Irreversible block of the mycelial-to-yeast phase transition of Histoplasma capsulatum.

p-Chloromercuriphenylsulfonic acid (PCMS), a sulfhydryl inhibitor, prevented the mycelial-to-yeast transition of the dimorphic fungal pathogen, Histoplasma capsulatum. The effect of PCMS was specific for the mycelial-to-yeast transformation; it had no effect on growth of either the yeast or mycelial forms or on the yeast-to-mycelial transition. The failure of PCMS-treated mycelia to transform to yeast was permanent and irreversible. PCMS-treated mycelia could not infect mice but could stimulate resistance to infection by a pathogenic strain of Histoplasma capsulatum. These results suggest a new general strategy for vaccine development in diseases caused by dimorphic pathogens.

MRPL53, a New Candidate Gene for Orofacial Clefting, Identified Using an eQTL Approach.

A valuable approach to understand how individual and population genetic differences can predispose to disease is to assess the impact of genetic variants on cellular functions (e.g., gene expression) of cell and tissue types related to pathological states. To understand the genetic basis of nonsyndromic cleft lip with or without cleft palate (NSCL/P) susceptibility, a complex and highly prevalent congenital malformation, we searched for genetic variants with a regulatory role in a disease-related tissue, the lip muscle (orbicularis oris muscle [OOM]), of affected individuals. From 46 OOM samples, which are frequently discarded during routine corrective surgeries on patients with orofacial clefts, we derived mesenchymal stem cells and correlated the individual genetic variants with gene expression from these cultured cells. Through this strategy, we detected significant cis-eQTLs (i.e., DNA variants affecting gene expression) and selected a few candidates to conduct an association study in a large Brazilian cohort (624 patients and 668 controls). This resulted in the discovery of a novel susceptibility locus for NSCL/P, rs1063588, the best eQTL for the MRPL53 gene, where evidence for association was mostly driven by the Native American ancestry component of our Brazilian sample. MRPL53 (2p13.1) encodes a 39S protein subunit of mitochondrial ribosomes and interacts with MYC, a transcription factor required for normal facial morphogenesis. Our study illustrates not only the importance of sampling admixed populations but also the relevance of measuring the functional effects of genetic variants over gene expression to dissect the complexity of disease phenotypes.

Electrophoretic analysis of Histoplasma capsulatum chromosomal DNA.

Seven chromosome-sized DNA molecules in the Downs strain of Histoplasma capsulatum were resolved by using chromosome-specific DNA probes in blot hybridizations of contour-clamped homogeneous electric field (CHEF) and field-inversion gel electrophoresis (FIGE) agarose gels. The sizes of the chromosomal DNA bands extended from that of the largest Saccharomyces cerevisiae chromosome to beyond that of the Schizosaccharomyces pombe chromosomes. Under our experimental conditions, the order of the five largest DNA bands was inverted in the FIGE gel relative to the CHEF gel, demonstrating a characteristic of FIGE whereby large DNA molecules may have greater rather than lesser mobility with increasing size. Comparison of the Downs strain with other H. capsulatum strains by CHEF and FIGE analysis revealed considerable variability in band mobility. The resolution of seven chromosome-sized DNA molecules in the Downs strain provides a minimum estimate of the chromosome number.

Morphological transition in the human fungal pathogen Histoplasma capsulatum.

Considerable information has accumulated recently about specific genes of Histoplasma capsulatum that are expressed during the process of adaptation when the organism undergoes morphological transition at the onset of infection. The study of these genes is crucial to identify targets for the development of novel antifungal agents.

Deuterium NMR investigation of an amphotericin B derivative in mechanically aligned lipid bilayers.

The methyl-d(3) amide derivative of the polyene antibiotic amphotericin B was synthesized, assayed for biological activity, incorporated into mechanically aligned bilayers of dipalmitoylphosphatidylcholine (DPPC), and examined by deuterium and phosphorus NMR. The amide derivative has a lesser, but qualitatively similar, biological activity relative to amphotericin B. Incorporation of the amide derivative and ergosterol into aligned DPPC bilayers resulted in a single, stable bilayer phase, as shown by phosphorus NMR of the DPPC headgroups. Deuterium NMR spectra revealed one major (2)H quadrupolar splitting and one major (2)H-(1)H dipolar splitting in the liquid-crystalline phase, consistent with a high degree of alignment and a single, averaged physical state for amphotericin B methyl-d(3) amide in the bilayer. Variations of the quadrupolar and dipolar splittings as a function of macroscopic sample orientation and temperature indicated that the amide derivative undergoes fast rotation about a motional axis that is parallel to the bilayer normal.

Mechanism of the inhibition by RNA of the RNA polymerases of Histoplasma capsulatum.

4 S RNA isolated from the dimorphic fungus Histoplasma capsulatum inhibited the DNA-dependent RNA polymerase activity of the yeast phase of this fungus. Inhibition was specific for initiation, and resulted from binding of the RNA to the enzyme. Among a variety of synthetic polynucleotides tested, only poly(G) and oligo(dG) were effective inhibitors, suggesting a role for guanines or guanine-rich sequences of RNA in the inhibition reaction.

