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Human milk is a complex mix of nutritional and bioactive components that provide complete nutrition for the infant. However, we lack a systematic knowledge of the factors shaping milk composition and how milk variation influences infant health. Here, we used multi-omic profiling to characterize interactions between maternal genetics, milk gene expression, milk composition, and the infant fecal microbiome in 242 exclusively breastfeeding mother-infant pairs. We identified 487 genetic loci associated with milk gene expression unique to the lactating mammary gland, including loci that impacted breast cancer risk and human milk oligosaccharide concentration. Integrative analyses uncovered connections between milk gene expression and infant gut microbiome, including an association between the expression of inflammation-related genes with IL-6 concentration in milk and the abundance ofBifidobacteriain the infant gut. Our results show how an improved understanding of the genetics and genomics of human milk connects lactation biology with maternal and infant health.

BACKGROUND Neonatal exposure to antibiotics, in the absence of infection, results in abnormal learning and memory in animals and is linked to changes in gut microbes. The relevance of early-life antibiotic exposure to brain function in humans is not known. METHODS Recognition memory was assessed at 1 month of age in 15 term-born infants exposed to antibiotics (with negative cultures) and 57 unexposed infants using event-related potentials (ERPs). Linear regression analysis, adjusting for covariates, was employed to compare groups with respect to ERP features representing early stimulus processing (P2 amplitude) and discrimination between mother and stranger voices. RESULTS Infants exposed to antibiotics exhibited smaller P2 amplitudes for both voice conditions (p = 0.001), with greatest reductions observed for mother's voice in frontal and central scalp regions (p

It is unclear whether gestational diabetes mellitus (GDM) alters breast milk composition. We prospectively examined associations of GDM status with concentrations of six potentially bioactive elements (glucose, insulin, C-reactive protein (CRP), interleukin-6 (IL-6), leptin, and adiponectin) in human milk. These were measured at both 1 and 3 months postpartum in 189 fully breastfeeding women. Mixed-effects linear regression assessed GDM status-related differences in these milk bioactives, adjusting for demographics, maternal factors, and diet. At 1 and 3 months postpartum, milk CRP was higher (1.46 ± 0.31 ng/mL; p < 0.001 and 1.69 ± 0.31 ng/mL; p < 0.001) in women with GDM than in women without GDM, whereas milk glucose (−5.23 ± 2.22 mg/dL; p = 0.02 and −5.70 ± 2.22; p = 0.01) and milk insulin (−0.38 ± 0.17 μIU/mL; p = 0.03 and −0.53 ± 0.17; p = 0.003) were lower in women with GDM. These significant associations remained similar after additional adjustment for maternal weight status and its changes. No difference was found for milk IL-6, leptin, and adiponectin. There was no evidence of association between these milk bioactive compounds and 1 h non-fasting oral glucose challenge serum glucose in the women without GDM. This prospective study provides evidence that potentially bioactive elements of human milk composition are altered in women with GDM.

PURPOSE: Human milk (HM) is a unique biological fluid that is enriched with a variety of factors, including microRNAs (miRNAs) that potentially provide both short- and long-term benefits to the infants. miRNAs are packaged within exosomes, making them bioavailable to infants. Gestational diabetes mellitus (GDM) may affect the abundance of exosomal miRNAs in HM, providing a mechanism for growth and adiposity variation in infants of mothers with GDM in early life. Therefore, the purposes of this study were to examine the impact of GDM on select miRNAs (miRNA-148a, miRNA-30b, miRNA-let-7a, and miRNA-let-7d) involved in metabolism and to examine the association of these miRNAs with measures of infant body composition in the first 6 months of life. METHODS: Milk samples were collected from a cohort of 94 mothers (62 mothers without GDM and 32 mothers with GDM) matched on body mass index strata at 1 month post partum. miRNA abundance was measured by real-time polymerase chain reaction. Linear regression models were used to examine potential differences in miRNA abundance in women with and without GDM, testing associations between miRNA abundance and infant growth and body composition measures from 1 to 6 months. FINDINGS: The abundances of miRNA-148a, miRNA-30b, miRNA-let-7a, and miRNA-let-7d were reduced in milk from mothers with GDM. Independent of GDM status, higher maternal diet quality was associated with increased abundance of each of the measured miRNAs. miRNA-148a was negatively associated with infant weight, percentage of body fat, and fat mass, whereas miRNA-30b was positively associated with infant weight and fat mass at 1 month of age. There was no association of milk miRNA-148a and miRNA-30b with infant weight at 1 month of age or with body composition measures at 3 months of age; however, miRNA-148a was negatively associated with infant weight at 6 months of age. IMPLICATIONS: If supported by randomized dietary supplementation or other intervention trials, HM miRNAs may be a therapeutic target to mitigate risk of metabolic outcomes in offspring of women with GDM.

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.

