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Subtractive Hybridization Analysis of Pneumocystis carinii Gene Activation Induced by Interaction With Lung Epithelial Cells and Matrix*

Theodore J. Kottom, MS; Andrew H. Limper, MD, FCCP
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*From the Mayo Clinic, Rochester, MN.

Correspondence to: Andrew H. Limper, MD, FCCP, 601-C Guggenheim Building, Mayo Clinic, Rochester, MN 55905; e-mail: limper.andrew@mayo.edu



Chest. 2002;121(3_suppl):78S-79S. doi:10.1378/chest.121.3_suppl.78S-a
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Pneumocystis carinii (PC) remains an important cause of life-threatening pneumonia in patients with AIDS and malignancies. The binding of PC to alveolar epithelial cells is an essential component in the establishment of infection. PC attachment to lung epithelial cells is associated with the outgrowth of organism extensions into the host and the subsequent proliferation of the organism. The mechanisms by which PC senses contact with host cells and becomes activated to complete its life cycle remain poorly defined. To address PC gene activation following adherence to mammalian surfaces, we employed a global genetic approach to identify the altered gene expression that potentially is involved in this process. Subtractive hybridization was used to isolate PC genes induced on PC binding to collagen, a substrate that supports the limited short-term proliferation of PC in the absence of host cells. Using this technique, we identified and cloned both a proposed fungal guanosine triphosphate-binding signaling protein (PCgtp1) and a PCste20 gene similar to Candida and Saccharomyces genes mediating pseudohyphal outgrowth and mating in those species. The expression of both PCste20 and PCgtp1 genes were specifically up-regulated by binding to collagen 1, fibronectin, and vitronectin, but not by interaction with uncoated or bovine serum albumin-coated surfaces. In addition, both genes exhibited significant expression following binding to alveolar epithelial cells (A549s) compared to plastic surfaces. Host-induced up-regulation of both PCste20 and PCgtp1 gene expression was limited to trophic forms of PC, with minimally altered expression in cysts. This is consistent with ultrastructural studies showing active adherence of PC trophic forms but not cysts to the lung epithelium. Using Ste20-deficient strains of Saccharomyces cerevisiae, we next heterologously expressed the PCste20 gene and demonstrated its activity in initiating mating and pseudohyphal growth. These studies indicate that PC trophic forms exhibit specific gene activation following the interaction with mammalian surfaces and alveolar cells causing the extension of pseudohyphae. Furthermore, this global genetic screen has provided the first functional evidence of a mating pathway being initiated by the interaction of the organisms with the host that results in PC proliferation. These studies further support the underlying tenet that the interaction of PC with the host epithelial cells is an integral component of the organism’s life cycle.

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