Limper, Andrew H.

ABSTRACT Cell wall β-glucans are highly conserved structural components of fungi that potently trigger inflammatory responses in an infected host. Identification of molecular mechanisms responsible for internalization and signaling of fungal β-glucans should enhance our understanding of innate immune responses to fungi. In this study, we demonstrated that internalization of fungal β-glucan particles requires actin polymerization but not participation of components of caveolar uptake mechanisms. Using fluorescence microscopy, we observed that uptake of 5-([4,6-dichlorotriazin-2-yl] amino)-fluorescein hydrochloride-Celite complex-labeled Saccharomyces cerevisiae β-glucan by RAW macrophages was substantially reduced in the presence of cytochalasin D, which antagonizes actin-mediated internalization pathways, but not by treatment with nystatin, which blocks caveolar uptake. Interestingly, β-glucan-induced NF-κB translocation, which is necessary for inflammatory activation, and tumor necrosis factor alpha production were both normal in the presence of cytochalasin D, despite defective internalization of β-glucan particles following actin disruption. Dectin-1, a major β-glucan receptor on macrophages, colocalized to phagocytic cups on macrophages and exhibited tyrosine phosphorylation after challenge with β-glucan particles. Dectin-1 localization and other membrane markers were not affected by treatment with cytochalasin D. Furthermore, dectin-1 receptors rather than Toll-like receptor 2 receptors were shown to be necessary for both efficient internalization of β-glucan particles and cytokine release in response to the fungal cell wall component.

Abstract Exuberant inflammatory responses are associated with respiratory failure during Pneumocystis pneumonia. Alveolar epithelial cells (AECs) promote Pneumocystis attachment and proliferation, but also contribute prominently to host cytokine-mediated inflammation during pneumonia. Recent investigations indicate that AECs produce macrophage inflammatory protein-2 (MIP-2) and tumor necrosis factor-α (TNF-α) following challenge with Pneumocystis carinii. Nuclear factor-κB (NF-κB) is a ubiquitous transcription factor critical for regulation of proinflammatory cytokine expression. Herein, we assess rat AEC NF-κB responses to challenge with a P. carinii β-glucan cell wall component (PCBG). Prominent nuclear translocation of p65 NF-κB was demonstrated following PCBG challenge. NF-κB activation was in part mediated through Protein Kinase C (PKC) signaling pathways. PCBG challenge of AECs was also shown to induce MIP-2 and TNF-α mRNA production, a response that was ameliorated by NF-κB inhibition. MIP-2 protein expression was also dramatically increased by PCBG challenge, in a manner that was significantly attenuated by both PKC and NF-κB inhibition. The data further demonstrate that AEC chemokine responses were not mediated by the recently described dectin-1 receptor, but instead involved participation of cell surface lactosylceramide. These data support a significant role for AECs in host responses during Pneumocystis pneumonia, and further indicate that β-glucan induces inflammatory cytokine production through NF-κB–dependent mechanisms.

ABSTRACT Pneumocystis species remain an important cause of life-threatening pneumonia in immunocompromised hosts, including those with AIDS. Responses of the organism to environmental cues both within the lung and elsewhere have been poorly defined. Herein, we report the identification of a cell wall biosynthesis kinase gene ( CBK1 ) homologue in Pneumocystis carinii , isolated by differential display PCR, that is expressed optimally at physiological pH (7 to 8) as opposed to more acidic environments. Expression of Pneumocystis CBK1 was also induced by contact with lung epithelial cells and extracellular matrix. Translation of this gene revealed extensive homology to other fungal CBK1 kinases. Pneumocystis CBK1 expression was equal in the cyst and trophic life forms of the organisms. We further demonstrate that Pneumocystis CBK1 expressed in cbk1Δ Saccharomyces cerevisiae cells restored defective cell wall separation during proliferation. Consistent with this, Pneumocystis CBK1 expression also stimulated transcription of the CTS1 chitinase in cbk1Δ mutant yeast cells, an event necessary for cell wall separation. In addition, Pneumocystis CBK1 cDNA supported normal mating projection formation in response to α-factor in the cbk1Δ yeast cells. Site-directed mutations of serine-303 and threonine-494, potential regulatory phosphorylation sites in Pneumocystis CBK1, abolished mating projection formation, indicating a role for these amino acid residues in CBK1 activity. These findings indicate that Pneumocystis CBK1 is an environmentally responsive gene that may function in signaling pathways necessary for cell growth and mating.