Surfaces within the neonatal intensive care unit (NICU), especially those handled frequently by hospital staff, provide sources of gut-colonizing bacteria for hospitalized infants, in addition to those acquired perinatally from maternal sources such as breastmilk. In comparison to bacteria, very little is known about potential sources of colonizing fungi in the NICU setting. Thus, the objective of this study was to characterize fungal communities (mycobiomes) of potential colonization sources for neonates hospitalized in a large university NICU. We hypothesized that the unit surfaces would contain different mycobiomes than those of human-associated (breastmilk) sources. We characterized mycobiomes of NICU surfaces of multiple individual patient care areas as well as those of breastmilk samples by sequencing the internal transcribed spacer region 2 (ITS2) of the fungal rDNA locus. We found that, across all samples, Candida and Saccharomyces species were the most prevalent taxa and had the greatest relative abundances. Breastmilk samples had significantly higher fungal alpha-diversities than NICU surface samples and fungal community compositions (beta diversities) differed significantly between the two sample types. Mycobiome compositions were predominantly driven by the relative abundances of three fungal taxa: Candida albicans, Candida parapsilosis, and Saccharomyces cerevisiae. In total, 21 individual fungal taxa showed significantly greater relative abundances in breastmilk as compared to NICU surfaces, with three being of particular interest to human health: Candida glabrata, Candida tropicalis, and Cryptococcus neoformans. Since no fungal DNA was detected when whole breastmilk was used as the DNA template, as opposed to breastmilk subjected to cell lysis during the DNA isolation procedure, our results indicate that DNA is from fungal cells and is not cell-free DNA. In summary, both NICU surfaces and human breastmilk harbor distinct fungal communities that could provide a source of fungi for the developing infant gut mycobiota. In particular, Candida and Saccharomyces species are abundant and prevalent for both of these potential sources that infants are exposed to.

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.

Candida parapsilosis is a common cause of invasive candidiasis, especially in premature infants, even surpassing Candida albicans as the most frequently identified Candida species in some newborn intensive care units. Whereas many molecular tools are available to facilitate the study of C. albicans, relatively few have been developed for C. parapsilosis. In this study, we show that plasmids harbouring green, yellow and mCherry fluorescent protein sequences, previously developed for expression in C. albicans, can be used to construct fluorescent fusion proteins in C. parapsilosis by PCR-mediated gene modification. Further, the strategy can be used in clinical isolates of C. parapsilosis, which are typically prototrophic, because the plasmids include NAT1, a dominant selectable trait that confers resistance to the antibiotic nourseothricin. Overall, these tools will be useful to yeast researchers who require the ability to visualize C. parapsilosis directly, e.g. in in vitro and in vivo infection models. In addition, this strategy can be used to generate fluorescence in other C. parapsilosis clinical isolates and to tag sequences of interest for protein localization studies. Lastly, the ability to express up to three different fluorescent proteins will allow researchers to visualize and differentiate C. parapsilosis and/or C. albicans clinical isolates from each other in mixed infection models.

Candida is a leading cause of late-onset sepsis in premature infants and is thought to invade the host via immature or damaged epithelial barriers. We previously showed that the hyphal form of Candida albicans invades and causes damage to premature intestinal epithelial cells (pIECs), whereas the non-hyphal Candida parapsilosis, also a fungal pathogen of neonates, has less invasion and damage abilities. In this study, we investigated the potential for C. parapsilosis to modulate pathogenic interactions of C. albicans with the premature intestine. While a mixed infection with two fungal pathogens may be expected to result in additive or synergistic damage to pIECs, we instead found that C. parapsilosis was able to protect pIECs from invasion and damage by C. albicans. C. albicans-induced pIEC damage was reduced to a similar extent by multiple different C. parapsilosis strains, but strains differed in their ability to inhibit C. albicans invasion of pIECs, with the inhibitory activity correlating with their adhesiveness for C. albicans and epithelial cells. C. parapsilosis cell-free culture fractions were also able to significantly reduce C. albicans adhesion and damage to pIECs. Furthermore, coadministration of C. parapsilosis cell-free fractions with C. albicans was associated with decreased infection and mortality in zebrafish. These results indicate that C. parapsilosis is able to reduce invasion, damage, and virulence functions of C. albicans. Additionally, the results with cellular and cell-free fractions of yeast cultures suggest that inhibition of pathogenic interactions between C. albicans and host cells by C. parapsilosis occurs via secreted molecules as well as by physical contact with the C. parapsilosis cell surface. We propose that non-invasive commensals can be used to inhibit virulence features of pathogens and deserve further study as a non-pharmacological strategy to protect the fragile epithelial barriers of premature infants.

Candida species, prevalent colonizers of the intestinal and genitourinary tracts, are the cause of the majority of invasive fungal infections in humans. Thus, molecular and genetic tools are needed to facilitate the study of their pathogenesis mechanisms. PCR-mediated gene modification is a straightforward and quick approach to generate epitope-tagged proteins to facilitate their detection. In particular, fluorescent protein (FP) fusions are powerful tools that allow visualization and quantitation of both yeast cells and proteins by fluorescence microscopy and immunoblotting, respectively. Plasmids containing FP encoding sequences, along with nutritional marker genes that facilitate the transformation of Candida species, have been generated for the purpose of FP construction and expression in Candida. Herein, we present a strategy for constructing a FP fusion in a Candida species. Plasmids containing the nourseothricin resistance transformation marker gene (NAT1) along with sequences for either green, yellow, or cherry FPs (GFP, YFP, mCherry) are used along with primers that include gene-specific sequences in a polymerase chain reaction (PCR) to generate a FP cassette. This gene-specific cassette has the ability to integrate into the 3'-end of the corresponding gene locus via homologous recombination. Successful in-frame fusion of the FP sequence into the gene locus of interest is verified genetically, followed by analysis of fusion protein expression by microscopy and/or immuno-detection methods. In addition, for the case of highly expressed proteins, successful fusions can be screened for primarily by fluorescence imaging techniques.

