High-frequency switching and strain variability at the site of infection was assessed in Il patients with acute Candida albicans vaginitis. By cloning cells directly from the site of infection, it was demonstrated that 4 of the 11 isolates contained multiple-switch phenotypes at the site of infection and that 9 of the 11 isolates were in a high-frequency mode of switching (10-2 to 10-3). Isolates could be separated into four general categories of switching repertoires. To demonstrate that multiple phenotypes at the site of a single infection represented the same strain, EcoRI digests of total cell DNA were separated on agarose gels, and Southern hybridization patterns with two cloned midrepeat sequences were compared. Candida albicans remains the most persistent and pervasive yeast pathogen in humans, capable of invading virtually every tissue of the body (2, 11). Not only is it the major cause of vaginal and oral yeast infections (6, 7, 9, 16, 17, 20), but it has evolved into a major systemic pathogen of compromised hosts (2, 3, 5, 10, 12). Recently, it was demonstrated that a common laboratory strain of C. albicans possesses a high-frequency switching system which can be distinguished by colony morphology on the proper agar substratum (14). Cells of this strain switch spontaneously to six general colony phenotypes at a combined frequency of ' The opaque sector (OS) and white sector (WS) were individually cloned.
Species and strain variabilities have been monitored during the history of a prolonged Candida infection in a single compromised bone marrow transplant patient by analyzing sugar assimilation patterns, highfrequency switching repertoires, and Southern blot hybridization patterns with two cloned mid-repeat sequences (Ca3 and Ca7) which are species specific for Candida albicans and one cloned mid-repeat sequence (Ctl3-8) which is species specific for Candida tropicalis. Evidence is presented that during the course of this infection (i) two strains of C. albicans and three strains of C. tropicalis were distinguished by their switching repertoires, Southern blot hybridization patterns, and sugar assimilation patterns; (ii) the three C. tropicalis strains were in a high-frequency mode of switching; (iii) two C. tropicalis strains coexisted in the blood and three C. tropicalis strains coexisted in the throat at different times during the history of the infection; (iv) amphotericin B treatment selectively removed one of two C. tropicalis strains coexisting in the blood and this strain exhibited greater susceptibility to amphotericin B in vitro (the remaining strain was subsequently removed from the blood by flucytosine treatment); and (v) both the strain removed from the blood by amphotericin B and the strain removed from the blood by flucytosine reappeared several days later at another site of infection. It is demonstrated for the first time that C. tropicalis is capable of high-frequency switching of colony morphology just as C. albicans is, that there is more than one strain-specific switching repertoire in C. tropicalis, and that a C. tropicalis mid-repeat sequence can be used for discriminating species and assessing strain relatedness, as previously demonstrated for C. albicans mid-repeat sequences.
Under the regime of pH-regulated dimorphism, stationary phase cells of the dimorphic yeast Candida albicans can be induced to form exclusively and synchronously ellipsoidal buds or elongate mycelia at the same temperature and in the same nutrient medium, the sole determinant of phenotype in this case being pH. Employing pH-regulated dimorphism, cells were pulse-labeled with [35S]-methionine during three consecutive intervals encompassing the preevagination period, the period including evagination and phenotypic commitment, and the post-evagination period. Labeled polypeptides were analyzed by 2D-PAGE. Of the 374 polypeptides examined, the majority (237) did not differ significantly in relative incorporation between the three pulse periods and were similar between budding and mycelium-forming populations. Sixty polypeptides were labeled at negligible or relatively low levels during the first pulse period, but at significantly higher levels during the second and third or third pulse periods. All but one were similar between budding and mycelium-forming populations. Seventeen polypeptides were synthesized at relatively high levels during the first pulse period, but at reduced or negligible levels during the second and third or third pulse periods. All but one were similar between budding and mycelium-forming populations. Only two polypeptides were found to be associated exclusively with mycelium-forming cultures, two associated exclusively with budding cultures, and two enriched significantly in budding cultures of wild-type cells. Employing a variant, MD20, which forms buds at both low and high pH, it was demonstrated that only one mycelium-associated polypeptide and only one bud-associated polypeptide are phenotype rather than pH-specific. Limits to this method of phenotype comparison are outlined, and the unusual similarity rather than dissimilarity in the programs of gene expression between the diverging populations considered in terms of phenotypic regulation.
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