Candida glabrata Binding to Candida albicans Hyphae Enables Its Development in Oropharyngeal Candidiasis

Pathogenic mechanisms of Candida glabrata in oral candidiasis, especially because of its inability to form hyphae, are understudied. Since both Candida albicans and C. glabrata are frequently co-isolated in oropharyngeal candidiasis (OPC), we examined their co-adhesion in vitro and observed adhesion of C. glabrata only to C. albicans hyphae microscopically. Mice were infected sublingually with C. albicans or C. glabrata individually, or with both species concurrently, to study their ability to cause OPC. Infection with C. glabrata alone resulted in negligible infection of tongues; however, colonization by C. glabrata was increased by co-infection or a pre-established infection with C. albicans. Furthermore, C. glabrata required C. albicans for colonization of tongues, since decreasing C. albicans burden with fluconazole also reduced C. glabrata. C. albicans hyphal wall adhesins Als1 and Als3 were important for in vitro adhesion of C. glabrata and to establish OPC. C. glabrata cell wall protein coding genes EPA8, EPA19, AWP2, AWP7, and CAGL0F00181 were implicated in mediating adhesion to C. albicans hyphae and remarkably, their expression was induced by incubation with germinated C. albicans. Thus, we found a near essential requirement for the presence of C. albicans for both initial colonization and establishment of OPC infection by C. glabrata.     

A Murine Model of Candida glabrata Vaginitis Shows No Evidence of an Inflammatory Immunopathogenic Response

Candida glabrata is the second most common organism isolated from women with vulvovaginal candidiasis (VVC), particularly in women with uncontrolled diabetes mellitus. However, mechanisms involved in the pathogenesis of C. glabrata-associated VVC are unknown and have not been studied at any depth in animal models. The objective of this study was to evaluate host responses to infection following efforts to optimize a murine model of C. glabrata VVC. For this, various designs were evaluated for consistent experimental vaginal colonization (i.e., type 1 and type 2 diabetic mice, exogenous estrogen, varying inocula, and co-infection with C. albicans). Upon model optimization, vaginal fungal burden and polymorphonuclear neutrophil (PMN) recruitment were assessed longitudinally over 21 days post-inoculation, together with vaginal concentrations of IL-1β, S100A8 alarmin, lactate dehydrogenase (LDH), and in vivo biofilm formation. Consistent and sustained vaginal colonization with C. glabrata was achieved in estrogenized streptozotocin-induced type 1 diabetic mice. Vaginal PMN infiltration was consistently low, with IL-1β, S100A8, and LDH concentrations similar to uninoculated mice. Biofilm formation was not detected in vivo, and co-infection with C. albicans did not induce synergistic immunopathogenic effects. This data suggests that experimental vaginal colonization of C. glabrata is not associated with an inflammatory immunopathogenic response or biofilm formation.     

Type I Interferons Promote Fatal Immunopathology by Regulating Inflammatory Monocytes and Neutrophils during Candida Infections

Invasive fungal infections by Candida albicans (Ca) are a frequent cause of lethal sepsis in intensive care unit patients. While a contribution of type I interferons (IFNs-I) in fungal sepsis remains unknown, these immunostimulatory cytokines mediate the lethal effects of endotoxemia and bacterial sepsis. Using a mouse model lacking a functional IFN-I receptor (Ifnar1^−/−), we demonstrate a remarkable protection against invasive Ca infections. We discover a mechanism whereby IFN-I signaling controls the recruitment of inflammatory myeloid cells, including Ly6C^hi monocytes and neutrophils, to infected kidneys by driving expression of the chemokines CCL2 and KC. Within kidneys, monocytes differentiate into inflammatory DCs but fail to functionally mature in Ifnar1^−/− mice, as demonstrated by the impaired upregulation of the key activation markers PDCA1 and iNOS. The increased activity of inflammatory monocytes and neutrophils results in hyper-inflammation and lethal kidney pathology. Pharmacological diminution of monocytes and neutrophils by treating mice with pioglitazone, a synthetic agonist of the nuclear receptor peroxisome proliferator-activated receptor-γ (PPAR-γ), strongly reduces renal immunopathology during Ca infection and improves mouse survival. Taken together, our data connect for the first time the sepsis-promoting functions of IFNs-I to the CCL2-mediated recruitment and the activation of inflammatory monocytes/DCs with high host-destructing potency. Moreover, our data demonstrate a therapeutic relevance of PPAR-γ agonists for microbial infectious diseases where inflammatory myeloid cells may contribute to fatal tissue damage.     

