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Highlights d Human fetuses in 2 nd trimester show T cell diversity with effector-memory phenotype d Fetal organs show diverse bacterial genera that can be cultured and propagated d Bacterial structures with mucin-like threads are visualized in 14-weeks EGA fetal gut d Fetal bacteria induce syngeneic memory T cell activation in fetal mLN T cells

We have characterized the role that the Msb2 protein plays in the fungal pathogen Candida albicans by the use of mutants defective in the putative upstream components of the HOG pathway. Msb2, in cooperation with Sho1, controls the activation of the Cek1 mitogen-activated protein kinase under conditions that damage the cell wall, thus defining Msb2 as a signaling element of this pathway in the fungus. Candida albicans is an important human fungal pathogen, causing infections that may represent a serious health problem. This yeast is found as a commensal in certain body locations (mainly, the vagina and tractointestinal duct) but is able to gain access to different organs under conditions of altered host immune defenses causing severe diseases. Dimorphism, the environmentally regulated differentiation program that allows this fungus to switch between a yeast-like-form (unicellular) and a filamentous form (multicellular) (25,59,95), is considered to play an important-albeit not exclusive-role (27,51,79,82,94) in the virulence of this fungus. Therefore, besides its clinical importance as an opportunistic pathogen, this microbe represents an interesting model of morphogenesis and differentiation in lower eukaryotes.

Living as a commensal, Candida albicans must adapt and respond to environmental cues generated by the mammalian host and by microbes comprising the natural flora. These signals have opposing effects on C. albicans, with host cues promoting the yeast-to-hyphal transition and bacteria-derived quorum-sensing molecules inhibiting hyphal development. Hyphal development is regulated through modulation of the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, and it has been postulated that quorum-sensing molecules can affect filamentation by inhibiting the cAMP pathway. Here, we show that both farnesol and 3-oxo-C 12 -homoserine lactone, a quorum-sensing molecule secreted by Pseudomonas aeruginosa, block hyphal development by affecting cAMP signaling; they both directly inhibited the activity of the Candida adenylyl cyclase, Cyr1p. In contrast, the 12-carbon alcohol dodecanol appeared to modulate hyphal development and the cAMP signaling pathway without directly affecting the activity of Cyr1p. Instead, we show that dodecanol exerted its effects through a mechanism involving the C. albicans hyphal repressor, Sfl1p. Deletion of SFL1 did not affect the response to farnesol but did interfere with the response to dodecanol. Therefore, quorum sensing in C. albicans is mediated via multiple mechanisms of action. Interestingly, our experiments raise the possibility that the Burkholderia cenocepacia diffusible signal factor, BDSF, also mediates its effects via Sfl1p, suggesting that dodecanol's mode of action, but not farnesol or 3-oxo-C 12 -homoserine lactone, may be used by other quorumsensing molecules.

Msb2 is a sensor protein in the plasma membrane of fungi. In the human fungal pathogen C. albicans Msb2 signals via the Cek1 MAP kinase pathway to maintain cell wall integrity and allow filamentous growth. Msb2 doubly epitope-tagged in its large extracellular and small cytoplasmic domain was efficiently cleaved during liquid and surface growth and the extracellular domain was almost quantitatively released into the growth medium. Msb2 cleavage was independent of proteases Sap9, Sap10 and Kex2. Secreted Msb2 was highly O-glycosylated by protein mannosyltransferases including Pmt1 resulting in an apparent molecular mass of >400 kDa. Deletion analyses revealed that the transmembrane region is required for Msb2 function, while the large N-terminal and the small cytoplasmic region function to downregulate Msb2 signaling or, respectively, allow its induction by tunicamycin. Purified extracellular Msb2 domain protected fungal and bacterial cells effectively from antimicrobial peptides (AMPs) histatin-5 and LL-37. AMP inactivation was not due to degradation but depended on the quantity and length of the Msb2 glycofragment. C. albicans msb2 mutants were supersensitive to LL-37 but not histatin-5, suggesting that secreted rather than cell-associated Msb2 determines AMP protection. Thus, in addition to its sensor function Msb2 has a second activity because shedding of its glycofragment generates AMP quorum resistance.

Like many organisms the fungal pathogen Candida albicans senses changes in the environmental CO2 concentration. This response involves two major proteins: adenylyl cyclase and carbonic anhydrase (CA). Here, we demonstrate that CA expression is tightly controlled by the availability of CO2 and identify the bZIP transcription factor Rca1p as the first CO2 regulator of CA expression in yeast. We show that Rca1p upregulates CA expression during contact with mammalian phagocytes and demonstrate that serine 124 is critical for Rca1p signaling, which occurs independently of adenylyl cyclase. ChIP-chip analysis and the identification of Rca1p orthologs in the model yeast Saccharomyces cerevisiae (Cst6p) point to the broad significance of this novel pathway in fungi. By using advanced microscopy we visualize for the first time the impact of CO2 build-up on gene expression in entire fungal populations with an exceptional level of detail. Our results present the bZIP protein Rca1p as the first fungal regulator of carbonic anhydrase, and reveal the existence of an adenylyl cyclase independent CO2 sensing pathway in yeast. Rca1p appears to regulate cellular metabolism in response to CO2 availability in environments as diverse as the phagosome, yeast communities or liquid culture.

