Carol A. Kumamoto

Candida albicans Biofilms Produce Antifungal-Tolerant Persister Cells

ABSTRACT Fungal pathogens form biofilms that are highly recalcitrant to antimicrobial therapy. The expression of multidrug resistance pumps in young biofilms has been linked to increased resistance to azoles, but this mechanism does not seem to underlie the resistance of mature biofilms that is a model of in vivo infection. The mechanism of drug resistance of mature biofilms remains largely unknown. We report that biofilms formed by the major human pathogen Candida albicans exhibited a strikingly biphasic killing pattern in response to two microbicidal agents, amphotericin B, a polyene antifungal, and chlorhexidine, an antiseptic, indicating that a subpopulation of highly tolerant cells, termed persisters, existed. The extent of killing with a combination of amphotericin B and chlorhexidine was similar to that observed with individually added antimicrobials. Thus, surviving persisters form a multidrug-tolerant subpopulation. Interestingly, surviving C. albicans persisters were detected only in biofilms and not in exponentially growing or stationary-phase planktonic populations. Reinoculation of cells that survived killing of the biofilm by amphotericin B produced a new biofilm with a new subpopulation of persisters. This suggests that C. albicans persisters are not mutants but phenotypic variants of the wild type. Using a stain for dead cells, rare dark cells were visible in a biofilm after amphotericin B treatment, and a bright and a dim population were physically sorted from this biofilm. Only the dim cells produced colonies, showing that this method allows the isolation of yeast persisters. Given that persisters formed only in biofilms, mutants defective in biofilm formation were examined for tolerance of amphotericin B. All of the known mutants affected in biofilm formation were able to produce normal levels of persisters. This finding indicates that attachment rather than formation of a complex biofilm architecture initiates persister formation. Bacteria produce multidrug-tolerant persister cells in both planktonic and biofilm populations, and it appears that yeasts and bacteria have evolved analogous strategies that assign the function of survival to a small part of the population. In bacteria, persisters are dormant cells. It remains to be seen whether attachment initiates dormancy that leads to the formation of fungal persisters. This study suggests that persisters may be largely responsible for the multidrug tolerance of fungal biofilms.

Contributions of hyphae and hypha‐co‐regulated genes to Candida albicans virulence

The fascinating ability of Candida albicans to undergo dramatic changes in cellular morphology has invited speculation that this plasticity in form contributes to the virulence of the organism. Molecular genetic analyses have confirmed this hypothesis and further demonstrated that genes that govern cellular morphology are co‐regulated with genes encoding conventional virulence factors such as proteases and adhesins. The transcriptional regulatory networks of C. albicans thus ensure that hyphae are produced concomitantly with virulence factors, resulting in cells that are adapted for invading the tissues of an immunocompromised host. Hyphae are able to exert mechanical force, aiding penetration of epithelial surfaces, and hyphae damage endothelial cells, aiding escape of C. albicans from the host bloodstream into deeper tissue. Hyphal morphogenesis is thus an integral part of the overall virulence strategy of C. albicans.

Filamentous growth of Candida albicans in response to physical environmental cues and its regulation by the unique CZF1 gene

Hyphal growth in the opportunistic fungal pathogen Candida albicans is believed to contribute to the virulence of the organism by promoting penetration of fungal cells into host tissue. In this study, stimulation of hyphal growth by a feature of the physical environment was demonstrated. Specifically, growth of cells embedded within a matrix promoted the formation of hyphae. The CZF1 gene, encoding a putative transcription factor, was shown to be involved in the regulation of hyphal growth under certain conditions, including embedded conditions. Ectopic expression of CZF1 in embedded cells promoted the rapid formation of hyphae. Elimination of CZF1 and CPH1, encoding a homologue of the Saccharomyces cerevisiae Ste12p transcription factor, led to a pronounced defect in filamentous growth of embedded cells. Elimination of CZF1 alone led to a moderate defect in hyphal growth under some conditions, including embedded conditions. Hyphal morphogenesis in response to matrix embedding may occur in the opportunistic pathogen, C. albicans, to promote invasion of fungal cells into host tissue.

Three pure chaperone proteins of Escherichia coli--SecB, trigger factor and GroEL--form soluble complexes with precursor proteins in vitro.