The role of galectin‐3 in phagocytosis of Candida albicans and Candida parapsilosis by human neutrophils

Candida albicans causes the majority of invasive candidiasis in immunocompromised adults while Candida parapsilosis is a leading cause of neonatal candidiasis. While much work has focused on how the immune system recognizes and responds to C. albicans, less is known about host interaction with C. parapsilosis. This study investigates the human neutrophil phagocytic response to these species. Neutrophils underwent phagocytosis of C. parapsilosis yeast and C. albicans hyphae much more efficiently than C. albicans yeast. Treatment of neutrophils with a galectin‐3 (gal3) blocking antibody inhibited phagocytosis of C. parapsilosis yeast and C. albicans hyphae, but not C. albicans yeast. The majority of neutrophil gal3 was expressed intracellularly and was secreted from neutrophils after treatment with C. parapsilosis mannan. When neutrophils were treated with exogenous gal3, phagocytosis of both C. albicans and C. parapsilosis yeast increased. Exposure of neutrophils to C. parapsilosis yeast increased phagocytosis of C. albicans yeast and was inhibited by gal3 blocking antibody. Taken together, these data indicate that gal3 secreted from neutrophils may act as a pro‐inflammatory autocrine/paracrine signal in neutrophil phagocytosis and suggest that gal3 has a unique role in neutrophil response to C. parapsilosis yeast and C. albicans hyphae distinct from C. albicans yeast.     

Candida albicans colonization and dissemination from the murine gastrointestinal tract: the influence of morphology and Th17 immunity

The ability of Candida albicans to cause disease is associated with its capacity to undergo morphological transition between yeast and filamentous forms, but the role of morphology in colonization and dissemination from the gastrointestinal (GI) tract remains poorly defined. To explore this, we made use of wild‐type and morphological mutants of C. albicans in an established model of GI tract colonization, induced following antibiotic treatment of mice. Our data reveal that GI tract colonization favours the yeast form of C. albicans, that there is constitutive low level systemic dissemination in colonized mice that occurs irrespective of fungal morphology, and that colonization is not controlled by Th17 immunity in otherwise immunocompetent animals. These data provide new insights into the mechanisms of pathogenesis and commensalism of C. albicans, and have implications for our understanding of human disease.     

Ribosomal protein S6 phosphorylation is controlled by TOR and modulated by PKA in Candida albicans

TOR and PKA signaling pathways control eukaryotic cell growth and proliferation. TOR activity in model fungi, such as Saccharomyces cerevisiae, responds principally to nutrients, e.g., nitrogen and phosphate sources, which are incorporated into the growing cell mass; PKA signaling responds to the availability of the cells' major energy source, glucose. In the fungal commensal and pathogen, Candida albicans, little is known of how these pathways interact. Here, the signal from phosphorylated ribosomal protein S6 (P‐S6) was defined as a surrogate marker for TOR‐dependent anabolic activity in C. albicans. Nutritional, pharmacologic and genetic modulation of TOR activity elicited corresponding changes in P‐S6 levels. The P‐S6 signal corresponded to translational activity of a GFP reporter protein. Contributions of four PKA pathway components to anabolic activation were then examined. In high glucose concentrations, only Tpk2 was required to upregulate P‐S6 to physiologic levels, whereas all four tested components were required to downregulate P‐S6 in low glucose. TOR was epistatic to PKA components with respect to P‐S6. In many host niches inhabited by C. albicans, glucose is scarce, with protein being available as a nitrogen source. We speculate that PKA may modulate TOR‐dependent cell growth to a rate sustainable by available energy sources, when monomers of anabolic processes, such as amino acids, are abundant.     

