Bacillus spp. and Clostridium spp. form a specialized cell type, called a spore, during a multistep differentiation process that is initiated in response to starvation. Spores are protected by a morphologically complex protein coat. The Bacillus anthracis coat is of particular interest because the spore is the infective particle of anthrax. We determined the roles of several B. anthracis orthologues of Bacillus subtilis coat protein genes in spore assembly and virulence. One of these, cotE, has a striking function in B. anthracis: it guides the assembly of the exosporium, an outer structure encasing B. anthracis but not B. subtilis spores. However, CotE has only a modest role in coat protein assembly, in contrast to the B. subtilis orthologue. cotE mutant spores are fully virulent in animal models, indicating that the exosporium is dispensable for infection, at least in the context of a cotE mutation. This has implications for both the pathophysiology of the disease and next-generation therapeutics. CotH, which directs the assembly of an important subset of coat proteins in B. subtilis, also directs coat protein deposition in B. anthracis. Additionally, however, in B. anthracis, CotH effects germination; in its absence, more spores germinate than in the wild type. We also found that SpoIVA has a critical role in directing the assembly of the coat and exosporium to an area around the forespore. This function is very similar to that of the B. subtilis orthologue, which directs the assembly of the coat to the forespore. These results show that while B. anthracis and B. subtilis rely on a core of conserved morphogenetic proteins to guide coat formation, these proteins may also be important for species-specific differences in coat morphology. We further hypothesize that variations in conserved morphogenetic coat proteins may play roles in taxonomic variation among species.

Glaucoma, the most frequent optic neuropathy, is a leading cause of blindness worldwide. Death of retinal ganglion cells (RGCs) occurs in all forms of glaucoma and accounts for the loss of vision, however the molecular mechanisms that cause RGC loss remain unclear. The pro-apoptotic molecule, Fas ligand, is a transmembrane protein that can be cleaved from the cell surface by metalloproteinases to release a soluble protein with antagonistic activity. Previous studies documented that constitutive ocular expression of FasL maintained immune privilege and prevented neoangeogenesis. We now show that FasL also plays a major role in retinal neurotoxicity. Importantly, in both TNFα triggered RGC death and a spontaneous model of glaucoma, gene-targeted mice that express only full-length FasL exhibit accelerated RGC death. By contrast, FasL-deficiency, or administration of soluble FasL, protected RGCs from cell death. These data identify membrane-bound FasL as a critical effector molecule and potential therapeutic target in glaucoma.

Bacterial spores possess a series of concentrically arranged protective structures that contribute to dormancy, survival and, ultimately, germination. One of these structures, the coat, is present in all spores. In Bacillus anthracis, however, the spore is surrounded by an additional, poorly understood, morphologically complex structure called the exosporium. Here, we characterize three previously discovered exosporium proteins called ExsFA (also known as BxpB), ExsFB (a highly related paralogue of exsFA/bxpB) and IunH (similar to an inosine-uridine-preferring nucleoside hydrolase). We show that in the absence of ExsFA/BxpB, the exosporium protein BclA accumulates asymmetrically to the forespore pole closest to the midpoint of the sporangium (i.e. the mother-cell-proximal pole of the forespore), instead of uniformly encircling the exosporium. ExsFA/BxpB may also have a role in coat assembly, as mutant spore surfaces lack ridges seen in wild-type spores and have a bumpy appearance. ExsFA/BxpB also has a modest but readily detected effect on germination. Nonetheless, an exsFA/bxpB mutant strain is fully virulent in both intramuscular and aerosol challenge models in Guinea pigs. We show that the pattern of localization of ExsFA/BxpB-GFP is a ring, consistent with a location for this protein in the basal layer of the exosporium. In contrast, ExsFB-GFP fluorescence is a solid oval, suggesting a distinct subcellular location for ExsFB-GFP. We also used these fusion proteins to monitor changes in the subcellular locations of these proteins during sporulation. Early in sporulation, both fusions were present throughout the mother cell cytoplasm. As sporulation progressed, GFP fluorescence moved from the mother cell cytoplasm to the forespore surface and formed either a ring of fluorescence, in the case of ExsFA/BxpB, or a solid oval of fluorescence, in the case of ExsFB. IunH-GFP also resulted in a solid oval of fluorescence. We suggest the interpretation that at least some ExsFB-GFP and IunH-GFP resides in the region between the coat and the exosporium, called the interspace. INTRODUCTIONThe bacilli and clostridia encompass a large number of species that produce highly resistant dormant cells, called spores, in response to stress (Fritze, 2004;Nicholson, 2002). Prominent among these species is Bacillus anthracis, which has played a central historical role in our understanding of the transmission of disease and the mechanisms of toxin action, and which has reemerged as a serious threat to human life in light of its utility as a biological weapon (Alibek, 1999;Koch, 1876;Mock & Fouet, 2001). Because the B. anthracis spore is the infectious particle for the disease anthrax, a deeper understanding of spore composition, assembly and function could be very useful in combating this threat.Abbreviations: AFM, atomic force microscopy; IFM, immunofluorescence microscopy; IM, intramuscular; TEM, thin-section electron microscopy.A supplementary table listing PCR primers used in this study is available with the online version of thi...

