Candida albicans Delays HIV-1 Replication in Macrophages

Macrophages are one of the most important HIV-1 target cells. Unlike CD4⁺ T cells, macrophages are resistant to the cytophatic effect of HIV-1. They are able to produce and harbor the virus for long periods acting as a viral reservoir. Candida albicans (CA) is a commensal fungus that colonizes the portals of HIV-1 entry, such as the vagina and the rectum, and becomes an aggressive pathogen in AIDS patients. In this study, we analyzed the ability of CA to modulate the course of HIV-1 infection in human monocyte-derived macrophages. We found that CA abrogated HIV-1 replication in macrophages when it was evaluated 7 days after virus inoculation. A similar inhibitory effect was observed in monocyte-derived dendritic cells. The analysis of the mechanisms responsible for the inhibition of HIV-1 production in macrophages revealed that CA efficiently sequesters HIV-1 particles avoiding its infectivity. Moreover, by acting on macrophages themselves, CA diminishes their permissibility to HIV-1 infection by reducing the expression of CD4, enhancing the production of the CCR5-interacting chemokines CCL3/MIP-1α, CCL4/MIP-1β, and CCL5/RANTES, and stimulating the production of interferon-α and the restriction factors APOBEC3G, APOBEC3F, and tetherin. Interestingly, abrogation of HIV-1 replication was overcome when the infection of macrophages was evaluated 2-3 weeks after virus inoculation. However, this reactivation of HIV-1 infection could be silenced by CA when added periodically to HIV-1-challenged macrophages. The induction of a silent HIV-1 infection in macrophages at the periphery, where cells are continuously confronted with CA, might help HIV-1 to evade the immune response and to promote resistance to antiretroviral therapy.     

Deficient Beta-Mannosylation of Candida albicans Phospholipomannan Affects the Proinflammatory Response in Macrophages

Candida albicans produces a complex glycosphingolipid called phospholipomannan (PLM), which is present on the cell-wall surface of yeast and shed upon contact with host cells. The glycan moiety of PLM is composed of β-mannosides with degrees of polymerization up to 19 in C. albicans serotype A. PLM from serotype B strains displays a twofold decrease in the length of the glycan chains. In this study we compared the proinflammatory activities of PLMs purified from C. albicans serotype A and serotype B strains and from a bmt6Δ mutant of C. albicans, whose PLM is composed of short truncated oligomannosidic chain. We found that PLMs activate caspase-1 in murine macrophage cell line J774 independent of the glycan chain length although IL-1β secretion is more intense with long glycan chain. None of the tested PLMs stimulate ROS production, indicating that caspase-1 activation may occur through a ROS-independent pathway. On the other hand, only long-chain oligomannosides present on PLM from serotype A strain (PLM-A) are able to induce TNF-α production in macrophages, a property that is not affect by blocking endocytosis through latrunculin A treatment. Finally, we demonstrate that soluble and not cell surface-bound galectin-3, is able to potentiate PLM-A-induced TNF-α production in macrophages. PLMs from C. albicans serotype B and from bmt6∆ mutant are not able to induce TNF-α production and galectin-3 pretreatment does not interfere with this result. In conclusion, we show here that PLMs are able to evoke a proinflammatory state in macrophage, which is in part dependent on their glycosylation status. Long-glycan chains favor interaction with soluble galectin-3 and help amplify inflammatory response.     

One Small Step for a Yeast - Microevolution within Macrophages Renders Candida glabrata Hypervirulent Due to a Single Point Mutation

Candida glabrata is one of the most common causes of candidemia, a life-threatening, systemic fungal infection, and is surpassed in frequency only by Candida albicans. Major factors contributing to the success of this opportunistic pathogen include its ability to readily acquire resistance to antifungals and to colonize and adapt to many different niches in the human body. Here we addressed the flexibility and adaptability of C. glabrata during interaction with macrophages with a serial passage approach. Continuous co-incubation of C. glabrata with a murine macrophage cell line for over six months resulted in a striking alteration in fungal morphology: The growth form changed from typical spherical yeasts to pseudohyphae-like structures – a phenotype which was stable over several generations without any selective pressure. Transmission electron microscopy and FACS analyses showed that the filamentous-like morphology was accompanied by changes in cell wall architecture. This altered growth form permitted faster escape from macrophages and increased damage of macrophages. In addition, the evolved strain (Evo) showed transiently increased virulence in a systemic mouse infection model, which correlated with increased organ-specific fungal burden and inflammatory response (TNFα and IL-6) in the brain. Similarly, the Evo mutant significantly increased TNFα production in the brain on day 2, which is mirrored in macrophages confronted with the Evo mutant, but not with the parental wild type. Whole genome sequencing of the Evo strain, genetic analyses, targeted gene disruption and a reverse microevolution experiment revealed a single nucleotide exchange in the chitin synthase-encoding CHS2 gene as the sole basis for this phenotypic alteration. A targeted CHS2 mutant with the same SNP showed similar phenotypes as the Evo strain under all experimental conditions tested. These results indicate that microevolutionary processes in host-simulative conditions can elicit adaptations of C. glabrata to distinct host niches and even lead to hypervirulent strains.     

