The opportunistic fungal pathogen Candida albicans is capable of adhering to the oral mucosa despite forces created by salivary flow. Although many fungal adhesion proteins have been identified, less is known about the temporal development of cell adhesion and biofilm growth in a flow environment. In this study, we use a flow system with real-time imaging of C. albicans cells as they adhere and grow. Rates of cell attachment and dispersion of C. albicans knockout strains of putative adhesins, transcription factors, and deletions with a hyperfilamentous phenotype were quantified during 18 h of biofilm development. Cell adhesion under flow is a multi-phase process initiated with cell rolling, then an initial firm attachment to the substrate occurs. After attachment, cells enter a growth phase where cells either commit to adherence or disperse. C. albicans Δ eap1 , Δ hwp2 , Δ hyr1 , and Δ ihd1 cells had significantly reduced initial attachment and subsequent adhesion, while Δ als1 /Δ als3 had no change in initial attachment but reduced adhesion maintenance. WT cells had increased adhesion during the late growth phase when hyphae were more highly expressed. Hyperfilamentous strains had 10-fold higher total biofilm growth, a result of significantly reduced detachment rates, showing that hyphal morphogenesis is important for adhesion maintenance in the developing biofilm. The rate of C. albicans biomass dispersion was most important for determining the density of the mature biomass. Adhesion maintenance was mediated in part by Ywp1, a protein previously thought to regulate dispersion, thus it functions as an adhesion maintenance protein in C. albicans .

is a newly identified species causing invasive candidemia and candidiasis. It has broad multidrug resistance (MDR) not observed for other pathogenic species. Histatin 5 (Hst 5) is a well-studied salivary cationic peptide with significant antifungal activity against and is an attractive candidate for treating MDR fungi, since antimicrobial peptides induce minimal drug resistance. We investigated the susceptibility of to Hst 5 and neutrophils, two first-line innate defenses in the human host. The majority of clinical isolates, including fluconazole-resistant strains, were highly sensitive to Hst 5: 55 to 90% of cells were killed by use of 7.5 μM Hst 5. Hst 5 was translocated to the cytosol and vacuole in cells; such translocation is required for the killing of by Hst 5. The inverse relationship between fluconazole resistance and Hst 5 killing suggests different cellular targets for Hst 5 than for fluconazole. showed higher tolerance to oxidative stress than, and higher survival within neutrophils, which correlated with resistance to oxidative stress Thus, resistance to reactive oxygen species (ROS) is likely one, though not the only, important factor in the killing of by neutrophils. Hst 5 has broad and potent candidacidal activity, enabling it to combat MDR strains effectively.

Non- albicans Candida species (NACS) are often isolated along with Candida albicans in cases of oropharyngeal candidiasis. C. albicans readily forms biofilms in conjunction with other oral microbiota including both bacteria and yeast. Adhesion between species is important to the establishment of these mixed biofilms, but interactions between C. albicans and many NACS are not well-characterized. We adapted a real-time flow biofilm model to study adhesion interactions and biofilm establishment in C. albicans and NACS in mono- and co-culture. Out of five NACS studied, only the filamenting species C. tropicalis and C. dubliniensis were capable of adhesion with C. albicans , while C. parapsilosis, C. lusitaniae, and C. krusei were not. Over the early phase (0–4 h) of biofilm development, both mono- and co-culture followed similar kinetics of attachment and detachment events, indicating that initial biofilm formation is not influenced by inter-species interactions. However, the NACS showed a preference for inter-species cell-cell interactions with C. albicans , and at later time points (5–11 h) we found that dual-species interactions impacted biofilm surface coverage. Dual-species biofilms of C. tropicalis and C. albicans grew more slowly than C. albicans alone, but achieved higher surface coverage than C. tropicalis alone. Biofilms of C. dubliniensis with C. albicans increased surface coverage more rapidly than either species alone. We conclude that dual culture biofilm of C. albicans with C. tropicalis or C. dubliniensis offers a growth advantage for both NACS. Furthermore, the growth and maintenance, but not initial establishment, of dual-species biofilms is likely facilitated by interspecies cell-cell adherence.

Host phagocytic cells are crucial players in initial defense against Candida albicans infection. C. albicans utilizes MAP kinases and Ras1 stress response signaling pathways to protect itself from killing by immune cells. In this study, we tested the importance of these pathways in C. albicans phagocytosis by neutrophils and subsequent phagosomal survival. Phagocytosis was influenced by C. albicans morphology, so hyphal length of >10 μm reduced the phagocytic index (PI) 2- to 3-fold in human neutrophils. Primary human neutrophils killed 81% of phagocytosed C. albicans, while primary mouse neutrophils killed 63% of yeasts. We found that both the C. albicans Cek1 and Hog1 pathways were required for survival of phagocytosed yeast, whereas deletion of C. albicans RAS1 resulted in an 84% increase in survival within neutrophils compared to that of the wild type (WT). The absence of Ras1 did not alter reactive oxygen species (ROS) production by C. albicans; however, phagocytosed C. albicans Δ/Δras1 cells reduced ROS release by neutrophils by 86%. Moreover, C. albicans Δ/Δras1 cells had increased resistance to hydrogen peroxide as a result of high levels of catalase activity. This phenotype was specific to Ras1, since these effects were not observed in the absence of its partner Cyr1 or with its downstream target Efg1. In addition, C. albicans Δ/Δras1 cells had a significantly increased resistance to nonoxidative killing by human neutrophil peptide 1 (HNP-1) that was reversed by restoring cellular cAMP levels. These data show that C. albicans Ras1 inactivation leads to fungal resistance to both oxidative and nonoxidative mechanisms of neutrophil phagosomal killing.

Formation of chlamydospores by Candida albicans was an established medical diagnostic test to confirm candidiasis before the molecular era. However, the functional role and pathological relevance of this in vitro morphological transition to pathogenesis in vivo remain unclear. We compared the physical properties of in vitro-induced chlamydospores with those of large C. albicans cells purified by density gradient centrifugation from Candida-infected mouse kidneys. The morphological and physical properties of these cells in kidneys of mice infected intravenously with wild type C. albicans confirmed that chlamydospores can form in infected kidneys. A previously reported chlamydospore-null Δisw2/Δisw2 mutant was used to investigate its role in virulence and chlamydospore induction. Virulence of the Δisw2/Δisw2 mutant strain was reduced 3.4-fold compared to wild type C. albicans or the ISW2 reconstituted strain. Altered host inflammatory reactions to the null mutant further indicate that ISW2 is a virulence factor in C. albicans. ISW2 deletion abolished chlamydospore formation within infected mouse kidneys, whereas the reconstituted strain restored chlamydospore formation in kidneys. Under chlamydospore inducing conditions in vitro, deletion of ISW2 significantly delayed chlamydospore formation, and those late induced chlamydospores lacked associated suspensor cells while attaching laterally to hyphae via novel spore-hypha septa. Our findings establish the induction of chlamydospores by C. albicans during mouse kidney colonization. Our results indicate that ISW2 is not strictly required for chlamydospores formation but is necessary for suspensor cell formation. The importance of ISW2 in chlamydospore morphogenesis and virulence may lead to additional insights into morphological differentiation and pathogenesis of C. albicans in the host microenvironment.