Mechanisms of phase variation of flagella and toxins in Clostridioides difficile

Clostridioides difficile is a Gram-positive, spore-forming obligate anaerobe that can cause antibiotic-associated intestinal disease and is the leading cause of hospital-acquired infections in the US. In spite of its significant burden to public health, many aspects of its physiology remain poorly understood. C. difficile produces two toxins, TcdA and TcdB, that are required for disease development. In addition, C. difficile synthesizes flagella that promote bacterial motility and intestinal colonization. The expression of flagellum and toxin genes is linked in vitro, therefore factors that regulate expression of the flagellar genes can influence both motility and toxin-mediated virulence. One of these factors is the “flagellar switch”, an invertible DNA sequence upstream of the flgB operon that mediates phase variation by site-specific DNA recombination, that controls the ON/OFF production of flagella and toxins. The main objectives of the research described in this dissertation were to characterize the mechanism by which the flagellar switch controls flagellar and toxin gene expression and to determine the importance of flagellum and toxin phase variation to C. difficile physiology and infection potential. In Chapter 2, we described a suppressor analysis that identified Rho factor in mediating phase variation via the flagellar switch. We found that Rho preferentially inhibits transcription of flg OFF mRNA and inhibits motility and toxin production in flg OFF bacteria. In addition, Rho was important for initial intestinal colonization in a mouse model of infection, which may be driven in part by sporulation and growth defects of rho mutants. In Chapter 3, we characterized C. difficile mutants with varying abilities to undergo flagellum and toxin phase variation. These mutants were either attenuated or fully “locked” for phase variation in vitro but indistinguishable from wildtype bacteria in other characteristics. We analyzed these mutants in a hamster model of acute C. difficile disease and found that preventing or inhibiting flagellar switch inversion led to differences in toxin production and bacterial burden but did not significantly alter disease outcome. This work greatly increased our understanding of a regulatory strategy with significant implications for C. difficile population heterogeneity and pathogenesis.Doctor of Philosoph

Intravital Imaging Reveals Divergent Cytokine and Cellular Immune Responses to Candida albicans and Candida parapsilosis

In modern medicine, physicians are frequently forced to balance immune suppression against immune stimulation to treat patients such as those undergoing transplants and chemotherapy. More-targeted therapies designed to preserve immunity and prevent opportunistic fungal infection in these patients could be informed by an understanding of how fungi interact with professional and nonprofessional immune cells in mucosal candidiasis. In this study, we intravitally imaged these host-pathogen dynamics during Candida infection in a transparent vertebrate model host, the zebrafish. Single-cell imaging revealed an unexpected partitioning of the inflammatory response between phagocytes and epithelial cells. Surprisingly, we found that in vivo cytokine profiles more closely match in vitro responses of epithelial cells rather than phagocytes. Furthermore, we identified a disconnect between canonical inflammatory cytokine production and phagocyte recruitment to the site of infection, implicating noncytokine chemoattractants. Our study contributes to a new appreciation for the specialization and cross talk among cell types during mucosal infection.Candida yeasts are common commensals that can cause mucosal disease and life-threatening systemic infections. While many of the components required for defense against Candida albicans infection are well established, questions remain about how various host cells at mucosal sites assess threats and coordinate defenses to prevent normally commensal organisms from becoming pathogenic. Using two Candida species, C. albicans and C. parapsilosis, which differ in their abilities to damage epithelial tissues, we used traditional methods (pathogen CFU, host survival, and host cytokine expression) combined with high-resolution intravital imaging of transparent zebrafish larvae to illuminate host-pathogen interactions at the cellular level in the complex environment of a mucosal infection. In zebrafish, C. albicans grows as both yeast and epithelium-damaging filaments, activates the NF-κB pathway, evokes proinflammatory cytokines, and causes the recruitment of phagocytic immune cells. On the other hand, C. parapsilosis remains in yeast morphology and elicits the recruitment of phagocytes without inducing inflammation. High-resolution mapping of phagocyte-Candida interactions at the infection site revealed that neutrophils and macrophages attack both Candida species, regardless of the cytokine environment. Time-lapse monitoring of single-cell gene expression in transgenic reporter zebrafish revealed a partitioning of the immune response during C. albicans infection: the transcription factor NF-κB is activated largely in cells of the swimbladder epithelium, while the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) is expressed in motile cells, mainly macrophages. Our results point to different host strategies for combatting pathogenic Candida species and separate signaling roles for host cell types

