SummaryIron, an essential element for almost every organism, serves as a regulatory signal for the expression of virulence determinants in many prokaryotic and eukaryotic pathogens. Using a custom Affymetrix GeneChip © representing the entire Candida albicans genome, we examined the changes in genome-wide gene expression in this opportunistic pathogen as a function of alterations in environmental concentrations of iron. A total of 526 open reading frame (ORF) transcripts are more highly expressed when the levels of available iron are low, while 626 ORF transcripts are more highly expressed in high-iron conditions. The transcripts dominantly affected by iron concentration range from those associated with cell-surface properties to others which affect mitochondrial function, iron transport and virulence-related secreted hydrolases. Moreover gene expression as assayed in DNA microarrays confirms and extends reports of alterations in cell-surface antigens and drug sensitivity correlated with iron availability. To understand how these genes and pathways might be regulated, we isolated a gene designated SFU1 that encodes a homologue of the Ustilago maydis URBS1, a transcriptional repressor of siderophore uptake/ biosynthesis. Comparisons between wild-type and SFU1 -null mutant strains revealed 139 potential target genes of Sfu1p; many of which are iron-responsive. Together, these results not only expand our understanding of global iron regulation in C . albicans , but also provide insights into the potential role of iron availability in C. albicans virulence.

Phase and antigenic variation are mechanisms used by microbial pathogens to stochastically change their cell surface composition. A related property, referred to as phenotypic switching, has been described for some pathogenic fungi. This phenomenon is best studied in Candida albicans, where switch phenotypes vary in morphology, physiology, and pathogenicity in experimental models. In this study, we report an application of a custom Affymetrix GeneChip representative of the entire C. albicans genome and assay the global expression profiles of white and opaque switch phenotypes of the WO-1 strain. Of 13,025 probe sets examined, 373 ORFs demonstrated a greater than twofold difference in expression level between switch phenotypes. Among these, 221 were expressed at a level higher in opaque cells than in white cells; conversely, 152 were more highly expressed in white cells. Affected genes represent functions as diverse as metabolism, adhesion, cell surface composition, stress response, signaling, mating type, and virulence. Approximately one-third of the differences between cell types are related to metabolic pathways, opaque cells expressing a transcriptional profile consistent with oxidative metabolism and white cells expressing a fermentative one. This bias was obtained regardless of carbon source, suggesting a connection between phenotypic switching and metabolic flexibility, where metabolic specialization of switch phenotypes enhances selection in relation to the nutrients available at different anatomical sites. These results extend our understanding of strategies used in microbial phase variation and pathogenesis and further characterize the unanticipated diversity of genes expressed in phenotypic switching.M any pathogenic bacteria, fungi, and protozoa have evolved strategies for alternative expression of surface-related phenotypes, a facility that enables their escape from immune surveillance and adaptation to changing environments. Antigenic variation, as displayed by African trypanosomes, Neisseria spp. and Borrelia spp., is a well studied example (1-4). Variation between phenotypes can be reversible and stochastic, leading to the expression of predefined traits which, when superimposed on classical environmentally responsive sensor mechanisms, extend the phenotypic diversity of the pathogen. In bacteria, many mechanisms have been described that lead to the expression of contingency loci that regulate expression of pili, flagella, adhesins, and surface-associated lipopolysaccharides and lipoproteins (5, 6).Strains of Candida albicans, the most important fungal pathogen of humans, are able to spontaneously and reversibly switch phenotypes at high frequency (7). Three different switching systems were first described by . C. albicans strain 3153A alternates between phenotypes distinguished by at least seven colony morphologies; conversion from the original smooth to other variant colony morphologies occurs at a combined frequency of 1.4 ϫ 10 Ϫ4 (11). Another system includes strains that switch between coloni...

We have previously shown that a mixture of three synthetic peptides (83.1, 55.1, 35.1), corresponding to fragments of the relative molecular mass 83,000 (83K), 55K and 35K Plasmodium falciparum merozoite-specific proteins, induces protection in Aotus triviroatus monkeys experimentally infected with P. falciparum. Here we describe two polymeric synthetic hybrid proteins based on these peptides that delay or suppress the development of parasitaemia in immunized human volunteers.

The ability to adhere to surfaces and develop as a multicellular community is an adaptation used by most microorganisms to survive in changing environments. Biofilm formation proceeds through distinct developmental phases and impacts not only medicine but also industry and evolution. In organisms such as the opportunistic pathogen Candida albicans, the ability to grow as biofilms is also an important mechanism for persistence, facilitating its growth on different tissues and a broad range of abiotic surfaces used in medical devices. The early stage of C. albicans biofilm is characterized by the adhesion of single cells to the substratum, followed by the formation of an intricate network of hyphae and the beginning of a dense structure. Changes in the transcriptome begin within 30 min of contact with the substrate and include expression of genes related to sulfur metabolism, in particular MET3, and the equivalent gene homologues of the Ribi regulon in Saccharomyces cerevisiae. Some of these changes are initiated early and maintained throughout the process; others are restricted to the earliest stages of biofilm formation. We identify here a potential alternative pathway for cysteine metabolism and the biofilm-associated expression of genes involved in glutathione production in C. albicans.

The Random Amplified Polymorphic DNA (RAPD) technique was used in the identification of a species-specific fragment of Mycobacterium bovis. A fragment of approximately 500 bp was amplified from the genome of 15 different M. bovis strains, including M. bovis BCG Pasteur, but was shown to be absent in 26 different mycobacteria and 20 different clinical isolates of Mycobacterium tuberculosis. When the fragment was used as a probe in a Southern blot analysis, several radioactive bands common to M. tuberculosis and M. bovis were observed. However, this fragment hybridized specifically to a 2900 bp EcoRl fragment in the M. bovis genome, but failed to hybridize in either M. tuberculosis or M. avium chromosomal DNA. Based on a partial nucleotide sequence of the 500 bp fragment, two oligonucleotide primers were designed and a PCR assay was developed. Using purified mycobacterial DNA samples, only M. bovis and M. bovis BCG rendered a unique amplification band. This PCR assay is able to detect down to 10 fg purified M. bovis DNA, which corresponds roughly to two bacilli. The assay is also useful for identifying the bacilli directly from uncultured biological samples, such as milk.

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A retrospective study was undertaken of local, regional, and distant recurrences in 346 patients with primary melanomas of tumor thickness less than 1.0 mm that were excised with margins of normal skin varying between 0.1 cm and 5.0 cm or more. Prospective histopathologic examination of 284 melanomas for the presence of microsatellites was also performed and their effect upon the frequency of local recurrence was studied. Margins of excision did not influence the frequency of local, regional, or distant metastases. Four recurrences of in situ superficial spreading melanoma occurred, however, when very narrow margins of excision (0.5 cm or less) were employed. Microsatellites were uncommon with tumors less than 3.0 mm in thickness (2.8% of all tumors of less than 3.0 mm in thickness, taken together), but relatively frequent in association with thicker tumors (37%). Melanomas with microsatellites were associated with a greater frequency of local clinical metastasis than those without (14% vs. 3%). Removal of more than 1.0 cm of normal skin around a melanoma of less than 1.0 mm in thickness does not further reduce rates of recurrence of any type. The use of margins of 0.5 cm or less for melanomas with a radial growth phase does appear to result in an increased frequency of local recurrence of the primary melanoma with an epidermal in situ component. These recurrences can be prevented by the removal of 1.0 cm of normal skin around such a melanoma. Microsatellites constitute a risk factor for local recurrence, but are a relatively uncommon phenomenon at tumor thickness less than 3.0 mm.