Michael B. Reed

A glycolipid of hypervirulent tuberculosis strains that inhibits the innate immune response

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-α 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.

DnaE2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis.

The presence of multiple copies of the major replicative DNA polymerase (DnaE) in some organisms, including important pathogens and symbionts, has remained an unresolved enigma. We postulated that one copy might participate in error-prone DNA repair synthesis. We found that UV irradiation of Mycobacterium tuberculosis results in increased mutation frequency in the surviving fraction. We identified dnaE2 as a gene that is upregulated in vitro by several DNA damaging agents, as well as during infection of mice. Loss of this protein reduces both survival of the bacillus after UV irradiation and the virulence of the organism in mice. Our data suggest that DnaE2, and not a member of the Y family of error-prone DNA polymerases, is the primary mediator of survival through inducible mutagenesis and can contribute directly to the emergence of drug resistance in vivo. These results may indicate a potential new target for therapeutic intervention.

Contribution of the Mycobacterium tuberculosis MmpL Protein Family to Virulence and Drug Resistance

ABSTRACT The genome sequence of Mycobacterium tuberculosis revealed the presence of 12 membrane proteins proposed to have a function in the transport of lipids. Insertional inactivation of 11 of these has revealed that only 1 (MmpL3) is apparently essential for viability. Five of these proteins are conserved within the genome of Mycobacterium leprae. The drug susceptibilities of these 11 mutants to a broad spectrum of agents are unaltered, suggesting that unlike their function in other organisms, these proteins do not play a significant role in intrinsic drug resistance. Each of these mutants was assessed for growth kinetics and lethality in a murine low-dose aerosol model of tuberculosis, and four were found to be impaired in one or both measures of virulence. Two of these, with mutations of MmpL4 and the previously characterized MmpL7, which transports phthiocerol dimycocerosate, were found to have both impaired growth kinetics and impaired lethality. Mutants with inactivation of MmpL8, which transports a precursor of the sulfatides, or MmpL11, which transports an unknown substrate, were found to establish infection normally but to be significantly attenuated for lethality in time-to-death studies. These studies support the concept that MmpL-mediated lipid secretion both contributes to the innate ability of the pathogen to survive intracellularly and also contributes directly to the host-pathogen dialogue that determines the ultimate outcome of infection.

Differential Monocyte Activation Underlies Strain-Specific Mycobacterium tuberculosis Pathogenesis

ABSTRACT 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.

Virulence of Selected Mycobacterium tuberculosis Clinical Isolates in the Rabbit Model of Meningitis Is Dependent on Phenolic Glycolipid Produced by the Bacilli

Infection with Mycobacterium tuberculosis in humans results in active disease in approximately 10% of immune-competent individuals, with the most-severe clinical manifestations observed when the bacilli infect the central nervous system (CNS). Here, we use a rabbit model of tuberculous meningitis to evaluate the severity of disease caused by the M. tuberculosis clinical isolates CDC1551, a highly immunogenic strain, and HN878 or W4, 2 members of the W/Beijing family of strains. Compared with infection with CDC1551, CNS infection with HN878 or W4 resulted in higher bacillary loads in the cerebrospinal fluid and brain, increased dissemination of bacilli to other organs, persistent levels of tumor necrosis factor-alpha , higher leukocytosis, and more-severe clinical manifestations. This pathogenic process is associated with the production by HN878 of a polyketide synthase-derived phenolic glycolipid (PGL), as demonstrated by reduced virulence in rabbits infected with an HN878 mutant disrupted in the pks1-15 gene, which is required for PGL synthesis.

The W-beijing lineage of Mycobacterium tuberculosis overproduces triglycerides and has the DosR dormancy regulon constitutively upregulated

The Beijing family of Mycobacterium tuberculosis strains has been associated with epidemic spread and an increased likelihood of developing drug resistance. The characteristics that predispose this family to such clinical outcomes have not been identified, although one potential candidate, the phenolic glycolipid PGL-tb, has been shown to mediate a fulminant lethal disease in mice and rabbits due to lipid-mediated immunosuppression. However, PGL-tb is not uniformly expressed throughout the Beijing lineage and may not be the only unique virulence trait associated with this family. In an attempt to define phenotypes common to all Beijing strains, we interrogated a carefully selected set of isolates representing the five extant lineages of the Beijing family. Comparison of lipid production in this set revealed that all Beijing strains accumulated large quantities of triacylglycerides in in vitro aerobic culture. This accumulation was found to be coincident with upregulation of Rv3130c, whose product was previously characterized as a triacylglyceride synthase. Rv3130c is a member of the DosR-controlled regulon of M. tuberculosis, and further examination revealed that several members of this regulon were upregulated throughout this strain family. The upregulation of the DosR regulon may confer an adaptive advantage for growth in microaerophilic or anaerobic environments encountered by the bacillus during infection and thus may be related to the epidemiological phenomena associated with this important strain lineage.

