Identification Of Regulatory Factors That Control Clostridium Difficile Sporulation and Germination

Clostridium difficile is a Gram-positive spore-forming strict anaerobe that can cause severe colitis in humans. C. difficile is best known as the leading cause of nosocomial-acquired diarrhea, particularly in people undergoing antibiotic therapies, since it is naturally resistant to most antibiotics. A clinical feature that makes C. difficile infection, or CDI, particularly difficult to treat is the organism\u27s inherent ability to resist antibiotic therapies while in its spore form. Since oxygen is toxic to C. difficile, spores are the major transmissive form; they are also resilient to most disinfectants, which makes them extremely difficult to eliminate to prevent additional infections. While over fifty years of studies on the spore-forming model organism Bacillus subtilis laid the foundation of how sporulation and germination occurs, little was known about how C. difficile regulates spore formation and/or what proteins are necessary for sporulation and germination processes. The work presented in this dissertation addresses how C. difficile regulates sporulation, identifies genes that are regulated during sporulation, and characterizes some key proteins that are required for either sporulation or germination. During the developmental process of sporulation, a cell divides into two asymmetrical compartments. In each compartment, specific transcriptional programs controlled by sporulation-specific sigma factors, drive the cell through a series of morphological events, culminating in the formation of a spore. Using genetic and cell biological techniques, we show that mutations in the genes encoding the master transcriptional regulator Spo0A and the sporulation-specific sigma factors σF, σE, σG, and σK block sporulation at various stages. Analysis of the mutants and wild type C. difficile strain using RNA-Sequencing identified genes regulated by a given sigma factor and revealed that the sigma factors control sporulation in a manner that differs from B. subtilis. Whereas the sporulation-specific sigma factor activity is regulated in a sequential manner involving cross talk between the different compartments in B. subtilis, C. difficile regulates these factors in a bifurcated manner, with less cross-compartment regulation. Guided by our RNA-Sequencing results, we constructed targeted gene mutations in spoIIQ and spoIIIA-H, which are important for forming a channel known as the \u27feeding tube\u27 in B. subtilis. We demonstrated that these proteins are necessary for maintaining forespore integrity, tethering the coat to the forespore, and engulfment. Using metabolic labeling, we show that while spoIIQ and spoIIIA mutants cannot finish the phagocytic-like process of engulfment, they are capable of transforming peptidoglycan, which is a necessary step for engulfment to occur. We also constructed a targeted gene mutation in a gene that is highly transcribed during sporulation, now known as gerS. We show that a gerS mutant cannot degrade cortex during germination and is required for SleC-mediated cortex hydrolysis, making GerS a novel regulator of C. difficile spore germination. Altogether, this research provides a framework for understanding how the pathogen C. difficile undergoes sporulation and is therefore capable of infecting humans. Further, our studies reveal important factors that mediate the essential process of engulfment during sporulation and an important factor that mediates cortex hydrolysis during germination. This work has demonstrated that C. difficile regulates sporulation and germination differently than what has previously been described in other Firmicutes

A Clostridium difficile-Specific, Gel-Forming Protein Required for Optimal Spore Germination

Clostridium difficile is a Gram-positive spore-forming obligate anaerobe that is a leading cause of antibiotic-associated diarrhea worldwide. In order for C. difficile to initiate infection, its aerotolerant spore form must germinate in the gut of mammalian hosts. While almost all spore-forming organisms use trans- membrane germinant receptors to trigger germination, C. difficile uses the pseu- doprotease CspC to sense bile salt germinants. CspC activates the related subtilisin-like protease CspB, which then proteolytically activates the cortex hy- drolase SleC. Activated SleC degrades the protective spore cortex layer, a step that is essential for germination to proceed. Since CspC incorporation into spores also depends on CspA, a related pseudoprotease domain, Csp family pro- teins play a critical role in germination. However, how Csps are incorporated into spores remains unknown. In this study, we demonstrate that incorporation of the CspC, CspB, and CspA germination regulators into spores depends on CD0311 (renamed GerG), a previously uncharacterized hypothetical protein. The reduced levels of Csps in gerG spores correlate with reduced responsiveness to bile salt germinants and increased germination heterogeneity in single-spore germination assays. Interestingly, asparagine-rich repeat sequences in GerG’s central region facilitate spontaneous gel formation in vitro even though they are dispensable for GerG-mediated control of germination. Since GerG is found exclusively in C. difficile, our results suggest that exploiting GerG function could represent a promising avenue for developing C. difficile-specific anti-infective therapies