A Virtual Infection Model Quantifies Innate Effector Mechanisms and Candida albicans Immune Escape in Human Blood

Candida albicans bloodstream infection is increasingly frequent and can result in disseminated candidiasis associated with high mortality rates. To analyze the innate immune response against C. albicans, fungal cells were added to human whole-blood samples. After inoculation, C. albicans started to filament and predominantly associate with neutrophils, whereas only a minority of fungal cells became attached to monocytes. While many parameters of host-pathogen interaction were accessible to direct experimental quantification in the whole-blood infection assay, others were not. To overcome these limitations, we generated a virtual infection model that allowed detailed and quantitative predictions on the dynamics of host-pathogen interaction. Experimental time-resolved data were simulated using a state-based modeling approach combined with the Monte Carlo method of simulated annealing to obtain quantitative predictions on a priori unknown transition rates and to identify the main axis of antifungal immunity. Results clearly demonstrated a predominant role of neutrophils, mediated by phagocytosis and intracellular killing as well as the release of antifungal effector molecules upon activation, resulting in extracellular fungicidal activity. Both mechanisms together account for almost of C. albicans killing, clearly proving that beside being present in larger numbers than other leukocytes, neutrophils functionally dominate the immune response against C. albicans in human blood. A fraction of C. albicans cells escaped phagocytosis and remained extracellular and viable for up to four hours. This immune escape was independent of filamentation and fungal activity and not linked to exhaustion or inactivation of innate immune cells. The occurrence of C. albicans cells being resistant against phagocytosis may account for the high proportion of dissemination in C. albicans bloodstream infection. Taken together, iterative experiment–model–experiment cycles allowed quantitative analyses of the interplay between host and pathogen in a complex environment like human blood.     

Virulence Factors and Anti Fungal Sensitivity Pattern of<Emphasis Type="Italic"> Candida</Emphasis> Sp. Isolated from HIV and TB Patients

The study comprised of 60 Candida spp., 50 isolates from HIV and TB positive individuals (immunocompromised) and 10 isolates from non-HIV and -TB patients (immunocompetent). Among the 60 Candidal isolates, 83.3% were identified as C. albicans, 11.6% as C. glabrata and rest 5% as C. krusei. There is no study in production pattern of extracellular enzymes of Candida spp. isolated from HIV and TB patients in comparison with non-HIV and -TB patients in India. The comparison of phospholipase activities showed that there was a significant difference between the groups at (P = 0.001). The non-HIV and -TB groups of C. glabrata and C. krusei did not show detectable phospholipase activity when compared to the HIV and TB groups. The mean difference in the phospholipase activities of these two groups was significant (P = <0.001). Candida spp. of both the groups do not possess the ability to hydrolyze gelatin. All the strains possessed the ability to show alpha haemolysis. Even though it had shown alpha haemolysis, the significant difference in haemolytic activity was observed only in C. albicans (P = <0.001). None of the isolates from the two groups possessed the ability to hydrolyze gelatin. In the resistance profile of Candida spp., C. albicans of HIV and TB groups had shown resistance to fluconazole, Itraconazole, ketaconazole, nystatin but showed 100% sensitivity towards amphotericin-B. The isolates of C. krusei and C. glabrata showed no resistance to any of the drugs tested. In the case of, non-HIV and -TB patients the resistance pattern was low.     

