Search Results (7)

Bacillus cereus is a spore-forming human pathogen that is a burden to the food chain. Dormant spores are highly resistant to harsh environmental conditions, but lose resistance after germination. In this study, we investigate the B. cereus spore proteome upon spore germination and outgrowth so as to obtain new insights into the molecular mechanisms involved. We used mass spectrometry combined with co-expression network analysis and obtained a unique global proteome view of the germination and outgrowth processes of B. cereus spores by monitoring 2211 protein changeovers. We are the first to examine germination and outgrowth models of B. cereus spores experimentally by studying the dynamics of germinant receptors, other proteins involved in spore germination and resistance, and coat and exosporium proteins. Furthermore, through the co-expression analysis of 1175 proteins identified with high quality data, germination proteome data were clustered into eight modules (termed black, blue, brown, green, red, turquoise, grey, and yellow), whose associated functions and expression profiles were investigated. Germination related proteins were clustered into blue and brown modules, the abundances of which decreased after finishing germination. In the brown and blue we identified 124 proteins that could be vital during germination. These proteins will be very interesting to study in future genetic studies regarding their function in spore revival in B. cereus. Full article

Clostridioides difficile is Gram-positive spore-former bacterium and the leading cause of nosocomial antibiotic-associated diarrhea. During disease, C. difficile forms metabolically dormant spores that persist in the host and contribute to recurrence of the disease. The outermost surface of C. difficile spores, termed the exosporium, plays an essential role in interactions with host surfaces and the immune system. The main exosporium proteins identified to date include three orthologues of the BclA family of collagen-like proteins, and three cysteine-rich proteins. However, how the underlying spore coat influences exosporium assembly remains unclear. In this work, we explore the contribution of spore coat proteins cotA and cotB, and the spore surface protein, CDIF630_02480, to the exosporium ultrastructure, formation of the polar appendage and the surface accessibility of exosporium proteins. Transmission electron micrographs of spores of insertional inactivation mutants demonstrate that while cotB contributes to the formation of thick-exosporium spores, cotA and CDIF630_02480 contribute to maintain proper thickness of the spore coat and exosporium layers, respectively. The effect of the absence of cotA, cotB and CDIF630_02480 on the surface accessibility of the exosporium proteins CdeA, CdeC, CdeM, BclA2 and BclA3 to antibodies was affected by the presence of the spore appendage, suggesting that different mechanisms of assembly of the exosporium layer might be implicated in each spore phenotype. Collectively, this work contributes to our understanding of the associations between spore coat and exosporium proteins, and how these associations affect the assembly of the spore outer layers. These results have implications for the development of anti-infecting agents targeting C. difficile spores. Full article

Bacillus cereus is a spore-forming ubiquitous bacterium notable as a food poisoning agent. Detection of B. cereus spores using selective media is laborious and non-specific. Herein, the quantitative detection of B. cereus spores was investigated with commercial antibodies and published aptamer sequences. Several detection reagents were screened for affinity to Bacillus collagen-like protein A (BclA), an abundant exosporium glycoprotein. Sensitivity and selectivity toward B. cereus spores were tested using immunoassays and multi-analyte profiling (xMAP). A recombinant antibody developed in llama against BclA protein showed B. cereus spore selectivity and sensitivity between 10² and 10⁵ spores/mL using xMAP. DNA aptamer sequences demonstrated sensitivity from 10³ to 10⁷ spores/mL and no cross-reaction to B. megaterium and B. subtilis. Selectivity for B. cereus spores was also demonstrated in a mixture of several diverse microorganisms and within a food sample with no compromise of sensitivity. As proof of concept for multiplexed measurement of human pathogens, B. cereus and three other microorganisms, E. coli, P. aeruginosa, and S. cerevisiae, were simultaneously detected using xMAP. These data support the development of a rapid, sensitive, and selective system for quantitation of B. cereus spores and multiplexed monitoring of human pathogens in complex matrices. Full article

