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.Candida albicans is an opportunistic pathogen that is a common member of the human oral and gastrointestinal microflora. Biofilms of C. albicans often form on indwelling devices, such as blood and urinary catheters and heart valves. These infections are essentially untreatable by antifungals and can develop into life-threatening disease with a mortality rate approaching 40%. The recalcitrance of these infections to antifungals is puzzling, since planktonic populations of the same strain can be susceptible to a range of antifungals, including azole compounds, echinocandins, and am...

SummaryThe 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 .Unlike highly specialized pathogens that express a single major virulence factor (e.g. Clostridium tetani ), the opportunistic fungal pathogen Candida albicans expresses a repertoire of activities that contribute to virulence. The sum of the effects of many Candida factors leads to the establishment of infection in a suitably compromised host. In some host niches, particular fungal proteins (e.g. adhesins) are important, while in other niches, these proteins are less important and other factors play a dominant role. By being adapted for growth in many possible host niches, C. albicans is able to cause the wide spectrum of clinical manifestations for which is it known.Discussions of C. albicans virulence factors usually include dimorphism, the ability to grow in more than one morphological form. C. albicans grows either as budding yeast cells or as filamentous hyphae (chains of elongated, parallel-sided cells lacking constrictions at the septa) or pseudohyphae (chains of variably elongated cells with constrictions at the septa). Because of their similar elongated shapes and the many conditions and regulators that are shared in common, the hyphal and pseudohyphal forms are often not distinguished in the literature; the more general term, 'filamentous forms', is used to refer to both. The two morphologies, however, are fundamentally different and can be distinguished by a number of characteristics. The distinctions between the two forms have been elegantly reviewed by Sudbery et al . (2004).Filamentation is believed to be important for virulence and thus, morphogenesis in this fungus has been a subject of considerable study. The potential roles of the hyphal form, in particular, in virulence have also been discussed frequently in the literature. While most mutants that fail to produce hyphae are compromised in virulence, several authors have pointed out that co-regulation of genes controlling hyphal morphogenesis with genes encoding virulence factors confounds the analysis (Kobayashi and Cutler, 1998;Brown, 2002;Gow et al ., 2002;Liu, 2002). Thus, it has been difficult to determine unequivoc...

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.

The identification of regulators, circuits, and target genes employed by the fungus Candida albicans to thrive in disparate niches in a mammalian host reveals interconnection between commensal and pathogenic lifestyles.

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.

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.

Intestinal functions are central to human physiology, health and disease. Options to study these functions with direct relevance to the human condition remain severely limited when using conventional cell cultures, microfluidic systems, organoids, animal surrogates or human studies. To replicate in vitro the tissue architecture and microenvironments of native intestine, we developed a 3D porous protein scaffolding system, containing a geometrically-engineered hollow lumen, with adaptability to both large and small intestines. These intestinal tissues demonstrated representative human responses by permitting continuous accumulation of mucous secretions on the epithelial surface, establishing low oxygen tension in the lumen, and interacting with gut-colonizing bacteria. The newly developed 3D intestine model enabled months-long sustained access to these intestinal functions in vitro, readily integrable with a multitude of different organ mimics and will therefore ensure a reliable ex vivo tissue system for studies in a broad context of human intestinal diseases and treatments.

In response to attachment to a surface, fungal cells produce biofilms, three-dimensional structures composed of cells surrounded by exopolymeric matrices. Surface attachment causes Candida albicans cells to enter a special physiological state in which they are highly resistant to antifungal drugs and express the drug efflux determinants CDR1, CDR2 and MDR1. C. albicans biofilms produced under different conditions differ in their cellular morphology and matrix content, which suggests that biofilms formed within a host, for example on indwelling medical devices, would also differ depending on the nature of the device and its location. The mechanisms by which surface attachment leads to biofilm formation are presently not understood.