Compositional and immunobiological analyses of extracellular vesicles released by Candida albicans

The release of extracellular vesicles (EV) by fungal organisms is considered an alternative transport mechanism to trans‐cell wall passage of macromolecules. Previous studies have revealed the presence of EV in culture supernatants from fungal pathogens, such as Cryptococcus neoformans, Histoplasma capsulatum, Paracoccidioides brasiliensis, Sporothrix schenckii, Malassezia sympodialis and Candida albicans. Here we investigated the size, composition, kinetics of internalization by bone marrow‐derived murine macrophages (MO) and dendritic cells (DC), and the immunomodulatory activity of C. albicans EV. We also evaluated the impact of EV on fungal virulence using the Galleria mellonella larvae model. By transmission electron microscopy and dynamic light scattering, we identified two populations ranging from 50 to 100 nm and 350 to 850 nm. Two predominant seroreactive proteins (27 kDa and 37 kDa) and a group of polydispersed mannoproteins were observed in EV by immunoblotting analysis. Proteomic analysis of C. albicans EV revealed proteins related to pathogenesis, cell organization, carbohydrate and lipid metabolism, response to stress, and several other functions. The major lipids detected by thin‐layer chromatography were ergosterol, lanosterol and glucosylceramide. Short exposure of MO to EV resulted in internalization of these vesicles and production of nitric oxide, interleukin (IL)‐12, transforming growth factor‐beta (TGF‐β) and IL‐10. Similarly, EV‐treated DC produced IL‐12p40, IL‐10 and tumour necrosis factor‐alpha. In addition, EV treatment induced the up‐regulation of CD86 and major histocompatibility complex class‐II (MHC‐II). Inoculation of G. mellonella larvae with EV followed by challenge with C. albicans reduced the number of recovered viable yeasts in comparison with infected larvae control. Taken together, our results demonstrate that C. albicans EV were immunologically active and could potentially interfere with the host responses in the setting of invasive candidiasis.     

Coccidioides Endospores and Spherules Draw Strong Chemotactic,Adhesive, and Phagocytic Responses by Individual Human Neutrophils

Coccidioides spp. are dimorphic pathogenic fungi whose parasitic forms cause coccidioidomycosis (Valley fever) in mammalian hosts. We use an innovative interdisciplinary approach to analyze one-on-one encounters between human neutrophils and two forms of Coccidioides posadasii. To examine the mechanisms by which the innate immune system coordinates different stages of the host response to fungal pathogens, we dissect the immune-cell response into chemotaxis, adhesion, and phagocytosis. Our single-cell technique reveals a surprisingly strong response by initially quiescent neutrophils to close encounters with C. posadasii, both from a distance (by complement-mediated chemotaxis) as well as upon contact (by serum-dependent adhesion and phagocytosis). This response closely resembles neutrophil interactions with Candida albicans and zymosan particles, and is significantly stronger than the neutrophil responses to Cryptococcus neoformans, Aspergillus fumigatus, and Rhizopus oryzae under identical conditions. The vigorous in vitro neutrophil response suggests that C. posadasii evades in vivo recognition by neutrophils through suppression of long-range mobilization and recruitment of the immune cells. This observation elucidates an important paradigm of the recognition of microbes, i.e., that intact immunotaxis comprises an intricate spatiotemporal hierarchy of distinct chemotactic processes. Moreover, in contrast to earlier reports, human neutrophils exhibit vigorous chemotaxis toward, and frustrated phagocytosis of, the large spherules of C. posadasii under physiological-like conditions. Finally, neutrophils from healthy donors and patients with chronic coccidioidomycosis display subtle differences in their responses to antibody-coated beads, even though the patient cells appear to interact normally with C. posadasii endospores.     