Clostridium botulinum produces the botulinum neurotoxin that causes botulism, a rare but potentially lethal paralysis. Endospores play an important role in the survival, transmission, and pathogenesis of C. botulinum. C. botulinum strains are very diverse, both genetically and ecologically. Group I strains are terrestrial, mesophilic, and produce highly heat-resistant spores, while Group II strains can be terrestrial (type B) or aquatic (type E) and are generally psychrotrophic and produce spores of moderate heat resistance. Group III strains are either terrestrial or aquatic, mesophilic or slightly thermophilic, and the heat resistance properties of their spores are poorly characterized. Here, we analyzed the sporulation dynamics in population, spore morphology, and other spore properties of 10 C. botulinum strains belonging to Groups I–III. We propose two distinct sporulation strategies used by C. botulinum Groups I–III strains, report their spore properties, and suggest a putative role for the exosporium in conferring high heat resistance. Strains within each physiological group produced spores with similar characteristics, likely reflecting adaptation to respective environmental habitats. Our work provides new information on the spores and on the population and single-cell level strategies in the sporulation of C. botulinum. Full article

The bacterium Moorella thermoacetica produces the most heat-resistant spores of any spoilage-causing microorganism known in the food industry. Previous work by our group revealed that the resistance of these spores to wet heat and biocides was lower when spores were produced at a lower temperature than the optimal temperature. Here, we used electron microcopy to characterize the ultrastructure of the coat of the spores formed at different sporulation temperatures; we found that spores produced at 55 °C mainly exhibited a lamellar inner coat tightly associated with a diffuse outer coat, while spores produced at 45 °C showed an inner and an outer coat separated by a less electron-dense zone. Moreover, misarranged coat structures were more frequently observed when spores were produced at the lower temperature. We then analyzed the proteome of the spores obtained at either 45 °C or 55 °C with respect to proteins putatively involved in the spore coat, exosporium, or in spore resistance. Some putative spore coat proteins, such as CotSA, were only identified in spores produced at 55 °C; other putative exosporium and coat proteins were significantly less abundant in spores produced at 45 °C. Altogether, our results suggest that sporulation temperature affects the structure and protein composition of M. thermoacetica spores. Full article

Clostridioides difficile is a Gram-positive, spore-forming bacterium that causes a severe intestinal infection. Spores of this pathogen enter in the human body through the oral route, interact with intestinal epithelial cells and persist in the gut. Once germinated, the vegetative cells colonize the intestine and produce toxins that enhance an immune response that perpetuate the disease. Therefore, spores are major players of the infection and ideal targets for new therapies. In this context, spore surface proteins of C. difficile, are potential antigens for the development of vaccines targeting C. difficile spores. Here, we report that the C-terminal domain of the spore surface protein BclA3, BclA3_CTD, was identified as an antigenic epitope, over-produced in Escherichia coli and tested as an immunogen in mice. To increase antigen stability and efficiency, BclA3_CTD was also exposed on the surface of B. subtilis spores, a mucosal vaccine delivery system. In the experimental conditions used in this study, free BclA3_CTD induced antibody production in mice and attenuated some C. difficile infection symptoms after a challenge with the pathogen, while the spore-displayed antigen resulted less effective. Although dose regimen and immunization routes need to be optimized, our results suggest BclA3_CTD as a potentially effective antigen to develop a new vaccination strategy targeting C. difficile spores. Full article

The tetrasaccharide (2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-α-d-glucopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→3)-α-l-rhamnopyranosyl-(1→2)-l-rhamnopyranose) from the major exosporium protein (BclA) of Bacillus anthracis has been proposed as a target for development of diagnostics and immune therapy or prophylaxis. While the immunodominant character of the anthrose residue has been previously elucidated, the role of the stereochemical configuration of the downstream rhamnose is unknown. Because the linkage of this residue to the GlcNAc bridging the glycan and the protein is lost during isolation of the tetrasaccharide, its α- and β-glycoforms have been synthesized. Herein, we prepared neoglycoconjugates from a series of fragments of the tetrasaccharide, including the complete α- and β-tetrasaccharide glycoforms, a 2-demethoxylated version of the α-tetrasaccharide, and the α- and β-trirhamnosides and CRM₁₉₇. By immunization of mice, we showed that the anti α- and β-tetrasaccharide serum equally recognized both glycoforms. In contrast the sera produced following immunization with the α- and β-trirhamnoside fragments exhibited higher recognition for their own antigens than for their anomeric counterparts. The anti α- and β-tetrasaccharide sera recognized Sterne spores in a comparable fashion. ΔBclA spores not expressing the major exosporium protein were also recognized by the same sera, while mutants that produced the carbohydrate antigen with deletion of either rhamnose or anthrose were not. The tetrasaccharide could, therefore, be expressed in proteins other than BlcA. This work proves that α- and β-tetrasaccharide are equally potent immunogens. Full article