Polarized cells reorient their direction of growth in response to environmental cues. In the fungus Candida albicans, the Rho-family small GTPase, Cdc42, is essential for polarized hyphal growth and Ca(2+) influx is required for the tropic responses of hyphae to environmental cues, but the regulatory link between these systems is unclear. In this study, the interaction between Ca(2+) influx and Cdc42 polarity-complex dynamics was investigated using hyphal galvanotropic and thigmotropic responses as reporter systems. During polarity establishment in an applied electric field, cathodal emergence of hyphae was lost when either of the two Cdc42 apical recycling pathways was disrupted by deletion of Rdi1, a guanine nucleotide dissociation inhibitor, or Bnr1, a formin, but was completely restored by extracellular Ca(2+). Loss of the Cdc42 GTPase activating proteins, Rga2 and Bem3, also abolished cathodal polarization, but this was not rescued by Ca(2+). Expression of GTP-locked Cdc42 reversed the polarity of hypha emergence from cathodal to anodal, an effect augmented by Ca(2+). The cathodal directional cue therefore requires Cdc42 GTP hydrolysis. Ca(2+) influx amplifies Cdc42-mediated directional growth signals, in part by augmenting Cdc42 apical trafficking. The Ca(2+)-binding EF-hand motif in Cdc24, the Cdc42 activator, was essential for growth in yeast cells but not in established hyphae. The Cdc24 EF-hand motif is therefore essential for polarity establishment but not for polarity maintenance.

Hyphae of filamentous fungi maintain generally linear growth over long distances. In Candida albicans, hyphae are able to reorient their growth in the direction of certain environmental cues. In previous work, the C. albicans bud-site selection proteins Rsr1 and Bud2 were identified as important for hyphae to maintain linear growth and were necessary for hyphal responses to directional cues in the environment (tropisms). To ask if hyphal directional responses are general functions of all yeast bud-site selection proteins, we studied the role of Rax2, ortholog of the Saccharomyces cerevisiae bud-site selection protein Rax2, in C. albicans hyphal morphogenesis. Rax2-YFP localized to the hyphal cell surface in puncta and at the hyphal tip in a crescent. Strains lacking Rax2 had hyphal morphologies that did not differ from control strains. In non-cued growth conditions, rax2 mutant strains had defects in both yeast (bud) and hyphal (branch) site selection and mutant hyphae exhibited non-linear growth trajectories as compared to control hyphae. In contrast, when encountering a directional environmental cue, hyphae lacking Rax2 retained the ability to reorient growth in response to both topographical (thigmotropism) and electric-field (galvanotropism) stimuli but exhibited a reduced ability to establish hyphal growth in the direction of a cathodal stimulus. In conclusion, these results indicate that C. albicans Rax2 is important for establishing sites of emergence of yeast and hyphal daughters and for maintaining the linearity of hyphal growth. In contrast to Rsr1 and Bud2, Rax2 is not involved in responses that require a reorientation of the direction of already established hyphal growth (tropisms). Thus, it appears that some hyphal directionality responses are separable in that they are mediated by a different set of polarity proteins.

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.

The early endocytic patch protein Sla2 is important for morphogenesis and growth rates inSaccharomyces cerevisiaeandCandida albicans,but the mechanism that connects these processes is not clear. Here we report that growth defects in cells lacking CaSLA2or ScSLA2are associated with a cell cycle delay that is influenced by Swe1, a morphogenesis checkpoint kinase. To establish how Swe1 monitors Sla2 function, we compared actin organization and cell cycle dynamics in strains lacking other components of early endocytic patches (Sla1 and Abp1) with those in strains lacking Sla2. Onlysla2strains had defects in actin cables, a known trigger of the morphogenesis checkpoint, yet all three strains exhibited Swe1-dependent phenotypes. Thus, Swe1 appears to monitor actin patch in addition to actin cable function. Furthermore, Swe1 contributed to virulence in a mouse model of disseminated candidiasis, implying a role for the morphogenesis checkpoint during the pathogenesis ofC. albicansinfections.

Candida parapsilosis is a common cause of invasive candidiasis, especially in premature infants, even surpassing Candida albicans as the most frequently identified Candida species in some newborn intensive care units. Whereas many molecular tools are available to facilitate the study of C. albicans, relatively few have been developed for C. parapsilosis. In this study, we show that plasmids harbouring green, yellow and mCherry fluorescent protein sequences, previously developed for expression in C. albicans, can be used to construct fluorescent fusion proteins in C. parapsilosis by PCR-mediated gene modification. Further, the strategy can be used in clinical isolates of C. parapsilosis, which are typically prototrophic, because the plasmids include NAT1, a dominant selectable trait that confers resistance to the antibiotic nourseothricin. Overall, these tools will be useful to yeast researchers who require the ability to visualize C. parapsilosis directly, e.g. in in vitro and in vivo infection models. In addition, this strategy can be used to generate fluorescence in other C. parapsilosis clinical isolates and to tag sequences of interest for protein localization studies. Lastly, the ability to express up to three different fluorescent proteins will allow researchers to visualize and differentiate C. parapsilosis and/or C. albicans clinical isolates from each other in mixed infection models.