The fungal cell wall is essential in maintaining cellular integrity and plays key roles in the interplay between fungal pathogens and their hosts. The PGA59 and PGA62 genes encode two short and related glycosylphosphatidylinositol-anchored cell wall proteins and their expression has been previously shown to be strongly upregulated when the human pathogen Candida albicans grows as biofilms. Using GFP fusion proteins, we have shown that Pga59 and Pga62 are cell-walllocated, N-and O-glycosylated proteins. The characterization of C. albicans pga59D/pga59D, pga62D/pga62D and pga59D/pga59D pga62D/pga62D mutants suggested a minor role of these two proteins in hyphal morphogenesis and that they are not critical to biofilm formation. Importantly, the sensitivity to different cell-wall-perturbing agents was altered in these mutants. In particular, simultaneous inactivation of PGA59 and PGA62 resulted in high sensitivity to Calcofluor white, Congo red and nikkomicin Z and in resistance to caspofungin. Furthermore, cell wall composition and observation by transmission electron microscopy indicated an altered cell wall structure in the mutant strains. Collectively, these data suggest that the cell wall proteins Pga59 and Pga62 contribute to cell wall stability and structure. INTRODUCTIONThe cell wall is an essential component of fungal cells, preserving cellular integrity and playing a central role in the interaction of fungi with their environment. This is particularly the case for pathogenic fungi such as the opportunistic yeast pathogen Candida albicans, where the cell wall has been shown to play central roles in adhesion, virulence, biofilm formation, infection and immunomodulation (Albrecht et al., 2006;Douglas, 2003;Netea et al., 2006;Richard et al., 2002b;Sundstrom, 2002). Because of the essential role of the cell wall in cellular integrity and fungal specificity of some enzymes involved in its biogenesis, it is a recognized target for the development of novel antifungals (e.g. echinocandins that target the b-1,3-glucan synthase) (Latge, 2007).The organization of the fungal cell wall has been mainly characterized in the yeasts Saccharomyces cerevisiae and C. albicans and in the filamentous fungus Aspergillus fumigatus (Klis et al., 2006;Latge, 2007;Lesage & Bussey, 2006;Ruiz-Herrera et al., 2006). The yeast cell wall has a bilayered structure. The inner part is composed of a network of b-1,3-glucan molecules linked by hydrogen bonds. These chains can be bound covalently to b-1,6-glucan molecules and to chitin chains. The outer part of the cell wall is composed mainly of mannoproteins (Klis et al., 2006Lesage & Bussey, 2006). Most proteins in the cell wall of ascomycetous yeasts are glycosylphosphatidylinositolanchored proteins (GPI-modified proteins) that become covalently linked to b-1,6-glucan through a remnant of their GPI anchor. As the b-1,6-glucan moiety can be linked to b-1,3-glucan or chitin, the cell wall GPI-modified proteins are strongly linked to the cell wall Klis et al., 2001;Richard & Plaine, 2007).GPI-...

Candida albicans is the most important fungal pathogen of humans, causing severe infections, especially in nosocomial and immunocompromised settings. However, it is also the most prevalent fungus of the normal human microbiome, where it shares its habitat with hundreds of trillions of other microbial cells. Despite weak organic acids (WOAs) being among the most abundant metabolites produced by bacterial microbiota, little is known about their effect on C. albicans. Here we used a sequencing-based profiling strategy to systematically investigate the transcriptional stress response of C. albicans to lactic, acetic, propionic, and butyric acid at several time points after treatment. Our data reveal a complex transcriptional response, with individual WOAs triggering unique gene expression profiles and with important differences between acute and chronic exposure. Despite these dissimilarities, we found significant overlaps between the gene expression changes induced by each WOA, which led us to uncover a core transcriptional response that was largely unrelated to other previously published C. albicans transcriptional stress responses. Genes commonly up-regulated by WOAs were enriched in several iron transporters, which was associated with an overall decrease in intracellular iron concentrations. Moreover, chronic exposure to any WOA lead to down-regulation of RNA synthesis and ribosome biogenesis genes, which resulted in significant reduction of total RNA levels and of ribosomal RNA in particular. In conclusion, this study suggests that gastrointestinal microbiota might directly influence C. albicans physiology via production of WOAs, with possible implications of how this fungus interacts with its host in both health and disease.