Search

ABSTRACT Spores of Bacillus subtilis are surrounded and protected by the coat and the crust, multi-layered structures mainly made of proteins and polysaccharides. These polysaccharides are covalently linked to some of the coat and crust proteins and influence some spore properties, such as surface adhesion and hydrophilicity. This study reports that a mutant strain lacking the spsA-L operon, encoding 11 enzymes involved in the synthesis of spore surface polysaccharides, produced spores exposing on their surface hydrophobic molecules that were responsible for the drastic reduction of hydrophilicity of the mutant spores. Biochemical and genetic data support the identification of the C35-terpenoid curcumene, a precursor of the spore-associated lipid sporulene, as the highly hydrophobic molecule present on the surface of mutant spores.IMPORTANCEBacterial spores are the most resistant cell forms on Earth. The metabolically quiescent spores withstand conditions that would be lethal for other cells, maintaining the capacity to sense the environment and respond to the presence of favorable conditions by germinating. Such remarkable resistance is also due to the complex layers that surround the spore cytoplasm and protect it against damaging factors. Altogether, the spore surface layers form a complex cell structure composed of proteins, polysaccharides, and, as highlighted by this study, also of lipids. Understanding the complexity of the spore surface and the specific molecules involved in its structure is an essential step for unraveling the mechanisms underlying the spore’s resistance to environmental assaults.

Clostridium sporogenes is an anaerobic spore-forming bacterium genetically related to Clostridium botulinum but lacks toxin genes. The sporulation mechanism and spore structures of anaerobic bacteria, including C. sporogenes, have not been comprehensively analyzed. Based on 16S rRNA gene analysis, it has been determined that C. sporogenes NBRC 14293 belongs to C. botulinum Group I. Moreover, SpoIVA is highly conserved in Bacillus and Clostridium species. Therefore, the aim of the present study is to investigate the mechanism of spore formation in C. sporogenes by performing a functional analysis of spoIVA encoding SpoIVA, a protein involved in the early development of the spore coat and cortex in Bacillus subtilis. Inactivation of spoIVA in C. sporogenes resulted in the loss of resistance of sporulating cells to lysozyme and heat treatments. Phase-contrast microscopy indicated that the inactivation of spoIVA caused the development of abnormal forespores and production of only a few immature spores. In the spoIVA mutant, abnormal swirl structures were detected in the mother cell using both phase-contrast and transmission electron microscopy. These swirls were stained with auramine O, pararosaniline hydrochloride, and 2-(4-aminophenyl)benzothiazole to examine the surface of mature spores of the wild-type strain. We found that the spore coat and exosporium proteins were misassembled and that they accumulated in the mother cells of the mutant. The results of this study indicate that SpoIVA is a spore morphogenetic protein, providing novel insights into spore morphogenesis in C. sporogenes.

ABSTRACT Proteins and glycoproteins that form the surface layers of the Bacillus spore assemble into semipermeable arrays that surround and protect the spore cytoplasm. Such layers, acting like molecular sieves, exclude large molecules but allow small nutrients (germinants) to penetrate. We report that CotG, a modular and abundant component of the Bacillus subtilis spore coat, controls spore permeability through its central region, formed by positively charged tandem repeats. These repeats act as spacers between the N and C termini of the protein, which are responsible for the interaction of CotG with at least one other coat protein. The deletion but not the replacement of the central repeats with differently charged repeats affects the spore resistance to lysozyme and the efficiency of germination—probably by reducing the coat permeability to external molecules. The presence of central repeats is a common feature of the CotG-like proteins present in most Bacillus species, and such a wide distribution of this protein family is suggestive of a relevant role for the structure and function of the Bacillus spore. IMPORTANCE Bacterial spores are quiescent cells extremely resistant to a variety of unphysiological conditions, including the presence of lytic enzymes. Such resistance is also due to the limited permeability of the spore surface, which does not allow lytic enzymes to reach the spore interior. This article proposes that the spore permeability in B. subtilis is mediated by CotG, a modular protein formed by a central region of repeats of positively charged amino acid acting as a “spacer” between the N and C termini. These, in turn, interact with other coat proteins, generating a protein layer whose permeability to external molecules is controlled by the distance between the N and C termini of CotG. This working model is most likely expandable to most sporeformers of the Bacillus genus, since they all have CotG-like proteins, not homologous to CotG of B. subtilis but similarly characterized by central repeats.

