pubmed.ncbi.nlm.nih.gov

Mechanisms of Surface Antigenic Variation in the Human Pathogenic Fungus Pneumocystis jirovecii - PubMed

  • ️Sun Jan 01 2017

Mechanisms of Surface Antigenic Variation in the Human Pathogenic Fungus Pneumocystis jirovecii

Emanuel Schmid-Siegert et al. mBio. 2017.

Abstract

Microbial pathogens commonly escape the human immune system by varying surface proteins. We investigated the mechanisms used for that purpose by Pneumocystis jirovecii This uncultivable fungus is an obligate pulmonary pathogen that in immunocompromised individuals causes pneumonia, a major life-threatening infection. Long-read PacBio sequencing was used to assemble a core of subtelomeres of a single P. jirovecii strain from a bronchoalveolar lavage fluid specimen from a single patient. A total of 113 genes encoding surface proteins were identified, including 28 pseudogenes. These genes formed a subtelomeric gene superfamily, which included five families encoding adhesive glycosylphosphatidylinositol (GPI)-anchored glycoproteins and one family encoding excreted glycoproteins. Numerical analyses suggested that diversification of the glycoproteins relies on mosaic genes created by ectopic recombination and occurs only within each family. DNA motifs suggested that all genes are expressed independently, except those of the family encoding the most abundant surface glycoproteins, which are subject to mutually exclusive expression. PCR analyses showed that exchange of the expressed gene of the latter family occurs frequently, possibly favored by the location of the genes proximal to the telomere because this allows concomitant telomere exchange. Our observations suggest that (i) the P. jirovecii cell surface is made of a complex mixture of different surface proteins, with a majority of a single isoform of the most abundant glycoprotein, (ii) genetic mosaicism within each family ensures variation of the glycoproteins, and (iii) the strategy of the fungus consists of the continuous production of new subpopulations composed of cells that are antigenically different.IMPORTANCEPneumocystis jirovecii is a fungus causing severe pneumonia in immunocompromised individuals. It is the second most frequent life-threatening invasive fungal infection. We have studied the mechanisms of antigenic variation used by this pathogen to escape the human immune system, a strategy commonly used by pathogenic microorganisms. Using a new DNA sequencing technology generating long reads, we could characterize the highly repetitive gene families encoding the proteins that are present on the cellular surface of this pest. These gene families are localized in the regions close to the ends of all chromosomes, the subtelomeres. Such chromosomal localization was found to favor genetic recombinations between members of each gene family and to allow diversification of these proteins continuously over time. This pathogen seems to use a strategy of antigenic variation consisting of the continuous production of new subpopulations composed of cells that are antigenically different. Such a strategy is unique among human pathogens.

Keywords: PCP; PacBio sequencing; Pneumocystis carinii; Pneumocystis jirovecii; adhesin; gene exchange; major surface glycoprotein; mosaicism; subtelomere; telomere exchange.

Copyright © 2017 Schmid-Siegert et al.

PubMed Disclaimer

Figures

FIG 1
FIG 1

Classification trees of P. jirovecii msg genes and Msg proteins. The different families are represented by color, and their characteristics are summarized in Table 1. A few unclassified outliers are in gray. The scale represents the number of mean substitutions per site. (a) RAxML DNA and PEP are maximum likelihood trees of nucleotide and amino acid sequences of the 61 genes with an exon larger than 1.6 kb. Members of family V were defined as the out-group (1,000 bootstraps). JACOP PEP is a hierarchical classification based on local sequence similarity, a method that does not rely on a particular multiple sequence alignment. (b) Maximum likelihood tree of the 61 genes with an exon larger than 1.6 kb plus 18 genes with an exon smaller than 1.6 kb. The sequences were trimmed from position 1540 of the first alignment up to their end and realigned to construct the tree (1,000 bootstraps). Seven of the 18 genes with an exon smaller than 1.6 kb constitute the msg family VI shown in brown, whereas the remaining 11 shown in black belong to the other msg families.

FIG 2
FIG 2

Diagrams of the structure of P. jirovecii msg genes and Msg proteins belonging to families I to VI. (a) Features of the msg genes of each family derived from the analysis of the full-length genes. The UCS and recombination between CRJE sequences are shown for family I. The approximate positions of PCR primers used for identification of the msg-I expressed genes linked to the UCS are shown by arrows (see note 4 in Text S1). (b) Features of Msg proteins of each family derived from the analyses of the full-length proteins. The 13 domains identified by MEME analysis are shown. The logos of these domains are shown in Fig. S4.