Characterization of CARE-1: Candida albicans repetitive element-1.

A middle repetitive DNA element, Candida albicans repetitive element-1 (CARE-1) has been isolated from the pathogenic yeast C. albicans. CARE-1 appears to be species-specific and constitutes approx. 0.045% of total C. albicans DNA, or a reiteration frequency of about two to twelve copies per haploid genome. The CARE-1 element has been detected on several C. albicans chromosomes separated by field-inversion gel electrophoresis, suggesting that the element is dispersed. Interstrain variation was observed in the number and distribution of hybridizing bands. The element is well conserved, since no nucleotide (nt) heterogeneity was observed when the sequences of two CARE-1 family members isolated from two different chromosomes (A and B) of C. albicans were compared. CARE-1 possesses 467 bp and is characterized by several stretches of A's and T's, short direct repeats and shows no significant homology to any known nt sequence.

Isolation, characterization, and sequencing of Candida albicans repetitive element 2.

A 1059-bp Sau3A fragment, designated Candida albicans repetitive element 2 (CARE-2), was isolated from the genome of the pathogenic yeast, C. albicans. CARE-2 DNA was detected on several C. albicans chromosomes separated by transverse alternating-field electrophoresis. A high degree of interstrain variation in the pattern of hybridizing bands were observed by Southern blot analysis, with a minimum of 10-14 copies of CARE-2 per strain. A low frequency of new CARE-2 polymorphisms was observed over time for three strains grown at 25 degrees C or 37 degrees C. No new CARE-2 polymorphisms were observed from two naturally occurring switch phenotypes. To localize repeated DNA, oligodeoxyribonucleotide probes, each representing a different region of CARE-2, were hybridized to genomic blots. A lower number of copies were observed 5' and 3' to a 600-bp region of CARE-2. Nucleotide (nt) sequence analysis of CARE-2 DNA shows the element is characterized by six perfect direct repeats 6 bp in length and shows no significant DNA similarity with any known nt sequence.

Cutaneous mycobacteriosis: analysis of 34 cases with a new classification of the disease.

Several points can be made from analysis of the published cases of cutaneous mycobacteriosis and those in our series: 1) mycobacterial cutaneous infections are probably more common than is reported-we collected 34 cases over a 10-year period; 2) most patients with cutaneous infections caused by nontuberculous mycobacteria have significant underlying disease; 3) there is a relative lack of classic histologic features in patients with cutaneous mycobacteriosis, and there appear to be diverse forms of clinical presentation; 4) a high index of suspicion is needed in evaluating patients with possible cutaneous mycobacteriosis, and appropriate cultures must be done to establish the diagnosis. In attempting to provide a practical classification of cutaneous mycobacteriosis which includes infection by nontuberculous mycobacteria, we propose the following grouping, which uses simple terms, avoids confusing nomenclature, and incorporates pathophysiologic descriptions and prognostic information: 1) Mycobacteriosis caused by inoculation from an exogenous source. 2) Cutaneous mycobacteriosis caused by spread from an endogenous source. Contiguous spread originates most often with osteomyelitis, but also occurs through autoinoculation of the perirectal, oral, or vaginal skin as organisms are passed or expectorated from pulmonary or genitourinary tuberculosis. 3) Cutaneous mycobacteriosis caused by hematogenous spread. This group includes lupus vulgaris, nodules and abscesses, and acute disease with hemorrhagic pustules. Some mycobacterioses will be difficult to classify when inoculation or hematogenous spread cannot be ruled out. However, the system of classification we have proposed should help clinicians understand and diagnose the diverse forms of cutaneous mycobacterial infections.

Demonstration of antigenemia in patients with invasive aspergillosis by solid phase (protein A-rich Staphylococcus aureus) radioimmunoassay.

A solid phase radioimmunoassay (RIA) was used to detect Aspergillus antigenemia in three patients, two with autopsy proved disseminated aspergillosis and one with a suspected infection. These RIA studies suggest that screening for antigenemia may be a specific and sensitive diagnostic test for disseminated aspergillosis.

Amphotericin B-resistant yeast infection in severely immunocompromised patients.

Systemic yeast infections are a major cause of morbidity and mortality in severely immunocompromised patients. The in vitro susceptibility to amphotericin B of 29 yeasts causing fungemia was examined in 26 patients undergoing allogeneic or autologous bone marrow transplantation and/or myelosuppressive chemotherapy. The minimal inhibitory concentrations (MICs) of amphotericin B observed with blood isolates from these patients were significantly higher than those observed with blood, sputum, or skin isolates from non-immunocompromised patients (p less than 0.01). All episodes (10 of 10) of bloodstream infection in immunocompromised patients caused by isolates with MICs greater than 0.8 micrograms/ml were fatal, versus eight of 17 episodes of bloodstream infection caused by yeasts with MICs of 0.8 micrograms/ml or less (p = 0.04). Although 15 of 26 patients received empiric treatment with amphotericin B before laboratory evidence of fungemia developed, the amphotericin B susceptibilities of their isolates were not significantly different from those of patients who had not received empiric amphotericin B treatment. It is concluded that yeast fungemia in severely immunocompromised patients is often caused by organisms resistant to the usual concentrations of amphotericin B obtainable in vivo, and that this finding is clinically significant.

