Pneumocystis carinii is an opportunistic fungus causing severe pneumonia in immune-compromised hosts. Recent evidence suggests that Pneumocystis exists as separate sex types, though definitive evidence is currently lacking. These studies were undertaken to determine whether Pneumocystis maintains functional meiotic control molecules, which are required for sexual life stages in eukaryotes. Using the Pneumocystis carinii Genome Project database, two partial sequences for meiotic control molecules were detected, namely, PCRan1, a presumptive meiotic control kinase, and PCMei2, a homologue to a primary activator of meiosis in Schizosaccharomyces pombe. Rapid amplification of cDNA ends was employed to obtain the full open reading frames and to further investigate the functions of these proteins. These presumptive meiotic control molecules were most homologous to molecules present in S. pombe (52% identical and 67% homologous for PCRan1 and 75% identical and 88% homologous for PCMei2 by BLAST analysis). Heterologous expression of these Pneumocystis meiotic genes in corresponding temperaturesensitive and knockout strains of S. pombe, respectively, further verified the functions of the PCRan1 and PCMei2 proteins. These proteins were further shown to control downstream components of the meiotic pathway in S. pombe. Lastly, in vitro kinase assays were used to determine that PCRan1p phosphorylates PCMei2p. These experiments represent the first characterization of any proteins in P. carinii involved in meiosis and indicate the presence of a conserved meiotic pathway in Pneumocystis. Elucidation of this pathway will be essential in gaining a greater understanding of this important opportunistic fungal pathogen.

Background: Blastomycosis is a potentially fatal infection caused by the fungus Blastomyces dermatitidis.During January 1 through March 5, 2006, twenty-one laboratory confirmed cases of blastomycosis were reported among residents of an endemic area in north-central Wisconsin; a striking increase compared with previous years. The objective of the study was to determine if an observed increase in blastomycosis among residents of an urban area in north-central Wisconsin was caused by a pointsource exposure and to identify its source. Methods:We compared epidemiologic features, and signs and symptoms of B. dermatitidis infection among 46 historic (1999)(2000)(2001)(2002)(2003)(2004)(2005) and 21 possible outbreak case patients. In addition, a case-control study was conducted to compare risk factors of the outbreak case patients with those of 64 age, gender, and geographically-matched control subjects. We conducted site inspections, evaluated meteorological data, genetically compared outbreak and non-outbreak isolates, and attempted environmental detection of B. dermatitidis using polymerase chain reaction, in vitro isolation, and in vivo isolation by tail vein injection of mice. Results:The unusual risk profile of this outbreak included: residence within non-rural city limits with limited time spent outdoors and an equivalent gender ratio and young median age among case patients consistent with common source rather than unrelated exposures. Thirteen of fourteen outbreakassociated clinical isolates of B. dermatitidis clustered in the same genetic group by PCR-RFLP analysis. Inspections near the cluster center suggested a yard waste collection site as the probable exposure source. B. dermatitidis nucleic acid was detected in one of 19 environmental samples. Environmental and meteorological conditions and material management practices were identified that may have facilitated growth and dispersal of B. dermatitidis conidia near this residential area. Conclusions:Results of our investigation of this large non-rural outbreak of blastomycosis suggest bioaerosol hazards may exist near yard waste collection and composting facilities, especially where pine tree litter is present, in B. dermatitidis endemic areas.

An immunosuppressed heart transplant recipient developed Exophiala jeanselmei infection on the second toe. After unsuccessful treatment with different antifungal drugs, the infection responded to a high-dose regimen of oral terbinafine (an antifungal agent not yet approved in the United States for use against the dematiaceous fungi) and warm packs. This is, to our knowledge, the only known case of successful terbinafine treatment of E. jeanselmei infection.

Blastomyces dermatitidis is the dimorphic fungal agent of blastomycosis, a disease that primarily affects humans and dogs. The clinical appearance of this mycosis is well characterized, but there is still little known about its environmental niche, having been isolated from nature only 21 times. We have developed a PCR-based assay to detect B. dermatitidis from soil samples using primers specific to a portion of the promoter region of the BAD1 virulence gene. An internal standard control, pTJV2, was constructed to validate the results from soil samples. Amplification of this control indicated adequate removal of ambient soil inhibitors. The PCR detection limits for the control plasmid and B. dermatitidis genomic DNA were 0.1 and 500 femtograms, respectively. No PCR cross-reactivity was observed against bacteria, actinomycetes, and 13 other fungi that were genetically related or found in the same geographic areas. In spiked soil samples, this method was sensitive to 304 copies of pTJV2 DNA and 8,450 live B. dermatitidis yeast cells. Three of eight natural soil samples from a dog kennel near Lexington, KY in which dogs suffered from blastomycosis were positive using the described method, demonstrating its utility in detecting B. dermatitidis in its natural surroundings.

Eukaryotic cell proliferation and phenotype are highly regulated by contact-dependent mechanisms. We have previously shown that the binding and interaction of the opportunistic fungal pathogen Pneumocystis carinii to lung epithelial cells and extracellular matrix proteins induces mRNA expression of both the mitogen-activated protein (MAP) kinase P. carinii Ste20 (PcSte20) and the cell wall-remodeling enzyme PcCbk1 (16). Herein, we report that in addition to PcSte20 mRNA expression being upregulated, Pneumocystis PcSte20 kinase activity is increased upon interacting with these same lung targets. This activity is also significantly suppressed by Clostridium difficile toxin B, a pan-specific inhibitor of small GTPases, demonstrating the potential role of a Cdc42-like molecule in this signaling cascade. We further observed that the PcSte20 kinase physically interacts with a specific region of the P. carinii cell wall biosynthesis kinase, PcCbk1, a downstream kinase important for mating projection formation and cell wall remodeling. This direct binding was mapped to a specific region of the PcCbk1 protein. We also demonstrated that PcSte20 obtained from whole P. carinii lysates has the ability to phosphorylate PcCbk1 after the organism interacts with lung epithelial cells and extracellular matrix components. These observations provide new insights into P. carinii signaling induced by interactions of this important opportunistic fungal pathogen with lung epithelial cells and matrix.