Role of CaECM25 in cell morphogenesis, cell growth and virulence in Candida albicans

Candida albicans is the most prominent opportunistic fungal pathogen in humans. Multiple factors are associated with the virulence of C. albicans, including morphogenesis, cell wall organization and growth rate. Here, we describe the identification and functional characterization of CaECM25, a gene that has not been reported before. We constructed Caecm25?/? mutants and investigated the role of the gene in morphogenesis, cell wall organization and virulence. CaECM25 deletion resulted in defects in cell separation, a slower growth rate, reduced filamentous growth and attenuated adherence to plastic surfaces. The Caecm25?/? mutant was also significantly less virulent than wild type when tested for systemic infection in mice. Therefore, CaECM25 plays important roles in morphogenesis, cell wall organization and virulence.     

Candida albicans ENO1 null mutants exhibit altered drug susceptibility, hyphal formation, and virulence

We previously showed that the expression of ENO1 (enolase) in the fungal pathogen Candida albicans is critical for cell growth. In this study, we investigate the contribution of the ENO1 gene to virulence. We conducted our functional study of ENO1 in C. albicans by constructing an eno1/eno1 null mutant strain in which both ENO1 alleles in the genome were knockouted with the SAT1 flipper cassette that contains the nourseothricin-resistance marker. Although the null mutant failed to grow on synthetic media containing glucose, it was capable of growth on media containing yeast extract, peptone, and non-fermentable carbon sources. The null mutant was more susceptible to certain antifungal drugs. It also exhibited defective hyphal formation, and was avirulent in BALB/c mice.     

HOS2 and HDA1 Encode Histone Deacetylases with Opposing Roles in Candida albicans Morphogenesis

Epigenetic mechanisms regulate the expression of virulence traits in diverse pathogens, including protozoan and fungi. In the human fungal pathogen Candida albicans, virulence traits such as antifungal resistance, white-opaque switching, and adhesion to lung cells are regulated by histone deacetylases (HDACs). However, the role of HDACs in the regulation of the yeast-hyphal morphogenetic transitions, a critical virulence attribute of C. albicans, remains poorly explored. In this study, we wished to determine the relevance of other HDACs on C. albicans morphogenesis. We generated mutants in the HDACs HOS1, HOS2, RPD31, and HDA1 and determined their ability to filament in response to different environmental stimuli. We found that while HOS1 and RPD31 have no or a more limited role in morphogenesis, the HDACs HOS2 and HDA1 have opposite roles in the regulation of hyphal formation. Our results demonstrate an important role for HDACs on the regulation of yeast-hyphal transitions in the human pathogen C. albicans.     

Methionine is required for cAMP‐PKA‐mediated morphogenesis and virulence of Candida albicans

Candida albicans is a major human fungal pathogen, causing superficial, as well as life‐threatening invasive infections. Therefore, it has to adequately sense and respond to the host defense by expressing appropriate virulence attributes. The most important virulence factor of C. albicans is the yeast‐to‐hyphae morphogenetic switch, which can be induced by numerous environmental cues, including the amino acid methionine. Here, we show an essential role for methionine permease Mup1 in methionine‐induced morphogenesis, biofilm formation, survival inside macrophages and virulence. Furthermore, we demonstrate that this process requires conversion of methionine into S‐adenosyl methionine (SAM) and its decarboxylation by Spe2. The resulting amino‐propyl group is then used for biosynthesis of polyamines, which have been shown to activate adenylate cyclase. Inhibition of the SPE2 SAM decarboxylase gene strongly impairs methionine‐induced morphogenesis on specific media and significantly delays virulence in the mouse systemic infection model system. Further proof of the connection between methionine uptake and initial metabolism and the cAMP‐PKA pathway was obtained by showing that both Mup1 and Spe2 are required for cAMP production in response to methionine. Our results suggest that amino acid transport and further metabolism are interesting therapeutic targets as inhibitors of this may prevent the morphogenetic switch, thereby preventing virulence.     

