Molecular Studies Of Human and Rodent Pneumocystis

Clinical Center

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Abstract

Our studies focus on improving the management of a lethal lung infection, Pneumocystis pneumonia. We are studying ways to improve the diagnosis, treatment, and prevention of this disease. We have developed and evaluated two typing techniques for human Pneumocystis. The first uses tandem repeats that occur in an intron of the major surface glycoprotein (Msg) gene, as we have previously reported. The second utilizes variation in the Msg gene family. By sequencing multiple Msg genes in a number of human and rodent Pneumocystis isolates, we have been able to demonstrate that the Msg repertoire in human Pneumocystis is very diverse, while the Msg repertoire in rat and mouse Pneumocystis is identical or very limited among different isolates. Using restriction fragment length polymorphism (RFLP) analysis, we have been able to demonstrate substantial diversity among endemic cases of PCP, that most or all isolates from individual outbreaks of PCP in renal transplant patients appear to be the same strain, indicating that recent infection is important, and that either host- to-host transmission has occurred or that all individuals were infected from a common source. We subsequently showed that the same isolate was responsible for an outbreak of PCP in renal transplant patients in Zurich, Switzerland, and Munich, Germany, though there was no easily identified epidemiologic link connecting the 2 sites. We have subsequently analyzed samples from an outbreak in Denmark and were able to show that 3 unique strains were responsible for this outbreak. We have applied next generation sequencing (NGS) methods to further understand the epidemiology of Pneumocystis infection. In a collaboration with investigators from Yale School of Medicine, we performed a molecular analysis related to an outbreak of PCP in their renal transplant center, and were able to demonstrate shared genotypes among patients, as well as evidence for infection with multiple strains in many patients, with up to 7 in one patient. We serve as a resource to the medical community when apparent outbreaks of PCP are identified. Most recently we have shown that multiple outbreaks in renal transplant patients are caused by Pneumocystis strains with mutations in the inosine monophosphate dehydrogenase (IMPDH) gene. IMPDH is targeted by mycophenolate, a drug used for immunosuppression to minimize graft rejection. Pneumocystis in patients receiving this drug incidentally develop mutations that confer resistance to mycophenolate, which appears to facilitate transmission to other transplant recipients. These studies help to better understand the epidemiology of Pneumocystis infection, and to identify methods to minimize transmission. In an effort spearheaded by Liang Ma and Ousmane Cisse (staff scientists) we utilized NGS sequencing to generate high quality sequence data that has permitted development of nearly complete draft genomes of 7 Pneumocystis species, including P. jirovecii, which infects humans. All genomes have lost ~40% of genes compared to other fungi, with only minor differences in these losses. Synteny mapping shows substantial chromosomal rearrangements, which may contribute to speciation. Our studies suggest that the hypothesis of co-evolution of Pneumocystis with its mammalian host species is inadequate to explain Pneumocystis speciation, with hybridization and host jumps likely contributing. Two distinct Pneumocystis species can infect dogs, as well as certain other host species. Pneumocystis has developed mechanisms to evade host innate immune responses, including inability to synthesize or degrade chitin, and inability to synthesize high mannose residues on proteins. Pneumocystis has also lost many other functions that highlight the probability that Pneumocystis lives almost exclusively in its hosts lung environment. We have recently characterized the centromeres of mouse and rat Pneumocystis using ChipSeq techniques. Centromeres in each species were regional rather than point centromeres, and spanned 4.8 to 8.0 kb. Each centromere sequence was unique to the genome, with no conserved DNA motif. Centromeres tend to have lower GC content than the rest of the genome, are not associated with repeats, and contain active genes, which is unusual for centromeres in other species. While the centromeres on each chromosome are unique, there are orthologous regions in both species, suggesting a common origin. Centromeres are more conserved than background genomic regions, suggesting positive selection. We are also examining Pneumocystis infection in a broad range of non-laboratory animals, including wild rats and mice, to have a better understanding of the diversity of strains infecting a given host species, as well as to further evaluate the current concept that there is strict mammalian host species specificity for each Pneumocystis species. Because of the lower organism load in these immunocompetent hosts, initial focus is on mitochondrial genes/genomes of Pneumocystis since there are multiple mitochondria per Pneumocystis organism, and the mitochondrial genome is relative small and thus easier to sequence in its entirety. We are examining genetic variation in isolates of Pneumocystis obtained from a broad range of sources to better understand the level of variability that can be seen in genes other than the Msg gene family, and to better understand the evolution of Pneumocystis species over time. Analysis of multiple isolates has suggested that strict co-evolution of Pneumocystis with its host species, which is the current prevailing hypothesis, is inadequate to explain the evolution of Pneumocystis species; bottlenecks are present in the evolutionary history of rat, mouse, and human Pneumocystis, and host jumps also appear to play a role. Our studies further suggest that the Msg superfamily plays a critical role in determining the very restricted host species specificity of Pneumocystis species. Given the large amount of the genome that each Pneumocystis species has devoted to the Msg superfamily, we undertook to better characterize the different subfamilies. This analysis has demonstrated that while the classic Msg family predominates in all species examined to date, there appear to be substantial differences in the number of variants of this subfamily in different Pneumocystis species, and further, that there are expansions of different subfamilies among different Pneumocystis species. These data suggest that the Msg subfamilies may be playing an important role in the adaptation of Pneumocystis to novel hosts. We are collaborating with structural biologists and electron microscopists at NIAID to help study Msg as well of other Pneumocystis proteins of potential biological relevance. These studies will hopefully lead to a better understanding of the biology of Pneumocystis and identify potential therapeutic targets.