Phthiocerol dimycocerosate transport is required for resisting interferon-gamma-independent immunity

Abstract

Nitric oxide (NO), which is an important component of immunity to Mycobacterium tuberculosis, has both cytotoxic and immune regulatory functions. We examined the way that this molecule interacts with M. tuberculosis in vivo by screening for bacterial mutations that alter growth in mice that are unable to produce inducible NO synthase (iNOS), the dominant source of NO during infection. We found that very few bacterial genes appeared to be specifically required for resistance to NO in vivo. Instead, mutations in several virulence factors caused greater attenuation in the absence of iNOS. Among these were mutants incapable of transporting the lipid phthiocerol dimycocerosate (PDIM). Although PDIM has been implicated in NO defense, this result indicates that PDIM has other roles during infection. We additionally found that PDIM transport is required for virulence in mice lacking interferon-gamma . Thus, PDIM is important for resisting an interferon-gamma-independent mechanism of immunity.

Figure 1

Comparison of growth in inducible nitric oxide synthase (iNOS)^−/− mice relative to wild-type (WT) mice and growth in WT mice relative to growth in vitro. Predicted WT growth rates are represented on the x axis as the ratio of transposon site hybridization (TraSH) probe from WT mice divided by probe from an in vitro pool [21]. The vertical dashed line represents the 0.4× cutoff value, which defines attenuating mutations. Predicted iNOS^−/− growth rates are represented on the y axis as TraSH ratios (4-week iNOS^−/− probe/4-week WT probe). The horizontal dashed lines represent values of 2.5× or 0.4×, which are the cutoff values for genes that are over or underrepresented in iNOS^−/− mice, respectively. Those genes which are significantly attenuated in WT/in vitro growth (in vivo/in vitro ratio <0.4; P < .05 by Student’s t test) and significantly 2.5× above or below 1 in the iNOS^−/−/WT experiment (P < .05 by Student’s t test) are indicated by ×. Genes required for phthiocerol dimycocerosate transport (drrABC, mmpL7, and lppX) are shown in green. Genes clearly required for esx1 secretion (Rv3614-Rv3616, Rv3868-Rv3871, esxA, esxB, and Rv3877) are shown in red [50].

Figure 2

Pooled growth of defined strains in wild-type (WT) and inducible nitric oxide synthase (iNOS)^−/− mice. Strains with defined mutations were mixed at roughly equal ratios. C57BL/6J (WT; circles) and iNOS^−/− mice (triangles) were injected intravenously with 10⁶ colony-forming units of the pooled strains. Mice were sacrificed at 1 day (closed symbols) and 28 days (open symbols) after infection, and surviving bacteria were recovered from spleens. Genomic DNA was extracted from pooled bacteria from each mouse, and triplicate quantitative real-time polymerase chain reactions using strain-specific primers were used to quantify the abundance of the indicated strains relative to the total pool, as assessed by sigA levels. Symbols show data from individual mice, and lines indicate mean values. Strains predicted by transposon site hybridization to replicate better (A) or worse (B) in iNOS^−/− relative to WT mice are shown next to a marked version of WT M. tuberculosis (Rv + JEB). Statistically significant differences between the log ratio of mutant to sigA levels after 4 weeks of infection in WT mice, compared with that after 4 weeks of infection in iNOS^−/− mice, were determined by 2-tailed, unpaired Student’s t test (*P < .005; **P < .001).

Figure 3

Mycobacterium tuberculosis strains lacking functional drrA have altered levels of phthiocerol (methoxy) and phthiodiolone (keto) dimycocerosate. A, Model of phthiocerol dimycocerosate (PDIM), showing phthiocerol (methoxy) and phthiodiolone (keto) forms [51]. B, Cells were labeled with [3-¹⁴C] propionic acid for 16 h, and crude lipid extracts were separated by thin-layer chromotography. Lane 1, wild-type H37Rv; lane 2, drrA::Tn.2; lane 3, drrA::Tn.2+JEBdrrA; lane 4, wild-type H37Rv; lane 5, drrA::Tn.1; lane 6, drrA::Tn.1+JEBdrrA.

Figure 4

Growth of the drrA mutant strain in murine macrophages. A, RAW 264.7 macrophages were infected at a multiplicity of infection of 1 with wild-type H37Rv (solid lines, closed squares) mixed with drrA::Tn.1 (dashed lines, open triangles) at a ratio of 6:1. Cells were lysed at 4 h (day 0), 3 days, and 5 days after infection. Colony-forming units (CFU) were determined for each strain. Data is represented as fold change in CFU from day 0; results are given as mean values (± standard deviation) for 6 replicates per condition. B, Macrophages were activated with interferon-γ (IFN-γ) beginning 24 h before infection and continuing throughout the course of the experiment. Infections were performed as described above. C, Macrophages were activated with IFN-γ and treated with the inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine beginning 24 h before infection and throughout the course of the experiment. Infections were performed as described above. D, Results from unactivated macrophages (solid lines, solid squares), IFN-γ–activated macrophages (dashed lines, solid circles), and IFN-γ–activated macrophages treated with aminoguanidine (dotted lines, open triangles) are represented as a ratio of drrA::Tn.1/wild-type H37Rv.

Figure 5

Growth of the drrA mutant strain in wild-type and interferon (IFN)-γ^−/− mice. A, Wild-type C57BL/6J and IFN-γ^−/− mice were injected intravenously with 10⁶ colony-forming units (CFU) of wild-type H37Rv (solid lines, solid squares) mixed with drrA::Tn.1 (dotted lines, open triangles) and drrA::Tn.1+JEBdrrA (solid lines, open circles) at a ratio of 9:5:1. Spleens and lungs were harvested at 1 and 21 days, and CFU were determined for each strain by plating on selective media. Because the drrA::Tn.1+JEBdrrA strain contained both antibiotic resistance markers, it was used at lower levels than were the other 2 strains. This ensured that the levels of the drrA::Tn.1+JEBdrrA strain would not dramatically affect the number of CFU on agar containing kanamycin (used to determine the levels of the drrA::Tn.1 strain) or agar without antibiotics (used to determine the levels of the wild-type strain). Results are given as the mean values (± standard deviation) for 3–6 mice per group. B, Results from A are represented as a ratio of drrA::Tn.1/drrA::Tn.1+JEBdrrA (or mutant/complemented strain) for day 1 (solid circles) and day 21 (open circles). Lines indicate the mean values. Statistically significant differences between the log ratio of drrA::Tn.1/drrA::Tn.1+JEBdrrA at days 1 and 21 were determined by 2-tailed, unpaired Student’s t test (*P < .05; **P < .005). Compl., complemented strain.