Inducible Expression of Spo0A as a Universal Tool for Studying Sporulation in Clostridium difficile

Clostridium difficile remains a leading nosocomial pathogen, putting considerable strain on the healthcare system. The ability to form endospores, highly resistant to environmental insults, is key to its persistence and transmission. However, important differences exist between the sporulation pathways of C. difficile and the model Gram-positive organism Bacillus subtilis. Amongst the challenges in studying sporulation in C. difficile is the relatively poor levels of sporulation and high heterogeneity in the sporulation process. To overcome these limitations we placed Ptet regulatory elements upstream of the master regulator of sporulation, spo0A, generating a new strain that can be artificially induced to sporulate by addition of anhydrotetracycline (ATc). We demonstrate that this strain is asporogenous in the absence of ATc, and that ATc can be used to drive faster and more efficient sporulation. Induction of Spo0A is titratable and this can be used in the study of the spo0A regulon both in vitro and in vivo, as demonstrated using a mouse model of C. difficile infection (CDI). Insights into differences between the sporulation pathways in B. subtilis and C. difficile gained by study of the inducible strain are discussed, further highlighting the universal interest of this tool. The Ptet-spo0A strain provides a useful background in which to generate mutations in genes involved in sporulation, therefore providing an exciting new tool to unravel key aspects of sporulation in C. difficile

Probing Clostridium difficile Infection in Complex Human Gut Cellular Models

Interactions of anaerobic gut bacteria, such as Clostridium difficile, with the intestinal mucosa have been poorly studied due to challenges in culturing anaerobes with the oxygen-requiring gut epithelium. Although gut colonization by C. difficile is a key determinant of disease outcome, precise mechanisms of mucosal attachment and spread remain unclear. Here, using human gut epithelial monolayers co-cultured within dual environment chambers, we demonstrate that C. difficile adhesion to gut epithelial cells is accompanied by a gradual increase in bacterial numbers. Prolonged infection causes redistribution of actin and loss of epithelial integrity, accompanied by production of C. difficile spores, toxins, and bacterial filaments. This system was used to examine C. difficile interactions with the commensal Bacteroides dorei, and interestingly, C. difficile growth is significantly reduced in the presence of B. dorei. Subsequently, we have developed novel models containing a myofibroblast layer, in addition to the epithelium, grown on polycarbonate or three-dimensional (3D) electrospun scaffolds. In these more complex models, C. difficile adheres more efficiently to epithelial cells, as compared to the single epithelial monolayers, leading to a quicker destruction of the epithelium. Our study describes new controlled environment human gut models that enable host–anaerobe and pathogen–commensal interaction studies in vitro

Increased sporulation underpins adaptation of Clostridium difficile strain 630 to a biologically–relevant faecal environment, with implications for pathogenicity

Abstract Clostridium difficile virulence is driven primarily by the processes of toxinogenesis and sporulation, however many in vitro experimental systems for studying C. difficile physiology have arguably limited relevance to the human colonic environment. We therefore created a more physiologically–relevant model of the colonic milieu to study gut pathogen biology, incorporating human faecal water (FW) into growth media and assessing the physiological effects of this on C. difficile strain 630. We identified a novel set of C. difficile–derived metabolites in culture supernatants, including hexanoyl– and pentanoyl–amino acid derivatives by LC-MSn. Growth of C. difficile strain 630 in FW media resulted in increased cell length without altering growth rate and RNA sequencing identified 889 transcripts as differentially expressed (p < 0.001). Significantly, up to 300–fold increases in the expression of sporulation–associated genes were observed in FW media–grown cells, along with reductions in motility and toxin genes’ expression. Moreover, the expression of classical stress–response genes did not change, showing that C. difficile is well–adapted to this faecal milieu. Using our novel approach we have shown that interaction with FW causes fundamental changes in C. difficile biology that will lead to increased disease transmissibility

A cortex-specific penicillin-binding protein contributes to heat resistance in Clostridioides difficile spores

