The p47 GTPases Igtp and Irgb10 map to the Chlamydia trachomatis susceptibility locus Ctrq-3 and mediate cellular resistance in mice

Abstract

Infections caused by the bacteria Chlamydia trachomatis contribute to diverse pathologies in a variety of human populations. We previously used a systemic model of C. trachomatis infection in mice to map three quantitative trait loci that influence in vivo susceptibility differences between the C57BL/6J and C3H/HeJ inbred strains of mouse. One of these quantitative trait loci, Ctrq-3, influences an IFN-gamma-dependent susceptibility difference in primary embryonic fibroblasts isolated from these strains. Here we use fine structure mapping in congenic fibroblasts carrying DNA from the susceptible parent to localize the effect of Ctrq-3 to a 1.2-megabase interval of genomic DNA that contains Irgb10 and Igtp, two members of the IFN-gamma-inducible p47 family of GTPases. This class of proteins has been widely implicated in resistance to intracellular pathogens in mice. We analyzed expression of Irgb10 and Igtp in parental and congenic embryonic fibroblasts treated with IFN-gamma and found that relatively resistant fibroblasts express more Irgb10 than relatively susceptible fibroblasts. However, we also found that abolishing the expression of either Irgb10 or Igtp increases susceptibility of embryonic fibroblasts to C. trachomatis. Thus, we conclude that, although a difference in Irgb10 expression is likely responsible for the effect of Ctrq-3 on susceptibility to C. trachomatis, both genes play a role in intracellular resistance to C. trachomatis.

Fig. 1.

Fine mapping of Ctrq-3. (A) A schematic showing breakpoints for various B6.C3H congenics. The curve at the top is a depiction of the linkage peak for Ctrq-3, including the shaded ±1.5 logarithm of odds support interval for this QTL (see ref. 14). Filled bars in the lower portion indicate C3H donor DNA, and open bars indicate recipient B6 DNA. Where breakpoints are not precisely mapped, the congenic interval giving the most conservative (i.e., largest) genetic interval is shown. Also, the congenic intervals have not been mapped beyond D11Mit20 proximally or D11Mit5 distally. The scale bar (in the upper right corner) corresponds to 10 megabases on the schematic of the chromosome. (B) The Chlamydia susceptibility phenotypes of the corresponding B6.C3H MEF lines. Indicated MEFs were pretreated in culture with IFN-γ and then infected with C. trachomatis L2 for 28 h before harvest. Each bar represents the mean of four different embryos. Bars with asterisks are significantly different from bars without asterisks (P < 0.05).

Fig. 2.

Physical map of Ctrq-3. The schematic shows the position and orientation of genes in the critical genetic interval for Ctrq-3 between D11Mit164 and D11Zbh12 on chromosome 11. All information was obtained from Build 36.1 of the mouse genome at www.ncbi.nlm.nih.gov/genome/guide/mouse. Regions of alternating black and white correspond to 50-kb intervals on the chromosome. The centromeric/proximal end of the interval is to the left, and the telomeric/distal end is to the right.

Fig. 3.

Protein sequence of Irgb10. A conceptual translation of B6 Irgb10 cDNA is shown. Coding polymorphisms between B6 and C3H are in bold, with the C3H residue indicated below the corresponding B6 residue. The highly conserved G1 (GXXXXG[K/M]S), G3 (DXXG), and G4 (TXXD) domains are underlined.

Fig. 4.

Expression of Irgb10 and Igtp after IFN-γ treatment. MEFs from indicated mouse strains were treated for 15 h with 10 units/ml IFN-γ and then harvested to test for mRNA expression of either Igtp (filled bars) or Irgb10 (striped bars). Data are expressed relative to B6. Each bar represents the mean of three separate experiments. Asterisks indicate that expression of the gene differs from the 9L line at P < 0.05.

Fig. 5.

Susceptibility of MEFs that are manipulated for Irgb10 and Igtp expression. (A) Effect of overexpressing alleles of Irgb10 or Igtp on susceptibility to C. trachomatis in MEFs. MEFs from the 2B congenic line were transduced with indicated vector and infected for 28 h with C. trachomatis L2 after pretreatment with IFN-γ. Bars with asterisks are significantly different from control (P < 0.05). (B) Effect of RNAi-mediated inhibition of Irgb10 expression on susceptibility in MEFs. B6 MEFs were transduced with indicated vector, pretreated with IFN-γ, and infected for 28 h with C. trachomatis L2; means are significantly different at P < 0.05. (C) Susceptibility of MEFs deleted for Igtp. Indicated MEFs were pretreated with IFN-γ and infected for 28 h with C. trachomatis L2; means for Igtp^+/− and Igtp^−/− are significantly different at P < 0.05. In each panel, bars represent the mean of at least three data points.