Fifty million new infections with Mycobacterium tuberculosis occur annually, claiming 2-3 million lives from tuberculosis worldwide. Despite the apparent lack of significant genetic heterogeneity between strains of M. tuberculosis, there is mounting evidence that considerable heterogeneity exists in molecules important in disease pathogenesis. These differences may manifest in the ability of some isolates to modify the host cellular immune response, thereby contributing to the observed diversity of clinical outcomes. Here we describe the identification and functional relevance of a highly biologically active lipid species-a polyketide synthase-derived phenolic glycolipid (PGL) produced by a subset of M. tuberculosis isolates belonging to the W-Beijing family that show 'hyperlethality' in murine disease models. Disruption of PGL synthesis results in loss of this hypervirulent phenotype without significantly affecting bacterial load during disease. Loss of PGL was found to correlate with an increase in the release of the pro-inflammatory cytokines tumour-necrosis factor-alpha and interleukins 6 and 12 in vitro. Furthermore, the overproduction of PGL by M. tuberculosis or the addition of purified PGL to monocyte-derived macrophages was found to inhibit the release of these pro-inflammatory mediators in a dose-dependent manner.

To understand how virulent mycobacteria subvert host immunity and establish disease, we examined the differential response of mice to infection with various human outbreak Mycobacterium tuberculosis clinical isolates. One clinical isolate, HN878, was found to be hypervirulent, as demonstrated by unusually early death of infected immune-competent mice, compared with infection with other clinical isolates. The differential effect on survival required lymphocyte function because severe combined immunodeficiency (SCID) mice infected with HN878 or other clinical isolates all died at the same rate. The hypervirulence of HN878 was associated with failure to induce M. tuberculosis-specific proliferation and IFN-␥ production by spleen and lymph node cells from infected mice. In addition, 2-to 4-fold lower levels of tumor necrosis factor-␣ (TNF-␣), IL-6, IL-12, and IFN-␥ mRNAs were observed in lungs of HN878-infected mice. IL-10, IL-4, and IL-5 mRNA levels were not significantly elevated in lungs of HN878 infected mice. In contrast, IFN-␣ mRNA levels were significantly higher in lungs of these mice. To further investigate the role of Type 1 IFNs, mice infected with HN878 were treated intranasally with purified IFN-␣͞␤. The treatment resulted in increased lung bacillary loads and even further reduced survival. These results suggest that the hypervirulence of HN878 may be due to failure of this strain to stimulate Th1 type immunity. In addition, the lack of development of Th1 immunity in response to HN878 appears to be associated with increased induction of Type 1 IFNs.

The role of type I interferons (IFNs) in the host response to bacterial infections is controversial. Here, we examined the role of IFN-alpha/beta in the murine response to infection with Mycobacterium tuberculosis, using wildtype mice, mice with impaired signaling through the type I IFN receptor (IFNAR), and mice treated to reduce levels of type I IFNs. In this study, we used virulent clinical isolates of M. tuberculosis, including HN878, W4, and CDC1551. Our results indicate that higher levels of type I IFNs are induced by the HN878 and W4 strains. Induction of type I IFNs was associated with lower levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin- 12 (IL-12) and reduced T cell activation, and associated with decreased survival of the mice infected with HN878 or W4 relative to infection with CDC1551. Infection of mice with HN878 and W4 was also associated with relatively higher levels of mRNA for a number of negative regulators of the Jak-Stat signaling pathway, such as suppressors of cytokine signaling (SOCS) 1, 4, and 5, CD45, protein inhibitor of activated Stat1 (PIAS1), protein tyrosine phosphatase nonreceptor type 1 (Ptpn1), and protein tyrosine phosphatase nonreceptor type substrate 1 (Ptpns1). Taken together, these results suggest that increased type I IFNs may be deleterious for survival of M. tuberculosis-infected mice in association with reduced Th1 immunity.

In vitro infection of monocytes with Mycobacterium tuberculosis HN878 and related W/Beijing isolates preferentially induced interleukin-4 (IL-4) and IL-13, which characterize Th2 polarized immunity. In contrast, CDC1551 induced more IL-12 and other molecules associated with phagocyte activation and Th1 protective immunity. The differential cytokine-chemokine response was mediated by extracted lipids, suggesting that these molecules regulate host responses to infection.

