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. IMPORTANCE 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.

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The intestinal pathogen Clostridioides difficile exhibits heterogeneity in motility and toxin production. This phenotypic heterogeneity is achieved through phase variation by site-specific recombination via the DNA recombinase RecV, which reversibly inverts the "flagellar switch" upstream of the flgB operon. A recV mutation prevents flagellar switch inversion and results in phenotypically locked strains. The orientation of the flagellar switch influences expression of the flgB operon post-transcription initiation, but the specific molecular mechanism is unknown. Here, we report the isolation and characterization of spontaneous suppressor mutants in the non-motile, non-toxigenic recV flg OFF background that regained motility and toxin production. The restored phenotypes corresponded with increased expression of flagellum and toxin genes. The motile suppressor mutants contained singlenucleotide polymorphisms (SNPs) in rho, which encodes the bacterial transcription terminator Rho factor. Analyses using transcriptional reporters indicate that Rho contributes to heterogeneity in flagellar gene expression by preferentially terminating transcription of flg OFF mRNA within the 5' leader sequence. Additionally, Rho is important for initial colonization of the intestine in a mouse model of infection, which may in part be due to the sporulation and growth defects observed in the rho mutants. Together these data implicate Rho factor as a regulator of gene expression affecting phase variation of important virulence factors of C. difficile.

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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. IMPORTANCE 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.