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

Bacterial spores produced by the Bacillales are composed of concentric shells, each of which contributes to spore function. Spores from all species possess a cortex and coat, but spores from many species possess additional outer layers. The outermost layer of Bacillus anthracis spores, the exosporium, is separated from the coat by a gap known as the interspace. Exosporium and interspace assembly remains largely mysterious. As a result, we have a poor understanding of the overarching mechanisms driving the assembly of one of the most ubiquitous cell types in nature. To elucidate the mechanisms directing exosporium assembly, we generated strains bearing mutations in candidate exosporium-controlling genes and analyzed the effect on exosporium formation. Biochemical and cell biological analyses argue that CotE directs the assembly of CotO into the spore and that CotO might be located at or close to the interior side of the cap. Taken together with data showing that CotE and CotO interact directly in vitro, we propose a model in which CotE and CotO are important components of a protein interaction network that connects the exosporium to the forespore during cap formation and exosporium elongation. Our data also suggest that the cap interferes with coat assembly at one pole of the spore, altering the pattern of coat deposition compared to the model organism Bacillus subtilis We propose that the difference in coat assembly patterns between these two species is due to an inherent flexibility in coat assembly, which may facilitate the evolution of spore outer layer complexity.IMPORTANCE 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.

An Engineered Synthetic Biologic Protects Against Clostridium difficile Infection.

Morbidity and mortality attributed to Clostridium difficile infection (CDI) have increased over the past 20 years. Currently, antibiotics are the only US FDA-approved treatment for primary C. difficile infection, and these are, ironically, associated with disease relapse and the threat of burgeoning drug resistance. We previously showed that non-toxin virulence factors play key roles in CDI, and that colonization factors are critical for disease. Specifically, a C. difficile adhesin, Surface Layer Protein A (SlpA) is a major contributor to host cell attachment. In this work, we engineered Syn-LAB 2.0 and Syn-LAB 2.1, two synthetic biologic agents derived from lactic acid bacteria, to stably and constitutively express a host-cell binding fragment of the C. difficile adhesin SlpA on their cell-surface. Both agents harbor conditional suicide plasmids expressing a codon-optimized chimera of the lactic acid bacterium's cell-wall anchoring surface-protein domain, fused to the conserved, highly adherent, host-cell-binding domain of C. difficile SlpA. Both agents also incorporate engineered biocontrol, obviating the need for any antibiotic selection. Syn-LAB 2.0 and Syn-LAB 2.1 possess positive biophysical and in vivo properties compared with their parental antecedents in that they robustly and constitutively display the SlpA chimera on their cell surface, potentiate human intestinal epithelial barrier function in vitro, are safe, tolerable and palatable to Golden Syrian hamsters and neonatal piglets at high daily doses, and are detectable in animal feces within 24 h of dosing, confirming robust colonization. In combination, the engineered strains also delay (in fixed doses) or prevent (when continuously administered) death of infected hamsters upon challenge with high doses of virulent C. difficile. Finally, fixed-dose Syn-LAB ameliorates diarrhea in a non-lethal model of neonatal piglet enteritis. Taken together, our findings suggest that the two synthetic biologics may be effectively employed as non-antibiotic interventions for CDI.

A Clostridium difficile Cell Wall Glycopolymer Locus Influences Bacterial Shape, Polysaccharide Production and Virulence.

Clostridium difficile is a diarrheagenic pathogen associated with significant mortality and morbidity. While its glucosylating toxins are primary virulence determinants, there is increasing appreciation of important roles for non-toxin factors in C. difficile pathogenesis. Cell wall glycopolymers (CWGs) influence the virulence of various pathogens. Five C. difficile CWGs, including PSII, have been structurally characterized, but their biosynthesis and significance in C. difficile infection is unknown. We explored the contribution of a conserved CWG locus to C. difficile cell-surface integrity and virulence. Attempts at disrupting multiple genes in the locus, including one encoding a predicted CWG exporter mviN, were unsuccessful, suggesting essentiality of the respective gene products. However, antisense RNA-mediated mviN downregulation resulted in slight morphology defects, retarded growth, and decreased surface PSII deposition. Two other genes, lcpA and lcpB, with putative roles in CWG anchoring, could be disrupted by insertional inactivation. lcpA- and lcpB- mutants had distinct phenotypes, implying non-redundant roles for the respective proteins. The lcpB- mutant was defective in surface PSII deposition and shedding, and exhibited a remodeled cell surface characterized by elongated and helical morphology, aberrantly-localized cell septae, and an altered surface-anchored protein profile. Both lcpA- and lcpB- strains also displayed heightened virulence in a hamster model of C. difficile disease. We propose that gene products of the C. difficile CWG locus are essential, that they direct the production/assembly of key antigenic surface polysaccharides, and thereby have complex roles in virulence.