Diverse studies of three cytoplasmic proteins of Escherichia coli‐‐SecB, trigger factor and GroEL‐‐have suggested that they can maintain precursor proteins in a conformation which is competent for membrane translocation. These proteins have been termed ‘chaperones’. Using purified chaperone proteins and precursor protein substrates, we find that each of these chaperones can stabilize proOmpA for translocation and for the translocation‐ATPase. These chaperones bind to proOmpA to form isolable complexes. SecB and GroEL will also form complexes with another exported protein, prePhoE. In contrast, these chaperones do not form stable complexes with a variety of soluble proteins such as SecA protein, bovine serum albumin, ovalbumin or ribonuclease A. While chaperones may transiently interact with soluble proteins to catalyze their folding, the stable interaction between chaperones and presecretory proteins, maintaining an open conformation which is essential for translocation, may commit these proteins to the secretion pathway.

The DsbA Signal Sequence Directs Efficient, Cotranslational Export of Passenger Proteins to the Escherichia coli Periplasm via the Signal Recognition Particle Pathway

The Escherichia coli cytoplasmic protein thioredoxin 1 can be efficiently exported to the periplasmic space by the signal sequence of the DsbA protein (DsbAss) but not by the signal sequence of alkaline phosphatase (PhoA) or maltose binding protein (MBP). Using mutations of the signal recognition particle (SRP) pathway, we found that DsbAss directs thioredoxin 1 to the SRP export pathway. When DsbAss is fused to MBP, MBP also is directed to the SRP pathway. We show directly that the DsbAss-promoted export of MBP is largely cotranslational, in contrast to the mode of MBP export when the native signal sequence is utilized. However, both the export of thioredoxin 1 by DsbAss and the export of DsbA itself are quite sensitive to even the slight inhibition of SecA. These results suggest that SecA may be essential for both the slow posttranslational pathway and the SRP-dependent cotranslational pathway. Finally, probably because of its rapid folding in the cytoplasm, thioredoxin provides, along with gene fusion approaches, a sensitive assay system for signal sequences that utilize the SRP pathway.

Self-Regulation of Candida albicans Population Size during GI Colonization

Interactions between colonizing commensal microorganisms and their hosts play important roles in health and disease. The opportunistic fungal pathogen Candida albicans is a common component of human intestinal flora. To gain insight into C. albicans colonization, genes expressed by fungi grown within a host were studied. The EFH1 gene, encoding a putative transcription factor, was highly expressed during growth of C. albicans in the intestinal tract. Counterintuitively, an efh1 null mutant exhibited increased colonization of the murine intestinal tract, a model of commensal colonization, whereas an EFH1 overexpressing strain exhibited reduced colonization of the intestinal tract and of the oral cavity of athymic mice, the latter situation modeling human mucosal candidiasis. When inoculated into the bloodstream of mice, both efh1 null and EFH1 overexpressing strains caused lethal infections. In contrast, other mutants are attenuated in virulence following intravenous inoculation but exhibited normal levels of intestinal colonization. Finally, although expression of several genes is dependent on transcription factor Efg1p during laboratory growth, Efg1p-independent expression of these genes was observed during growth within the murine intestinal tract. These results show that expression of EFH1 regulated the level of colonizing fungi, favoring commensalism as opposed to candidiasis. Also, different genes are required in different host niches and the pathway(s) that regulates gene expression during host colonization can differ from well-characterized pathways used during laboratory growth.

Preprotein transfer to the Escherichia coli translocase requires the co‐operative binding of SecB and the signal sequence to SecA

In Escherichia coli, precursor proteins are targeted to the membrane‐bound translocase by the cytosolic chaperone SecB. SecB binds to the extreme carboxy‐terminus of the SecA ATPase translocase subunit, and this interaction is promoted by preproteins. The mutant SecB proteins, L75Q and E77K, which interfere with preprotein translocation in vivo, are unable to stimulate in vitro translocation. Both mutants bind proOmpA but fail to support the SecA‐dependent membrane binding of proOmpA because of a marked reduction in their binding affinities for SecA. The stimulatory effect of preproteins on the interaction between SecB and SecA exclusively involves the signal sequence domain of the preprotein, as it can be mimicked by a synthetic signal peptide and is not observed with a mutant preprotein (Δ8proOmpA) bearing a non‐functional signal sequence. Δ8proOmpA is not translocated across wild‐type membranes, but the translocation defect is suppressed in inner membrane vesicles derived from a prlA4 strain. SecB reduces the translocation of Δ8proOmpA into these vesicles and almost completely prevents translocation when, in addition, the SecB binding site on SecA is removed. These data demonstrate that efficient targeting of preproteins by SecB requires both a functional signal sequence and a SecB binding domain on SecA. It is concluded that the SecB–SecA interaction is needed to dissociate the mature preprotein domain from SecB and that binding of the signal sequence domain to SecA is required to ensure efficient transfer of the preprotein to the translocase.

