Michael C. Lorenz

Evolution of pathogenicity and sexual reproduction in eight Candida genomes.

Candida species are the most common cause of opportunistic fungal infection worldwide. Here we report the genome sequences of six Candida species and compare these and related pathogens and non-pathogens. There are significant expansions of cell wall, secreted and transporter gene families in pathogenic species, suggesting adaptations associated with virulence. Large genomic tracts are homozygous in three diploid species, possibly resulting from recent recombination events. Surprisingly, key components of the mating and meiosis pathways are missing from several species. These include major differences at the mating-type loci (MTL); Lodderomyces elongisporus lacks MTL, and components of the a1/α2 cell identity determinant were lost in other species, raising questions about how mating and cell types are controlled. Analysis of the CUG leucine-to-serine genetic-code change reveals that 99% of ancestral CUG codons were erased and new ones arose elsewhere. Lastly, we revise the Candida albicans gene catalogue, identifying many new genes.

A Human-Curated Annotation of the Candida albicans Genome

Recent sequencing and assembly of the genome for the fungal pathogen Candida albicans used simple automated procedures for the identification of putative genes. We have reviewed the entire assembly, both by hand and with additional bioinformatic resources, to accurately map and describe 6,354 genes and to identify 246 genes whose original database entries contained sequencing errors (or possibly mutations) that affect their reading frame. Comparison with other fungal genomes permitted the identification of numerous fungus-specific genes that might be targeted for antifungal therapy. We also observed that, compared to other fungi, the protein-coding sequences in the C. albicans genome are especially rich in short sequence repeats. Finally, our improved annotation permitted a detailed analysis of several multigene families, and comparative genomic studies showed that C. albicans has a far greater catabolic range, encoding respiratory Complex 1, several novel oxidoreductases and ketone body degrading enzymes, malonyl-CoA and enoyl-CoA carriers, several novel amino acid degrading enzymes, a variety of secreted catabolic lipases and proteases, and numerous transporters to assimilate the resulting nutrients. The results of these efforts will ensure that the Candida research community has uniform and comprehensive genomic information for medical research as well as for future diagnostic and therapeutic applications.

The Fungal Pathogen Candida albicans Autoinduces Hyphal Morphogenesis by Raising Extracellular pH

ABSTRACT pH homeostasis is critical for all organisms; in the fungal pathogen Candida albicans, pH adaptation is critical for virulence in distinct host niches. We demonstrate that beyond adaptation, C. albicans actively neutralizes the environment from either acidic or alkaline pHs. Under acidic conditions, this species can raise the pH from 4 to >7 in less than 12 h, resulting in autoinduction of the yeast-hyphal transition, a critical virulence trait. Extracellular alkalinization has been reported to occur in several fungal species, but under the specific conditions that we describe, the phenomenon is more rapid than previously observed. Alkalinization is linked to carbon deprivation, as it occurs in glucose-poor media and requires exogenous amino acids. These conditions are similar to those predicted to exist inside phagocytic cells, and we find a strong correlation between the use of amino acids as a cellular carbon source and the degree of alkalinization. Genetic and genomic approaches indicate an emphasis on amino acid uptake and catabolism in alkalinizing cells. Mutations in four genes, STP2, a transcription factor regulating amino acid permeases, ACH1 (acetyl-coenzyme A [acetyl-CoA] hydrolase), DUR1,2 (urea amidolyase), and ATO5, a putative ammonia transporter, abolish or delay neutralization. The pH changes are the result of the extrusion of ammonia, as observed in other fungi. We propose that nutrient-deprived C. albicans cells catabolize amino acids as a carbon source, excreting the amino nitrogen as ammonia to raise environmental pH and stimulate morphogenesis, thus directly contributing to pathogenesis. IMPORTANCE Candida albicans is the most important fungal pathogen of humans, causing disease at multiple body sites. The ability to switch between multiple morphologies, including a rounded yeast cell and an elongated hyphal cell, is a key virulence trait in this species, as this reversible switch is thought to promote dissemination and tissue invasion in the host. We report here that C. albicans can actively alter the pH of its environment and induce its switch to the hyphal form. The change in pH is caused by the release of ammonia from the cells produced during the breakdown of amino acids. This phenomenon is unprecedented in a human pathogen and may substantially impact host physiology by linking morphogenesis, pH adaptation, carbon metabolism, and interactions with host cells, all of which are critical for the ability of C. albicans to cause disease. Candida albicans is the most important fungal pathogen of humans, causing disease at multiple body sites. The ability to switch between multiple morphologies, including a rounded yeast cell and an elongated hyphal cell, is a key virulence trait in this species, as this reversible switch is thought to promote dissemination and tissue invasion in the host. We report here that C. albicans can actively alter the pH of its environment and induce its switch to the hyphal form. The change in pH is caused by the release of ammonia from the cells produced during the breakdown of amino acids. This phenomenon is unprecedented in a human pathogen and may substantially impact host physiology by linking morphogenesis, pH adaptation, carbon metabolism, and interactions with host cells, all of which are critical for the ability of C. albicans to cause disease.