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Bacillus anthracis is the causative agent of anthrax, and the spore form of the bacterium represents the infectious particle introduced into a host. The spore is surrounded by an exosporium, a loose-fitting membrane composed of proteins and carbohydrates from which hair-like projections extend. These projections are composed mainly of BclA (Bacillus-collagen-like protein of B. anthracis). To date, exact roles of the exosporium structure and BclA protein remain undetermined. We examined differences in spore binding of wild-type Ames and a bclA mutant of B. anthracis to bronchial epithelial cells as well as to the following other epithelial cells: A549, CHO, and Caco-2 cells; the IMR-90 fibroblast line; and human umbilical vein vascular endothelium cells. The binding of wild-type Ames spores to bronchial epithelial cells appeared to be a dose-dependent, receptor-ligand-mediated event. There were similar findings for the bclA mutant, with an additional nonspecific binding component likely leading to significantly more adherence to all nonprofessional phagocytic cell types. In contrast, we detected no difference in adherence and uptake of spores by macrophages for either the wild-type Ames or the bclA mutant strain. These results suggest that one potential role of the BclA fibers may be to inhibit nonspecific interactions between B. anthracis spores with nonprofessional phagocytic cells and thus direct the spores towards uptake by macrophages during initiation of infection in mammals.Bacillus anthracis, a gram-positive, spore-forming bacillus, is the causative agent of anthrax (11,25). The spore structure of B. anthracis consists of overlapping layers called the core, cortex, coat, and exosporium (9). The central interior of a spore, known as the core, houses the chromosome. The cortex is a thick layer of peptidoglycan that surrounds the core (27), which is then further enveloped by the spore coat (1). The outermost structure of the B. anthracis spore is a loose-fitting exosporium (12). Approximately 20 exosporium-associated proteins and glycoproteins have been identified from analyses of B. anthracis and Bacillus cereus spores (4,5,29,(35)(36)(37)(38)(39). The exosporium membrane projects hair-like fibers (12) of which the major component is the BclA glycoprotein (35, 37).Fibers of the B. anthracis exosporium were once believed to be important for virulence or adherence to host cells (20). However, several studies have demonstrated that the fibers and exosporium are not necessary for full virulence of B. anthracis. Upon identifying the BclA protein, Sylvestre et al. (37) examined the effect of a bclA mutation on the virulence of the attenuated Sterne strain of B. anthracis. In this first study, when spores were administered subcutaneously to mice, no appreciable differences in virulence were observed between the parental Sterne and bclA mutant strains (37). We recently showed that by using a fully virulent strain of B. anthracis, an Ames bclA mutant retains complete virulence in mouse and guinea pig models of anthr...

The BclA protein is the immunodominant epitope on the surface of Bacillus anthracis spores; however, its roles in pathogenesis are unclear. We constructed a BclA deletion mutant (bclA) of the fully virulent Ames strain. This derivative retained full virulence in several small-animal models of infection despite the bclA deletion.

Endosomal Toll-like receptors (TLRs) play an important role in systemic autoimmune diseases such as SLE, where DNA- and RNA-associated autoantigens activate autoreactive B cells through TLR9- and TLR7-dependent pathways. Nevertheless, TLR9-deficient autoimmune prone mice develop more severe clinical disease, while TLR7-deficient and TLR7/9-double deficient autoimmune-prone mice develop less severe disease. To determine whether the regulatory activity of TLR9 is B cell intrinsic, we have now directly compared the functional properties of autoantigen activated WT, TLR9-deficient and TLR7-deficient B cells, in an experimental system where proliferation depends on BCR/TLR co-engagement. In vitro, TLR9-deficient cells are less dependent on survival factors for a sustained proliferative response than either WT or TLR7-deficient cells. The TLR9-deficient cells also preferentially differentiate toward the plasma cell lineage, as indicated by expression of CD138, sustained expression of IRF4, and other molecular markers of plasma cells. In vivo, autoantigen-activated TLR9-deficient cells give rise to greater numbers of autoantibody producing cells. Our results identify distinct roles for TLR7 and TLR9 in the differentiation of autoreactive B cells that explain the capacity of TLR9 to limit, and TLR7 to promote, the clinical features of SLE.

In mice that fail to express the phagolysosomal endonuclease, DNase II, and the type I IFN receptor, excessive accrual of undegraded DNA results in a STING-dependent, TLR-independent inflammatory arthritis. These double knockout (DKO) mice develop additional indications of systemic autoimmunity, including anti-nuclear autoantibodies and splenomegaly, not found in Unc93b1−/− DKO mice and therefore TLR-dependent. The DKO autoantibodies predominantly detect RNA-associated autoantigens, commonly targeted in TLR7-dominated SLE-prone mice. To determine whether an inability of TLR9 to detect endogenous DNA could explain the absence of dsDNA-reactive autoantibodies in DKO mice, we used a novel class of bifunctional autoantibodies, IgM/DNA DVD-Ig™ molecules, to activate B cells through a BCR/TLR9-dependent mechanism. DKO B cells could not respond to the IgM/DNA DVD-Ig™ molecule, despite a normal response to both anti-IgM and CpG ODN 1826. Thus DKO B cells only respond to RNA-associated ligands because DNase II-mediated degradation of self-DNA is required for TLR9 activation.