The protein kinase Ire1 has a Hac1-independent essential role in iron uptake and virulence of Candida albicans.

Protein kinases play central roles in virtually all signaling pathways that enable organisms to adapt to their environment. Microbial pathogens must cope with severely restricted iron availability in mammalian hosts to invade and establish themselves within infected tissues. To uncover protein kinase signaling pathways that are involved in the adaptation of the pathogenic yeast Candida albicans to iron limitation, we generated a comprehensive protein kinase deletion mutant library of a wild-type strain. Screening of this library revealed that the protein kinase Ire1, which has a conserved role in the response of eukaryotic cells to endoplasmic reticulum stress, is essential for growth of C. albicans under iron-limiting conditions. Ire1 was not necessary for the activity of the transcription factor Sef1, which regulates the response of the fungus to iron limitation, and Sef1 target genes that are induced by iron depletion were normally upregulated in ire1Δ mutants. Instead, Ire1 was required for proper localization of the high-affinity iron permease Ftr1 to the cell membrane. Intriguingly, iron limitation did not cause increased endoplasmic reticulum stress, and the transcription factor Hac1, which is activated by Ire1-mediated removal of the non-canonical intron in the HAC1 mRNA, was dispensable for Ftr1 localization to the cell membrane and growth under iron-limiting conditions. Nevertheless, expression of a pre-spliced HAC1 copy in ire1Δ mutants restored Ftr1 localization and rescued the growth defects of the mutants. Both ire1Δ and hac1Δ mutants were avirulent in a mouse model of systemic candidiasis, indicating that an appropriate response to endoplasmic reticulum stress is important for the virulence of C. albicans. However, the specific requirement of Ire1 for the functionality of the high-affinity iron permease Ftr1, a well-established virulence factor, even in the absence of endoplasmic reticulum stress uncovers a novel Hac1-independent essential role of Ire1 in iron acquisition and virulence of C. albicans.

Rapid proliferation due to better metabolic adaptation results in full virulence of a filament-deficient Candida albicans strain.

The ability of the fungal pathogen Candida albicans to undergo a yeast-to-hypha transition is believed to be a key virulence factor, as filaments mediate tissue damage. Here, we show that virulence is not necessarily reduced in filament-deficient strains, and the results depend on the infection model used. We generate a filament-deficient strain by deletion or repression of EED1 (known to be required for maintenance of hyphal growth). Consistent with previous studies, the strain is attenuated in damaging epithelial cells and macrophages in vitro and in a mouse model of intraperitoneal infection. However, in a mouse model of systemic infection, the strain is as virulent as the wild type when mice are challenged with intermediate infectious doses, and even more virulent when using low infectious doses. Retained virulence is associated with rapid yeast proliferation, likely the result of metabolic adaptation and improved fitness, leading to high organ fungal loads. Analyses of cytokine responses in vitro and in vivo, as well as systemic infections in immunosuppressed mice, suggest that differences in immunopathology contribute to some extent to retained virulence of the filament-deficient mutant. Our findings challenge the long-standing hypothesis that hyphae are essential for pathogenesis of systemic candidiasis by C. albicans.

Clinical Candida albicans Vaginal Isolates and a Laboratory Strain Show Divergent Behaviors during Macrophage Interactions.

Typically, established lab strains are widely used to study host-pathogen interactions. However, to better reflect the infection process, the experimental use of clinical isolates has come more into focus. Here, we analyzed the interaction of multiple vaginal isolates of the opportunistic fungal pathogen Candida albicans, the most common cause of vulvovaginal candidiasis in women, with key players of the host immune system: macrophages. We tested several strains isolated from asymptomatic or symptomatic women with acute and recurrent infections. While all clinical strains showed a response similar to the commonly used lab strain SC5314 in various in vitro assays, they displayed remarkable differences during interaction with macrophages. This coincided with significantly reduced ß-glucan exposure on the cell surface, which appeared to be a shared property among the tested vaginal strains for yeast extract/peptone/dextrose-grown cells, which is partly lost when the isolates faced vaginal niche-like nutrient conditions. However, macrophage damage, survival of phagocytosis, and filamentation capacities were highly strain-specific. These results highlight the high heterogeneity of C. albicans strains in host-pathogen interactions, which have to be taken into account to bridge the gap between laboratory-gained data and disease-related outcomes in an actual patient.IMPORTANCE Vulvovaginal candidiasis is one of the most common fungal infections in humans with Candida albicans as the major causative agent. This study is the first to compare clinical vaginal isolates of defined patient groups in their interaction with macrophages, highlighting the vastly different outcomes in comparison to a laboratory strain using commonly applied virulence-determining assays.

Significant Differences in Host-Pathogen Interactions Between Murine and Human Whole Blood.

