Recombinant urease and urease DNA of Coccidioides immitis elicit an immunoprotective response against coccidioidomycosis in mice.

Coccidioides immitis antigens which stimulate a T helper cell 1 (Th1) pathway of host immune response are considered to be essential components of a vaccine against coccidioidomycosis. Recombinant urease (rURE) and recombinant heat shock protein 60 (rHSP60) of C. immitis were expressed in Escherichia coli and tested as vaccine candidates in BALB/c mice. A synthetic oligodeoxynucleotide which contained unmethylated CpG dinucleotides and was previously shown to enhance a murine Th1 response was used as an immunoadjuvant. T cells isolated from the spleens and lymph nodes of the rURE- and rHSP60-immune mice showed in vitro proliferative responses to the respective recombinant protein, but only those T lymphocytes from rURE-immunized mice revealed markedly elevated levels of expression of selected Th1-type cytokine genes. BALB/c mice immunized subcutaneously with rURE and subsequently challenged by the intraperitoneal (i.p.) route with a lethal inoculum of C. immitis arthroconidia demonstrated a significant reduction in the level of C. immitis infection compared to control animals. rHSP60 was much less effective as a protective antigen. Evaluation of cytokine gene expression in lung tissue and levels of recombinant urease-specific immunoglobulins (immunoglobulin G1 [IgG1] versus IgG2a) in murine sera at 12 days after challenge provided additional evidence that immunization with rURE stimulated a Th1 response to the pathogen. Urease was further evaluated by expression of the URE gene in a mammalian plasmid vector (pSecTag2A.URE) which was used to immunize mice by the intradermal route. In this case, 82% of the vector construct-immunized animals survived more than 40 days after i.p. infection, compared to only 10% of the mice immunized with the vector alone. In addition, 87% of the pSecTag2A.URE-immunized survivors had sterile lungs and spleens. These data support the need for further evaluation of the C. immitis urease as a candidate vaccine against coccidioidomycosis.

Cloning and expression of the gene which encodes a tube precipitin antigen and wall-associated beta-glucosidase of Coccidioides immitis.

We report the structure and expression of the Coccidioides immitis BGL2 gene which encodes a previously characterized 120-kDa glycoprotein of this fungal respiratory pathogen. The glycoprotein is recognized by immunoglobulin M tube precipitin (TP) antibody present in sera of patients with coccidioidomycosis, a reaction which has been used for serodiagnosis of early coccidioidal infection. The deduced amino acid sequence of BGL2 shows 12 potential N glycosylation sites and numerous serine-threonine-rich regions which could function as sites for O glycosylation. In addition, the protein sequence includes a domain which is characteristic of family 3 glycosyl hydrolases. Earlier biochemical studies of the purified 120-kDa TP antigen revealed that it functions as a beta-glucosidase (EC 3.2.1.21). Its amino acid sequence shows high homology to several other reported fungal beta-glucosidases which are members of the family 3 glycosyl hydrolases. Results of previous studies have also suggested that the 120-kDa beta-glucosidase participates in wall modification during differentiation of the parasitic cells (spherules) of C. immitis. In this study we showed that expression of the BGL2 gene is elevated during isotropic growth of spherules and the peak of wall-associated BGL2 enzyme activity correlates with this same phase of parasitic cell differentiation. These data support our hypothesis that the 120-kDa beta-glucosidase plays a morphogenetic role in the parasitic cycle of C. immitis.

Acne: a review of immunologic and microbiologic factors.

Acne vulgaris is a self-limiting skin disorder seen primarily in adolescents, whose aetiology appears to be multifactorial. The four main aetiological factors are hypercornification of the pilosebaceous duct, increased sebum production, colonization with Propionibacterium acnes, and subsequently the production of inflammation. Considerable investigation has addressed the immunologic reaction to extracellular products produced by the acne-causing organism, P acnes. The immunologic response involves both humoral and cell-mediated pathways. Further research should clarify the role of complement, cytotoxins, and neutrophils in this acne-forming response.