Surface-layer protein A (SlpA) is a major contributor to host-cell adherence of Clostridium difficile.

Clostridium difficile is a leading cause of antibiotic-associated diarrhea, and a significant etiologic agent of healthcare-associated infections. The mechanisms of attachment and host colonization of C. difficile are not well defined. We hypothesize that non-toxin bacterial factors, especially those facilitating the interaction of C. difficile with the host gut, contribute to the initiation of C. difficile infection. In this work, we optimized a completely anaerobic, quantitative, epithelial-cell adherence assay for vegetative C. difficile cells, determined adherence proficiency under multiple conditions, and investigated C. difficile surface protein variation via immunological and DNA sequencing approaches focused on Surface-Layer Protein A (SlpA). In total, thirty-six epidemic-associated and non-epidemic associated C. difficile clinical isolates were tested in this study, and displayed intra- and inter-clade differences in attachment that were unrelated to toxin production. SlpA was a major contributor to bacterial adherence, and individual subunits of the protein (varying in sequence between strains) mediated host-cell attachment to different extents. Pre-treatment of host cells with crude or purified SlpA subunits, or incubation of vegetative bacteria with anti-SlpA antisera significantly reduced C. difficile attachment. SlpA-mediated adherence-interference correlated with the attachment efficiency of the strain from which the protein was derived, with maximal blockage observed when SlpA was derived from highly adherent strains. In addition, SlpA-containing preparations from a non-toxigenic strain effectively blocked adherence of a phylogenetically distant, epidemic-associated strain, and vice-versa. Taken together, these results suggest that SlpA plays a major role in C. difficile infection, and that it may represent an attractive target for interventions aimed at abrogating gut colonization by this pathogen.

Carbohydrate-based Clostridium difficile vaccines.

Clostridium difficile is responsible for thousands of deaths each year and a vaccine would be welcomed, especially one that would disrupt bacterial maintenance, colonization and persistence in carriers and convalescent patients. Structural explorations at the University of Guelph (ON, Canada) discovered that C. difficile may express three phosphorylated polysaccharides, named PSI, PSII and PSIII; this review captures our recent efforts to create vaccines based on these glycans, especially PSII, the common antigen that has precipitated immediate attention. The authors describe the design and immunogenicity of vaccines composed of raw polysaccharides and conjugates thereof. So far, it has been observed that anti-PSII antibodies can be raised in farm animals, mice and hamster models; humans and horses carry anti-PSII IgA and IgG antibodies from natural exposure to C. difficile, respectively; phosphate is an indispensable immunogenic epitope and vaccine-induced PSII antibodies recognize PSII on C. difficile outer surface.

Clostridium difficile infection: toxins and non-toxin virulence factors, and their contributions to disease establishment and host response.

Clostridium difficile infection is the leading cause of antibiotic- and healthcare-associated diarrhea, and its containment and treatment imposes a significant financial burden, estimated to be over $3 billion in the USA alone. Since the year 2000, CDI epidemics/outbreaks have occurred in North America, Europe and Asia. These outbreaks have been variously associated with, or attributed to, the emergence of Clostridium difficile strains with increased virulence, an increase in resistance to commonly used antimicrobials such as the fluoroquinolones, or host susceptibilities, including the use of gastric acid suppressants, to name a few. Efforts to elucidate C. difficile pathogenic mechanisms have been hampered by a lack of molecular tools, manipulatable animal models, and genetic intractability of clinical C. difficile isolates. However, in the past 5 y, painstaking efforts have resulted in the unraveling of multiple C. difficile virulence-associated pathways and mechanisms. We have recently reviewed the disease, its associated risk factors, transmission and interventions (Viswanathan, Gut Microbes 2010). This article summarizes genetics, non-toxin virulence factors, and host-cell biology associated with C. difficile pathogenesis as of 2011, and highlights those findings/factors that may be of interest as future intervention targets.

Clostridium difficile carbohydrates: glucan in spores, PSII common antigen in cells, immunogenicity of PSII in swine and synthesis of a dual C. difficile-ETEC conjugate vaccine.

