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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. more...

Humans are colonized by diverse fungi (mycobiome), which have received much less study to date than colonizing bacteria. We know very little about the succession of fungal colonization in early life and whether it may relate to long-term health. To better understand fungal colonization and its sources, we studied the skin, oral, and anal mycobiomes of healthy term infants and the vaginal and anal mycobiomes of their mothers. Generally, infants were colonized by few fungal taxa, and fungal alpha diversity did not increase over the first month of life. There was no clear community maturation over the first month of life, regardless of body site. Key body-site-specific taxa, but not overall fungal community structures, were impacted by birth mode. Thus, additional studies to characterize mycobiome acquisition and succession throughout early life are needed to form a foundation for research into the relationship between mycobiome development and human disease., With the advent of next-generation sequencing and microbial community characterization, we are beginning to understand the key factors that shape early-life microbial colonization and associated health outcomes. Studies characterizing infant microbial colonization have focused mostly on bacteria in the microbiome and have largely neglected fungi (the mycobiome), despite their relevance to mucosal infections in healthy infants. In this pilot study, we characterized the skin, oral, and anal mycobiomes of infants over the first month of life (n = 17) and the anal and vaginal mycobiomes of mothers (n = 16) by internal transcribed spacer 2 (ITS2) amplicon sequencing. We found that infant mycobiomes differed by body site, with the infant mycobiomes at the anal sites being different from those at the skin and oral sites. The relative abundances of body site-specific taxa differed by birth mode, with significantly more Candida albicans fungi present on the skin of vaginally born infants on day 30 and significantly more Candida orthopsilosis fungi present in the oral cavity of caesarean section-born infants throughout the first month of life. We found the mycobiomes within individual infants to be variable over the first month of life, and vaginal birth did not result in infant mycobiomes that were more similar to the mother’s vaginal mycobiome. Therefore, although vertical transmission of specific fungal isolates from mother to infant has been reported, it is likely that other sources (environment, other caregivers) also contribute to early-life mycobiome establishment. Thus, future longitudinal studies of mycobiome and bacterial microbiome codevelopment, with dense sampling from birth to beyond the first month of life, are warranted. IMPORTANCE Humans are colonized by diverse fungi (mycobiome), which have received much less study to date than colonizing bacteria. We know very little about the succession of fungal colonization in early life and whether it may relate to long-term health. To better understand fungal colonization and its sources, we studied the skin, oral, and anal mycobiomes of healthy term infants and the vaginal and anal mycobiomes of their mothers. Generally, infants were colonized by few fungal taxa, and fungal alpha diversity did not increase over the first month of life. There was no clear community maturation over the first month of life, regardless of body site. Key body-site-specific taxa, but not overall fungal community structures, were impacted by birth mode. Thus, additional studies to characterize mycobiome acquisition and succession throughout early life are needed to form a foundation for research into the relationship between mycobiome development and human disease. more...

The extremely elongated morphology of fungal hyphae is dependent on the cell's ability to assemble and maintain polarized growth machinery over multiple cell cycles. The different morphologies of the fungus Candida albicans make it an excellent model organism in which to study the spatiotemporal requirements for constitutive polarized growth and the generation of different cell shapes. In C. albicans , deletion of the landmark protein Rsr1 causes defects in morphogenesis that are not predicted from study of the orthologous protein in the related yeast Saccharomyces cerevisiae , thus suggesting that Rsr1 has expanded functions during polarized growth in C. albicans . Here, we show that Rsr1 activity localizes to hyphal tips by the differential localization of the Rsr1 GTPase-activating protein (GAP), Bud2, and guanine nucleotide exchange factor (GEF), Bud5. In addition, we find that Rsr1 is needed to maintain the focused localization of hyphal polarity structures and proteins, including Bem1, a marker of the active GTP-bound form of the Rho GTPase, Cdc42. Further, our results indicate that tip-localized Cdc42 clusters are associated with the cell's ability to express a hyphal transcriptional program and that the ability to generate a focused Cdc42 cluster in early hyphae (germ tubes) is needed to maintain hyphal morphogenesis over time. We propose that in C. albicans , Rsr1 “fine-tunes” the distribution of Cdc42 activity and that self-organizing (Rsr1-independent) mechanisms of polarized growth are not sufficient to generate narrow cell shapes or to provide feedback to the transcriptional program during hyphal morphogenesis. more...

Candida albicans , the most prevalent fungal pathogen of humans, causes superficial mycoses, invasive mucosal infections, and disseminated systemic disease. Many studies have shown an intriguing association between C. albicans morphogenesis and the pathogenesis process. For example, hyphal cells have been observed to penetrate host epithelial cells at sites of wounds and between cell junctions. Ras- and Rho-type GTPases regulate many morphogenetic processes in eukaryotes, including polarity establishment, cell proliferation, and directed growth in response to extracellular stimuli. We found that the C. albicans Ras-like GTPase Rsr1p and its predicted GTPase-activating protein Bud2p localized to the cell cortex, at sites of incipient daughter cell growth, and provided landmarks for the positioning of daughter yeast cells and hyphal cell branches, similar to the paradigm in the model yeast Saccharomyces cerevisiae . However, in contrast to S. cerevisiae , CaRsr1p and CaBud2p were important for morphogenesis: C. albicans strains lacking Rsr1p or Bud2p had abnormal yeast and hyphal cell shapes and frequent bends and promiscuous branching along the hypha and were unable to invade agar. These defects were associated with abnormal actin patch polarization, unstable polarisome localization at hyphal tips, and mislocalized septin rings, consistent with the idea that GTP cycling of Rsr1p stabilizes the axis of polarity primarily to a single focus, thus ensuring normal cell shape and a focused direction of polarized growth. We conclude that the Rsr1p GTPase functions as a polarity landmark for hyphal guidance and may be an important mediator of extracellular signals during processes such as host invasion. more...

The Candida albicans INT1 gene is important for hyphal morphogenesis, adherence, and virulence (C. Gale, C. Bendel, M. McClellan, M. Hauser, J. M. Becker, J. Berman, and M. Hostetter, Science 279:1355-1358, 1998). The ability to switch between yeast and hyphal morphologies is an important virulence factor in this fungal pathogen. When INT1 is expressed in Saccharomyces cerevisiae, cells grow with a filamentous morphology that we exploited to gain insights into how C. albicans regulates hyphal growth. In S. cerevisiae, INT1-induced filamentous growth was affected by a small subset of actin mutations and a limited set of actin-interacting proteins including Sla2p, an S. cerevisiae protein with similarity in its C terminus to mouse talin. Interestingly, while SLA2 was required for INT1-induced filamentous growth, it was not required for polarized growth in response to several other conditions, suggesting that Sla2p is not required for polarized growth per se. The morphogenesis checkpoint, mediated by Swe1p, contributes to INT1-induced filamentous growth; however, epistasis analysis suggests that Sla2p and Swe1p contribute to INT1-induced filamentous growth through independent pathways. The C. albicans SLA2 homolog (CaSLA2) complements S. cerevisiae sla2Delta mutants for growth at 37 degrees C and INT1-induced filamentous growth. Furthermore, in a C. albicans Casla2/Casla2 strain, hyphal growth did not occur in response to either nutrient deprivation or to potent stimuli, such as mammalian serum. Thus, through analysis of INT1-induced filamentous growth in S. cerevisiae, we have identified a C. albicans gene, SLA2, that is required for hyphal growth in C. albicans. more...