A Klebsiella pneumoniae Regulatory Mutant Has Reduced Capsule Expression but Retains Hypermucoviscosity

Klebsiella pneumoniae continues to be a substantial public health threat due to its ability to cause health care-associated and community-acquired infections combined with its ability to acquire antibiotic resistance. Novel therapeutics are needed to combat this pathogen, and a greater understanding of its virulence factors is required for the development of new drugs. A key virulence factor for K. pneumoniae is the capsule, and community-acquired hypervirulent strains produce a capsule that causes hypermucoidy. We report here a novel capsule regulator, RmpC, and provide evidence that capsule production and the hypermucoviscosity phenotype are distinct processes. Infection studies showing that this and other capsule regulator mutants have a range of phenotypes indicate that additional virulence factors are in their regulons. These results shed new light on the mechanisms controlling capsule production and introduce targets that may prove useful for the development of novel therapeutics for the treatment of this increasingly problematic pathogen.The polysaccharide capsule is an essential virulence factor for Klebsiella pneumoniae in both community-acquired hypervirulent strains as well as health care-associated classical strains that are posing significant challenges due to multidrug resistance. Capsule production is known to be transcriptionally regulated by a number of proteins, but very little is known about how these proteins collectively control capsule production. RmpA and RcsB are two known regulators of capsule gene expression, and RmpA is required for the hypermucoviscous (HMV) phenotype in hypervirulent K. pneumoniae strains. In this report, we confirmed that these regulators performed their anticipated functions in the ATCC 43816 derivative, KPPR1S: rcsB and rmpA mutants are HMV negative and have reduced capsule gene expression. We also identified a novel transcriptional regulator, RmpC, encoded by a gene near rmpA. The ΔrmpC strain has reduced capsule gene expression but retains the HMV phenotype. We further showed that a regulatory cascade exists in which KvrA and KvrB, the recently characterized MarR-like regulators, and RcsB contribute to capsule regulation through regulation of the rmpA promoter and through additional mechanisms. In a murine pneumonia model, the regulator mutants have a range of colonization defects, suggesting that they regulate virulence factors in addition to capsule. Further testing of the rmpC and rmpA mutants revealed that they have distinct and overlapping functions and provide evidence that HMV is not dependent on overproduction of capsule. This distinction will facilitate a better understanding of HMV and how it contributes to enhanced virulence of hypervirulent strains

Epigenomic characterization of Clostridioides difficile finds a conserved DNA methyltransferase that mediates sporulation and pathogenesis

International audienceClostridioides difficile is a leading cause of health care-associated infections. Although significant progress has been made in the understanding of its genome, the epigenome of C. difficile and its functional impact has not been systematically explored. Here, we performed a comprehensive DNA methylome analysis of C. difficile using 36 human isolates and observed great epigenomic diversity. We discovered an orphan DNA methyltransferase with a well-defined specificity whose corresponding gene is highly conserved across our dataset and in all ∼300 global C. difficile genomes examined. Inactivation of the methyltransferase gene negatively impacted sporulation, a key step in C. difficile disease transmission, consistently supported by multi-omics data, genetic experiments, and a mouse colonization model. Further experimental and transcriptomic analysis also suggested that epigenetic regulation is associated with cell length, biofilm formation, and host colonization. These findings provide a unique epigenetic dimension to characterize medically relevant biological processes in this critical pathogen. This work also provides a set of methods for comparative epigenomics and integrative analysis, which we expect to be broadly applicable to bacterial epigenomics studies