Asc1 Supports Cell-Wall Integrity Near Bud Sites by a Pkc1 Independent Mechanism

Background

The yeast ribosomal protein Asc1 is a WD-protein family member. Its mammalian ortholog, RACK1 was initially discovered as a receptor for activated protein C kinase (PKC) that functions to maintain the active conformation of PKC and to support its movement to target sites. In the budding yeast though, a connection between Asc1p and the PKC signaling pathway has never been reported.

Methodology/Principal Findings

In the present study we found that asc1-deletion mutant (asc1Δ) presents some of the hallmarks of PKC signaling mutants. These include an increased sensitivity to staurosporine, a specific Pkc1p inhibitor, and susceptibility to cell-wall perturbing treatments such as hypotonic- and heat shock conditions and zymolase treatment. Microscopic analysis of asc1Δ cells revealed cell-wall invaginations near bud sites after exposure to hypotonic conditions, and the dynamic of cells'' survival after this stress further supports the involvement of Asc1p in maintaining the cell-wall integrity during the mid-to late stages of bud formation. Genetic interactions between asc1 and pkc1 reveal synergistic sensitivities of a double-knock out mutant (asc1Δ/pkc1Δ) to cell-wall stress conditions, and high basal level of PKC signaling in asc1Δ. Furthermore, Asc1p has no effect on the cellular distribution or redistribution of Pkc1p at optimal or at cell-wall stress conditions.

Conclusions/Significance

Taken together, our data support the idea that unlike its mammalian orthologs, Asc1p acts remotely from Pkc1p, to regulate the integrity of the cell-wall. We speculate that its role is exerted through translation regulation of bud-site related mRNAs during cells'' growth.     

<Emphasis Type="Italic">Candida albicans SUR7</Emphasis> contributes to secretion,biofilm formation,and macrophage killing

Background  

Candida albicans SUR7 has been shown to be required for plasma membrane organization and cell wall synthesis, but its role in virulence is not known. Using a bioinformatics strategy, we previously identified several novel putative secretion pathway proteins potentially involved in virulence, including the C. albicans homolog of the Saccharomyces cerevisiae endocytosis-related protein Sur7p. We therefore generated a C. albicans sur7Δ null mutant and examined its contribution to key virulence attributes.     

Towards non‐invasive monitoring of pathogen–host interactions during Candida albicans biofilm formation using in vivo bioluminescence

Candida albicans is a major human fungal pathogen causing mucosal and deep tissue infections of which the majority is associated with biofilm formation on medical implants. Biofilms have a huge impact on public health, as fungal biofilms are highly resistant against most antimycotics. Animal models of biofilm formation are indispensable for improving our understanding of biofilm development inside the host, their antifungal resistance and their interaction with the host immune defence system. In currently used models, evaluation of biofilm development or the efficacy of antifungal treatment is limited to ex vivo analyses, requiring host sacrifice, which excludes longitudinal monitoring of dynamic processes during biofilm formation in the live host. In this study, we have demonstrated for the first time that non‐invasive, dynamic imaging and quantification of in vitro and in vivo C. albicans biofilm formation including morphogenesis from the yeast to hyphae state is feasible by using growth‐phase dependent bioluminescent C. albicans strains in a subcutaneous catheter model in rodents. We have shown the defect in biofilm formation of a bioluminescent bcr1 mutant strain. This approach has immediate applications for the screening and validation ofantimycotics under in vivo conditions, for studying host–biofilm interactions in different transgenic mouse models and for testing the virulence of luminescent C. albicans mutants, hereby contributing to a better understanding of the pathogenesis of biofilm‐associated yeast infections.     

Comprehensive genetic analysis of adhesin proteins and their role in virulence of Candida albicans

Candida albicans is a microbial fungus that exists as a commensal member of the human microbiome and an opportunistic pathogen. Cell surface-associated adhesin proteins play a crucial role in C. albicans’ ability to undergo cellular morphogenesis, develop robust biofilms, colonize, and cause infection in a host. However, a comprehensive analysis of the role and relationships between these adhesins has not been explored. We previously established a CRISPR-based platform for efficient generation of single- and double-gene deletions in C. albicans, which was used to construct a library of 144 mutants, comprising 12 unique adhesin genes deleted singly, and every possible combination of double deletions. Here, we exploit this adhesin mutant library to explore the role of adhesin proteins in C. albicans virulence. We perform a comprehensive, high-throughput screen of this library, using Caenorhabditis elegans as a simplified model host system, which identified mutants critical for virulence and significant genetic interactions. We perform follow-up analysis to assess the ability of high- and low-virulence strains to undergo cellular morphogenesis and form biofilms in vitro, as well as to colonize the C. elegans host. We further perform genetic interaction analysis to identify novel significant negative genetic interactions between adhesin mutants, whereby combinatorial perturbation of these genes significantly impairs virulence, more than expected based on virulence of the single mutant constituent strains. Together, this study yields important new insight into the role of adhesins, singly and in combinations, in mediating diverse facets of virulence of this critical fungal pathogen.     

