Autophagy Gene Variant IRGM −261T Contributes to Protection from Tuberculosis Caused by Mycobacterium tuberculosis but Not by M. africanum Strains

The human immunity-related GTPase M (IRGM) has been shown to be critically involved in regulating autophagy as a means of disposing cytosolic cellular structures and of reducing the growth of intracellular pathogens in vitro. This includes Mycobacterium tuberculosis, which is in agreement with findings indicating that M. tuberculosis translocates from the phagolysosome into the cytosol of infected cells, where it becomes exposed to autophagy. To test whether IRGM plays a role in human infection, we studied IRGM gene variants in 2010 patients with pulmonary tuberculosis (TB) and 2346 unaffected controls. Mycobacterial clades were classified by spoligotyping, IS6110 fingerprinting and genotyping of the pks1/15 deletion. The IRGM genotype −261TT was negatively associated with TB caused by M. tuberculosis (OR 0.66, CI 0.52–0.84, Pnominal 0.0009, Pcorrected 0.0045) and not with TB caused by M. africanum or M. bovis (OR 0.95, CI 0.70–1.30. P 0.8). Further stratification for mycobacterial clades revealed that the protective effect applied only to M. tuberculosis strains with a damaged pks1/15 gene which is characteristic for the Euro-American (EUAM) subgroup of M. tuberculosis (OR 0.63, CI 0.49–0.81, Pnominal 0.0004, Pcorrected 0.0019). Our results, including those of luciferase reporter gene assays with the IRGM variants −261C and −261T, suggest a role for IRGM and autophagy in protection of humans against natural infection with M. tuberculosis EUAM clades. Moreover, they support in vitro findings indicating that TB lineages capable of producing a distinct mycobacterial phenolic glycolipid that occurs exclusively in strains with an intact pks1/15 gene inhibit innate immune responses in which IRGM contributes to the control of autophagy. Finally, they raise the possibility that the increased frequency of the IRGM −261TT genotype may have contributed to the establishment of M. africanum as a pathogen in the West African population

Variant G57E of Mannose Binding Lectin Associated with Protection against Tuberculosis Caused by Mycobacterium africanum but not by M. tuberculosis

Structural variants of the Mannose Binding Lectin (MBL) cause quantitative and qualitative functional deficiencies, which are associated with various patterns of susceptibility to infectious diseases and other disorders. We determined genetic MBL variants in 2010 Ghanaian patients with pulmonary tuberculosis (TB) and 2346 controls and characterized the mycobacterial isolates of the patients. Assuming a recessive mode of inheritance, we found a protective association between TB and the MBL2 G57E variant (odds ratio 0.60, confidence interval 0.4–0.9, P 0.008) and the corresponding LYQC haplotype (Pcorrected 0.007) which applied, however, only to TB caused by M. africanum but not to TB caused by M. tuberculosis. In vitro, M. africanum isolates bound recombinant human MBL more efficiently than did isolates of M. tuberculosis. We conclude that MBL binding may facilitate the uptake of M. africanum by macrophages, thereby promoting infection and that selection by TB may have favoured the spread of functional MBL deficiencies in regions endemic for M. africanum

Contents

This publication was made possible through support provided by the U.S. Agency for International Development under the terms of Contract No. GPO-C-00-03-00002-00. The opinions expressed herein are those of the authors and do not necessarily reflect the views of the U.S. Agency for International Development

CTLA4 Autoimmunity-Associated Genotype Contributes to Severe Pulmonary Tuberculosis in an African Population

The gene of Cytotoxic T Lymphocyte-associated Antigen 4 (CTLA4), a negative regulator of T lymphocytes, contains a singlenucleotide polymorphism (SNP) at position +6230A-.G (ct60A-.G), which has been found associated with several autoimmune diseases and appears to reduce T-cell inhibitory activity. In Ghana, West Africa, we compared the frequencies of CTLA4 +6230 A/G and 6 haplotype-tagging SNPs in 2010 smear-positive, HIV-negative patients with pulmonary tuberculosis (TB) and 2346 controls matched for age, gender and ethnicity. We found no difference in allele frequencies between cases and controls. However, +6230A and a distinct CTLA4 haplotype and a diplotype comprising the +6230A allele were significantly less frequent among cases with large opacities in chest radiographs compared to those with small ones (Pcorrected [cor] = 0.002, Pcor = 0.00045, P = 0.0005, respectively). This finding suggests that an increased T-cell activit

<i>CTLA4</i> haplotype frequencies in TB cases stratified for the sizes of opacities and cavities in chest radiographs¹.

1<p>Seven tagging SNPs reported to appropriately indicate <i>CTLA4</i> haplotypes in sub-Saharan Africans (Ref. 13) analysed by the Haplo.score software assuming a recessive mode of inheritance.</p>2<p>P values obtained testing an overall association between haplotypes and the phenotype.</p>3<p>Global P values corrected for multiple testing by 20,000 permutations.</p>4<p>Frequency; only haplotypes with a frequency>0.01 are shown.</p>5<p>P values of haplotype-specific associations.</p>6<p>Simulated haplotype-specific P values corrected for multiple testing by 20,000 permutations.</p>7<p>Score statistic of the haplo.score software.</p

Pairwise linkage disequilibrium (r²) based on 9 <i>CTLA4</i> SNPs using HaploView 4.0 software.

<p>The figure depicts the strong LD of variants −1577/+6230 and of variants −1147/−1661. The positions relative to the ATG start codon of SNPs are given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006307#pone-0006307-t002" target="_blank">Table 2</a>.</p

Primers and sensor/anchor oligonucleotides used for <i>CTLA4</i> genotyping¹.

1<p>Performed by dynamic allele specific hybridisation with fluorescence resonance energy transfer (FRET) in a LightTyper©.</p>2<p>F, forward primer; R, reverse primer.</p>3<p>S, sensor; A, anch.</p

Frequencies and distributions of SNPs studied¹.

1<p>The χ2 test was applied to test for deviations from Hardy-Weinberg equilibrium.</p>2<p>Minor allele frequency.</p>3<p>P value for a deviation from Hardy-Weinberg equilibrium.</p