Clostridium difficile is responsible for severe diarrhea in humans that may cause death. Spores are the infectious form of C. difficile, which germinate into toxin-producing vegetative cells in response to bile acids. Recently, we discovered that C. difficile cells possess three complex polysaccharides (PSs), named PSI, PSII, and PSIII, in which PSI was only associated with a hypervirulent ribotype 027 strain, PSII was hypothesized to be a common antigen, and PSIII was a water-insoluble polymer. Here, we show that (i) C. difficile spores contain, at least in part, a D-glucan, (ii) PSI is not a ribotype 027-unique antigen, (iii) common antigen PSII may in part be present as a low molecular weight lipoteichoic acid, (iv) selective hydrolysis of PSII yields single PSII repeat units, (v) the glycosyl diester-phosphate linkage affords high flexibility to PSII, and (vi) that PSII is immunogenic in sows. Also, with the intent of creating a dual anti-diarrheal vaccine against C. difficile and enterotoxin Escherichia coli (ETEC) infections in humans, we describe the conjugation of PSII to the ETEC-associated LTB enterotoxin.

Clostridium perfringens alpha toxin is produced in the intestines of broiler chicks inoculated with an alpha toxin mutant.

Poultry necrotic enteritis (NE) is caused by specific strains of Clostridium perfringens, most of which are type A. The role of alpha toxin (CPA) in NE has been called into question by the finding that an engineered cpa mutant retains full virulence in vivo[9]. This is in contrast to the finding that immunization with CPA toxoids protects against NE. We confirmed the earlier findings, in that 14-day-old Cornish × Rock broiler chicks challenged with a cpa mutant developed lesions compatible with NE in >90% of birds inoculated with the mutant. However, CPA was detected in amounts ranging from 10 to >100 ng per g of gut contents and mucosa in birds inoculated with the cpa mutant, the wildtype strain from which the mutant was constructed, and our positive control strain. There was a direct relationship between lesion severity and amount of CPA detected (R = 0.89-0.99). These findings suggest that the role of CPA in pathogenesis of NE requires further investigation.

Human hypervirulent Clostridium difficile strains exhibit increased sporulation as well as robust toxin production.

Toxigenic Clostridium difficile strains produce two toxins (TcdA and TcdB) during the stationary phase of growth and are the leading cause of antibiotic-associated diarrhea. C. difficile isolates of the molecular type NAP1/027/BI have been associated with severe disease and hospital outbreaks worldwide. It has been suggested that these "hypervirulent" strains produce larger amounts of toxin and that a mutation in a putative negative regulator (TcdC) allows toxin production at all growth phases. To rigorously explore this possibility, we conducted a quantitative examination of the toxin production of multiple hypervirulent and nonhypervirulent C. difficile strains. Toxin gene (tcdA and tcdB) and toxin gene regulator (tcdR and tcdC) expression was also monitored. To obtain additional correlates for the hypervirulence phenotype, sporulation kinetics and efficiency were measured. In the exponential phase, low basal levels of tcdA, tcdB, and tcdR expression were evident in both hypervirulent and nonhypervirulent strains, but contrary to previous assumptions, toxin levels were below the detectable thresholds. While hypervirulent strains displayed robust toxin production during the stationary phase of growth, the amounts were not significantly different from those of the nonhypervirulent strains tested; further, total toxin amounts were directly proportional to tcdA, tcdB, and tcdR gene expression. Interestingly, tcdC expression did not diminish in stationary phase, suggesting that TcdC may have a modulatory rather than a strictly repressive role. Comparative genomic analyses of the closely related nonhypervirulent strains VPI 10463 (the highest toxin producer) and 630 (the lowest toxin producer) revealed polymorphisms in the tcdR ribosome binding site and the tcdR-tcdB intergenic region, suggesting that a mechanistic basis for increased toxin production in VPI 10463 could be increased TcdR translation and read-through transcription of the tcdA and tcdB genes. Hypervirulent isolates produced significantly more spores, and did so earlier, than all other isolates. Increased sporulation, potentially in synergy with robust toxin production, may therefore contribute to the widespread disease now associated with hypervirulent C. difficile strains.

Spore-forming Bacilli and Clostridia in human disease.