Role of a Candida albicans P1-Type ATPase in Resistance to Copper and Silver Ion Toxicity

Copper ion homeostasis is complicated in that copper is an essential element needed for a variety of cellular processes but is toxic at excess levels. To identify Candida albicans genes that are involved in resistance to copper ion toxicity, a library containing inserts of C. albicans genomic DNA was used to complement the copper sensitivity phenotype of a Saccharomyces cerevisiae cup1Delta strain that is unable to produce Cup1p, a metallothionein (MT) responsible for high-level copper ion resistance. A P1-type ATPase (CPx type) that is closely related to the human Menkes and Wilson disease proteins was cloned. The gene encoding this pump was termed CRD1 (for copper resistance determinant). A gene encoding a 76-amino-acid MT similar to higher eukaryotic MTs in structure was also cloned, and the gene was termed CRD2. Transcription of the CRD1 gene was found to increase upon growth with increasing copper levels, while the CRD2 mRNA was expressed at a constant level. Strains with the CRD1 gene disrupted were extremely sensitive to exogenous copper and failed to grow in medium containing 100 microM CuSO(4). These crd1 strains also exhibited increased sensitivity to silver and cadmium, indicating that Crd1p is somewhat promiscuous with respect to metal ion transport. Although strains with the CRD2 gene disrupted showed reduced growth rate with increasing copper concentration, the crd2 mutants eventually attained wild-type levels of growth, demonstrating that CRD2 is less important for resistance to copper ion toxicity. Crd1p is the first example of a eukaryotic copper pump that provides the primary source of cellular copper resistance, and its ability to confer silver resistance may enhance the prevalence of C. albicans as a nosocomial pathogen.

Inflammation and gastrointestinal Candida colonization

Candida organisms commonly colonize the human gastrointestinal tract as a component of the resident microbiota. Their presence is generally benign. Recent studies, however, show that high level Candida colonization is associated with several diseases of the gastrointestinal tract. Further, results from animal models argue that Candida colonization delays healing of inflammatory lesions and that inflammation promotes colonization. These effects may create a vicious cycle in which low-level inflammation promotes fungal colonization and fungal colonization promotes further inflammation. Both inflammatory bowel disease and gastrointestinal Candida colonization are associated with elevated levels of the pro-inflammatory cytokine IL-17. Therefore, effects on IL-17 levels may underlie the ability of Candida colonization to enhance inflammation. Because Candida is a frequent colonizer, these effects have the potential to impact many people.

Adaptations of Candida albicans for Growth in the Mammalian Intestinal Tract

ABSTRACT Although the fungus Candida albicans is a commensal colonizer of humans, the organism is also an important opportunistic pathogen. Most infections caused by C. albicans arise from organisms that were previously colonizing the host as commensals, and therefore successful establishment of colonization is a prerequisite for pathogenicity. To elucidate fungal activities that promote colonization, an analysis of the transcription profile of C. albicans cells recovered from the intestinal tracts of mice was performed. The results showed that within the C. albicans colonizing population, cells expressed genes characteristic of the laboratory-grown exponential phase and genes characteristic of post-exponential-phase cells. Thus, gene expression both promoted the ability to grow rapidly (a characteristic of exponential-phase cells) and enhanced the ability to resist stresses (a characteristic of post-exponential-phase cells). Similarities in gene expression in commensal colonizing cells and cells invading host tissue during disease were found, showing that C. albicans cells adopt a particular cell surface when growing within a host in both situations. In addition, transcription factors Cph2p and Tec1p were shown to regulate C. albicans gene expression during intestinal colonization.