Time between Inoculations and Karyotype Forms ofPneumocystis cariniif. sp.cariniiInfluence Outcome of Experimental Coinfections in Rats

Abstract

The prevalence of Pneumocystis carinii pneumonia (PCP) in humans caused by more than a single genotype has been reported to range from 10 to 67%, depending on the method used for detection (3, 19). Most coinfections were associated with primary rather than recurrent disease. To better understand the factors influencing the development of coinfections, the time periods between inoculations and the genotype of the infecting organisms were evaluated in the chronically immunosuppressed-inoculated rat model of PCP. P. carinii f. sp. carinii infecting rats differentiated by karyotypic profiles exhibit the same low level of genetic divergence manifested by organisms infecting humans. P. carinii f. sp. carinii karyotype forms 1, 2, and 6 were inoculated into immunosuppressed rats, individually and in dual combinations, spaced 0, 10, and 20 days apart. Infections comprised of both organism forms resulted from admixtures inoculated at the same time. In contrast, coinfections did not develop in most rats, where a 10-or 20-day gap was inserted between inoculations; only the first organism form inoculated was detected by pulsed-field gel electrophoresis in the resultant infection. Organism burdens were reduced with combinations of forms 1 and 2 spaced 20 days apart but not in rats inoculated with forms 1 and 6. A role for the host response in the elimination of the second population and in reduction of the organism burden was suggested by the lack of direct killing of forms 1 and 2 in an in vitro ATP assay, by reduction of the burden by autoclaved organisms, and by the specific reactions of forms 1 and 2 but not forms 1 and 6. These studies showed that the time between inoculations was critical in establishing coinfections and P. carinii f. sp. carinii karyotype profiles were associated with differences in biological responses. This model provides a useful method for the study of P. carinii coinfections and their transmission in humans.Organisms termed Pneumocystis carinii were placed in the Fungal kingdom more than 10 years ago on the basis of gene sequence comparisons (14, 29), yet much of their basic biological processes remains poorly understood, due in large part to their poor growth outside the mammalian lung environment (11). Recent genetic comparisons (32), as well as animal inoculation studies (16) and antigenic analyses (2, 15), provide strong evidence that "P. carinii" is actually a family of organisms that, although related, exhibit species specificity among its mammalian hosts.Comparisons at five genetic loci suggest three levels of genetic divergence are present among P. carinii organism populations (30). The highest level of divergence, class III, was observed among Pneumocystis populations isolated from different mammalian hosts and ranged from 5 to 50% divergence in nucleotide sequence at these selected loci, with the internal transcribed sequence (ITS) regions being most divergent. Class II divergence ranged from 4 to 7% for all genes, with a 20 to 30% difference at the ITS regions. Organisms demonstrating ...

Lung Epithelial Cells and Extracellular Matrix Components Induce Expression of Pneumocystis carinii STE20 , a Gene Complementing the Mating and Pseudohyphal Growth Defects of ste20 Mutant Yeast

“…Preparations containing detectable bacteria or other fungi were discarded. Nitrocellulose membranes with separated P. carinii chromosomes were provided by Melanie T. Cushion, University of Cincinnati (4). Saccharomyces cerevisiae strains L5366 (STE20ϩ wild type, MATa/␣ ura3-52/ura3-52 in the Sigma 1278b strain background), L5624 (MATa/␣ ste20:: TRP1/ste20::TRP1 ura3-52/ura3-52 trp1::hisG/trp1::hisG in the Sigma 1278b strain background), and JKY40 (MATa ste20::TRP1 ura3-52 leu2-3,112 his3⌬200 or his3-11,15 trp1⌬63 ade2 lys2::FUS1-lacZ in the S288c strain background) were used as described previously.…”

“…A 371-bp PCSTE20 probe was generated between bp 507 and 878 of PCSTE20. Southern hybridization was performed with digested P. carinii genomic DNA or whole P. carinii chromosomes separated by contour-clamped homogeneous electric field electrophoresis as described previously (4,21,22). To verify that PCSTE20 expression was induced upon P. carinii binding to collagen, RNA was isolated from P. carinii incubated for 2.0 h under the exact conditions used for subtractive hybridization.…”

“…4C to E). Thus, the enhanced expression of 4. Adherence of P. carinii to A549 alveolar epithelial cells specifically induces expression of PCSTE20 and PCMAPK, which participate in fungal mating pathways.…”

Rapid PCR–Single-Strand Conformation Polymorphism Method To Differentiate and Estimate Relative Abundance of Pneumocystis carinii Special Forms Infecting Rats

“…carinii and P. carinii f. sp. ratti are not uncommon (4,9) and constitute a useful model for coinfections, which also frequently occur in humans (3,11). Because these two special forms are genetically and antigenically distinct, it is important to determine if rats used for experimental studies harbor one or both forms, as well as to estimate the relative abundance of the forms in case of a coinfection.…”

“…DNA samples were prepared from organisms obtained from individual Sprague-Dawley or Long Evans rat lungs (n ϭ 20) and analyzed by contour-clamped homogeneous electric field (CHEF) analysis as described previously (3). CHEF analysis identified the special form(s) present in each rat and provided an estimation of the relative abundance of each special form in case of a coinfection.…”