BACKGROUND Human milk oligosaccharides (HMOs) are a highly abundant, diverse group of unique glycans that are postulated to promote the development of a protective bacterial microbiota in the intestine and prevent adhesive and invasive interactions of pathogenic bacteria with mucosal epithelia. Candida albicans, a prevalent fungal colonizer of the neonatal gut, causes the majority of fungal disease in premature infants and is highly associated with life-threatening intestinal disorders. OBJECTIVE The objective of the current study was to test the hypothesis that HMOs protect human premature intestinal epithelial cells (pIECs) from invasion by C. albicans. METHODS To study fungal invasion, a quantitative immunocytochemical assay was used to distinguish invading from noninvading C. albicans cells in the presence and absence of HMOs. To understand how HMOs affect C. albicans invasion of pIECs, the expression of C. albicans virulence traits that are important for invasiveness (hyphal morphogenesis and ability to associate with host cells) were quantified. RESULTS Treatment with HMOs reduced invasion of pIECs by C. albicans in a dose-dependent manner by 14-67%, with a physiologic concentration (15mg/mL) of HMOs causing a 52% reduction in invasion (P < 0.05). The decreased invasive ability of C. albicans was associated with hyphal lengths that were ∼30% shorter (P < 0.05), likely because of a delay in the induction of hyphal morphogenesis after inoculation of yeast onto pIECs, which correlated with a 23% reduction in the combined expression level of hyphal-specific genes (P < 0.05). In addition, HMOs caused a 40% decrease in the number of C. albicans cells able to associate with pIECs at the time of hyphal induction (P < 0.05). CONCLUSIONS These results, obtained with the use of a primary pIEC model, indicate that HMOs reduce virulence characteristics of C. albicans and suggest a role for HMOs in protecting the premature infant intestine from invasion and damage by C. albicans hyphae.

Fungi grow with a variety of morphologies: oval yeast cells, chains of elongated cells called pseudohyphae and long, narrow, tube-like filaments called hyphae. In filamentous fungi, hyphal growth is strongly polarised to the tip and is mediated by the Spitzenkörper, which acts as a supply centre to concentrate the delivery of secretory vesicles to the tip. In the budding yeast Saccharomyces cerevisiae, polarised growth is mediated by the polarisome, a surface cap of proteins that nucleates the formation of actin cables delivering secretory vesicles to the growing tip. The human fungal pathogen, Candida albicans, can grow in all three morphological forms. Here we show the presence of a Spitzenkörper at the tip of C. albicans hyphae as a ball-like localisation of secretory vesicles, together with the formin Bni1 and Mlc1, an ortholog of an S. cerevisiae myosin regulatory light chain. In contrast, in C. albicans yeast cells, pseudohyphae and hyphae Spa2 and Bud6, orthologs of S. cerevisiae polarisome components, as well as the master morphology regulator Cdc42, localise predominantly, but not exclusively, to a surface cap resembling the polarisome of S. cerevisiae yeast cells. A small amount of Cdc42 also localises to the Spitzenkörper. Furthermore, we show differences in the genetic and cytoskeletal requirements, and cell cycle dynamics of polarity determinants in yeast, pseudohyphae and hyphae. These results, together with the cytological differences between the cell types, suggest that the Spitzenkörper and polarisome are distinct structures, that the polarisome and Spitzenkörper coexist in hyphae, and that polarised growth in hyphae is driven by a fundamentally different mechanism to that in yeast and pseudohyphae.

Proteins that enter the secretory pathway play important roles in virulence and pathogenesis in Candida albicans, but our understanding of the trafficking of these proteins is in its early stages. In Saccharomyces cerevisiae, dominant negative alleles of YPT1 and SEC4 interrupt secretory traffic at pre- and post-Golgi steps, respectively. We therefore used a dominant negative genetic approach to examine the intracellular trafficking of several proteins associated with virulence or azole resistance. When the dominant negative ypt1(N121I) allele of C. albicans was overexpressed, yellow-fluorescent protein (YFP) tagged forms of two plasma membrane transporters (Cdrlp and Ftrlp) and the vacuolar membrane ABC transporter Mltlp accumulated in intracellular structures that appeared related to the ER, but localization of Cdc10p and Int1p was unaffected. When the dominant negative sec4(S28N) allele of C. albicans was overexpressed, Cdrlp and Ftrlp accumulated intracellularly, and localization of Mltlp, Cdc10p and Int1p was unaffected. These results imply that (i) Cdrlp and Ftrlp are transported to the plasma membrane by the general secretory pathway, (ii) Mlt1p enters the secretory pathway but is diverted to the vacuole at an early post-Golgi step, and (iii) like Cdc10p, Int1p does not enter the general secretory pathway.