Increased NOD2-mediated recognition of N-glycolyl muramyl dipeptide

Peptidoglycan-derived muramyl dipeptide (MDP) activates innate immunity via the host sensor NOD2. Although MDP is N-acetylated in most bacteria, mycobacteria and related Actinomycetes convert their MDP to an N-glycolylated form through the action of N-acetyl muramic acid hydroxylase (NamH). We used a combination of bacterial genetics and synthetic chemistry to investigate whether N-glycolylation of MDP alters NOD2-mediated immunity. Upon infecting macrophages with 12 bacteria, tumor necrosis factor (TNF) α secretion was NOD2 dependent only with mycobacteria and other Actinomycetes (Nocardia and Rhodococcus). Disruption of namH in Mycobacterium smegmatis obrogated NOD2-mediated TNF secretion, which could be restored upon gene complementation. In mouse macrophages, N-glycolyl MDP was more potent than N-acetyl MDP at activating RIP2, nuclear factor κB, c-Jun N-terminal kinase, and proinflammatory cytokine secretion. In mice challenged intraperitoneally with live or killed mycobacteria, NOD2-dependent immune responses depended on the presence of bacterial namH. Finally, N-glycolyl MDP was more efficacious than N-acetyl MDP at inducing ovalbumin-specific T cell immunity in a model of adjuvancy. Our findings indicate that N-glycolyl MDP has a greater NOD2-stimulating activity than N-acetyl MDP, consistent with the historical observation attributing exceptional immunogenic activity to the mycobacterial cell wall.

Major Mycobacterium tuberculosis Lineages Associate with Patient Country of Origin

ABSTRACT Over recent years, there has been an increasing acknowledgment of the diversity that exists among Mycobacterium tuberculosis clinical isolates. To facilitate comparative studies aimed at deciphering the relevance of this diversity to human disease, an unambiguous and easily interpretable method of strain classification is required. Presently, the most effective means of assigning isolates into a series of unambiguous lineages is the method of Gagneux et al. (S. Gagneux et al., Proc. Natl. Acad. Sci. USA 103:2869-2873, 2006) that involves the PCR-based detection of large sequence polymorphisms (LSPs). In this manner, isolates are classified into six major lineages, the majority of which display a high degree of geographic restriction. Here we describe an independent replicate of the Gagneux study carried out on 798 isolates collected over a 6-year period from mostly foreign-born patients resident on the island of Montreal, Canada. The original trends in terms of bacterial genotype and patient ethnicity are remarkably conserved within this Montreal cohort, even though the patient distributions between the two populations are quite distinct. In parallel with the LSP analysis, we also demonstrate that “clustered” tuberculosis (TB) cases defined through restriction fragment length polymorphism (RFLP) analysis (for isolates with ≥6 IS6110 copies) or RFLP in combination with spoligotyping (for isolates with <6 IS6110 copies) do not stray across the LSP-defined lineage boundaries. However, our data also demonstrate the poor discriminatory power of either RFLP or spoligotyping alone for these low-IS6110-copy-number isolates. We believe that this independent validation of the LSP method should encourage researchers to adopt this system in investigations aimed at elucidating the role of strain variation in TB.

In Vivo Phenotypic Dominance in Mouse Mixed Infections with Mycobacterium tuberculosis Clinical Isolates

Clinical isolates of Mycobacterium tuberculosis demonstrate significant heterogeneity in virulence potential in animal models of infection. Isolate CDC1551, for example, has previously been described in mouse survival studies as being hypovirulent, and isolate HN878 has been described as being hypervirulent. Observed differences in this mouse infection experiment have been proposed to reflect differential engagement of the host immune response. To assess whether this is a local or a systemic effect, C57BL/6 mice were infected simultaneously with mixtures of CDC1551 and HN878 in varying ratios and were monitored for mycobacterial growth kinetics, strain proportions during infection, and mouse survival. Strain mixtures that contained primarily HN878 grew more quickly during the first 5 weeks of infection and were more lethal for mice, and HN878 was enriched during in vivo growth. The absolute number of implanted HN878 bacilli at infection correlated inversely with mouse survival and was independent of concomitant infection with CDC1551. In infections of nonactivated mouse macrophages, HN878 grew more quickly. However, phagocyte preactivation reduced and equalized the growth rate of both strains. These results suggest that HN878 exerts a dominant immunosuppressive effect limited to the granuloma in which it is contained.

Massive Gene Duplication Event among Clinical Isolates of the Mycobacterium tuberculosis W/Beijing Family

As part of our effort to uncover the molecular basis for the phenotypic variation among clinical Mycobacterium tuberculosis isolates, we have previously reported that isolates belonging to the W/Beijing lineage constitutively overexpress the DosR-regulated transcriptional program. While generating dosR knockouts in two independent W/Beijing sublineages, we were surprised to discover that they possess two copies of dosR. This dosR amplification is part of a massive genomic duplication spanning 350 kb and encompassing >300 genes. In total, this equates to 8% of the genome being present as two copies. The presence of IS6110 elements at both ends of the region of duplication, and in the novel junction region, suggests that it arose through unequal homologous recombination of sister chromatids at the IS6110 sequences. Analysis of isolates representing the major M. tuberculosis lineages has revealed that the 350-kb duplication is restricted to the most recently evolved sublineages of the W/Beijing family. Within these isolates, the duplication is partly responsible for the constitutive dosR overexpression phenotype. Although the nature of the selection event giving rise to the duplication remains unresolved, its evolution is almost certainly the result of specific selective pressure(s) encountered inside the host. A preliminary in vitro screen has failed to reveal a role of the duplication in conferring resistance to common antitubercular drugs, a trait frequently associated with W/Beijing isolates. Nevertheless, this first description of a genetic remodeling event of this nature for M. tuberculosis further highlights the potential for the evolution of diversity in this important global pathogen.