Polymorphic clusters of pathogen resistance genes (Jonathan Howard)
Clustered genes may arise as natural selection brings together individual components of advantageous haplotypes, but they also arise through the elementary chromosome dynamics of gene duplication and cluster expansion. Once in place, gene clustering provides novel opportunities for the rapid generation of sequence variants by cis and trans gene conversion-like processes. Many such diversified and polymorphic gene clusters are associated with resistance against microbial pathogens, presumably because the dynamics of host-pathogen interactions favour rapid genetic diversification. The effects of these processes are plain to see in R-gene clusters in plants and in the Major Histocompatibility Complex in vertebrates. Our group is interested in the analysis of such clusters, especially in the mouse. The motive in basic science is to understand the functional basis of gene divergence and polymorphism in such systems. A second, more general technical issue, however, is to develop fast and labour-saving bioinformatic methods of resolving clustered genes in re-sequenced genomes. Most such gene clusters remain unresolved at present because the short sequence reads associated with NGS prevent linear arrays from being assembled correctly.
Our work is presently focused on the clusters of genes encoding the large, interferon-inducible GTPases of the IRG family in the mouse genome. IRG proteins mediate resistance in mice against a phylogenetically incoherent group of intracellular pathogens that enter cells by non-phagocytic mechanisms. At present we are aware of one protozoal pathogen, Toxoplasma gondii (Fig. 1), two closely related bacterial pathogens, Chlamydia trachomatis and Chlamydia psittaci, and one microsporidian fungus, Encephalitozoon cuniculi, that are resisted by the IRG mechanism.
Toxoplasma gondii in the cytoplasm of a mouse fibroblast induced with interferon-g. The parasitophorous vacuole membrane is being attacked by IRG proteins and is ruffled and damaged.
In the case of T. gondii, the IRG resistance mechanism of the mouse is confronted by an array of polymorphic virulence mechanisms. Best understood is a family of kinases and pseudokinases, members of which form active complexes that target the IRG resistance proteins and inactivate them by phosphorylation of specific threonine residues. Likewise, the IRG family is extremely polymorphic in wild mice, and certain haplotypes are highly resistant to T. gondii strains that are highly virulent in other mouse hosts. It now seems almost certain that resistance and virulence are parameters whose definition is only possible when both partners of an individual infection are considered. Thus a mouse strain highly resistant to a T. gondii strain known to be highly virulent in a second host mouse, may be susceptible to a second T. gondii strain, and this strain may prove avirulent in a third host mouse. We are interested in examining the reality of this scenario in the wild setting, and are collecting wild mice from a number of localities. Our analysis involves sequencing the IRG transcriptomes of multiple wild mice. All wild mice examined so far are heterozygous at one or more of the three clusters of IRG genes and the polymorphism of some IRG genes is at least as great as members of the MHC family, both in the number of alleles and in the complexity of the sequence polymorphism (Fig 2).
Figure 2: The clusters of IRG genes on mouse chromosomes 11 and 18 from laboratory and wild mice. Each coloured block represents one coding unit of an IRG protein, and different colours indicate deviations from the canonical sequence of the C57BL/6 mouse. The numbers of amino acid substitutions relative to C57BL/6 are shown in white numerals on each coloured block. The first group, of inbred laboratory strains, are markedly similar to one another, especially on chromosome 11, as a result of a dramatic selective sweep that must have occurred during the early domestication of the mouse (unpublished). The wild-derived and wild haplotypes are extremely diverse in terms of protein sequence and number. To date, no two wild mice have the same IRG protein complement.
To complement this genetic analysis we are also beginning to isolate wild strains of intracellular parasites, including T. gondii, that can be recovered from the tissues of mice captured in the wild. Our current effort is to correlate the alleles of genes of the IRG system with the genotypes of parasites strains tolerated by the host. The goal of the project is to identify the parameters that maintain such a complex polymorphic system in the wild.
The rapid analysis of IRG genes in multiple transcriptomes has been made possible by a number of small tricks. In essence the problem is easy because the genes we are looking at, albeit highly variable, belong to a homologous series. As the number of sequences accumulates, so the precision of the search improves. Jingtao Li (who publishes under the name Jingtao Lilue) is the bioinformaticist in the group. His experience with the IRG gene clusters has led him to an extended interest in other polymorphic gene clusters and he is now pioneering new tactics for the correct assembly of these complex arrays from genomic resequencing data.
Publications (since 2012)
Gazzinelli RT, Mendonça-Neto R, Lilue J, Howard J, Sher A.(2014) Innate resistance against Toxoplasma gondii: an evolutionary tale of mice, cats, and men. Cell Host Microbe.15(2):132-8. doi: 10.1016/j.chom.01.004. PMID: 24528860
Lilue, J, Mueller, UB, Steinfeldt,T, Howard, JC (2013) Toxoplasma gondii and the mouse; reciprocal virulence and resistance polymorphism. eLIFE 10.7554/eLife.01298
Springer HM, Schramm M, Taylor GA, Howard JC. (2013). Irgm1 (LRG-47), a Regulator of Cell-Autonomous Immunity, Does Not Localize to Mycobacterial or Listerial Phagosomes in IFN-γ-Induced Mouse Cells. J Immunol 191(4):1765-74 doi: 10.4049/jimmunol.1300641.
Spekker K, Leineweber M, Degrandi D, Ince V, Brunder S, Schmidt SK, Stuhlsatz S, Howard JC, Schares G, Degistirici O, Meisel R, Sorg RV, Seissler J, Hemphill A, Pfeffer K, Däubener W. (2012) Antimicrobial effects of murine mesenchymal stromal cells directed against Toxoplasma gondii and Neospora caninum: role of immunity-related GTPases (IRGs) and guanylate-binding proteins (GBPs). Med Microbiol Immunol. 202(3):197-206. doi: 10.1007/s00430-012-0281-y.PMID: 23269418
Fentress SJ, Steinfeldt T, Howard JC, Sibley LD. (2012) The arginine-rich N-terminal domain of ROP18 is necessary for vacuole targeting and virulence of Toxoplasma gondii. Cell Microbiol. 2012 Dec;14(12):1921-33. doi: 10.1111/cmi.12022. PMID: 22906355
Fleckenstein, M, Reese, ML, Boothroyd, JC, Howard, JC, Steinfeldt, T. (2012) A Toxoplasma gondii pseudokinase inhibits host IRG resistance proteins. PloS Biology. PLoS Biol 10(7): e1001358. doi:10.1371/journal.pbio.1001358
Reid, AJ, Vermont, SJ, Cotton, JA, Harris, D, Hill-Cawthorne, GA, Könen-Waisman, S, Latham, S, Mourier, T, Norton, R, Quail, M, Sanders, M, Shanmugam, D, Sohal, A, Wasmuth, J, Brunk, B, Grigg, M, Howard, JC, Parkinson, J, Roos, DS, Trees, AJ, Berriman, M, Pain, A & Wastling, JM (2012) Comparative genomics of Coccidian parasites differing in host range and transmission strategy. PLoS Pathogens (3): e1002567. doi:10.1371/journal.ppat.1002567