Clostridium difficile, a Gram positive, anaerobic, spore-forming bacterium is an emergent pathogen and the most common cause of nosocomial diarrhea. Although transmission of C. difficile is mediated ...by contamination of the gut by spores, the regulatory cascade controlling spore formation remains poorly characterized. During Bacillus subtilis sporulation, a cascade of four sigma factors, σ(F) and σ(G) in the forespore and σ(E) and σ(K) in the mother cell governs compartment-specific gene expression. In this work, we combined genome wide transcriptional analyses and promoter mapping to define the C. difficile σ(F), σ(E), σ(G) and σ(K) regulons. We identified about 225 genes under the control of these sigma factors: 25 in the σ(F) regulon, 97 σ(E)-dependent genes, 50 σ(G)-governed genes and 56 genes under σ(K) control. A significant fraction of genes in each regulon is of unknown function but new candidates for spore coat proteins could be proposed as being synthesized under σ(E) or σ(K) control and detected in a previously published spore proteome. SpoIIID of C. difficile also plays a pivotal role in the mother cell line of expression repressing the transcription of many members of the σ(E) regulon and activating sigK expression. Global analysis of developmental gene expression under the control of these sigma factors revealed deviations from the B. subtilis model regarding the communication between mother cell and forespore in C. difficile. We showed that the expression of the σ(E) regulon in the mother cell was not strictly under the control of σ(F) despite the fact that the forespore product SpoIIR was required for the processing of pro-σ(E). In addition, the σ(K) regulon was not controlled by σ(G) in C. difficile in agreement with the lack of pro-σ(K) processing. This work is one key step to obtain new insights about the diversity and evolution of the sporulation process among Firmicutes.
Endospores formed by Bacillus, Clostridia, and related genera are encased in a protein shell called the coat. In many species, including B. subtilis, the coat is the outermost spore structure, and in ...other species, such as the pathogenic organisms B. anthracis and B. cereus, the spore is encased in an additional layer called the exosporium. Both the coat and the exosporium have roles in protection of the spore and in its environmental interactions. Assembly of both structures is a function of the mother cell, one of two cellular compartments of the developing sporangium. Studies in B. subtilis have revealed that the timing of coat protein production, the guiding role of a small group of morphogenetic proteins, and several types of posttranslational modifications are essential for the fidelity of the assembly process. Assembly of the exosporium requires a set of novel proteins as well as homologues of proteins found in the outermost layers of the coat and of some of the coat morphogenetic factors, suggesting that the exosporium is a more specialized structure of a multifunctional coat. These and other insights into the molecular details of spore surface morphogenesis provide avenues for exploitation of the spore surface layers in applications for biotechnology and medicine.
Endosporulation is an ancient bacterial developmental program that culminates with the differentiation of a highly resistant endospore. In the model organism Bacillus subtilis, gene expression in the ...forespore and in the mother cell, the two cells that participate in endospore development, is governed by cell type-specific RNA polymerase sigma subunits. σ(F) in the forespore, and σ(E) in the mother cell control early stages of development and are replaced, at later stages, by σ(G) and σ(K), respectively. Starting with σ(F), the activation of the sigma factors is sequential, requires the preceding factor, and involves cell-cell signaling pathways that operate at key morphological stages. Here, we have studied the function and regulation of the sporulation sigma factors in the intestinal pathogen Clostridium difficile, an obligate anaerobe in which the endospores are central to the infectious cycle. The morphological characterization of mutants for the sporulation sigma factors, in parallel with use of a fluorescence reporter for single cell analysis of gene expression, unraveled important deviations from the B. subtilis paradigm. While the main periods of activity of the sigma factors are conserved, we show that the activity of σ(E) is partially independent of σ(F), that σ(G) activity is not dependent on σ(E), and that the activity of σ(K) does not require σ(G). We also show that σ(K) is not strictly required for heat resistant spore formation. In all, our results indicate reduced temporal segregation between the activities of the early and late sigma factors, and reduced requirement for the σ(F)-to-σ(E), σ(E)-to-σ(G), and σ(G)-to-σ(K) cell-cell signaling pathways. Nevertheless, our results support the view that the top level of the endosporulation network is conserved in evolution, with the sigma factors acting as the key regulators of the pathway, established some 2.5 billion years ago upon its emergence at the base of the Firmicutes Phylum.
Abstract
Bacteria of the Firmicutes phylum are able to enter a developmental pathway that culminates with the formation of highly resistant, dormant endospores. Endospores allow environmental ...persistence, dissemination and for pathogens, are also infection vehicles. In both the model Bacillus subtilis, an aerobic organism, and in the intestinal pathogen Clostridioides difficile, an obligate anaerobe, sporulation mobilizes hundreds of genes. Their expression is coordinated between the forespore and the mother cell, the two cells that participate in the process, and is kept in close register with the course of morphogenesis. The evolutionary mechanisms by which sporulation emerged and evolved in these two species, and more broadly across Firmicutes, remain largely unknown. Here, we trace the origin and evolution of sporulation using the genes known to be involved in the process in B. subtilis and C. difficile, and estimating their gain-loss dynamics in a comprehensive bacterial macroevolutionary framework. We show that sporulation evolution was driven by two major gene gain events, the first at the base of the Firmicutes and the second at the base of the B. subtilis group and within the Peptostreptococcaceae family, which includes C. difficile. We also show that early and late sporulation regulons have been coevolving and that sporulation genes entail greater innovation in B. subtilis with many Bacilli lineage-restricted genes. In contrast, C. difficile more often recruits new sporulation genes by horizontal gene transfer, which reflects both its highly mobile genome, the complexity of the gut microbiota, and an adjustment of sporulation to the gut ecosystem.
