biofilm-associated P. mirabilis 9B-m cells, MALDI-TOF MS analysis was performed. The spectra of lipids A (Fig. 5) showed heterogeneity and had an identical pattern of ions indicating a common structure of both examined lipid A samples. The interpretation of the observed ions was in agreement with previously published data [34]. The most abundant ion at m/z 1824.02 represented the main population in the P. mirabilis 9B-m lipid A, corresponding to

Analysis of TNF-α secretion
Since LPS is considered as a classic pro-inflammatory stimulus that leads to TNF-α release, we checked whether the described differences observed in LPSs from both: planktonic and sessile cells influenced the level of TNF-α secretion by THP-1 monocytic cell-line-derived macrophages (results shown in Fig. 6, detailed information under the chart).
LPSs obtained from both planktonic and biofilm-associated P. mirabilis 9B-m cells stimulated THP-1 maturated macrophages much more weakly than, used as a positive control, E. coli O55:B5 LPS. No significant differences (p > 0.05) in the TNF-α concentration were observed between LPSs derived from P. mirabilis 9B-m cells cultured as free-floating and sessile microorganisms (irrespective of the used LPS concentration).

Discussion
It has been demonstrated that many bacteria exhibit different properties while growing in liquid media or on a solid surface as biofilm [35], but little is known about the influence of the growth type on the modification of LPS of Gram-negative microorganisms. Most data refer to P. aeruginosa strains and point to the alteration in both OPS and more internal LPS areas [22,36,37]. P. mirabilis bacilli often form biofilm during urinary tract infection (especially on the intraluminal catheter surface). There is no information how changes of the lifestyle modify the synthesis of LPS of Proteus spp. cells. To the best of our knowledge, our report is the first to indicate that this outer membrane heteropolymer may undergo modifications when Proteus bacteria mode of life is changed.
We have demonstrated that LPS of the P. mirabilis 9B-m clinical strain undergoes some alterations depending on planktonic or biofilm lifestyle. The investigated P. mirabilis strain represents the described Proteus O11 serotype, which seems to be one of the most frequently isolated from patients [38], also in central Poland [30]. Such high frequency of isolation of Proteus spp. strains possessing an O-antigen identical with that of the investigated strain indicates that the chemical structure of OPS of these strains may be beneficial during macroorganism colonization. P. mirabilis 9B-m strain intensively forms biofilm-in the group of 30 clinical isolates pre-selected for the study (including 12 clinical Proteus O11 isolates, as well as 10 and 8 strains representing O78 and O3 serotypes, respectively), it is one of the three strains form biofilm most efficiently (data not shown). At the same time, the structure of the O-specific part of its LPS was established [30]; thus, this strain was the best candidate for the presented research.
Using the silver-staining technique we showed that in response to changes in the lifestyle, the investigated strain synthesized LPS containing an O-antigen exhibiting a different molecular mass (Fig. 1). In sessile cells, there was observed a reduced number of long-chain O-specific units, whereas in planktonic cells (also those reverted from biofilm), the full-length OPS phenotype was visible. These findings indicate that the observed changes are growth state dependent.
This conclusion was confirmed by the results of Western blotting (Fig. 2), where only a weak reaction was seen within the fraction of the biofilm-associated LPS corresponding to the molecules with a long-chain OPS. The shortening of the LPS polysaccharide chains in sessile cells was also observed in the studies of other opportunistic pathogens like P. aeruginosa or food-borne Cronobacter sakazakii [22,36,37,39] as well as in the environmental nitrogen-fixing Bradyrhizobium japonicum strains [40].
The serological results suggested that the LPS of P. mirabilis 9B-m biofilm-living strain could also undergo structural modifications, because changes in its serospecificity were observed. Various reaction titres were found when the native (non-adsorbed) O-antiserum was checked in ELISA with the LPS from planktonic or sessile cells. Using the serum adsorbed by biomass of planktonic cells Such bilateral abolition of reactivity did not take place when biofilm-forming bacteria were used for adsorption (Table 1). In our previously reported investigations concerning serological classification of Proteus spp. strains, we have demonstrated several times that some pool of antibodies remaining in the serum are observed when slight changes in the O-antigen structure or modifications of the LPS core region take place [41][42][43][44]. Likewise, in the Western-blotting technique, the differences between the investigated types of LPSs were revealed (Fig. 2). Anticore antibodies in the O-specific serum (against planktonic P. mirabilis 9B-m cells) used in the study have no ability to bind the epitopes located in the prevalent fast-migrating molecules of the LPS from sessile cells. This clearly demonstrate that serospecificity of these epitopes in biofilmassociated LPS is changed that pointed to differences in the chemical structure of LPS in the biofilm in the reference to planktonic mode of growth. NMR spectroscopy revealed that OPSs from the planktonic and biofilm-associated cells possessed the same structure (Fig. 3); therefore, the form of growth of the P. mirabilis 9B-m strain had no effect on the chemistry of its OPS. The obtained results indicate that changes observed in the slow-migrating fraction of biofilm-associated LPS using silver staining (Fig. 1) as well as Western-blotting technique (Fig. 2) were related only to the loss of longchain OPS, but not to the modification of the chemical structure of this region.
