Expression and complexity of the PRT1 multigene family of Pneumocystis carinii

Abstract

Pneumocystis carinii has a multigene family, PRT1, that encodes proteins with homology to KEX2-like proteases. PRT1 genes cluster with MSG genes near the telomeres and, like MSG, PRT1 proteins seem to be surface-expressed. The clustering of PRT1 and MSG genes suggested that expression of the two multigene families might be coordinated. Studying gene expression in P. carinii has been hampered by the lack of a culture system, and by lack of clonality in P. carinii populations in naturally infected rats, the host of this fungus. Heterogeneity can be reduced, however, by low-dose intratracheal inoculation, which can produce P. carinii populations dominated by organisms derived from a single progenitor. To study PRT1 expression, nude rats were inoculated with approximately 10 P. carinii each. The clonality of the P. carinii populations from inoculated rats was assessed by analysis of the UCS locus, a site in the genome that is known to be very heterogeneous in naturally infected rats, but nearly homogeneous in rats infected by low-dose intratracheal inoculation. Each of the populations had the same MSG gene at the UCS locus in at least 80 % of the organisms. To investigate PRT1 gene expression, RNA was amplified using primers that amplify numerous PRT1 genes. Seventy-four cloned cDNAs were sequenced, including at least 12 clones from each population of P. carinii. Many differently expressed PRT1 sequences were identified in each population, and a total of 45 different sequences were detected. However, the same PRT1 sequence was present in 15 of 74 plasmids and was found in 3 of the 5 P. carinii populations, suggesting that some PRT1 genes may be either more commonly expressed or expressed at a higher level. These data show that many members of the PRT1 gene family can be expressed in populations of P. carinii derived from few progenitors and suggest that the regulation of this family is different from that governing expression of the MSG gene family.

Differential Macrophage Polarization from Pneumocystis in Immunocompetent and Immunosuppressed Hosts: Potential Adjunctive Therapy during Pneumonia

“…These samples were washed, and RNA was isolated as described. Quantitative PCR was performed to enumerate the P. carinii using the Bio-Rad iCycler with primers to the P. carinii prt1 multigene (spanning P. carinii prot1 and prot3) (61). Amplifications of unknown samples were compared to standard samples containing rat P. carinii prt1 RNA, and the resulting measure of RNA copies was proportioned to the net P. carinii burden (62).…”

Pneumocystis murinaInfection and Cigarette Smoke Exposure Interact To Cause Increased Organism Burden, Development of Airspace Enlargement, and Pulmonary Inflammation in Mice

“…This immune response, either through a direct injury or indirectly via activation of a protease cascade, may have led to the physiologic and morphological changes that we measured. It is also possible that there is a direct effect of the organism itself (e.g., activation of a protease cascade by a P. murina-specific protease [1,3,26,41]). These data strongly support a recent report examining primates infected with simian immunodeficiency virus after prolonged colonization with P. macaca (36).…”

Hypobaric hypoxia-related impairment of pulmonary surfactant proteins A and D did not favourPneumocystis cariniiFrenkel 1999 growth in non-immunocompromised rats

“…Other experimental models were used to address this question. In a transgenic SP-D-overexpression mouse model, which developed PcP after antibody-induced CD4 + -cell depletion and inoculation of P. murina Keely et al 2004, it was observed that both P. murina infection and lung inflammatory changes were increased (Vuk-Pavlovic et al, 2006). With regard with SP-A, in the pulmonary lavage fluid of SP-A deficient mice with corticosteroid-induced PcP were observed higher concentrations of TNF-α, IFN-γ and MIP-2 than in wild-type mice (Linke et al, 2005).…”

“…The rats of all groups, except of group 2, were infected by non-surgical endotracheal route with P. carinii organisms (Ambrose et al, 2004) obtained from corticosteroid-treated Wistar rats . Parasite extraction from tissue lung and purification were performed as previously (Aliouat et al, 1993).…”