Bacterial endospores are the most resistant cell type known to humans, as they are able to withstand extremes of temperature, pressure, chemical injury, and time. They are also of interest because ...the endospore is the infective particle in a variety of human and livestock diseases. Endosporulation is characterized by the morphogenesis of an endospore within a mother cell. Based on the genes known to be involved in endosporulation in the model organism Bacillus subtilis, a conserved core of about 100 genes was derived, representing the minimal machinery for endosporulation. The core was used to define a genomic signature of about 50 genes that are able to distinguish endospore-forming organisms, based on complete genome sequences, and we show this 50-gene signature is robust against phylogenetic proximity and other artifacts. This signature includes previously uncharacterized genes that we can now show are important for sporulation in B. subtilis and/or are under developmental control, thus further validating this genomic signature. We also predict that a series of polyextremophylic organisms, as well as several gut bacteria, are able to form endospores, and we identified 3 new loci essential for sporulation in B. subtilis: ytaF, ylmC, and ylzA. In all, the results support the view that endosporulation likely evolved once, at the base of the Firmicutes phylum, and is unrelated to other bacterial cell differentiation programs and that this involved the evolution of new genes and functions, as well as the cooption of ancestral, housekeeping functions.
Clostridioides difficile Sporulation Serrano, Mónica; Martins, Diogo; Henriques, Adriano O
Advances in experimental medicine and biology,
2024, Letnik:
1435
Journal Article
Recenzirano
Some members of the Firmicutes phylum, including many members of the human gut microbiota, are able to differentiate a dormant and highly resistant cell type, the endospore (hereinafter spore for ...simplicity). Spore-formers can colonize virtually any habitat and, because of their resistance to a wide variety of physical and chemical insults, spores can remain viable in the environment for long periods of time. In the anaerobic enteric pathogen Clostridioides difficile the aetiologic agent is the oxygen-resistant spore, while the toxins produced by actively growing cells are the main cause of the disease symptoms. Here, we review the regulatory circuits that govern entry into sporulation. We also cover the role of spores in the infectious cycle of C. difficile in relation to spore structure and function and the main control points along spore morphogenesis.
Assembly of the Bacillus subtilis spore coat involves over 80 proteins which self‐organize into a basal layer, a lamellar inner coat, a striated electrodense outer coat and a more external crust. ...CotB is an abundant component of the outer coat. The C‐terminal moiety of CotB, SKRB, formed by serine‐rich repeats, is polyphosphorylated by the Ser/Thr kinase CotH. We show that another coat protein, CotG, with a central serine‐repeat region, SKRG, interacts with the C‐terminal moiety of CotB and promotes its phosphorylation by CotH in vivo and in a heterologous system. CotG itself is phosphorylated by CotH but phosphorylation is enhanced in the absence of CotB. Spores of a strain producing an inactive form of CotH, like those formed by a cotG deletion mutant, lack the pattern of electrondense outer coat striations, but retain the crust. In contrast, deletion of the SKRB region, has no major impact on outer coat structure. Thus, phosphorylation of CotG by CotH is a key factor establishing the structure of the outer coat. The presence of the cotB/cotH/cotG cluster in several species closely related to B. subtilis hints at the importance of this protein phosphorylation module in the morphogenesis of the spore surface layers.
CotB and CotG are abundant components of the Bacillus subtilis spore coat, both possessing a series of serine‐/lysine‐rich repeats (SKR). CotG interacts with CotB promoting its polyphosphorylation in the SKR region by the CotH kinase. CotG, itself phosphorylated by CotH, is a key determinant of the striated pattern of the outer coat. Conservancy of the cotB/cotH/cotG cluster suggests that protein phosphorylation is an important mechanism in the morphogenesis of the spore surface across species.
Toxin production and sporulation are key determinants of pathogenesis in
. Toxins cause the clinical manifestation of clostridial diseases, including diarrhea and colitis, tissue damage, and systemic ...effects on the nervous system. Spores ensure long-term survival and persistence in the environment, act as infectious agents, and initiate the host tissue colonization leading to infection. Understanding the interplay between toxin production and sporulation and their coordination in bacterial cells and cultures provides novel intervention points for controlling the public health and food safety risks caused by clostridial diseases. We demonstrate environmentally driven cellular heterogeneity in botulinum neurotoxin and spore production in
type E populations and discuss the biological rationale of toxin and spore production in the pathogenicity and ecology of
. The results invite to reassess the epidemiology of botulism and may have important implications in the risk assessment and risk management strategies in food processing and human and animal health.
The human pathogenic bacteria Bacillus cereus, Bacillus anthracis and the entomopathogenic Bacillus thuringiensis form spores encased in a protein coat surrounded by a balloon-like exosporium. These ...structures mediate spore interactions with its environment, including the host immune system, control the transit of molecules that trigger germination and thus are essential for the spore life cycle. Formation of the coat and exosporium has been traditionally visualized by transmission electronic microscopy on fixed cells. Recently, we showed that assembly of the exosporium can be directly observed in live B. cereus cells by super resolution-structured illumination microscopy (SR-SIM) using the membrane MitoTrackerGreen (MTG) dye. Here, we demonstrate that the different steps of coat formation can also be visualized by SR-SIM using MTG and SNAP-cell TMR-star dyes during B. cereus sporulation. We used these markers to characterize a subpopulation of engulfment-defective B. cereus cells that develops at a suboptimal sporulation temperature. Importantly, we predicted and confirmed that synthesis and accumulation of coat material, as well as synthesis of the σK-dependent protein BxpB, occur in cells arrested during engulfment. These results suggest that, unlike the well-studied model organism Bacillus subtilis, the activity of σK is not strictly linked to the state of forespore development in B. cereus.
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