Murine infection models are widely used to study systemic candidiasis caused by C. albicans. Whole-blood models can help to elucidate host-pathogens interactions and have been used for several Candida species in human blood. We adapted the human whole-blood model to murine blood. Unlike human blood, murine blood was unable to reduce fungal burden and more substantial filamentation of C. albicans was observed. This coincided with less fungal association with leukocytes, especially neutrophils. The lower neutrophil number in murine blood only partially explains insufficient infection and filamentation control, as spiking with murine neutrophils had only limited effects on fungal killing. Furthermore, increased fungal survival is not mediated by enhanced filamentation, as a filament-deficient mutant was likewise not eliminated. We also observed host-dependent differences for interaction of platelets with C. albicans, showing enhanced platelet aggregation, adhesion and activation in murine blood. For human blood, opsonization was shown to decrease platelet interaction suggesting that complement factors interfere with fungus-to-platelet binding. Our results reveal substantial differences between murine and human whole-blood models infected with C. albicans and thereby demonstrate limitations in the translatability of this ex vivo model between hosts.

Kallikrein Cleaves C3 and Activates Complement.

The human plasma contact system is an immune surveillance system activated by the negatively charged surfaces of bacteria and fungi and includes the kallikrein-kinin, the coagulation, and the fibrinolytic systems. Previous work shows that the contact system also activates complement, and that plasma enzymes like kallikrein, plasmin, thrombin, and FXII are involved in the activation process. Here, we show for the first time that kallikrein cleaves the central complement component C3 directly to yield active components C3b and C3a. The cleavage site within C3 is identical to that recognized by the C3 convertase. Also, kallikrein-generated C3b forms C3 convertases, which trigger the C3 amplification loop. Since kallikrein also cleaves factor B to yield Bb and Ba, kallikrein alone can trigger complement activation. Kallikrein-generated C3 convertases are inhibited by factor H; thus, the kallikrein activation pathway merges with the amplification loop of the alternative pathway. Taken together, these data suggest that activation of the contact system locally enhances complement activation on cell surfaces. The human pathogenic microbe Candida albicans activates the contact system in normal human serum. However, C. albicans immediately recruits factor H to the surface, thereby evading the alternative and likely kallikrein-mediated complement pathways.

A family of glutathione peroxidases contributes to oxidative stress resistance in Candida albicans.

Candida albicans is a well-adapted human commensal but is also a facultative pathogen that can cause superficial and systemic infections. Its remarkable capacity to thrive within the human host relies on its ability to adapt and respond to the local environment of different niches. C. albicans is able to cope with oxidative stress in a coordinated fashion via upregulation of different protective mechanisms. Here, we unravel the role of a family of glutathione peroxidase (GPx), designated Gpx31, Gpx32, and Gpx33, in oxidative stress resistance. We show that GPx activity in C. albicans is induced upon exposure to peroxides and that this enzymatic activity is required for full resistance to oxidative stress. The GPx activity relies on the presence of GPX31, with no apparent contribution from GPX32 and GPX33 during in vitro short-term (3 h) exposure to peroxides. However, a triple gpx31-33Δ/Δ mutant exhibited a more pronounced sensitivity than a single gpx31Δ/Δ mutant on solid media in the presence of oxidants, suggesting that GPX32 and GPX33 may be involved in long-term adaptation to oxidative stress. Interestingly, reintegration of a single allele of GPX31 was sufficient to restore the wild-type phenotype in both the single and triple mutants. We found that mutants lacking GPX31-33 were more susceptible to killing by phagocytic cells, suggesting that GPxs are required for full resistance to innate immune effector cells. Despite the sensitivity to oxidative stress and phagocytes, these mutants were not affected in their virulence in the chicken embryo model of candidiasis.

Cellular responses of Candida albicans to phagocytosis and the extracellular activities of neutrophils are critical to counteract carbohydrate starvation, oxidative and nitrosative stress.

Neutrophils are key players during Candida albicans infection. However, the relative contributions of neutrophil activities to fungal clearance and the relative importance of the fungal responses that counteract these activities remain unclear. We studied the contributions of the intra- and extracellular antifungal activities of human neutrophils using diagnostic Green Fluorescent Protein (GFP)-marked C. albicans strains. We found that a carbohydrate starvation response, as indicated by up-regulation of glyoxylate cycle genes, was only induced upon phagocytosis of the fungus. Similarly, the nitrosative stress response was only observed in internalised fungal cells. In contrast, the response to oxidative stress was observed in both phagocytosed and non-phagocytosed fungal cells, indicating that oxidative stress is imposed both intra- and extracellularly. We assessed the contributions of carbohydrate starvation, oxidative and nitrosative stress as antifungal activities by analysing the resistance to neutrophil killing of C. albicans mutants lacking key glyoxylate cycle, oxidative and nitrosative stress genes. We found that the glyoxylate cycle plays a crucial role in fungal resistance against neutrophils. The inability to respond to oxidative stress (in cells lacking superoxide dismutase 5 or glutathione reductase 2) renders C. albicans susceptible to neutrophil killing, due to the accumulation of reactive oxygen species (ROS). We also show that neutrophil-derived nitric oxide is crucial for the killing of C. albicans: a yhb1Δ/Δ mutant, unable to detoxify NO•, was more susceptible to neutrophils, and this phenotype was rescued by the nitric oxide scavenger carboxy-PTIO. The stress responses of C. albicans to neutrophils are partially regulated via the stress regulator Hog1 since a hog1Δ/Δ mutant was clearly less resistant to neutrophils and unable to respond properly to neutrophil-derived attack. Our data indicate that an appropriate fungal response to all three antifungal activities, carbohydrate starvation, nitrosative stress and oxidative stress, is essential for full wild type resistance to neutrophils.