Molecular Studies Of Human and Rodent Pneumocystis

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Abstract

We have developed and evaluated two typing techniques for human Pneumocystis. The first uses tandem repeats that occur in an intron of the 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 Pneumocystis isolates, we have been able to demonstrate that recombination occurs in human Pneumocystis. These results led to studies that demonstrated 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. Based on these studies, we have developed a restriction fragment length polymorphism (RFLP) typing assay for human Pneumocystis, and have been able to demonstrate substantial diversity among human isolates. Using RFLP analysis, we have been able to demonstrate 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. To address this further, we examined samples from an outbreak of PCP in Japan. We found that while a single organism was responsible for the outbreak there as well, it was different from the European strain based on RFLP analysis. We have subsequently analyzed samples from an outbreak in Denmark and were able to show that 3 unique strains were responsible for this outbreak. In addition to RFLP, we are using multi-locus sequence typing (MLST). MLST is the most commonly used method for typing Pneumocystis in other laboratories, and this will allow us to compare the results of these two typing methods. We are also exploring sequencing larger regions of the Pneumocystis genome to be able to better examine the phylogenetic relationships among different isolates. In addition, we have recently applied next generation sequencing (NGS) methods to further understand the epidemiology of Pneumocystis infection. Multiple studies, including from our group, have shown that most infections are caused by multiple strains of Pneumocystis, with up to 7 or more strains present in a single infection. NGS of even short regions can identify polymorphisms that result from infection with multiple strains. In a collaboration with investigators from Yale School of Medicine, we performed the 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. We serve as a resource to the medical community when apparent outbreaks of PCP are identified: we periodically get requests from clinicians to examine clinical isolates of Pneumocystis and will perform RFLP analysis or MLST to help inform the clinical team on whether the isolates appear to be the same strain, suggesting an outbreak had occurred. These studies should help to better understand the epidemiology of Pneumocystis infection. In collaboration with investigators at the Broad Institute and Leidos Biomedical Research, Inc., we sequenced the genome of Pneumocystis species from different hosts. The major difficulty with this project was obtaining DNA of sufficient purity to allow next generation sequencing, since the organism cannot be cultured. In an effort spearheaded by Liang Ma (staff scientist) we were able to purify Pneumocystis DNA from infected rat, mouse, and human lungs sufficiently to allow NGS sequencing to generate high quality sequence data that has permitted development of nearly complete draft genomes of all 3 Pneumocystis species (P. carinii, P. murina, P. jirovecii). RNA sequencing in parallel has allowed identification of P. murina and P. carinii transcripts and characterization of the genes encoded by this organism. Through analysis of these data, we have been able to demonstrate that all 3 Pneumocystis species have a highly condensed genome compared to other fungi, and that 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 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 bottlenecks are present in the evolution of rat, mouse, and human Pneumocystis, and that host jumps may have occurred, rather than co- evolution of Pneumocystis with its host species, which is the current prevailing hypothesis. In efforts being led by Liang Ma and Ousmane Cisse, we have undertaking to sequence the genomes of other Pneumocystis species, including those infecting dogs, macaques, pigs and rabbits, as well as a second species that infects rats (P. wakefieldiae). The availability of additional genomes, especially primate genomes, will provide a more robust dataset to examine the evolution of Pneumocystis species and potentially will provide insights into the genes that play a role in determining the very restricted host species specificity of Pneumocystis species. 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. This involves requesting samples from a variety of investigators world-wide, which Liang Ma has been accomplishing very successfully. Because of the lower organism load in these immunocompetent hosts, initial focus is on mitochondrial genes/genomes of Pneumocystis since there are multiple mictochondria per Pneumocystis organism, and the mitochondrial genome is relative small and thus easier to sequence in its entirety. Availability of these genome should allow us to better understand the biology of this family of organisms, should potentially allow identification of metabolic pathways that need to be complemented to successfully culture the organism, and should facilitate the identification of important antigens and pathogenic factors. We tried to replicate a published method for culturing human Pneumocystis but were unable to do so. 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.