Mutations in Alternative Carbon Utilization Pathways in Candida albicans Attenuate Virulence and Confer Pleiotropic Phenotypes

ABSTRACT The interaction between Candida albicans and cells of the innate immune system is a key determinant of disease progression. Transcriptional profiling has revealed that C. albicans has a complex response to phagocytosis, much of which is similar to carbon starvation. This suggests that nutrient limitation is a significant stress in vivo, and we have shown that glyoxylate cycle mutants are less virulent in mice. To examine whether other aspects of carbon metabolism are important in vivo during an infection, we have constructed strains lacking FOX2 and FBP1, which encode key components of fatty acid β-oxidation and gluconeogenesis, respectively. As expected, fox2Δ mutants failed to utilize several fatty acids as carbon sources. Surprisingly, however, these mutants also failed to grow in the presence of several other carbon sources, whose assimilation is independent of β-oxidation, including ethanol and citric acid. Mutants lacking the glyoxylate enzyme ICL1 also had more severe carbon utilization phenotypes than were expected. These results suggest that the regulation of alternative carbon metabolism in C. albicans is significantly different from that in other fungi. In vivo, fox2Δ mutants show a moderate but significant reduction in virulence in a mouse model of disseminated candidiasis, while disruption of the glyoxylate cycle or gluconeogenesis confers a severe attenuation in this model. These data indicate that C. albicans often encounters carbon-poor conditions during growth in the host and that the ability to efficiently utilize multiple nonfermentable carbon sources is a virulence determinant. Consistent with this in vivo requirement, C. albicans uniquely regulates carbon metabolism in a more integrated manner than in Saccharomyces cerevisiae, such that defects in one part of the machinery have wider impacts than expected. These aspects of alternative carbon metabolism may then be useful as targets for therapeutic intervention.

Enterococcus faecalis Inhibits Hyphal Morphogenesis and Virulence of Candida albicans

ABSTRACT The Gram-positive bacterium Enterococcus faecalis and the fungus Candida albicans are both found as commensals in many of the same niches of the human body, such as the oral cavity and gastrointestinal (GI) tract. However, both are opportunistic pathogens and have frequently been found to be coconstituents of polymicrobial infections. Despite these features in common, there has been little investigation into whether these microbes affect one another in a biologically significant manner. Using a Caenorhabditis elegans model of polymicrobial infection, we discovered that E. faecalis and C. albicans negatively impact each others virulence. Much of the negative effect of E. faecalis on C. albicans was due to the inhibition of C. albicans hyphal morphogenesis, a developmental program crucial to C. albicans pathogenicity. We discovered that the inhibition was partially dependent on the Fsr quorum-sensing system, a major regulator of virulence in E. faecalis. Specifically, two proteases regulated by Fsr, GelE and SerE, were partially required. Further characterization of the inhibitory signal revealed that it is secreted into the supernatant, is heat resistant, and is between 3 and 10 kDa. The substance was also shown to inhibit C. albicans filamentation in the context of an in vitro biofilm. Finally, a screen of an E. faecalis transposon mutant library identified other genes required for suppression of C. albicans hyphal formation. Overall, we demonstrate a biologically relevant interaction between two clinically important microbes that could affect treatment strategies as well as impact our understanding of interkingdom signaling and sensing in the human-associated microbiome.