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.

Abstract Alkaline glutaraldehyde (GTA) is a high‐level chemical disinfectant/sterilant and has a broad microbial kill spectrum. The precise antimicrobial mechanism of GTA remains debated. GTA kill times are extremely variable across different organisms, illustrating the need for a better understanding of GTA kill mechanisms related to different organisms. A commonly proposed GTA kill mechanism suggests that it works by cross‐linking accessible primary amines on important surface proteins. If true, the antimicrobial activity of GTA may directly correlate to the number of these available functional groups. Bacillus species form highly resistant bacterial endospores that are commonly used as one of the most stringent test organisms for disinfection and sterilization. In this study, we compared the log reduction times of alkaline GTA on spores from 4 Bacillus species to fluorescent profiles generated using Alexa Fluor™ amine‐reactive dyes. GTA kill times were also compared to mean spore coat thicknesses as measured with scanning electron microscopy (SEM). Fluorescence values generated from bound amine‐reactive dye showed a strong, positive correlation to GTA susceptibility, as measured by GTA 6‐log10 reduction times. Spore coat thickness also showed a strong, positive correlation to reduction time values. Results support the hypothesis that GTA kill times are directly related to the number of available primary amines on bacterial endospores. Results also indicated that the killing efficacy of GTA may be influenced by its ability to penetrate the spore coat to reach additional targets, suggesting that damaging important biomolecules beyond surface proteins may be involved in GTA killing mechanisms.

ABSTRACT Probiotic Bacillales are effective in controlling pathogens. Live probiotic bacteria improve the composition of the gastrointestinal microbiota, leading to a reduction in pathogen colonization. However, it remains largely unknown how probiotics regulate the host’s immunologic responses and protect the host from parasitic infection. In this study, we addressed whether Bacillales were effective against Haemonchus contortus, a parasitic nematode that infects small ruminants worldwide. Using 16S rRNA sequencing, we found that Bacillales were largely depleted in the abomasal microbiota of sheep infected with H. contortus. We constructed a recombinant Bacillus subtilis named rBSCotB-HcG that express the glyceraldehyde-3-phosphate dehydrogenase of H. contortus (HcGAPDH) on its spore surface using the Bacillus subtilis spore coat protein B (CotB) as a carrier. Mice receiving rBSCotB-HcG orally showed strong Th1-dominated immune responses. More importantly, sheep administered BSCotB-HcG per os showed increasing proliferation of the peripheral blood mononucleates, elevated anti-HcGAPDH IgG in sera, and higher anti-HcGAPDH sIgA in the intestinal mucus than the control sheep. The average weight gain of H. contortus-infected sheep treated with rBSCotB-HcG (Hc+rBSCotB-HcG) was 48.73% greater than that of unvaccinated sheep. Furthermore, these Hc+rBSCotB-HcG sheep had fewer eggs per gram of feces by 84.1% and adult worms by 71.5%. They also demonstrated greatly lessened abomasal damage by H. contortus with an abundance of probiotic species in the abomasal microbiota. Collectively, our data unequivocally demonstrate the protective roles of CotB-HcGAPDH-expressing B. subtilis spores in against H. contortus infection and showed great potential of using probiotic-based strategy in controlling parasitic nematodes of socioeconomic importance in general. IMPORTANCE Initial analyses of the abomasal microbiota of sheep using 16S rRNA sequencing suggested that probiotic bacteria played a protective role in against H. contortus infection. A recombinant Bacillus subtilis expressing a fusion protein CotB-HcGAPDH on its spore’s surface induced strong Th1 immune response in a murine model. The same probiotic recombinant, upon only one oral application, protected sheep against H. contortus infection by reducing egg shedding and decreasing adult worm loads of the parasite and increasing body weight gain of infected sheep. Both Th1 and Th2 immune responses were evident in these immunized sheep.