Comparison of the core region structures of LPSs from planktonic and sessile cells revealed changes in the chemistry of that inner part of the P. mirabilis 9B-m LPS. We have observed that when bacteria grow in biofilm, the core region in their LPS gains an additional oligosaccharide (ion peaks at m/z 1978.54 and 1847.48, Fig. 4c). As was mentioned above, none of the currently known core oligosaccharides in the genus Proteus was characterized by this kind of peaks. Moreover, this is the first information on the extension of the core part of LPS in bacterial cells forming a biofilm. Most literature data available indicate that truncation of the outer core in LPS is connected with the sessile mode of bacterial growth, and significantly influences biofilm formation. Isogenic mutants of the P. aeruginosa PAO1 wild strain, with a truncated outer core, produced LPS lacking the long-chain OPS in biofilm. The study also demonstrated that mutants without OPS showed increased cohesion (cell-to-cell adherence) and adhesion to the glass, which resulted in a more intensive formation of biofilms [45]. This confirmed an earlier observation indicating the loss of polysaccharide chains in LPS of a P. aeruginosa biofilm-forming strain [46]. The importance of the modification of the outer core for the intensification of the biofilm formation has also been demonstrated in Campylobacter jejuni cells, the species which does not possess the O-antigen in LPS, as well as in series of deep rough mutants of E. coli. In the both case, mutants characterized by the significant shortening of the core oligosaccharide formed biofilm more intensively than the parental strain [47,48]. All the above observations indicate that the modification of the LPS core region during biofilm development resulted from the strain's increased ability to adhere through the loss of OPS. Although in most cases, the reduction of OPS in LPS was probably due to the truncation of the core region, in the case of the investigated P. mirabilis strain, a decrease in the number of full-length O-specific chains attached to the core may have resulted from the emergence of additional oligosaccharide in this region. Further analyses including the determination of the exact structure of the new core oligosaccharide, and the use of a mutant lacking this component would allow elucidating the role played by this additional oligosaccharide during the bacterial biofilm development.
Literature data also show that modification of LPS in biofilm can be related to the lipid A part [22,49]. In the biofilm formed by P. aeruginosa, the changes in lipid A molecules are manifested by increased hydroxylation of the secondary fatty acid chains [22] and in Escherichia coli by increased palmitoylation [49]. In the first case, the observed modification led to a slightly higher inflammatory response: TNF-α and interleukin-6 (Il-6) production by human monocytes. Palmitoylation observed in the last case may have enhanced resistance to some antimicrobials and increase survival of bacteria growing in biofilm (in a rat model of catheter infection). MALDI-TOF MS analysis of lipids A derived from planktonic as well as biofilmassociated P. mirabilis 9B-m strain showed no differences in the structures of these molecules (Fig. 5). Moreover, the results were confronted with those obtained by Sidorczyk and co-workers [34] for lipid A of Proteus mirabilis Remutant and two other P. mirabilis clinical isolates (data not showed), and again, no alteration was found. This may indicate that in P. mirabilis cells, changes in the form of growth do not affect the lipid A synthesis. Interestingly, we have also found no increase in the level of palmitoylation in lipids A under the investigated conditions. The lack of the lipid A modification proved in chemical analyses in the P. mirabilis 9B-m planktonic and biofilm-associated strain (and in mentioned two other clinical strains-data not showed) was also reflected by in the results of TNF-α production by THP-1 monocytic cell-line-derived macrophages. In the studies on P. aeruginosa strains [22], it was shown that modifications of lipid A occurring during these microorganisms grow in the biofilm and resulted in changes in the TNF-α secretion. In contrast, in our study, no statistically significant differences were observed in the level of this cytokine produced in response to the presence of LPS from both growth states of the P. mirabilis 9B-m isolate (as well as of the two other clinical P. mirabilis strains mentioned above-data not shown).
In conclusion, we have shown that in P. mirabilis, the changes of the form of cells growth may induce alterations in the LPS synthesis. These modifications may affect the most external part of LPS (O-specific part), and the core region but do not concern lipid A part.
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Study of the association of 17 lipid-related gene polymorphisms with coronary heart disease