Disseminated histoplasmosis presenting as an acute tenosynovitis.

Disseminated histoplasmosis is usually a multifocal process with a wide variety of clinical presentations. Despite frequent bone marrow involvement, overt bone and joint disease is uncommon and isolated synovial involvement is extremely rare. We describe in this report an unusual case of disseminated histoplasmosis presenting as acute tenosynovitis. To our knowledge, this is only the second reported case of synovial involvement by H. capsulatum without a concomitant osseous lesion.

Disseminated coccidioidomycosis in a patient with the acquired immune deficiency syndrome.

Disseminated coccidioidomycosis is a recognized but infrequent accompaniment of the acquired immune deficiency syndrome (AIDS). A patient with AIDS, Pneumocystis carinii pneumonia, and disseminated coccidioidomycosis occurring outside of an endemic area is described. Fungal infection presented atypically with progressive thoracic adenopathy and the development of cold soft tissue abscesses. As is often the case with AIDS, serologic testing proved to be unreliable and tissue biopsy the only means of accurate diagnosis.

Cutaneous protothecosis. Successful treatment with amphotericin B.

A patient had cutaneous protothecosis because of the alga-like organism, Prototheca wickerhamii. In vitro sensitivity tests showed that the organism was sensitive to amphotericin B, and was treated successfully with this polyene antibiotic. As with treatment of some fungal infections, a clinical response was achieved when therapy with low doses of amphotericin B was given during a short period of time. The basis of the amphotericin B response may have been due to a combination of its immunostimulatory and antibiotic properties.

Molecular genetics and the diagnostic mycology laboratory.

The laboratory diagnoses or confirmation of fungal diseases are extremely important considerations in the proper and efficient management of patients experiencing these infections. When an infectious process is suspected, suitable material must be collected and submitted to the pathologist for microscopic examination and to the diagnostic laboratory for culture. The practice of modern medicine dictates that the turn-around time be reduced to a minimum so that specific therapy can be rapidly instituted. Unfortunately, the classical procedures used in processing specimens for isolation and identification of fungi implicated in these diseases are technically time consuming and on occasion labor-intensive. More importantly, fungi are slow growing and cultures are frequently held for 3 to 4 weeks before they are discarded as negative. With the exception of coccidioidomycosis, most attempts to develop serological tests for rapid diagnosis of fungal infections have been hampered by poor specificity caused by immunologic cross reactivity. To some extent, such problems can be circumvented by application of molecular biological techniques to these problems. For example, procedures have been developed that allow efficient screening of DNA expression libraries to identify useful recombinant antigens and production of selected antigens in quantity. This is potentially a practical approach using molecular biological techniques for development of sensitive, specific and practical reagents useful in the serological diagnosis of fungal infections. In addition, nucleic acid hybridization techniques based on the ability of complementary nucleic acid strands to specifically align and associate to form stable double-stranded complexes have been put to use to develop confirmation tests for a variety of important fungal isolates.(ABSTRACT TRUNCATED AT 250 WORDS)

A temperature-sensitive strain of Histoplasma capsulatum has an altered delta 9-fatty acid desaturase gene.

We have isolated and characterized the delta 9-desaturase gene (Ole1), which codes for a key enzyme involved in regulating membrane fluidity in animal cells and microorganisms, from two strains of Histoplasma capsulatum, one that is temperature-tolerant (G217B) and the other temperature-susceptible (Downs). These pathogenic fungi are dimorphic in that they undergo a morphologic transition from the mycelial to yeast-like form when the temperature of incubation is switched from 25 to 37 degrees C or when they infect a susceptible host. The coding sequences of the two genes, both containing an intron of 93 nucleotides, are virtually identical and analogous to the delta 9-desaturase gene of Saccharomyces cerevisiae and those of the rat, mouse and human. Ole1 transcription of the thermotolerant G217B and thermosensitive Downs strains is similar in yeast phase cells and during the temperature shift down from 34, 37, or 40 to 25 degrees C (yeast-to-mycelia transition). Nevertheless, the delta 9-desaturase gene is transcriptionally inactive in mycelia of G217B at 25 degrees C while it is actively transcribed in the Downs strain at the same temperature. These results are in agreement with the finding that membranes of the Downs strain have a higher level of oleic acid. The differential expression of delta 9-desaturase genes is discussed in relationship to differences in thermosensitivity in the fungal isolates and in regulating the level of expression of heat shock genes.