ABSTRACT Pneumocystis carinii causes severe pneumonia in immunocompromised hosts. The binding of P. carinii to alveolar epithelial cells and extracellular matrix constituents such as fibronectin and vitronectin is a central feature of infection, which initiates proliferation of the organism. Herein, we demonstrate that P. carinii binding to lung cells specifically alters the gene expression of the organism, regulating fungal growth. Subtractive hybridization was performed to isolate P. carinii genes expressed following binding to mammalian extracellular matrix constituents. P. carinii STE20 ( PCSTE20 ), a gene participating in mating and pseudohyphal growth of other fungi, was identified following adherence to the extracellular matrix constituents fibronectin, vitronectin, collagen, and lung epithelial cells. The expression of PCSTE20 and a related P. carinii mitogen-activated protein kinase (MAPK) kinase gene, also implicated in signaling of mating, were both specifically upregulated by binding to matrix protein. The expression of general cyclin-dependent kinases and other MAPKs not involved in mating pathways were not altered by organism binding. PCSTE20 expression was also strongly enhanced following organism attachment to A549 lung epithelial cells. When expressed in a Saccharomyces cerevisiae ste20Δ mutant, PCSTE20 suppressed defects in both mating and pseudohyphal growth. These findings are consistent with the observed proliferation and filopodial extension of Pneumocystis organisms adherent to the epithelium in the lungs of immunocompromised hosts. PCSTE20 expression appears to represent a significant component in the regulation of the life cycle of this intractable opportunistic pathogen.

ABSTRACT Fluorescein isothiocyanate-labeled monoclonal antibodies specific for fungal melanin were used in this study to visualize melanin-like components of the Pneumocystis carinii cell wall. A colorimetric enzyme assay confirmed these findings. This is the first report of melanin-like pigments in Pneumocystis .

ABSTRACTPneumocystis cariniiremains an important and potentially fatal cause of opportunistic pneumonia. Animal studies reveal that substantial quantities of surfactant protein D (SP-D) accumulate in the airspaces duringP. cariniipneumonia and are particularly abundant in aggregates of organisms. Due to the multimeric structure of SP-D, we hypothesized that SP-D mediates aggregation of the organism. From previous clinical studies it is known that aggregated organisms are conspicuous in sections of lung tissue and bronchoalveolar lavage (BAL) fluids of humans with activeP. cariniipneumonia. Herein, we observe that SP-D levels increased at least fourfold in BAL fluids of patients withP. cariniipneumonia. Next, a spectrophotometric sedimentation assay was developed to assess the aggregation ofP. cariniiin vitro by SP-D.P. cariniiorganisms were first stripped with glutathione to remove bound SP-D and subsequently incubated in the presence of SP-D and 2 mM calcium.P. cariniiincubated with natural SP-D (10 μg/ml) containing dodecamers and higher-order forms exhibited aggregation and enhanced sedimentation compared to that of glutathione-strippedP. carinii. Aggregation was also enhanced by the concentrated supernatant of rat BAL fluid, and this effect was abolished by the selective removal of SP-D from the lavage fluid.P. cariniiaggregation was reduced by maltose, mannose, and EDTA, consistent with the role of the SP-D C-type lectin domain (CRD) in the aggregation event. Comparisons of different molecular forms of SP-D showed that dodecamers—but not trimeric subunits—mediate optimal aggregation ofP. carinii. Aggregation ofP. cariniiby SP-D was shown to be responsible for the impaired phagocytosis of the organisms by alveolar macrophages. Thus, SP-D-mediated aggregation ofP. cariniimay represent one means by which the organism avoids elimination by the host.

Abstract Pneumocystis carinii (PC) causes severe pneumonia in immunocompromised patients. PC is intrinsically resistant to treatment with azole antifungal medications. The enzyme lanosterol 14α-demethylase (Erg11) is the target for azole antifungals. We cloned PCERG11 and compared its sequence to Erg11 proteins present in azole-resistant organisms, and performed chromosomal and Northern blot analysis for PCERG11. Of 13 potential sites which could confer resistance to azoles, two were identical to azole-resistant Candida. By site-directed mutagenesis we changed these two sites in PCERG11 to those present in azole-sensitive Candida to generate PCERG11-SDM (E113D, T125K). We tested the susceptibility of ERG11 deletion strains of Saccharomyces cerevisiae (SC) expressing PCERG11, PCERG11-SDM, and wild-type SCERG11 to three azole antifungals: fluconazole, itraconazole, and voriconazole. PCERG11 required a 2.2-fold higher dose of voriconazole and 3.5-fold higher dose of fluconazole than SCERG11 for a 50% reduction in growth. No difference was observed in the sensitivity to itraconazole. PCERG11-SDM has increased sensitivity to fluconazole and voriconazole, but not itraconazole. We believe that the molecular structure of the lanosterol 14α-demethylase encoded by PCERG11 confers inherent resistance to azole antifungals and plays an integral part in the overall resistance of this PC to azole therapy.