An essential role for phosphatidylinositol 3‐kinase in the inhibition of phagosomal maturation,intracellular survival and virulence in Candida glabrata

The yeast class III phosphoinositide 3‐kinase (PI3K) that catalyses production of the lipid signalling molecule, phosphatidylinositol‐3‐phosphate, is primarily implicated in vesicle‐mediated transport and autophagy. In this study, we identified, through a genetic screen, the Candida glabrata CgVPS15 gene, an orthologue of the Saccharomyces cerevisiae PI3K regulatory subunit‐encoding open reading frame (ORF) to be required for impairment of phagosomal maturation in human macrophages. We also disrupted catalytic subunit of the C. glabrata PI3K complex, CgVps34, and found it to be pivotal to arrest mature phagolysosome biogenesis. Further, deletion of either CgVPS15 or CgVPS34 rendered C. glabrata cells hyperadherent to epithelial cells and susceptible to the antimicrobial arsenal of primary murine and cultured human macrophages and diverse stresses. Despite no growth retardation at 37°C, Cgvps15Δ and Cgvps34Δ mutants were severely virulence attenuated in mice. We demonstrate that trafficking and/or processing of the vacuolar lumenal hydrolase, carboxypeptidase Y, and the major adhesin, Epa1, rely on PI3K regulatory mechanisms in C. glabrata. By disrupting autophagy‐related PI3K complex genes, we show that C. glabrata PI3K‐impeded phagolysosomal acidification is primarily owing to its role in cellular trafficking events. Altogether, our findings underscore the essentiality of PI3K signalling in modulation of host immune response, intracellular survival and virulence in C. glabrata.     

Disruption of Sphingolipid Biosynthesis Blocks Phagocytosis of Candida albicans

The ability of phagocytes to clear pathogens is an essential attribute of the innate immune response. The role of signaling lipid molecules such as phosphoinositides is well established, but the role of membrane sphingolipids in phagocytosis is largely unknown. Using a genetic approach and small molecule inhibitors, we show that phagocytosis of Candida albicans requires an intact sphingolipid biosynthetic pathway. Blockade of serine-palmitoyltransferase (SPT) and ceramide synthase-enzymes involved in sphingolipid biosynthesis- by myriocin and fumonisin B1, respectively, impaired phagocytosis by phagocytes. We used CRISPR/Cas9-mediated genome editing to generate Sptlc2-deficient DC2.4 dendritic cells, which lack serine palmitoyl transferase activity. Sptlc2^-/- DC2.4 cells exhibited a stark defect in phagocytosis, were unable to bind fungal particles and failed to form a normal phagocytic cup to engulf C. albicans. Supplementing the growth media with GM1, the major ganglioside present at the cell surface, restored phagocytic activity of Sptlc2^-/- DC2.4 cells. While overall membrane trafficking and endocytic pathways remained functional, Sptlc2^-/- DC2.4 cells express reduced levels of the pattern recognition receptors Dectin-1 and TLR2 at the cell surface. Consistent with the in vitro data, compromised sphingolipid biosynthesis in mice sensitizes the animal to C. albicans infection. Sphingolipid biosynthesis is therefore critical for phagocytosis and in vivo clearance of C. albicans.     

Carbon source‐induced reprogramming of the cell wall proteome and secretome modulates the adherence and drug resistance of the fungal pathogen Candida albicans

The major fungal pathogen Candida albicans can occupy diverse microenvironments in its human host. During colonization of the gastrointestinal or urogenital tracts, mucosal surfaces, bloodstream, and internal organs, C. albicans thrives in niches that differ with respect to available nutrients and local environmental stresses. Although most studies are performed on glucose‐grown cells, changes in carbon source dramatically affect cell wall architecture, stress responses, and drug resistance. We show that growth on the physiologically relevant carboxylic acid, lactate, has a significant impact on the C. albicans cell wall proteome and secretome. The regulation of cell wall structural proteins (e.g. Cht1, Phr1, Phr2, Pir1) correlated with extensive cell wall remodeling in lactate‐grown cells and with their increased resistance to stresses and antifungal drugs, compared with glucose‐grown cells. Moreover, changes in other proteins (e.g. Als2, Gca1, Phr1, Sap9) correlated with the increased adherence and biofilm formation of lactate‐grown cells. We identified mating and pheromone‐regulated proteins that were exclusive to lactate‐grown cells (e.g. Op4, Pga31, Pry1, Scw4, Yps7) as well as mucosa‐specific and other niche‐specific factors such as Lip4, Pga4, Plb5, and Sap7. The analysis of the corresponding null mutants confirmed that many of these proteins contribute to C. albicans adherence, stress, and antifungal drug resistance. Therefore, the cell wall proteome and secretome display considerable plasticity in response to carbon source. This plasticity influences important fitness and virulence attributes known to modulate the behavior of C. albicans in different host microenvironments during infection.     