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To study the role of MmpL8-mediated lipid transport in sulfatide biogenesis, we insertionally inactivated the mmpL8 gene in Mycobacterium tuberculosis. Characterization of this strain showed that the synthesis of mature sulfolipid SL-1 was interrupted and that a more polar sulfated molecule, termed SL-N, accumulated within the cell. Purification of SL-N and structural analysis identified this molecule as a family of 2,3-diacyl-␣,␣-D-trehalose-2-sulfates. This structure suggests that transport and biogenesis of SL-1 are coupled and that the final step in sulfatide biosynthesis may be the extracellular esterification of two trehalose 6-positions with hydroxyphthioceranic acids. To assess the effect of the loss of this anionic surface lipid on virulence, we infected mice via aerosol with the MmpL8 mutant and found that, although initial replication rates and containment levels were identical, compared with the wild type, a significant attenuation of the MmpL8 mutant strain in time-to-death was observed. Early in infection, differential expression of cytokines and cytokine receptors revealed that the mutant strain less efficiently suppresses key indicators of a Th1-type immune response, suggesting an immunomodulatory role for sulfatides in the pathogenesis of tuberculosis.

Mycobacterium tuberculosis possesses a diversity of potential virulence factors including complex branched lipids such as the phenolic glycolipid PGL-tb. PGL-tb expression by the clinical M. tuberculosis isolate HN878 has been associated with a less efficient Th1 response and increased virulence in mice and rabbits. It has been suggested that the W-Beijing family is the only group of M. tuberculosis strains with an intact pks1-15 gene, required for the synthesis of PGL-tb and capable of producing PGL-tb. We have found that some strains with an intact pks1-15 do not produce PGL-tb while others may produce a variant of PGL-tb. We examined the early host cytokine response to infection with these strains in vitro to better understand the effect of PGL-tb synthesis on immune responses. In addition, we generated a PGL-tb-producing H37Rv in order to determine the effect of PGL-tb production on the host immune response during infection by a strain normally devoid of PGL-tb synthesis. We observed that PGL-tb production by clinical M. tuberculosis isolates affected cytokine production differently depending on the background of the strain. Importantly, while ectopic PGL-tb production by H37Rv suppressed the induction of several pro-and anti-inflammatory cytokines in vitro in human monocytes, it did not lead to increased virulence in infected mice and rabbits. Collectively, our data indicate that, while PGL-tb may play a role in the immunogenicity and/or virulence of M. tuberculosis, it probably acts in concert with other bacterial factors which seem to be dependent on the background of the strain.

Mycobacterium tuberculosis has a relatively high resistance to killing by hydrogen peroxide and organic peroxides. Resistance may be mediated by mycobacterial catalase-peroxidase (KatG) and possibly by alkyl hydroperoxide reductase (AhpC). To determine the interrelationship between sensitivity to H2O2, catalase and peroxidase activities, and bacillary growth rates measured both intracellularly in human monocytes and in culture medium, we examined one laboratory strain, two clinical isolates, and three recombinant strains of M. tuberculosis with differing levels of KatG and AhpC. Five of the mycobacterial strains had intracellular doubling times of 27 to 32 h, while one KatG-deficient clinical isolate (ATCC 35825) doubled in ∼76 h. Killing of mycobacteria by exogenously added H2O2 was more pronounced for intracellular bacilli than for those bacilli derived from disrupted monocytes. Strains with no detectable KatG expression or catalase activity were relatively sensitive to killing (43 to 67% killing) by exogenous H2O2. However, once even minimal catalase activity was present, mycobacterial catalase activity over a 10-fold range (0.56 to 6.2 U/mg) was associated with survival of 85% of the bacilli. Peroxidase activity levels correlated significantly with resistance of the mycobacterial strains to H2O2-mediated killing. An endogenous oxidative burst induction by 4β-phorbol 12β-myristate 13α-acetate treatment of infected monocytes reduced the viability of the KatG null strain (H37Rv Inhr) but not the KatG-overexpressing strain [H37Rv(pMH59)]. These results suggest that mycobacterial resistance to oxidative metabolites (including H2O2 and other peroxides) may be an important mechanism of bacillary survival within the host phagocyte.