ABSTRACT Probiotic Bacillales are effective in controlling pathogens. Live probiotic bacteria improve the composition of the gastrointestinal microbiota, leading to a reduction in pathogen colonization. However, it remains largely unknown how probiotics regulate the host’s immunologic responses and protect the host from parasitic infection. In this study, we addressed whether Bacillales were effective against Haemonchus contortus, a parasitic nematode that infects small ruminants worldwide. Using 16S rRNA sequencing, we found that Bacillales were largely depleted in the abomasal microbiota of sheep infected with H. contortus. We constructed a recombinant Bacillus subtilis named rBSCotB-HcG that express the glyceraldehyde-3-phosphate dehydrogenase of H. contortus (HcGAPDH) on its spore surface using the Bacillus subtilis spore coat protein B (CotB) as a carrier. Mice receiving rBSCotB-HcG orally showed strong Th1-dominated immune responses. More importantly, sheep administered BSCotB-HcG per os showed increasing proliferation of the peripheral blood mononucleates, elevated anti-HcGAPDH IgG in sera, and higher anti-HcGAPDH sIgA in the intestinal mucus than the control sheep. The average weight gain of H. contortus-infected sheep treated with rBSCotB-HcG (Hc+rBSCotB-HcG) was 48.73% greater than that of unvaccinated sheep. Furthermore, these Hc+rBSCotB-HcG sheep had fewer eggs per gram of feces by 84.1% and adult worms by 71.5%. They also demonstrated greatly lessened abomasal damage by H. contortus with an abundance of probiotic species in the abomasal microbiota. Collectively, our data unequivocally demonstrate the protective roles of CotB-HcGAPDH-expressing B. subtilis spores in against H. contortus infection and showed great potential of using probiotic-based strategy in controlling parasitic nematodes of socioeconomic importance in general. IMPORTANCE Initial analyses of the abomasal microbiota of sheep using 16S rRNA sequencing suggested that probiotic bacteria played a protective role in against H. contortus infection. A recombinant Bacillus subtilis expressing a fusion protein CotB-HcGAPDH on its spore’s surface induced strong Th1 immune response in a murine model. The same probiotic recombinant, upon only one oral application, protected sheep against H. contortus infection by reducing egg shedding and decreasing adult worm loads of the parasite and increasing body weight gain of infected sheep. Both Th1 and Th2 immune responses were evident in these immunized sheep.

Bacillus subtilis endospores are exceptionally resistant cells encircled by two protective layers: a petidoglycan layer, termed the cortex, and the spore coat, a proteinaceous layer. The formation of both structures depends upon the proper assembly of a basement coat layer, which is composed of two proteins, SpoIVA and SpoVM. The present work examines the interactions of SpoIVA and SpoVM with coat proteins recruited to the spore surface during the early stages of coat assembly. We showed that the alanine racemase YncD associates with two morphogenetic proteins, SpoIVA and CotE. Mutant spores lacking the yncD gene were less resistant against wet heat and germinated to a greater extent than wild-type spores in the presence of micromolar concentrations of l-alanine. In seeking a link between the coat and cortex formation, we investigated the interactions between SpoVM and SpoIVA and the proteins essential for cortex synthesis and found that SpoVM interacts with a penicillin-binding protein, SpoVD, and we also demonstrated that SpoVM is crucial for the proper localization of SpoVD. This study shows that direct contacts between coat morphogenetic proteins with a complex of cortex-synthesizing proteins could be one of the tools by which bacteria couple cortex and coat formation.