The relative contribution of resident pulmonary alveolar macrophage and inflammatory polymorphonuclear neutrophils in host resistance to pulmonary infection by Candida albicans

Cortisone (CA) or cyclophosphamide (Cy) treatment of mice was used to investigate the relative contributions of pulmonary alveolar macrophages (PAM) and inflammatory neutrophils (PMN) in the initial defense against intratracheal challenge (IT) with Candida albicans. Mice treated with either CA or Cy were susceptible to IT challenge with 10–100 x less C. albicans than were untreated mice. Untreated mice rapidly eliminated C. albicans from their lungs with the majority of the organisms being cleared within three hours of challenge. Mice treated with CA initially cleared some of the C. albicans but were unable to clear all the C. albicans as did the untreated mice. Mice treated with Cy were unable to clear C. albicans from their lungs. Candida albicans did not disseminate from the lungs of untreated mice, while in both of the treated groups, C. albicans disseminated to the liver, spleen, brain and kidneys, rapidly killing the treated hosts. Analysis of the changes in cells in lung lavage fluids collected at various times after C. albicans challenge, revealed that large numbers of PMN accumulated in the lungs of both untreated and CA-treated mice, whereas PMN were virtually undetectable in lavage fluids from Cy-treated mice. Resident PAM from untreated mice were able to kill approximately 70 % of 10⁵ C. albicans in a 3 hr in vitro killing assay. By contrast, at similar effector: target ratios, resident PAM from Cy-treated mice killed only about 20% of the inoculum and resident PAM from CA-treated mice were unable to kill C. albicans. PMNs from both untreated and CA-treated mice killed approximately 70% of 10⁵ C. albicans in vitro. The data indicates that both PAM and PMN were critical to the initial clearance of C. albicans from pulmonary tissue. The accumulation of PMN in the lungs appeared to be required for the complete clearance of C. albicans from the lungs yet was not sufficient to inhibit dissemination of C. albicans from the lungs in CA-treated mice. The presence of PAM with in vitro candidacidal abilities appeared to be required for both the clearance of C. albicans and inhibition of dissemination of C. albicans from the lungs. Compromise of either PAM or PMN function can lead to increased pulmonary susceptibility to C. albicans.     

Smoking increases oral mucosa susceptibility to Candida albicans infection via the Nrf2 pathway: In vitro and animal studies

Smoking and Candida albicans (C. albicans) infection are risk factors for many oral diseases. Several studies have reported a close relationship between smoking and the occurrence of C. albicans infection. However, the exact underlying mechanism of this relationship remains unclear. We established a rat infection model and a C. albicans-Leuk1 epithelial cell co-culture model with and without smoke exposure to investigate the mechanism by which smoking contributes to C. albicans infection. Oral mucosa samples from healthy individuals and patients with oral leucoplakia were also analysed according to their smoking status. Our results indicated that smoking induced oxidative stress and redox dysfunction in the oral mucosa. Smoking-induced Nrf2 negatively regulated the NLRP3 inflammasome, impaired the oral mucosal defence response and increased the oral mucosa susceptibility to C. albicans. The results suggest that the Nrf2 pathway could be involved in the pathogenesis of oral diseases by mediating an antioxidative response to cigarette smoke exposure and suppressing host immunity against C. albicans.