Protein O-mannosyltransferases (Pmt proteins) initiate O-mannosylation of secretory proteins. The PMT gene family of the human fungal pathogen Candida albicans consists of PMT1 and PMT6, as well as three additional PMT genes encoding Pmt2, Pmt4 and Pmt5 isoforms described here. Both PMT2 alleles could not be deleted and growth of conditional strains, containing PMT2 controlled by the MET3- or tetOScHOP1-promoters, was blocked in non-permissive conditions, indicating that PMT2 is essential for growth. A homozygous pmt4 mutant was viable, but synthetic lethality of pmt4 was observed in combination with pmt1 mutations. Hyphal morphogenesis of a pmt4 mutant was defective under aerobic induction conditions, yet increased in embedded or hypoxic conditions, suggesting a role of Pmt4p-mediated O-glycosylation for environment-specific morphogenetic signalling. Although a PMT5 transcript was detected, a homozygous pmt5 mutant was phenotypically silent. All other pmt mutants showed variable degrees of supersensitivity to antifungals and to cell wall-destabilizing agents. Cell wall composition was markedly affected in pmt1 and pmt4 mutants, showing a significant decrease in wall mannoproteins. In a mouse model of haematogenously disseminated infection, PMT4 was required for full virulence of C. albicans. Functional analysis of the first complete PMT gene family in a fungal pathogen indicates that Pmt isoforms have variable and specific roles for in vitro and in vivo growth, morphogenesis and antifungal resistance.

Recent studies highlight the importance of intestinal fungal microbiota in the development of human disease. Infants, in particular, are an important population in which to study intestinal microbiomes because microbial community structure and dynamics during this formative window of life have the potential to influence host immunity and metabolism. When compared to bacteria, much less is known about the early development of human fungal communities, owing partly to their lower abundance and the relative lack of established molecular and taxonomic tools for their study. Herein, we describe the development, validation, and use of complementary amplicon-based genomic strategies to characterize infant fungal communities and provide quantitative information about Candida, an important fungal genus with respect to intestinal colonization and human disease. Fungal communities were characterized from 11 infant fecal samples using primers that target the internal transcribed spacer (ITS) 2 locus, a region that provides taxonomic discrimination of medically relevant fungi. Each sample yielded an average of 27,553 fungal sequences and Candida albicans was the most abundant species identified by sequencing and quantitative PCR (qPCR). Low numbers of Candida krusei and Candida parapsilosis sequences were observed in several samples, but their presence was detected by species-specific qPCR in only one sample, highlighting a challenge inherent in the study of low-abundance organisms. Overall, the sequencing results revealed that infant fecal samples had fungal diversity comparable to that of bacterial communities in similar-aged infants, which correlated with the relative abundance of C. albicans. We conclude that targeted sequencing of fungal ITS2 amplicons in conjunction with qPCR analyses of specific fungi provides an informative picture of fungal community structure in the human intestinal tract. Our data suggests that the infant intestine harbors diverse fungal species and is consistent with prior culture-based analyses showing that the predominant fungus in the infant intestine is C. albicans.

This chapter describes the major Candida albicans morphologies and the current understanding of the cell biological and cell cycle features that distinguish them. It highlights recent insights into how cell cycle regulators influence the formation of hypha-specific cellular features in particular. Since morphogenesis and cell cycle regulation have been studied most extensively in C. albicans, the chapter primarily focuses on work in C. albicans. The important distinction between yeast and pseudohyphae is that pseudohyphae spend more time in G2 phase of the cell cycle than yeast cells , and they continue to elongate during this time. There has long been a controversy as to how pseudohyphae are related to true hyphae. Initial models suggested that yeast cells, pseudohyphae, and true hyphae reside along a continuum. Later, based on differences in cell cycle dynamics and subcellular structures, it was proposed that pseudohyphae and hyphae represent two distinct morphological states, with pseudohyphae being more like yeast form growth with respect to cell cycle progression and cell biological markers. Recent work has shed light on cell biological features associated with cell cycle progression in chlamydospores and is discussed in theis chapter. In the C. albicans genome sequence, there are three G1 cyclins (Ccn1, Cln3, and Hgc1) and two G2 or B-type/mitotic cyclins (Clb2 and Clb4) that are predicted to associate with Cdc28.

The ability to switch between yeast and hyphal morphologies is an important virulence factor for the opportunistic pathogenCandida albicans. Although the kinetics of appearance of the filamentous ring that forms at the incipient septum differ in yeast and cells forming hyphae (germ tubes) ( Soll and Mitchell, 1983 ), the molecular mechanisms that regulate this difference are not known. Int1p, a C. albicans gene product with similarity in its C terminus to Saccharomyces cerevisiae Bud4p, has a role in hyphal morphogenesis. Here we report that in S. cerevisiae, Int1p expression results in the growth of highly polarized cells with delocalized chitin and defects in cytokinesis and bud-site selection patterns, phenotypes that are also seen in S. cerevisiae septin mutant strains. Expression of high levels of Int1p in S. cerevisiae generated elaborate spiral-like structures at the periphery of the polarized cells that contained septins and Int1p. In addition, Int1p coimmunoprecipitated with the Cdc11p and Cdc12p septins, and Cdc12p is required for the establishment and maintenance of these Int1p/septin spirals. Although Swe1p kinase contributes to INT1-induced filamentous growth inS. cerevisiae, it is not required for the formation of ectopic Int1p/septin structures. In C. albicans, Int1p was important for the axial budding pattern and colocalized with Cdc3p septin in a ring at the mother-bud neck of yeast and pseudohyphal cells. Under conditions that induce hyphae, both Cdc3p and Int1p localized to a ring distal to the junction of the mother cell and germ tube. Thus, placement of the Int1p/septin ring with respect to the mother–daughter cell junction distinguishes yeast/pseudohyphal growth from hyphal growth in C. albicans.