Modulation of Phagosomal pH by Candida albicans Promotes Hyphal Morphogenesis and Requires Stp2p, a Regulator of Amino Acid Transport

Candida albicans, the most important fungal pathogen of humans, has a unique interaction with macrophages in which phagocytosis induces a switch from the yeast to hyphal form, allowing it to escape by rupturing the immune cell. While a variety of factors induce this switch in vitro, including neutral pH, it is not clear what triggers morphogenesis within the macrophage where the acidic environment should inhibit this transition. In vitro, C. albicans grown in similar conditions in which amino acids are the primary carbon source generate large quantities of ammonia to raise the extracellular pH and induce the hyphal switch. We show here that C. albicans cells neutralize the macrophage phagosome and that neutral pH is a key inducer of germination in phagocytosed cells by using a mutant lacking STP2, a transcription factor that regulates the expression of multiple amino acid permeases, that is completely deficient in alkalinization in vitro. Phagocytosed stp2Δ mutant cells showed significant reduction in hypha formation and escaped from macrophages less readily compared to wild type cells; as a result stp2Δ mutant cells were killed at a higher rate and caused less damage to RAW264.7 macrophages. Stp2p-regulated import leads to alkalinization of the phagosome, since the majority of the wild type cells fail to co-localize with acidophilic dyes, whereas the stp2Δ mutant cells were located in acidic phagosomes. Furthermore, stp2Δ mutant cells were able to form hyphae and escape from neutral phagosomes, indicating that the survival defect in these cells was pH dependent. Finally, these defects are reflected in an attenuation of virulence in a mouse model of disseminated candidiasis. Altogether our results suggest that C. albicans utilizes amino acids to promote neutralization of the phagosomal pH, hyphal morphogenesis, and escape from macrophages.

CTA4 Transcription Factor Mediates Induction of Nitrosative Stress Response in Candida albicans

ABSTRACT This work has identified regulatory elements in the major fungal pathogen Candida albicans that enable response to nitrosative stress. Nitric oxide (NO) is generated by macrophages of the host immune system and commensal bacteria, and the ability to resist its toxicity is one adaptation that promotes survival of C. albicans inside the human body. Exposing C. albicans to NO induces upregulation of the flavohemoglobin Yhb1p. This protein confers protection by enzymatically converting NO to harmless nitrate, but it is unknown how C. albicans is able to detect NO in its environment and thus initiate this defense only as needed. We analyzed this problem by incrementally mutating the YHB1 regulatory region to identify a nitric oxide-responsive element (NORE) that is required for NO sensitivity. Five transcription factor candidates of the Zn(II)2-Cys6 family were then isolated from crude whole-cell extracts by using magnetic beads coated with this DNA element. Of the five, only deletion of the CTA4 gene prevented induction of YHB1 transcription during nitrosative stress and caused growth sensitivity to the NO donor dipropylenetriamine NONOate; Cta4p associates in vivo with NORE DNA from the YHB1 regulatory region. Deletion of CTA4 caused a small but significant decrease in virulence. A CTA4-dependent putative sulfite transporter encoded by SSU1 is also implicated in NO response, but C. albicans ssu1 mutants were not sensitive to NO, in contrast to findings in Saccharomyces cerevisiae. Cta4p is the first protein found to be necessary for initiating NO response in C. albicans.