Propionibacterium acnes: interaction with complement and development of an enzyme-linked immunoassay for the detection of antibody.

OBJECTIVE: To characterize the immune response to Propionibacterium acnes in acne patients. DESIGN: Comparison of serologic responses in acne and normal patients using counterimmunoelectrophoresis for antibody and an enzyme-linked immunosorbent assay (ELISA) to detect immunoglobulin G (IgG) antibody. SETTING: The serum of acne and nonacne patients from the Dermatology Clinic at the Medical College of Ohio was utilized for analysis. RESULTS: Using counterimmunoelectrophoresis, antibody was detected in 13 of 20 acne patients. The antigen was detectable as an anion in the barbital buffer at pH 8.2, strongly suggesting a carbohydrate component. By ELISA, the antibody proved to be IgG, and the bacteria and its water-soluble fractions were capable of fixing complement. CONCLUSIONS: The primary instigator of inflammation in acne vulgaris is an immunologic reaction to extracellular products of P. acnes. The immunologic response involves both humoral and cell-mediated pathways. The antibodies to P. acnes have not been characterized fully, although they are largely of the IgG class. We have further characterized the dominant antigen to have a carbohydrate component.

Clinical isolates of Candida guilliermondii include Candida fermentati.

Clinical isolates of Candida guilliermondii that were investigated by isoenzyme and randomly amplified polymorphic DNA analyses represented two distinct species. The two species were distinguished on the basis of delayed fermentation of galactose. The larger group of isolates was closely related to the anamorph C. guilliermondii ATCC 6260T (T = type strain) and its teleomorph, Yamadazyma (= Pichia) guilliermondii ATCC 46036T. The remaining group, whose members fermented galactose, was very similar to Candida fermentati CBS 2022, which had for many years been placed in synonymy with C. guilliermondii. Three additional groups were represented by individual strains; these strains included C. guilliermondii var. soya ATCC 20216, which was found to represent Yamadazyma ohmeri. The type strain of Y. guilliermondii is redefined.

Random amplified polymorphic DNA analysis of culture collection strains of Candida species.

Random amplified polymorphic DNA (RAPD) profiles, obtained when screening culture collection strains of Candida spp., showed several species to be highly heterogeneous. The RAPD-defined groups were as follows: C. catenulata, two groups among five strains; Debaryomyces hansenii (anamorph C. famata), three groups among five strains; C. sake, three groups among three strains; C. intermedia, two groups among two strains; C. rugosa, two groups among three strains and C. parapsilosis, three groups among four strains. The five strains of Issatchenkia orientalis (anamorph C. krusei) belonged to a single RAPD-defined group. The two strains of the teleomorphic yeast Arxiozyma telluris had distinct RAPD profiles but neither resembled profiles of its anamorph, C. pintolopesii. The two varieties of C. pintolopesii had different RAPD profiles. Researchers are cautioned that organisms with the same name may not be closely related.

Random amplified polymorphic DNA for strain delineation within Candida tropicalis.

Candida tropicalis DNA was used as a template in a polymerase chain reaction (PCR) utilizing a 10-mer primer to generate random amplified polymorphic DNA (RAPD). RAPD patterns associated with 25 primers were obtained for six epidemiologically-unrelated isolates, then a subset of six primers were selected to screen a panel of 18 isolates of C. tropicalis and six isolates of Candida paratropicalis, a species that resembles C. tropicalis but has a sucrose-negative phenotype. The panel, which included nine epidemiologically-related isolates from an outbreak of sternal wound infections, was typed without knowledge of each isolate's origin. The RAPD profiles of the epidemiologically-related isolates were identical to very similar; in contrast, the profiles of most unrelated isolates showed more dissimilarity. While RAPD profiles of C. albicans and Candida parapsilosis differed substantially from those of C. tropicalis, the profiles obtained for C. paratropicalis were consistent with it being a variant of C. tropicalis.

Three distinct genotypes within Candida parapsilosis from clinical sources.

