Infants exposed to antibiotics after birth have altered recognition memory responses at one month of age.

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 < 0.04). Infants exposed to antibiotics showed larger P2 amplitudes to stranger's as compared to mother's voice, a reversal of the typical response exhibited by unexposed infants. Abnormal ERP responses did not consistently correlate with increased inflammatory cytokines within the antibiotic-exposed group. CONCLUSIONS: Otherwise healthy infants exposed to antibiotics soon after birth demonstrated altered auditory processing and recognition memory responses, supporting the possibility of a microbiota-gut-brain axis in humans during early life. IMPACT: Infants exposed to antibiotics after birth demonstrate altered auditory processing and recognition memory responses at 1 month of age. Preclinical models support a role for gut microbiomes in modulating brain function and behavior, particularly in developing brains. This study is one of the first to explore the relevance of these findings for human infants. The findings of this study have implications for the management and follow-up of at-risk infants with exposure to gut-microbiome disrupting factors and lay foundation for future studies to further characterize the short- and long-term effects of gut microbiome perturbation on brain development.

Breastmilk and NICU surfaces are potential sources of fungi for infant mycobiomes.

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.

Infant fungal communities: current knowledge and research opportunities.

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.

Cdc42 GTPase dynamics control directional growth responses.

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.

PCR-mediated gene modification strategy for construction of fluorescent protein fusions in Candida parapsilosis.

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.

Human Milk Oligosaccharides Inhibit Candida albicans Invasion of Human Premature Intestinal Epithelial Cells.

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.

Rsr1 focuses Cdc42 activity at hyphal tips and promotes maintenance of hyphal development in Candida albicans.

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.

Assembly, stability, and dynamics of the infant gut microbiome are linked to bacterial strains and functions in mother's milk.

The establishment of the gut microbiome in early life is critical for healthy infant development. Although human milk is recommended as the sole source of nutrition for the human infant, little is known about how variation in milk composition, and especially the milk microbiome, shapes the microbial communities in the infant gut. Here, we quantified the similarity between the maternal milk and the infant gut microbiome using 507 metagenomic samples collected from 195 mother-infant pairs at one, three, and six months postpartum. We found that the microbial taxonomic overlap between milk and the infant gut was driven by bifidobacteria, in particular by B. longum. Infant stool samples dominated by B. longum also showed higher temporal stability compared to samples dominated by other species. We identified two instances of strain sharing between maternal milk and the infant gut, one involving a commensal (B. longum) and one a pathobiont (K. pneumoniae). In addition, strain sharing between unrelated infants was higher among infants born at the same hospital compared to infants born in different hospitals, suggesting a potential role of the hospital environment in shaping the infant gut microbiome composition. The infant gut microbiome at one month compared to six months of age was enriched in metabolic pathways associated with de-novo molecule biosynthesis, suggesting that early colonisers might be more versatile and metabolically independent compared to later colonizers. Lastly, we found a significant overlap in antimicrobial resistance genes carriage between the mother's milk and their infant's gut microbiome. Taken together, our results suggest that the human milk microbiome has an important role in the assembly, composition, and stability of the infant gut microbiome.

Rax2 is important for directional establishment of growth sites, but not for reorientation of growth axes, during Candida albicans hyphal morphogenesis.

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.

Importance of human milk for infants in the clinical setting: Updates and mechanistic links.

INTRODUCTION: Human milk (HM) is the optimal source of nutrition for infants and has been implicated in multiple aspects of infant health. Although much of the existing literature has focused on the individual components that drive its nutrition content, examining HM as a biological system is needed for meaningful advancement of the field. Investigation of the nonnutritive bioactive components of HM and the maternal, infant, and environmental factors which affect these bioactives is important to better understand the importance of HM provision to infants. This information may inform care of clinical populations or infants who are critically ill, hospitalized, or who have chronic diseases and may benefit most from receiving HM. METHODS: In this narrative review, we reviewed literature examining maternal and infant influences on HM composition with a focus on studies published in the last 10 years that were applicable to clinical populations. RESULTS: We found multiple studies examining HM components implicated in infant immune and gut health and neurodevelopment. Additional work is needed to understand how donor milk and formula may be used in situations of inadequate maternal HM. Furthermore, a better understanding of how maternal factors such as maternal genetics and metabolic health influence milk composition is needed. CONCLUSION: In this review, we affirm the importance of HM for all infants, especially clinical populations. An understanding of how HM composition is modulated by maternal and environmental factors is important to progress the field forward with respect to mechanistic links between HM biology and infant health outcomes.

Human Cytomegalovirus in breast milk is associated with milk composition, the infant gut microbiome, and infant growth.