Objective Blood lipids are well-known risk factors for coronary heart disease (CHD). The aim of this study was to explore the association between 17 lipid-related gene polymorphisms and CHD. Methods The current study examined with 784 CHD cases and 739 non-CHD controls. Genotyping was performed on the MassARRAY iPLEX® assay platform. Results Our analyses revealed a significant association of APOE rs7259620 with CHD (genotype: χ2=6.353, df=2, p=0.042; allele: χ2=5.05, df=1, p=0.025; recessive model: χ2=5.57, df=1, p=0.018). A further gender-based subgroup analysis revealed significant associations of APOE rs7259620 and PPAP2B rs72664392 with CHD in males (genotype: χ2=8.379, df=2, p=0.015; allele: χ2=5.190, df=1, p=0.023; recessive model: χ2=19.3, df=1, p<0.0001) and females (genotype: χ2=9.878, df=2, p=0.007), respectively. Subsequent breakdown analysis by age showed that CETP rs4783961, MLXIPL rs35493868, and PON2 rs12704796 were significantly associated with CHD among individuals younger than 55 years of age (CETP rs4783961: χ2=8.966, df=1, p=0.011 by genotype; MLXIPL rs35493868: χ2=4.87, df=1, p=0.027 by allele; χ2=4.88, df=1, p=0.027 by dominant model; PON2 rs12704796: χ2=6.511, df=2, p=0.039 by genotype; χ2=6.210, df=1, p=0.013 by allele; χ2=5.03, df=1, p=0.025 by dominant model). Significant allelic association was observed between LEPR rs656451 and CHD among individuals older than 65 years of age (χ2=4.410, df=1, p=0.036). Conclusion Our study revealed significant associations of APOE, PPAP2B, CETP, MLXIPL, PON2, and LEPR gene polymorphisms with CHD among the Han Chinese.

Introduction
Coronary heart disease (CHD) is characterized by atherosclerosis, which leads to vascular stenosis and occlusion. Dyslipidemia is known as a risk factor for CHD (1). Blood lipids have been reported to predict the risk of CHD (2,3), which encouraged us to examine the association of lipid-related gene polymorphisms with CHD (4).
On the basis of previous studies, the aim of this study was to assess the association of 17 lipid-related gene polymorphisms with CHD in the Han Chinese.

Sample collection
A total of 784 CHD cases and 739 non-CHD controls were enrolled in the current study. The enrolled cases were classified on the basis of the findings of standardized coronary angiography according to the Seldinger's method (18). The classification details have been reported in our previous studies (19)(20)(21). The study protocol was approved by the ethical committees of Ningbo First Hospital and Ningbo University. Written informed consent was obtained from all the participants.
Single nucleotide polymorphism (SNP) genotyping DNA extraction and quantification were performed as previously described (22,23). Genotyping was performed on the Mas-sARRAY iPLEX ® assay platform (Sequenom, San Diego, CA, USA). The primer sequences and the details of the selected SNPs are presented in Table 1.

