Genetic and antigenic diversity in Pneumocystis jirovecii

Pneumocystis jirovecii is a human specific, atypical fungus with a worldwide distribution that causes disease in immunocompromised individuals. The fungus proliferates in the lungs where it binds to epithelial alveolar cells, provoking severe pneumonia, denoted pneumocystis pneumonia (PCP). As there is no in vitro culture system for the organisms, and no morphological means to differentiate between P. jirovecii strains, we have used molecular tools to study the fungus in patient samples. For this purpose we have been targeting different loci in the P. jirovecii genome (ITS, DHPS and MSG) to address different aspects of P. jirovecii infections.

The nucleotide sequence of the internal transcribed spacers (ITS) in P. jirovecii has been useful for isolate genotyping. We investigated the genetic diversity in Sweden by analyzing 408 cloned ITS sequences, from 64 clinical specimens. Several globally common haplotypes (combination of ITS1 and ITS2) and one local ITS2 were found. No correlations between certain haplotypes and patient characteristics or geographical associations were uncovered. In this context, a model describing the genealogic relationships of the strains was presented. During this process, we found that the typing system was generating artifactual sequences. We established a set of criteria to determine “bona fide” haplotypes, and optimized the typing method to avoid the generation of artifactual recombinants. These improved tools have enabled a more correct assessment of the overestimated genetic diversity of P. jirovecii populations.

Trimethoprim-sulfamethoxazole (TMP-SMX) is the most widely used drug for prevention and treatment of PCP. Non-synonymous substitutions in the dihydropteroate synthase (DHPS) gene of P. jirovecii have been found to be associated with sulpha exposure. It has been suggested that this is the result of the fungus developing resistance towards the drug. We conducted a study to investigate the presence of P. jirovecii DHPS mutations in the Swedish population and 104 specimens from patients with a suspected PCP were screened. All of the specimens (100%) showed a wild-type DHPS pattern, suggesting that there is no, or a very low prevalence of DHPS mutations in the country.

One surface molecule of P. jirovecii with a probable key function in the colonization of the alveoli and in immune evasion is the major surface glycoprotein (MSG). The MSGs are encoded by the msg-gene family, and transcription is limited to a single msg-gene located in a unique expression site. To investigate the expressed msg-genes and the extent of the variability of the MSG antigen, we analyzed msg-genes located at the expression site. First, we analyzed a short segment of the 5’-end of the msg-genes in 13 patient samples. Second, we extended these studies to two full-length msg-sequences from two different patients. We concluded, from these analyses, that there is considerable variation in the potentially expressed MSG-proteins, but that a substantial amount of conservation can be found in the msg-gene family, even in samples of unrelated origins.

In conclusion, the complexity of P. jirovecii populations has been overestimated but typing fidelity can easily be improved. The numbers of ITS haplotypes in Sweden are restricted, and a model depicting the relationships between strains is proposed. Furthermore, P. jirovecii DHPS mutations are very rare or possibly even absent in Sweden. Finally, the expressed msg-genes display both a remarkable variation and conservation.

List of scientific papers

I. Beser J, Hagblom P and Fernandez V. Frequent in vitro recombination in internal transcribed spacers 1 and 2 during genotyping of Pneumocystis jirovecii. J Clin Microbiol. 2007 Mar;45(3):881-6.
https://doi.org/10.1128/JCM.02245-06

II. Beser J, Botero-Kleiven S, Lebbad M, Hagblom P and Fernandez V. A limited number of ITS haplotypes defines the diversity of Pneumocystis jirovecii strains in Sweden. Infect Genet Evol. 2011 Jul;11(5):948-54.
https://doi.org/10.1016/j.meegid.2011.03.004

III. Beser J, Dini L, Botero-Kleiven S, Krabbe M, Lindh J and Hagblom P. Absence of dihydropteroate synthase gene mutations in Pneumocystis jirovecii isolated from Swedish patients. Med Mycol. 2011 Jul 6. [Epub ahead of print]
https://doi.org/10.3109/13693786.2011.593051

IV. Beser J, Joannin N, Botero-Kleiven S, Lindh J and Hagblom P. Variation of the expressed major surface glycoprotein in Pneumocystis jirovecii. [Manuscript]