We have developed a set of plasmids containing fluorescent protein cassettes for use in PCR-mediated gene tagging in Candida albicans. We engineered YFP and CFP variants of the GFP sequence optimized for C. albicans codon usage. The fluorescent protein sequences, linked to C. albicans auxotrophic marker sequences, were amplified by PCR and transformed directly into yeast. Gene-specific sequence was incorporated into the PCR primers, such that the tag-cassette integrates by homologous recombination at the 3′-end of the gene of interest. This technique was used to tag Cdc3 and Tub1 with GFP, YFP and CFP, which were readily visualized by fluorescence microscopy and localized as expected. In addition, Tub1–YFP and Cdc3–CFP were visualized in the same cells. Thus, this technique directs one-step construction of multiple fluorescent protein fusions, facilitating the study of protein co-expression and co-localization in C. albicans cells in vivo. Copyright © 2001 John Wiley & Sons, Ltd.

The Candida albicans gene INT1 is associated with epithelial adhesion, hyphal formation, and virulence. C. albicans strains carrying two, one, or no functional INT1 alleles were used to assess the association between mortality and C. albicans persistence in the liver and kidney of intravenously inoculated mice. Mice were injected with 10(5) C. albicans CAF2 (parent strain, INT1/INT1), C. albicans CAG3 (homozygous disruptant, Int1/int1), or C. albicans CAG5 (heterozygous reintegrant, int1/int1 + INT1). Mortality was monitored and mice were sacrificed on Days 1, 7, 14, and 21 for quantitative analysis of kidney and liver microbes, with histologic analysis of these tissues as well. Mortality was highest for mice injected with the wild-type strain CAF2 (INT1/INT1) and lowest for mice injected with the homozygous disruptant CAG3 (int/int1). Yeast were readily cleared from the liver of all mice injected with any of the three C. albicans strains. Although the mutant strains CAG3 and CAG5 are defective for hyphal formation in vitro, there was histological evidence of abundant hyphal formation in the renal pelvis of mice injected with these strains. Compared to the wild-type strain, mutant strains were associated with reduced mortality but increased C. albicans persistence in the kidney. Thus, the absolute ability to form hyphae in the kidney did not appear to modulate either C. albicans-induced mortality or the course of progressive infection in the kidney. In addition, reduced virulence was paradoxically associated with increased, not decreased, persistence of C. albicans in the kidney.

Life-threatening gastrointestinal (GI) diseases of prematurity are highly associated with systemic candidiasis. This implicates the premature GI tract as an important site for invasion by Candida. Invasive interactions of Candida spp. with immature enterocytes have heretofore not been analyzed. Using a primary immature human enterocyte line, we compared the ability of multiple isolates of different Candida spp. to penetrate, injure, and induce a cytokine response from host cells. Of all the Candida spp. analyzed, C. albicans had the greatest ability to penetrate and injure immature enterocytes and to elicit interleukin-8 (IL-8) release (p < 0.01). In addition, C. albicans was the only Candida spp. to form filamentous hyphae when in contact with immature enterocytes. Similarly, a C. albicans mutant with defective hyphal morphogenesis and invasiveness had attenuated cytotoxicity for immature enterocytes (p < 0.003). Thus, hyphal morphogenesis correlates with immature enterocyte penetration, injury and inflammatory responses. Furthermore, variability in enterocyte injury was observed among hyphal-producing C. albicans strains suggesting that individual organism genotypes also influence host-pathogen interactions. Overall, the finding that Candida spp. differed in their interactions with immature enterocytes implicates that individual spp. may employ different pathogenesis mechanisms.

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.

The recent availability of genome sequence information for the opportunistic pathogen Candida albicans has greatly facilitated the ability to perform genetic manipulations in this organism. Two important molecular tools for studying gene function are regulatable promoters for generating conditional mutants and fluorescent proteins for determining the subcellular localization of fusion gene products. We describe a set of plasmids containing promoter-GFP cassettes (P(MET3)-GFP, P(GAL1)-GFP, and P(PCK1)-GFP), linked to a selectable nutritional marker gene (URA3). PCR-mediated gene modification generates gene-specific promoter, or gene-specific promoter-GFP, fusions at the 5'-end of the gene of interest. One set of primers can be used to generate three strains expressing a native protein of interest, or an amino-terminal GFP-tagged version, from three different regulatable promoters. Thus, these promoter cassette plasmids facilitate construction of conditional mutant strains, overexpression alleles and/or inducible amino-terminal GFP fusion proteins.

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.