Statistical analysis
Genotype and allele distributions were compared between the two groups using the Chi-squared test. Differences were

Results
As shown in the Table 2, 17 SNPs were detected in the upstream regions of lipid-related genes. LEPR rs6656451 was located in the upstream region of a transcript isoform. Among the tested SNPs, APOE rs7259620 was significantly associated with CHD [genotype p=0.042 (df=2), allele p=0.025 (df=1), OR (95% ADIPOQ rs266729, VLDLR rs7852409, and PPAP2B rs72664392 were excluded from further analyses since their genotypic distributions did not meet HWE in the controls (data not shown). In addition, the association of the remaining 12 SNPs with CHD could not be evaluated in the total samples (p>0.05).
Age-based subgroup analyses revealed that CETP rs4783961, MLXIPL rs35493868, and PON2 rs12704796 were significantly as-  Table 4). In addition, LEPR rs656451 was associated with CHD in participants older than 65 years of age (χ 2 =4.410, df=1, p=0.036 by allele, Table 4). No other SNPs were associated with CHD in the age-based subgroup analyses.

Discussion
In the present study, we examined the association of 17 lipidrelated SNPs with CHD among 784 CHD cases and 739 non-CHD controls. We identified a male-specific association of APOE rs7259620 with CHD. Meanwhile, we also found a significant association of PON2 rs12704796 with CHD among participants younger than 55 years of age. On the genotypic level, we identify a significant association of CHD with PPAP2B rs72664392 in females and CETP rs4783961 in participants younger than 55 years of age. On the allelic level, we identified a significant association of CHD with MLXIPL rs35493868 in participants younger than 55 years of age and LEPR rs6656451 in participants older than 65 years of age.
Previous studies have indicated that APOE is significantly associated with CHD. APOE ε2 was shown to reduce the risk of CHD by 20% (24), whereas ε4 was shown to increase the risk of CHD by approximately 42% compared with ε3/ε3 genotype (25). Epidemiological evidence has shown that males are at a higher risk of CHD than females worldwide (26). Gender disparity has been found in APOE-related cardiovascular disease (27). In the previous studies, we have shown that CHD risk was gender-dependent in the Han Chinese and that APOE rs4420638 polymorphism was significantly associated with increased CHD risk in male Han Chinese (28). This observation might be explained by the differences of hormonal profiles, smoking status, alcohol-drinking, occupation, and dietary habits between males and females (29,30). In the present study, we also identified a novel genetic variant of APOE associated with CHD in males.
PPAR2B is a negative regulator of inflammatory cytokines, leucocyte adhesion, cell survival, and migration in human primary aortic endothelial cells (31), suggesting that PPAR2B can protect blood vessel against inflammation (32). Mechanosensitive PPAP2B plays a critical role in promoting anti-inflammatory phenotype and maintaining the vascular integrity of endothelial monolayer under atheroprotective flow (33). However, discrepancies exist regarding the association of PPAP2B with CHD (34). PPAP2B rs1759752 is associated with increased CHD risk in males, while PPAP2B rs12566304 is associated with a decreased CHD risk in females (34). Other studies have shown that PPAP2B rs17114036-A is associated with CHD (35,36). In contrast, PPAP2B rs17114036 is not associated with CHD after adjustments for gender (16,35). Here, we identified a novel   polymorphism (rs72664392) in PPAP2B promoter associated with CHD in females. This finding could be partly explained by the particular genetic background. Aging is a pivotal risk factor for CHD (37,38). The incidence of CHD in people younger than 40 years of age is 0.6%, and it increases two-fold or more with every 10-year increase in age (39). High adiponectin concentration has been shown to be associated with a lower risk of CHD in people younger 65 years of age (40). In people younger than 55 years of age, PON2 rs12704796-A has been shown to increase the risk of CHD by 43.1%, whereas MLXIPL rs35493868-G has been shown to reduce the risk of CHD by 38.1%. In addition, LEPR rs6656451-T has been reported to reduce the risk of CHD by 45.5% among people older than 65 years of age.

Study limitations
Our results did not demonstrate a significant association of 11 of the tested SNPs with CHD. A power analysis revealed that these SNPs showed a minimal or moderate power to detect a significant association in the current study (power=0.074-0.425). In addition, several SNPs did not present reliable association results in genderand age-based subgroup analyses since their genotype distributions did not meet HWE in the controls. Future association study of these SNPs with CHD is warranted in other cohorts.

Conclusion
Our study demonstrated the gender-or age-dependent association of six SNPs (APOE rs7259620, PPAP2B rs72664392, CETP rs4783961, PON2 rs12704796, MLXIPL rs35493868, and LEPR rs6656451) CHD in Han Chinese population. However, future replication is required to validate our findings.