Background Sporulation is a complex cell differentiation programme shared by many members of the Firmicutes, the end result of which is a highly resistant, metabolically inert spore that can survive harsh environmental insults. Clostridioides difficile spores are essential for transmission of disease and are also required for recurrent infection. However, the molecular basis of sporulation is poorly understood, despite parallels with the well-studied Bacillus subtilis system. The spore envelope consists of multiple protective layers, one of which is a specialised layer of peptidoglycan, called the cortex, that is essential for the resistant properties of the spore. We set out to identify the enzymes required for synthesis of cortex peptidoglycan in C. difficile. Methods Bioinformatic analysis of the C. difficile genome to identify putative homologues of Bacillus subtilis spoVD was combined with directed mutagenesis and microscopy to identify and characterise cortex-specific PBP activity. Results Deletion of CDR20291_2544 (SpoVDCd) abrogated spore formation and this phenotype was completely restored by complementation in cis. Analysis of SpoVDCd revealed a three domain structure, consisting of dimerization, transpeptidase and PASTA domains, very similar to B. subtilis SpoVD. Complementation with SpoVDCd domain mutants demonstrated that the PASTA domain was dispensable for formation of morphologically normal spores. SpoVDCd was also seen to localise to the developing spore by super-resolution confocal microscopy. Conclusions We have identified and characterised a cortex specific PBP in C. difficile. This is the first characterisation of a cortex-specific PBP in C. difficile and begins the process of unravelling cortex biogenesis in this important pathogen

Clostridium difficile infection.

Infection of the colon with the Gram-positive bacterium Clostridium difficile is potentially life threatening, especially in elderly people and in patients who have dysbiosis of the gut microbiota following antimicrobial drug exposure. C. difficile is the leading cause of health-care-associated infective diarrhoea. The life cycle of C. difficile is influenced by antimicrobial agents, the host immune system, and the host microbiota and its associated metabolites. The primary mediators of inflammation in C. difficile infection (CDI) are large clostridial toxins, toxin A (TcdA) and toxin B (TcdB), and, in some bacterial strains, the binary toxin CDT. The toxins trigger a complex cascade of host cellular responses to cause diarrhoea, inflammation and tissue necrosis - the major symptoms of CDI. The factors responsible for the epidemic of some C. difficile strains are poorly understood. Recurrent infections are common and can be debilitating. Toxin detection for diagnosis is important for accurate epidemiological study, and for optimal management and prevention strategies. Infections are commonly treated with specific antimicrobial agents, but faecal microbiota transplants have shown promise for recurrent infections. Future biotherapies for C. difficile infections are likely to involve defined combinations of key gut microbiota

Regulation of <i>Clostridium difficile</i> Spore Formation by the SpoIIQ and SpoIIIA Proteins

<div><p>Sporulation is an ancient developmental process that involves the formation of a highly resistant endospore within a larger mother cell. In the model organism <i>Bacillus subtilis</i>, sporulation-specific sigma factors activate compartment-specific transcriptional programs that drive spore morphogenesis. σ^G activity in the forespore depends on the formation of a secretion complex, known as the “feeding tube,” that bridges the mother cell and forespore and maintains forespore integrity. Even though these channel components are conserved in all spore formers, recent studies in the major nosocomial pathogen <i>Clostridium difficile</i> suggested that these components are dispensable for σ^G activity. In this study, we investigated the requirements of the SpoIIQ and SpoIIIA proteins during <i>C</i>. <i>difficile</i> sporulation. <i>C</i>. <i>difficile spoIIQ</i>, <i>spoIIIA</i>, and <i>spoIIIAH</i> mutants exhibited defects in engulfment, tethering of coat to the forespore, and heat-resistant spore formation, even though they activate σ^G at wildtype levels. Although the <i>spoIIQ</i>, <i>spoIIIA</i>, and <i>spoIIIAH</i> mutants were defective in engulfment, metabolic labeling studies revealed that they nevertheless actively transformed the peptidoglycan at the leading edge of engulfment. <i>In vitro</i> pull-down assays further demonstrated that <i>C</i>. <i>difficile</i> SpoIIQ directly interacts with SpoIIIAH. Interestingly, mutation of the conserved Walker A ATP binding motif, but not the Walker B ATP hydrolysis motif, disrupted SpoIIIAA function during <i>C</i>. <i>difficile</i> spore formation. This finding contrasts with <i>B</i>. <i>subtilis</i>, which requires both Walker A and B motifs for SpoIIIAA function. Taken together, our findings suggest that inhibiting SpoIIQ, SpoIIIAA, or SpoIIIAH function could prevent the formation of infectious <i>C</i>. <i>difficile</i> spores and thus disease transmission.</p></div