FIG 3
FIG 3

Diagrams of 10 representative P. jirovecii assembled subtelomeres. The other 27 assembled subtelomeres are shown in Fig. S6. The msg genes are described in detail in Table S1. The attribution of the contigs to the chromosomes previously described using flanking non-msg genes is described in Table S3 at

http://www.chuv.ch/microbiologie/en/imu_home/imu-recherche/imu-research-groups/imu-research-phauser/imu-supplementary_data.htm

. The number of subclones obtained from the generic PCR amplifying msg-I genes linked to the UCS is indicated close to the asterisk of one msg-I gene of contig 55 (see note 4 in Text S1). The functions/products of 4 of the 20 non-msg genes are known (see Table S3): gene 6, thiamine pyrophosphokinase; gene 9, amidophosphoribosyltransferase; gene 15, 60S ribosomal protein L28; and gene 16, potassium-sodium efflux P-type ATPase.

FIG 4
FIG 4

Examples of detection of potential mosaic genes. (a) Mosaic gene msg32. (a1) The set of 11 full-length msg-I genes was analyzed using the Recombination Analysis Tool. This method measures genetic distances in windows sliding along the MSA. The genetic distance scores of the putative parent genes at the middle of each window are plotted against the position in the mosaic gene. The predicted recombination site is at position ca. 600, at the crossover of the curves. The second screening method, Bellerophon, which is based on a similar analysis, identified a recombination event at position 392. (a2) Analysis of the mosaic gene msg32 with its putative parent genes together with the randomly chosen gene msg84 of the same family using the more sensitive method TOPALi, based on the hidden Markov model. This method analyzes only four sequences at a time and calculates the probabilities of the three possible tree topologies at each residue of the MSA. A recombination event is also detected at positions ca. 400 to 600, but many other recombination events are predicted. (b) Mosaic gene msg79. This gene shares an almost identical fragment of 947 bp with its putative parent, msg7 (see alignment in Fig. S8c). (b1) The set of 11 full-length msg-II genes was analyzed using the Recombination Analysis Tool. The predicted recombination sites are at positions ca. 400, 1300, 2100, and 3100. The Bellerophon method did not identify this mosaic gene. (b2) Analysis of the mosaic gene msg79 with its putative parent genes together with the randomly chosen gene msg85 of the same family using TOPALi based on the hidden Markov model. Recombination events are also detected at positions ca. 400, 1500, and 3100, but not at 2100, and other recombination events are predicted.

FIG 5
FIG 5

Telomere exchange model for swapping the msg-I expressed gene through a single recombination between CRJE sequences. One exchanged telomere is shown in red. Subpopulations of cells expressing a potentially new mosaic msg-I gene are generated over time and may then multiply. Polycistronic expression of two msg-I genes is shown in the second subpopulation generated (see the text).

Similar articles

Cited by

References

    1. Cushion MT, Smulian AG, Slaven BE, Sesterhenn T, Arnold J, Staben C, Porollo A, Adamczak R, Meller J. 2007. Transcriptome of Pneumocystis carinii during fulminate infection: carbohydrate metabolism and the concept of a compatible parasite. PLoS One 2:e423. doi:10.1371/journal.pone.0000423. - DOI - PMC - PubMed
    1. Cushion MT, Stringer JR. 2010. Stealth and opportunism: alternative lifestyles of species in the fungal genus Pneumocystis. Annu Rev Microbiol 64:431–452. doi:10.1146/annurev.micro.112408.134335. - DOI - PubMed
    1. Hauser PM. 2014. Genomic insights into the fungal pathogens of the genus Pneumocystis: obligate biotrophs of humans and other mammals. PLoS Pathog 10:e1004425. doi:10.1371/journal.ppat.1004425. - DOI - PMC - PubMed
    1. Ma L, Chen Z, Huang DW, Kutty G, Ishihara M, Wang H, Abouelleil A, Bishop L, Davey E, Deng R, Deng X, Fan L, Fantoni G, Fitzgerald M, Gogineni E, Goldberg JM, Handley G, Hu X, Huber C, Jiao X, Jones K, Levin JZ, Liu Y, Macdonald P, Melnikov A, Raley C, Sassi M, Sherman BT, Song X, Sykes S, Tran B, Walsh L, Xia Y, Yang J, Young S, Zeng Q, Zheng X, Stephens R, Nusbaum C, Birren BW, Azadi P, Lempicki RA, Cuomo CA, Kovacs JA. 2016. Genome analysis of three Pneumocystis species reveals adaptation mechanisms to life exclusively in mammalian hosts. Nat Commun 7:10740. doi:10.1038/ncomms10740. - DOI - PMC - PubMed
    1. Brown GD, Denning DW, Gow NAR, Levitz SM, Netea MG, White TC. 2012. Hidden killers: human fungal infections. Sci Transl Med 4:165rv13. doi:10.1126/scitranslmed.3004404. - DOI - PubMed

MeSH terms

Substances