Background: In neonates with necrotizing enterocolitis/spontaneous intestinal perforation, Candida species are frequent blood/peritoneal fluid isolates. Relatively little is known about host (immature enterocyte)/pathogen interactions. Design and population: The goal of this study is to understand how Candida invades the neonatal GI tract. We hypothesize that Candida species differ in their invasive potential and mechanism of penetration. Material and methods: The ability of multiple Candida species to injure and penetrate GI epithelium was compared, using a premature human enterocyte cell in culture. Epithelial injury was determined using a colorimetric assay of lactate dehydrogenase (LDH) released from epithelial cells infected with Candida species. Immunocytochemical analysis was performed, using an anti-Candida antibody conjugated to the red-fluorescing Alexa 568, to distinguish penetrating from non-penetrating fungal cells. Results: C. albicans caused the most cell damage (53.9% of max LDH released), C. parapsilosis (6.4%, p = 0.034), C. dubliniensis (2.2%, p = 0.043), C. tropicalis (1.5%. p = 0.045), and C. glabrata (0%, p = 0.021). There was no difference in LDH when comparing C. parapsilosis to the other non-albicans species ( p = 0.18–0.52). Microscopically, C. albicans was intraepithelial more frequently. Only C. albicans formed abundant hyphae and the hyphal filament most often penetrated the enterocytes. Conclusions: Infection of immature neonatal enterocytes with C. albicans resulted in significantly more injury than the other Candida species tested. Injury was associated with penetration of the epithelial cell by fungal hyphae. We speculate that the preferential ability of C. albicans to cause cell penetration and damage is linked to its ability to form hyphae.

Background. Increased intestinal colonization with Candida albicans is believed to be a major factor predisposing immunocompromised and postsurgical patients to systemic candidiasis, although the mechanisms facilitating C. albicans colonization remain unclear. Because previous studies have linked the C. albicans INT1 gene to filament formation, epithelial adherence, and mouse virulence, experiments were designed to evaluate the effect of INT1 on intestinal colonization. Materials and Methods. Mice were orally inoculated with either the parent strain (CAF2, INT/INT1), an int1 heterozygote (CAG1, INT1/int1), an int1 homozygote (CAG3, int1/int1), or a reintegrant (CAG5, int1/int1 + INT1), and sacrificed 3 and 7 days later for quantitative analysis of cecal C. albicans. Results. Following oral inoculation with 103C. albicans, only small numbers of each strain were recovered from the cecal flora of normal mice. However, in mice pretreated with oral antibiotics, cecal colonization of each strain was increased (P < 0.01). In addition, cecal colonization was reduced for all int1 mutant strains compared with the parent strain (P < 0.05). By light microscopy, all four C. albicans strains were easily observed in the ileal lumen as both budding yeast and filamentous forms, although only occasional yeast forms appeared adherent to the intestinal epithelium. Conclusions. C. albicans readily colonized and replicated in the ceca of antibiotic-treated mice. The presence of two functional copies of INT1 appeared to facilitate C. albicans cecal colonization, suggesting that intestinal colonization may be another virulence factor associated with INT1 and that the gene product may be an attractive target to control C. albicans intestinal colonization.

Adhesion and the ability to form filaments are thought to contribute to the pathogenicity of Candida albicans , the leading cause of fungal disease in immunocompromised patients. Int1p is a C. albicans surface protein with limited similarity to vertebrate integrins. INT1 expression in Saccharomyces cerevisiae was sufficient to direct the adhesion of this normally nonadherent yeast to human epithelial cells. Furthermore, disruption of INT1 in C. albicans suppressed hyphal growth, adhesion to epithelial cells, and virulence in mice. Thus, INT1 links adhesion, filamentous growth, and pathogenicity in C. albicans and Int1p may be an attractive target for the development of antifungal therapies.

The existence of integrin-like proteins in Candida albicans has been postulated because monoclonal antibodies to the leukocyte integrins alpha M and alpha X bind to blastospores and germ tubes, recognize a candidal surface protein of approximately 185 kDa, and inhibit candidal adhesion to human epithelium. The gene alpha INT1 was isolated from a library of C. albicans genomic DNA by screening with a cDNA probe from the transmembrane domain of human alpha M. The predicted polypeptide (alpha Int1p) of 188 kDa contains several motifs common to alpha M and alpha X: a putative I domain, two EF-hand divalent cation-binding sites, a transmembrane domain, and a cytoplasmic tail with a single tyrosine residue. An internal RGD tripeptide is also present. Binding of anti-peptide antibodies raised to potential extracellular domains of alpha Int1p confirms surface localization in C. albicans blastopores. By Southern blotting, alpha INT1 is unique to C. albicans. Expression of alpha INT1 under control of a galactose-inducible promoter led to the production of germ tubes in haploid Saccharomyces cerevisiae and in the corresponding ste12 mutant. Germ tubes were not observed in haploid yeast transformed with vector alone, in transformants expressing a galactose-inducible gene from Chlamydomonas, or in transformants grown in the presence of glucose or raffinose. Transformants producing alpha Int1p bound an anti-alpha M monoclonal antibody and exhibited enhanced aggregation. Studies of alpha Int1p reveal novel roles for primitive integrin-like proteins in adhesion and in STE12-independent morphogenesis.