Candida albicans Suppresses Nitric Oxide Generation from Macrophages via a Secreted Molecule

Macrophages and neutrophils generate a potent burst of reactive oxygen and nitrogen species as a key aspect of the antimicrobial response. While most successful pathogens, including the fungus Candida albicans, encode enzymes for the detoxification of these compounds and repair of the resulting cellular damage, some species actively modulate immune function to suppress the generation of these toxic compounds. We report here that C. albicans actively inhibits macrophage production of nitric oxide (NO). NO production was blocked in a dose-dependent manner when live C. albicans were incubated with either cultured or bone marrow-derived mouse macrophages. While filamentous growth is a key virulence trait, yeast-locked fungal cells were still capable of dose-dependent NO suppression. C. albicans suppresses NO production from macrophages stimulated by exposure to IFN-γ and LPS or cells of the non-pathogenic Saccharomyces cerevisiae. The NO inhibitory activity was produced only when the fungal cells were in direct contact with macrophages, but the compound itself was secreted into the culture media. LPS/IFNγ stimulated macrophages cultured in cell-free conditioned media from co-cultures showed reduced levels of iNOS enzymatic activity and lower amounts of iNOS protein. Initial biochemical characterization of this activity indicates that the inhibitor is a small, aqueous, heat-stable compound. In summary, C. albicans actively blocks NO production by macrophages via a secreted mediator; these findings expand our understanding of phagocyte modulation by this important fungal pathogen and represent a potential target for intervention to enhance antifungal immune responses.     

Pathways Regulating Cytosolic Phospholipase A2 Activation and Eicosanoid Production in Macrophages by Candida albicans

Resident tissue macrophages are activated by the fungal pathogen Candida albicans to release eicosanoids, which are important modulators of inflammation and immune responses. Our objective was to identify the macrophage receptors engaged by C. albicans that mediate activation of group IVA cytosolic phospholipase A₂ (cPLA₂α), a regulatory enzyme that releases arachidonic acid (AA) for production of prostaglandins and leukotrienes. A comparison of peritoneal macrophages from wild type and knock-out mice demonstrates that the β-glucan receptor Dectin-1 and MyD88 regulate early release of AA and eicosanoids in response to C. albicans. However, cyclooxygenase 2 (COX2) expression and later phase eicosanoid production are defective in MyD88^−/− but not Dectin-1^−/− macrophages. Furthermore, C. albicans-stimulated activation of MAPK and phosphorylation of cPLA₂α on Ser-505 are regulated by MyD88 and not Dectin-1. In contrast, Dectin-1 mediates MAPK activation, cPLA₂α phosphorylation, and COX2 expression in response to particulate β-glucan suggesting that other receptors engaged by C. albicans preferentially mediate these responses. Results also implicate the mannan-binding receptor Dectin-2 in regulating cPLA₂α. C. albicans-stimulated MAPK activation and AA release are blocked by d-mannose and Dectin-2-specific antibody, and overexpression of Dectin-2 in RAW264.7 macrophages enhances C. albicans-stimulated MAPK activation, AA release, and COX2 expression. In addition, calcium mobilization is enhanced in RAW264.7 macrophages overexpressing Dectin-1 or -2. The results demonstrate that C. albicans engages both β-glucan and mannan-binding receptors on macrophages that act with MyD88 to regulate the activation of cPLA₂α and eicosanoid production.     

Shewanella oneidensis MR-1 Restores Menaquinone Synthesis to a Menaquinone-Negative Mutant

The mechanisms underlying the use of insoluble electron acceptors by metal-reducing bacteria, such as Shewanella oneidensis MR-1, are currently under intensive study. Current models for shuttling electrons across the outer membrane (OM) of MR-1 include roles for OM cytochromes and the possible excretion of a redox shuttle. While MR-1 is able to release a substance that restores the ability of a menaquinone (MK)-negative mutant, CMA-1, to reduce the humic acid analog anthraquinone-2,6-disulfonate (AQDS), cross-feeding experiments conducted here showed that the substance released by MR-1 restores the growth of CMA-1 on several soluble electron acceptors. Various strains derived from MR-1 also release this substance; these include mutants lacking the OM cytochromes OmcA and OmcB and the OM protein MtrB. Even though strains lacking OmcB and MtrB cannot reduce Fe(III) or AQDS, they still release a substance that restores the ability of CMA-1 to use MK-dependent electron acceptors, including AQDS and Fe(III). Quinone analysis showed that this released substance restores MK synthesis in CMA-1. This ability to restore MK synthesis in CMA-1 explains the cross-feeding results and challenges the previous hypothesis that this substance represents a redox shuttle that facilitates metal respiration.     