Three genetically distinct groups of Candida parapsilosis were detected among clinical isolates. These were distinguishable on the basis of isoenzyme profiles and DNA sequences of internally transcribed spacer (ITS) sequences flanking the 5.8S RNA gene. In an investigation of 45 strains, including 32 clinical isolates from Texas, C. parapsilosis group I composed the majority of the common clinical isolates. The type strain of C. parapsilosis was a member of this group. The 10 group II isolates were indistinguishable from group I strains when tested with the API 20C kit. The two group III isolates differed from those in groups I and II by being D-xylitol positive by the API 20C kit; however, isolates in all groups assimilated D-xylitol from broth. Isoenzyme profiles excluded the close relationship of any of these groups to Lodderomyces elongisporus, which is a teleomorphic yeast that has a physiological profile similar to that of C. parapsilosis. Although there were insignificant differences in the ITS2 rDNA sequences, comparisons of the ITS1 sequences revealed several differences. A sequence analysis of ITS1 in which missing bases were counted as mismatches showed the following similarities: group I versus group II, 87.7%; group I versus group III, 82.1%; group II versus group III, 84.5%. Also, the activity of secreted proteinase showed differences among the three groups, with many group I isolates having moderate to high activity. The degree of susceptibility to antifungal agents, amphotericin B, ketoconazole, and 5-fluorocytosine, could not be used to determine an isolate's group.

Comparison of three typing methods for clinical and environmental isolates of Aspergillus fumigatus.

To evaluate procedures used for epidemiologic analysis of outbreaks of aspergillosis, we analyzed a collection of 35 Aspergillus fumigatus isolates using three typing methods: isoenzyme analysis (IEA), random amplified polymorphic DNA (RAPD) analysis, and restriction endonuclease analysis (REA). Twenty-one isolates were from a single hospital, with four isolates coming from different patients. Three clinical isolates came from a different hospital, and 11 clinical or environmental isolates were derived from a culture collection. With IEA, the patterns of alkaline phosphatase, esterase, and catalase discriminated nine types. In contrast, 22 types were obtained with five different RAPD primers, and 21 types could be detected with three of these (R108, R151, and UBC90). Restriction endonuclease analysis of genomic DNA, digested with either XbaI, XhoI, or SalI, detected 3, 17, and 13 different REA types, respectively, and 22 types were identified by combining the data from the XhoI and SalI REAs. Twenty-eight types were obtainable with a combination of REA, IEA, and RAPD patterns. Overall, the results pointed to substantial genetic variation among the isolates. Though two isolates had markedly distinct genotypes, their morphologic features and exoantigens were consistent with their being A. fumigatus. The analysis will help in planning epidemiologic studies of aspergillosis.

Unrelatedness of groups of yeasts within the Candida haemulonii complex.

Isoenzyme and protein profiles of clinical isolates initially identified as Candida haemulonii demonstrated the presence of two distinct groups. DNA relatedness studies with representative cultures confirmed the presence of two species. Physiological features that can be used to separate two groups within C. haemulonii are reported.

Comparison of pulsed-field gel electrophoresis with isoenzyme profiles as a typing system for Candida tropicalis.

Candida species are important nosocomial pathogens, particularly in immunocompromised and critically ill patients. A variety of methods have been used to differentiate strains, but an optimal system has not been established. We compared methods for typing a panel of nine related isolates of Candida tropicalis from an outbreak of sternal wound infections as well as four unrelated control isolates of this species. (The genetic relationships of the nine isolates in the panel had been confirmed previously by restriction fragment analysis.) Typing was undertaken without knowledge of an isolate's origin. Karyotyping by contour-clamped homogeneous electric field (CHEF) gel electrophoresis failed to distinguish between outbreak and control isolates. However, when chromosome-sized DNA was digested with SfiI, EagI, SacII, or NaeI and the fragments were separated by CHEF electrophoresis, the outbreak isolates were readily identified. The isoenzyme profiles of the outbreak isolates were identical and were distinctly different from those of the control isolates. While both isoenzyme profiles and the modified CHEF procedure were discriminatory, the latter is recommended as a relatively convenient and reproducible technique for comparison of types of C. tropicalis.