Disruption of Homocitrate Synthase Genes in <Emphasis Type="Italic">Candida albicans</Emphasis> Affects Growth But Not Virulence

Two genes, LYS21 and LYS22, encoding isoforms of homocitrate synthase, an enzyme catalysing the first committed step in the lysine biosynthetic pathway, were disrupted in Candida albicans using the SAT1 flipper strategy. The double null lys21Δ/lys22Δ mutant lacked homocitrate synthase activity and exhibited lysine auxotrophy in minimal media that could be fully rescued by the addition of 0.5–0.6 mM l-lysine. On the other hand, its virulence in vivo in the model of disseminated murine candidiasis appeared identical to that of the mother, wild-type strain. These findings strongly question a possibility of exploitation of homocitrate synthase and possibly also other enzymes of the lysine biosynthetic pathway as targets in chemotherapy of disseminated fungal infections.     

Effect of 2, 4-di-tert-butylphenol on growth and biofilm formation by an opportunistic fungus Candida albicans

Candida albicans, an opportunistic pathogen, has been known to form hypoxic biofilms on medical devices which in turn confers resistance towards antifungals, resulting in subsequent therapeutic failures. Inclusion of anti-biofilm agents in the control of infections is a topic of current interest in developing potential anti-infectives. The in vitro anti-fungal and anti-biofilm efficacy of 2,4-di-tert-butyl phenol [DTBP] was evaluated in this study, which revealed the potential fungicidal action of DTBP at higher concentrations where fluconazole failed to act completely. DTBP also inhibited the production of hemolysins, phospholipases and secreted aspartyl proteinase which are the crucial virulence factors required for the invasion of C. albicans. Various anti-biofilm assays and morphological observations revealed the efficacy of DTBP in both inhibiting and disrupting biofilms of C. albicans. Inhibition of hyphal development, a key process that aids in initial adhesion of C. albicans, was observed, and this could be a mechanism for the anti-biofilm activity of DTBP.     

Extensive functional redundancy in the regulation of Candida albicans drug resistance and morphogenesis by lysine deacetylases Hos2, Hda1, Rpd3 and Rpd31

Current treatment efforts for fungal infections are hampered by the limited availability of antifungal drugs and by the emergence of drug resistance. A powerful strategy to enhance the efficacy of antifungal drugs is to inhibit the molecular chaperone Hsp90. Hsp90 governs drug resistance, morphogenesis and virulence in a leading fungal pathogen of humans, Candida albicans. Our previous work with Saccharomyces cerevisiae established acetylation as a novel mechanism of posttranslational control of Hsp90 function in fungi. We implicated lysine deacetylases (KDACs) as key regulators of resistance to the most widely deployed class of antifungals, the azoles, in both S. cerevisiae and C. albicans. Here, we demonstrate high levels of functional redundancy among the KDACs that are important for regulating Hsp90 function. We identify Hos2, Hda1, Rpd3 and Rpd31 as the KDACs mediating azole resistance and morphogenesis in C. albicans. Furthermore, we identify lysine 30 and 271 as critical acetylation sites on C. albicans Hsp90, and substitutions at these residues compromise Hsp90 function. Finally, we show that pharmacological inhibition of KDACs phenocopies pharmacological inhibition of Hsp90 and abrogates Hsp90‐dependent azole resistance in numerous Candida species. This work illuminates new facets to the impact of KDACs on fungal drug resistance and morphogenesis, provides important insights into the divergence of the C. albicans Hsp90 regulatory network and reveals new targets for development of antifungal drugs.