ABSTRACT We developed a rapid thermocycling, real-time detection (also known as real-time PCR) method for the detection of Legionella species directly from clinical specimens. This method uses the LightCycler (Roche Molecular Biochemicals, Indianapolis, Ind.) and requires approximately 1 to 2 h to perform. Both a Legionella genus PCR assay and Legionella pneumophila species-specific PCR assay were designed. A total of 43 archived specimens from 35 patients were evaluated, including 19 bronchoalveolar lavage (BAL) specimens and 24 formalin-fixed, paraffin-embedded open lung biopsy specimens. Twenty-five of the specimens were culture-positive for Legionella (9 BAL specimens and 16 tissue specimens). BAL specimens were tested by LightCycler PCR (LC-PCR) methods and by a direct fluorescent antibody (DFA) assay, which detects L. pneumophila serogroups 1 to 6 and several other Legionella species. Tissue sections were tested by the two LC-PCR methods, by DFA, by an in situ hybridization (ISH) assay, specifically designed to detect L. pneumophila , and by Warthin-Starry (WS) staining. The results were compared to the “gold standard” method of bacterial culture. With BAL specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by Legionella genus LC-PCR, 100 and 100%; Legionella genus detection by DFA assay, 33 and 100%; and L. pneumophila detection by L. pneumophila species-specific LC-PCR, 100 and 100%. With open lung biopsy specimens the following assays yielded the indicated sensitivities and specificities, respectively: Legionella genus detection by LC-PCR 68.8 and 100%; Legionella genus detection by DFA assay, 44 and 100%; Legionella genus detection by WS staining, 63 and 100%; L. pneumophila species-specific detection by LC-PCR, 17 and 100%; and L. pneumophila species-specific detection by ISH, 100 and 100%. The analytical sensitivity of both LC-PCR assays was <10 CFU/reaction. LC-PCR is a reliable method for the direct detection of Legionella species from BAL specimens. The Legionella genus LC-PCR assay could be performed initially; if positive, L. pneumophila species-specific LC-PCR could then be performed (if species differentiation is desired). The speed with which the LC-PCR procedure can be performed offers significant advantages over both culture-based methods and conventional PCR techniques. In contrast, for the methods evaluated, culture was the best for detecting multiple Legionella species in lung tissue. WS staining, Legionella genus LC-PCR, and L. pneumophila species-specific ISH were useful as rapid tests with lung tissue.

ABSTRACTPneumocystis cariniiremains an important opportunistic fungal pathogen causing life-threatening pneumonia in patients with AIDS and malignancy. Currently, little is known about how the organism adapts to environmental stresses and maintains its cellular integrity. We recently discovered an open reading frame approximately 600 bp downstream of the region codingGSC-1, a gene mediating β-glucan cell wall synthesis inP. carinii. The predicted amino acid sequence of this new gene, termedP. carinii PHR1,exhibited 38% homology toSaccharomyces cerevisiae GAS1, a glycosylphosphatidylinositol-anchored protein essential to maintaining cell wall integrity, and 37% homology toCandida albicans PHR1/PHR2, pH-responsive genes encoding proteins recently implicated in cross-linking β-1,3- and β-1,6-glucans. In view of its homology to these related fungal genes, the pH-dependent expression ofP. carinii PHR1was examined. As inC. albicans,P. carinii PHR1expression was repressed under acidic conditions but induced at neutral and more alkaline pH.PHR1-related proteins have been implicated in glucan cell wall stability under various environmental conditions. Although difficulties withP. cariniiculture and transformation have traditionally limited assessment of gene function in the organism itself, we have successfully used heterologous expression ofP. cariniigenes in related fungi to address functional correlates ofP. carinii-encoded proteins. Therefore, the potential role ofP. carinii PHR1in cell wall integrity was examined by assessing its ability to rescue anS. cerevisiae gas1mutant with absent endogenous Phr1p-like activity. Interestingly,P. carinii PHR1DNA successfully restored proliferation ofS. cerevisiae gas1mutants under lethal conditions of cell wall stress. These results indicate thatP. carinii PHR1encodes a protein responsive to environmental pH and capable of mediating fungal cell wall integrity.