Genotypic identification and characterization of species and strains within the genus Candida by using random amplified polymorphic DNA.

Random amplified polymorphic DNA (RAPD) was used to better characterize the genotypic relatedness among medically important Candida species. By using short oligomer primers (10-mers) with arbitrarily chosen sequences in the polymerase chain reaction, distinctive and reproducible sets of polymerase chain reaction products were observed for isolates of C. albicans, C. lusitaniae, C. tropicalis, and Torulopsis (Candida) glabrata. The RAPD analysis differentiated a physiologically homogeneous panel of C. parapsilosis into three distinct groups and showed genetic diversity within C. haemulonii. Intraspecies DNA-length polymorphisms were seen for RAPD profiles derived from different isolates of each species. Analysis of RAPDs from a panel of C. albicans, which included 16 laboratory derivatives of two reference strains, showed that the profiles of unrelated strains differed and that the derivatives of each reference strain were identifiable. Minor differences in the RAPD profiles, suggestive of mutations that had occurred during the long-term maintenance of the strains, were detected. Because of its ease and reliability, RAPD analysis should be useful in providing genotypic characters for taxonomic descriptions, for confirming the identities of stock isolates, for typing Candida species in epidemiologic investigations, and for use in the rapid identification of pathogenic fungi.

Mushroom poisoning by Chlorophyllum molybdites in the Midwest United States. Cases and a review of the syndrome.

The paper describes two incidents of poisoning by the mushroom Chlorophyllum molybdites and reviews the literature covering this organism, a common agent of mushroom poisoning in many countries and the most common cause of mushroom poisoning in North America. Both poisoning incidents occurred in adults and were associated with severe gastrointestinal symptoms including profuse diarrhea, vomiting and intestinal pain. In each case, hospitalization was required. An unusual aspect of one case was the development of signs and symptoms suggestive of muscarine poisoning. The review includes a description of the mushroom, the geographic distribution of cases, the signs and symptoms of poisoning and its treatment, the toxic principles, particularly susceptible populations, and the variations in response associated with cooking C. molybdites and with individual idiosyncrasies. For identification of C. molybdites, the reader is alerted to the inappropriateness of some books, including many written in Europe, and is warned of the occasional finding of sterile mushrooms that lack its characteristic green basidiospores. Spores, as allergens, are discussed and simple rules are given for eaters of wild mushrooms.

Strain delineation and epidemiology of Candida (Clavispora) lusitaniae.

Electrophoretic karyotype (EK) patterns, determined by using contour-clamped homogeneous pulsed-field electrophoresis, and isoenzyme (IZ) profiles were evaluated as methods for strain delineation among 35 isolates of Candida lusitaniae recovered from 15 patients. All isolates were identified to the species level by using conventional morphologic and physiologic criteria, and the identification was confirmed by gas-liquid chromatography analysis of the cellular fatty acids. The isolates were then typed without knowledge of the patient source. The IZ profiles showed all isolates to be closely related. Fifteen EK patterns were found; each pattern was restricted to isolates recovered from a single patient. In contrast, on the basis of heterogeneity in phosphatases, beta-glucosidases, esterases, and catalases, 10 IZ profiles were found; 4 were shared by isolates recovered from more than one patient. Multiple isolates from six patients were analyzed, and for each patient, a single EK- and IZ-defined type was found. The types of isolates obtained from two patients, after the emergence of resistance to amphotericin B, remained the same as the types of isolates obtained earlier. The data suggest that a patient becomes colonized by a single strain of C. lusitaniae which may disseminate to multiple sites, that the colonizing strain can persist during the patient's hospitalization, and that it may develop resistance to amphotericin B. Both EK patterns and IZ profiles can be used to delineate strains of C. lusitaniae, but the EK pattern provides more discriminatory power.

Isoenzyme changes in Candida albicans during domestication.