Defects in Assembly of the Extracellular Matrix Are Responsible for Altered Morphogenesis of a Candida albicans phr1 Mutant

Analysis of Candida albicans cells using antibodies directed against Gas1p/Ggp1p, Saccharomyces cerevisiae homolog of Phr1p, revealed that Phr1p is a glycoprotein of about 88 kDa whose accumulation increases with the rise of external pH. This polypeptide is present both in the yeast form and during germ tube induction. In the Phr1⁻ cells at pH 8 the solubility of glucans in alkali is greatly affected. In the parental strain the alkali-soluble/-insoluble glucan ratio shows a 50% decrease at pH 8 with respect to pH 4.5, whereas in the null mutant it is unchanged, indicating the lack of a polymer cross-linker activity induced by the rise of pH. The mutant has a sixfold increase in chitin level and is hypersensitive to calcofluor. Consistently with a role of chitin in strengthening the cell wall, Phr1⁻ cells are more sensitive to nikkomycin Z than the parental strain.     

Gluconeogenesis in Candida albicans

According to different metabolic situations in various stages of Candida albicans pathogenesis the regulation of carbohydrate metabolism was investigated. We report the genetic characterization of all major C. albicans gluconeogenic and glyoxylate cycle genes (fructose-1,6-bisphosphatase, PEP carboxykinase, malate synthase and isocitrate lyase) which were isolated after functional complementation of the corresponding Saccharomyces cerevisiae deletion mutants. Remarkably, the regulation of the heterologously expressed C. albicans gluconeogenic and glyoxylate cycle genes was similar to that of the homologous S. cerevisiae genes. A C. albicans DeltaCafbp1 deletion strain failed to utilize non-fermentable carbon sources but hyphal growth was not affected. Our results show that regulation of gluconeogenesis in C. albicans is similar to that of S. cerevisiae and that the current knowledge on how gluconeogenesis is regulated will facilitate the physiological understanding of C. albicans.     

念珠菌性阴道炎小鼠模型的建立

目的建立念珠菌定植的阴道感染小鼠模型。方法先用林可霉素溶液(浓度为100mg/ml)冲洗小鼠阴道,再将念珠菌接种到小鼠阴道内。结果模型组小鼠阴道内主要细菌数量乳杆菌显著下降(P<0.01),出现阴道红肿、分泌物多呈块状和阴道黏膜充血等念珠菌阴道炎的典型症状。结论通过抗生素处理后,再在小鼠阴道内接种念珠菌,念珠菌能在小鼠阴道内定植,即建立了念珠菌性阴道炎的小鼠模型。     

Derepressed Hyphal Growth and Reduced Virulence in a VH1 Family-related Protein Phosphatase Mutant of the Human Pathogen Candida albicans

Mitogen-activated protein (MAP) kinases are pivotal components of eukaryotic signaling cascades. Phosphorylation of tyrosine and threonine residues activates MAP kinases, but either dual-specificity or monospecificity phosphatases can inactivate them. The Candida albicans CPP1 gene, a structural member of the VH1 family of dual- specificity phosphatases, was previously cloned by its ability to block the pheromone response MAP kinase cascade in Saccharomyces cerevisiae. Cpp1p inactivated mammalian MAP kinases in vitro and acted as a tyrosine-specific enzyme. In C. albicans a MAP kinase cascade can trigger the transition from the budding yeast form to a more invasive filamentous form. Disruption of the CPP1 gene in C. albicans derepressed the yeast to hyphal transition at ambient temperatures, on solid surfaces. A hyphal growth rate defect under physiological conditions in vitro was also observed and could explain a reduction in virulence associated with reduced fungal burden in the kidneys seen in a systemic mouse model. A hyper-hyphal pathway may thus have some detrimental effects on C. albicans cells. Disruption of the MAP kinase homologue CEK1 suppressed the morphological effects of the CPP1 disruption in C. albicans. The results presented here demonstrate the biological importance of a tyrosine phosphatase in cell-fate decisions and virulence in C. albicans.     

Adhesins in Candida albicans.

The adherent properties of Candida albicans blastoconidia and germ tubes have long been appreciated, but little is known about the mechanisms responsible for adherence. Recently, three genes, ALA1, ALS1 and HWP1, encoding proteins with adherent properties and motifs consistent with linkage to the beta-1, 6-glucan of C. albicans cell walls have provided insight into the topology of protein adhesins. Hwp1, a developmentally regulated adhesin of germ tubes and hyphae, attaches to buccal epithelial cells by an unconventional, transglutaminase-mediated mechanism of adhesion.     

Chromosome instability in Candida albicans

Candida albicans maintains genetic diversity by random chromosome alterations, and this diversity allows utilization of various nutrients. Although the alterations seem to occur spontaneously, their frequencies clearly depend on environmental factors. In addition, this microorganism survives in adverse environments, which cause lethality or inhibit growth, by altering specific chromosomes. A reversible loss or gain of one homolog of a specific chromosome in this diploid organism was found to be a prevalent means of adaptation. We found that loss of an entire chromosome is required because it carries multiple functionally redundant negative regulatory genes. The unusual mode of gene regulation in Candida albicans implies that genes in this organism are distributed nonrandomly over chromosomes.