Effects of Alteration of the Candida albicans Gene INT1 on Cecal Colonization in Orally Innoculated Mice

A Novel Localization Pattern of Septins in Saccharomyces Cerevisiae Cells Expressing the Candida Albicans Gene INT1

Virulence in Candida albicans correlates with adhesion to human epithelium. The C. albicans gene INT1 encodes a putative adhesin (Int1p) homologous to the mammalian integrins αM/αX. The nonpathogenic, nonadhesive, nonfilamentous yeast S. cerevisiae does not contain INT1. When INT1 is expressed in S. cerevisiae, germ tube formation and adhesion to human epithelium parallels that of C. albicans. Because there are multiple mediators of candidal adhesion to epithelium, we sought to map the adhesive domains of Int1p in nonadherent yeast S. cerevisiae. Sequence analysis of INT1 predicts a 188 kD polypeptide including several motifs common to αM/αX [an I domain, two divalent cation binding sites (CBS1& CBS2), a transmembrane domain (TM) and a cytoplasmic tail with a single tyrosine residue (CYTO)], as well as an internal RGD site not found in integrins. INT1 constructs involving deletions/substitutions of these sites were subcloned into a yeast shuttle vector (pBM272) and expressed in S. cerevisiae YPH500 under the control of a galactose-inducible promoter. All transformed yeast strains showed normal growth curves in standard and selective media. All mutants, save the RGD deletion, formed INT1-dependent germ tubes when grown in galactose. Western blot analysis using Int1p specific antibodies revealed a protein of 200 ± 20 kD in all transformants but the RGD mutant. Epithelial adhesion of S. cerevisiae expressing INT1 and each mutant gene was determined. Adhesion of the yeast expressing the CBS2 construct was reduced but not significantly different from that expressing native INT1(24.9±4.4 vs 32.6±1.4). Specific adhesion was significantly reduced for S. cerevisiae expressing the TMCYTO (14.9 ± 2.4) and RGD (12.5 ± 2.5) constructs (p

Virulence of the yeast Candida albicans has been correlated with its ability to adhere to host epithelium and to undergo a morphologic switch from the yeast phase to various filamentous forms: germ tubes, pseudohyphae and hyphae. The C. albicans gene INT1 encodes a putative adhesin (Int1p) with structural similarity to vertebrate integrins that induces the formation of germ tubes when expressed in the budding yeast Saccharomyces cerevisiae (PNAS 1996). Because multiple adhesins mediate adherence of C. albicans to biologic sites such as epithelial cells, endothelial cells, or platelets, we tested the ability of Int1p to confer adhesive capacity on the non-adherent yeast, S. cerevisiae. We show that expression of Int1p in S. cerevisiae enables these normally non-adherent yeast cells to adhere to mammalian epithelial cells (HeLa cells). This adhesion is Int1p specific: polyclonal antibodies recognizing the EF-hand and RGD motifs of Int1p block yeast cell adhesion to HeLa cells, while pre-immune IgG does not. Epithelial adhesion of S. cerevisiae expressing Int1p approached that of the C. albicans strain from which INT1 was cloned. We also demonstrate that INT1 is involved in the hyphal growth of C. albicans. When both copies of INT1 are disrupted in C. albicans, hyphal growth on solid medium is suppressed; reintegration of INT1 into its native chromosomal locus restores the hyphal phenotype. Thus,INT1 expression is sufficient to induce both epithelial adhesion and a morphologic switch to filamentous growth in S. cerevisiaeand INT1 is required for morphogenesis in C. albicans. INT1 is the first candidal gene implicated in two steps of candidal pathogenesis: adhesion and morphogenesis. By analogy with mammalian integrins, adhesion of C. albicans cells via Int1p to an epithelial ligand may provide a signal that initiates the morphologic switch to hyphal growth. If this is the case, strategies to interrupt the interaction of Int1p with its epithelial ligands may avert the pathologic correlates of morphogenesis in the host.

MONOALLELIC DISRUPTION OF α INT1 REDUCES ANTIBODY BINDING, ADHESION, AND GERM TUBE FORMATION IN C. ALBICANS . • 1014

Results of a study are presented in which 129 grade 10 male and female students were measured by batteries from three individual-difference domains: the Comprehensive Ability Battery (CAB), the High School Personality Questionnaire (HSPQ), and the School Motivation Analysis Test (SMAT). Four ability measures from the CAB found previously to be maximally predictive of academic achievement, and three factor scales from each of the personality temperament trait (HSPQ) and motivational trait (SMAT) domains, isolated by stepwise multiple regression methods, are dealt with in detail. Correlations between measures of these traits or of simple linear composites of these measures, on the one hand, and year-end grades in various academic courses, on the other, are presented separately by sex. Information from the personality and motivational trait domains is shown to add significantly to the already-efficient prediction obtained from the ability variables.

The microbes colonizing the infant gastrointestinal tract have been implicated in later-life disease states such as allergies and obesity. Recently, the medical research community has begun to realize that very early colonization events may be most impactful on future health, with the presence of key taxa required for proper immune and metabolic development. However, most studies to date have focused on bacterial colonization events and have left out fungi, a clinically important sub-population of the microbiota. A number of recent findings indicate the importance of host-associated fungi (the mycobiota) in adult and infant disease states, including acute infections, allergies, and metabolism, making characterization of early human mycobiota an important frontier of medical research. This review summarizes the current state of knowledge with a focus on factors influencing infant mycobiota development and associations between early fungal exposures and health outcomes. We also propose next steps for infant fungal mycobiome research, including longitudinal studies of mother–infant pairs while monitoring long-term health outcomes, further exploration of bacterium–fungus interactions, and improved methods and databases for mycobiome quantitation.