Many Gram-positive spore-forming bacteria in the Firmicute phylum are important members of the human commensal microbiota, which, in rare cases, cause opportunistic infections. Other spore-formers, however, have evolved to become dedicated pathogens that can cause a striking variety of diseases. Despite variations in disease presentation, the etiologic agent is often the spore, with bacterially produced toxins playing a central role in the pathophysiology of infection. This review will focus on the specific diseases caused by spores of the Clostridia and Bacilli.

B cell development in GALT: role of bacterial superantigen-like molecules.

Intestinal bacteria drive the formation of lymphoid tissues, and in rabbit, bacteria also promote development of the preimmune Ab repertoire and positive selection of B cells in GALT. Previous studies indicated that Bacillus subtilis promotes B cell follicle formation in GALT, and we investigated the mechanism by which B. subtilis stimulates B cells. We found that spores of B. subtilis and other Bacillus species, including Bacillus anthracis, bound rabbit IgM through an unconventional, superantigen-like binding site, and in vivo, surface molecules of B. anthracis spores promoted GALT development. Our study provides direct evidence that B cell development in GALT may be driven by superantigen-like molecules, and furthermore, that bacterial spores modulate host immunity.

Bacillus anthracis and Bacillus subtilis spore surface properties and transport.

Effective decontamination of environments contaminated by Bacillus spores remains a significant challenge since Bacillus spores are highly resistant to killing and could plausibly adhere to many non-biological as well as biological surfaces. Decontamination of Bacillus spores can be significantly improved if the chemical basis of spore adherence is understood. In this research, we investigated the surface adhesive properties of Bacillus subtilis and Bacillus anthracis spores. The spore thermodynamic properties obtained from contact angle measurements indicated that both species were monopolar with a preponderance of electron-donating potential. This was also the case for spores of both species missing their outer layers, due to mutation. Transport of wild type and mutant spores of these two species was further analyzed in silica sand under unsaturated water conditions. A two-region solute transport model was used to simulate the spore transport with the assumption that the spore retention occurred within the immobile region only. Bacillus spore adhesion to the porous media was related to the interactions between the spores and the porous media. Our data indicated that spore surface structures played important roles in spore surface properties, since mutant spores missing outer layers had different surface thermodynamic and transport properties as compared to wild type spores. The changes in surface thermodynamic properties were further evidenced by infrared spectroscopy analysis.

Roles of the Bacillus anthracis spore protein ExsK in exosporium maturation and germination.

The Bacillus anthracis spore is the causative agent of the disease anthrax. The outermost structure of the B. anthracis spore, the exosporium, is a shell composed of approximately 20 proteins. The function of the exosporium remains poorly understood and is an area of active investigation. In this study, we analyzed the previously identified but uncharacterized exosporium protein ExsK. We found that, in contrast to other exosporium proteins, ExsK is present in at least two distinct locations, i.e., the spore surface as well as a more interior location underneath the exosporium. In spores that lack the exosporium basal layer protein ExsFA/BxpB, ExsK fails to encircle the spore and instead is present at only one spore pole, indicating that ExsK assembly to the spore is partially dependent on ExsFA/BxpB. In spores lacking the exosporium surface protein BclA, ExsK fails to mature into high-molecular-mass species observed in wild-type spores. These data suggest that the assembly and maturation of ExsK within the exosporium are dependent on ExsFA/BxpB and BclA. We also found that ExsK is not required for virulence in murine and guinea pig models but that it does inhibit germination. Based on these data, we propose a revised model of exosporium maturation and assembly and suggest a novel role for the exosporium in germination.

Localization and assembly of proteins comprising the outer structures of the Bacillus anthracis spore.

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.

Characterization of a Bacillus anthracis spore coat-surface protein that influences coat-surface morphology.

Bacterial spores are encased in a multilayered proteinaceous shell, called the coat. In many Bacillus spp., the coat protects against environmental assault and facilitates germination. In Bacillus anthracis, the spore is the etiological agent of anthrax, and the functions of the coat likely contribute to virulence. Here, we characterize a B. anthracis spore protein, called Cotbeta, which is encoded only in the genomes of the Bacillus cereus group. We found that Cotbeta is synthesized specifically during sporulation and is assembled onto the spore coat surface. Our analysis of a cotbeta null mutant in the Sterne strain reveals that Cotbeta has a role in determining coat-surface morphology but does not detectably affect germination. In the fully virulent Ames strain, a cotbeta null mutation has no effect on virulence in a murine model of B. anthracis infection.

Morphogenesis of the Bacillus anthracis spore.

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.