Role of Acetyl Coenzyme A Synthesis and Breakdown in Alternative Carbon Source Utilization in Candida albicans

ABSTRACT Acetyl coenzyme A (acetyl-CoA) is the central intermediate of the pathways required to metabolize nonfermentable carbon sources. Three such pathways, i.e., gluconeogenesis, the glyoxylate cycle, and β-oxidation, are required for full virulence in the fungal pathogen Candida albicans. These processes are compartmentalized in the cytosol, mitochondria, and peroxosomes, necessitating transport of intermediates across intracellular membranes. Acetyl-CoA is trafficked in the form of acetate by the carnitine shuttle, and we hypothesized that the enzymes that convert acetyl-CoA to/from acetate, i.e., acetyl-CoA hydrolase (ACH1) and acetyl-CoA synthetase (ACS1 and ACS2), would regulate alternative carbon utilization and virulence. We show that C. albicans strains depleted for ACS2 are unviable in the presence of most carbon sources, including glucose, acetate, and ethanol; these strains metabolize only fatty acids and glycerol, a substantially more severe phenotype than that of Saccharomyces cerevisiae acs2 mutants. In contrast, deletion of ACS1 confers no phenotype, though it is highly induced in the presence of fatty acids, perhaps explaining why acs2 mutants can utilize fatty acids. Strains lacking ACH1 have a mild growth defect on some carbon sources but are fully virulent in a mouse model of disseminated candidiasis. Both ACH1 and ACS2 complement mutations in their S. cerevisiae homolog. Together, these results show that acetyl-CoA metabolism and transport are critical for growth of C. albicans on a wide variety of nutrients. Furthermore, the phenotypic differences between mutations in these highly conserved genes in S. cerevisiae and C. albicans support recent findings that significant functional divergence exists even in fundamental metabolic pathways between these related yeasts.

Alternative splicing and subfunctionalization generates functional diversity in fungal proteomes.

Alternative splicing is commonly used by the Metazoa to generate more than one protein from a gene. However, such diversification of the proteome by alternative splicing is much rarer in fungi. We describe here an ancient fungal alternative splicing event in which these two proteins are generated from a single alternatively spliced ancestral SKI7/HBS1 gene retained in many species in both the Ascomycota and Basidiomycota. While the ability to express two proteins from a single SKI7/HBS1 gene is conserved in many fungi, the exact mechanism by which they achieve this varies. The alternative splicing was lost in Saccharomyces cerevisiae following the whole-genome duplication event as these two genes subfunctionalized into the present functionally distinct HBS1 and SKI7 genes. When expressed in yeast, the single gene from Lachancea kluyveri generates two functionally distinct proteins. Expression of one of these proteins complements hbs1, but not ski7 mutations, while the other protein complements ski7, but not hbs1. This is the first known case of subfunctionalization by loss of alternative splicing in yeast. By coincidence, the ancestral alternatively spliced gene was also duplicated in Schizosaccharomyces pombe with subsequent subfunctionalization and loss of splicing. Similar subfunctionalization by loss of alternative splicing in fungi also explains the presence of two PTC7 genes in the budding yeast Tetrapisispora blattae, suggesting that this is a common mechanism to preserve duplicate alternatively spliced genes.

The bZIP Transcription Factor Rca1p Is a Central Regulator of a Novel CO2 Sensing Pathway in Yeast

Like many organisms the fungal pathogen Candida albicans senses changes in the environmental CO2 concentration. This response involves two major proteins: adenylyl cyclase and carbonic anhydrase (CA). Here, we demonstrate that CA expression is tightly controlled by the availability of CO2 and identify the bZIP transcription factor Rca1p as the first CO2 regulator of CA expression in yeast. We show that Rca1p upregulates CA expression during contact with mammalian phagocytes and demonstrate that serine 124 is critical for Rca1p signaling, which occurs independently of adenylyl cyclase. ChIP-chip analysis and the identification of Rca1p orthologs in the model yeast Saccharomyces cerevisiae (Cst6p) point to the broad significance of this novel pathway in fungi. By using advanced microscopy we visualize for the first time the impact of CO2 build-up on gene expression in entire fungal populations with an exceptional level of detail. Our results present the bZIP protein Rca1p as the first fungal regulator of carbonic anhydrase, and reveal the existence of an adenylyl cyclase independent CO2 sensing pathway in yeast. Rca1p appears to regulate cellular metabolism in response to CO2 availability in environments as diverse as the phagosome, yeast communities or liquid culture.