Isoenzyme analysis was performed on multiple strains of two commonly used reference cultures of Candida albicans (B311 and NCPF3153). Whereas strains originating from C. albicans B311 showed no variation in isoenzyme profiles, some strains derived from NCPF3153 were identical to B311 strains but others showed variation in their glucose-6-phosphate dehydrogenase isoenzymes. The results are compared with those from previous analyses with these strains and show that C. albicans can undergo genetic alterations during prolonged maintenance in laboratories.

Distinction of deep versus superficial clinical and nonclinical isolates of Trichosporon beigelii by isoenzymes and restriction fragment length polymorphisms of rDNA generated by polymerase chain reaction.

Fifteen clinical and environmental strains of Trichosporon beigelii were analyzed for similarities by using morphological features, biochemical profiles based on carbon compound assimilation and uric acid utilization, isoenzyme electrophoresis, and restriction fragment length polymorphisms of a segment of genes coding for rRNA expanded with the polymerase chain reaction. The findings suggest that strains that cause invasive disease are distinct from the superficial and the nonclinical isolates and that isolates from the skin and mucosae represent a number of different organisms, including some environmental forms. The study shows that T. beigelii is a complex of genetically distinct organisms and that more than one type is found in clinical samples.

Application of multidimensional scaling in numerical taxonomy: analysis of isoenzyme types of Candida species.

Multidimensional scaling (MDS) was applied to the numerical taxonomy of Candida species based on isoenzyme profiles. Multidimensional scaling uses proximity measures to generate a spatial configuration of points in multidimensional space where distances between points reflect similarity among types. The biochemical profiles of 35 types of Candida species based on 26 tests consisting of isoenzymes of alpha-glucosidase, alkaline phosphatase, glucose-6-phosphate dehydrogenase, malate dehydrogenase, isocitrate dehydrogenase, and superoxide dismutase were analyzed. Cluster analysis of MDS, using the Euclidean distance as a proximity measure, separated C. tropicalis and C. paratropicalis from C. albicans and C. stellatoidea. Stepwise multiple linear regression revealed the isoenzyme tests which influenced each of the MDS dimensions. MDS was able to reduce the dimensionality of the test profile.

Isoenzyme biotypes of Candida species.

Isoenzyme profiles were obtained following polyacrylamide gel electrophoresis of crude extracts of Candida albicans and C. tropicalis. The two species were separated by distinct isoenzyme patterns. Within each species, variations were found for several isoenzymes. This allowed the development of a method for biotyping these fungi. Isoenzyme patterns of the sucrose-negative variants "C. stellatoidea" and "C. paratropicalis Baker, Salkin, Pincus, and D'Amato" were obtained and subjected to cluster analysis. This procedure failed to place the variants into clusters that were clearly distinct from the conventional sucrose-positive strains. All sucrose-negative strains were found to have alpha-glucosidase activity. There was an almost complete lack of heterogeneity in the isoenzyme patterns of 11 C. stellatoidea type I strains.

Protein and enzyme electrophoresis profiles of selected Candida species.

The cellular protein profiles and malate dehydrogenases, superoxide dismutases, alkaline phosphatases, and esterases from whole cell extracts of Candida spp. were studied with polyacrylamide gel electrophoresis. We investigated isolates that differed in their ability to assimilate sucrose as the sole carbon source. The protein and enzyme patterns of Candida tropicalis and its sucrose-negative variant "Candida paratropicalis Baker, Salkin, Pincus et D'Amato" were indistinguishable. Although the cellular protein and superoxide dismutase patterns of Candida albicans and its sucrose-negative variant "Candida stellatoidea" were quite similar, differences were noted in the profiles of the other enzymes studied. In addition, the C. stellatoidea isolates were found to be separable, on the basis of their enzyme profiles, into the same two types that have been reported by Kwon-Chung et al. (K.J. Kwon-Chung, B.L. Wickes, and W.G. Merz, Infect. Immun. 56:1814-1819, 1988).