Human cytomegalovirus (CMV) is a highly prevalent herpesvirus that is often transmitted to the neonate via breast milk. Postnatal CMV transmission can have negative health consequences for preterm and immunocompromised infants, but any effects on healthy term infants are thought to be benign. Furthermore, the impact of CMV on the composition of the hundreds of bioactive factors in human milk has not been tested. Here, we utilize a cohort of exclusively breastfeeding full term mother-infant pairs to test for differences in the milk transcriptome and metabolome associated with CMV, and the impact of CMV in breast milk on the infant gut microbiome and infant growth. We find upregulation of the indoleamine 2,3- dioxygenase (IDO) tryptophan-to-kynurenine metabolic pathway in CMV+ milk samples, and that CMV+ milk is associated with decreased Bifidobacterium in the infant gut. Our data indicate a complex relationship between milk CMV, milk kynurenine, and infant growth; with kynurenine positively correlated, and CMV viral load negatively correlated, with infant weight-for-length at 1 month of age. These results suggest CMV transmission, CMV-related changes in milk composition, or both may be modulators of full term infant development.

Human milk variation is shaped by maternal genetics and impacts the infant gut microbiome.

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 of Bifidobacteria in 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.

Gestational Diabetes Mellitus Is Associated with Differences in Human Milk Hormone and Cytokine Concentrations in a Fully Breastfeeding United States Cohort.

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.

Gestational Diabetes Mellitus Is Associated with Altered Abundance of Exosomal MicroRNAs in Human Milk.

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.

Bacterial, fungal, and interkingdom microbiome features of exclusively breastfeeding dyads are associated with infant age, antibiotic exposure, and birth mode.

The composition and function of early life gut bacterial communities (microbiomes) have been proposed to modulate health for the long term. In addition to bacteria, fungi (mycobiomes) also colonize the early life gut and have been implicated in health disorders such as asthma and obesity. Despite the potential importance of mycobiomes in health, there has been a lack of study regarding fungi and their interkingdom interactions with bacteria during infancy. The goal of this study was to obtain a more complete understanding of microbial communities thought to be relevant for the early life programming of health. Breastmilk and infant feces were obtained from a unique cohort of healthy, exclusively breastfeeding dyads recruited as part of the Mothers and Infants Linked for Healthy Growth (MILk) study with microbial taxa characterized using amplicon-based sequencing approaches. Bacterial and fungal communities in breastmilk were both distinct from those of infant feces, consistent with niche-specific microbial community development. Nevertheless, overlap was observed among sample types (breastmilk, 1-month feces, 6-month feces) with respect to the taxa that were the most prevalent and abundant. Self-reported antibacterial antibiotic exposure was associated with micro- as well as mycobiome variation, which depended upon the subject receiving antibiotics (mother or infant), timing of exposure (prenatal, peri- or postpartum), and sample type. In addition, birth mode was associated with bacterial and fungal community variation in infant feces, but not breastmilk. Correlations between bacterial and fungal taxa abundances were identified in all sample types. For infant feces, congruency between bacterial and fungal communities was higher for older infants, consistent with the idea of co-maturation of bacterial and fungal gut communities. Interkingdom connectedness also tended to be higher in older infants. Additionally, higher interkingdom connectedness was associated with Cesarean section birth and with antibiotic exposure for microbial communities of both breastmilk and infant feces. Overall, these results implicate infant age, birth mode, and antibiotic exposure in bacterial, fungal and interkingdom relationship variation in early-life-relevant microbiomes, expanding the current literature beyond bacteria.

Candida species differ in their interactions with immature human gastrointestinal epithelial cells.

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 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 use different pathogenesis mechanisms.

SLA2 mutations cause SWE1-mediated cell cycle phenotypes in Candida albicans and Saccharomyces cerevisiae.

The early endocytic patch protein Sla2 is important for morphogenesis and growth rates in Saccharomyces cerevisiae and Candida albicans, but the mechanism that connects these processes is not clear. Here we report that growth defects in cells lacking CaSLA2 or ScSLA2 are 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. Only sla2 strains 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 of C. albicans infections.

An internal polarity landmark is important for externally induced hyphal behaviors in Candida albicans.

Directional growth is a function of polarized cells such as neurites, pollen tubes, and fungal hyphae. Correct orientation of the extending cell tip depends on signaling pathways and effectors that mediate asymmetric responses to specific environmental cues. In the hyphal form of the eukaryotic fungal pathogen Candida albicans, these responses include thigmotropism and galvanotropism (hyphal turning in response to changes in substrate topography and imposed electrical fields, respectively) and penetration into semisolid substrates. During vegetative growth in C. albicans, as in the model yeast Saccharomyces cerevisiae, the Ras-like GTPase Rsr1 mediates internal cellular cues to position new buds in a prespecified pattern on the mother cell cortex. Here, we demonstrate that Rsr1 is also important for hyphal tip orientation in response to the external environmental cues that induce thigmotropic and galvanotropic growth. In addition, Rsr1 is involved in hyphal interactions with epithelial cells in vitro and its deletion diminishes the hyphal invasion of kidney tissue during systemic infection. Thus, Rsr1, an internal polarity landmark in yeast, is also involved in polarized growth responses to asymmetric environmental signals, a paradigm that is different from that described for the homologous protein in S. cerevisiae. Rsr1 may thereby contribute to the pathogenesis of C. albicans infections by influencing hyphal tip responses triggered by interaction with host tissues.

Development of the Human Mycobiome over the First Month of Life and across Body Sites.

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.

Generation of Fluorescent Protein Fusions in Candida Species.

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.