Pilar Domenech

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.

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.

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.

Regulation of catalase–peroxidase (KatG) expression, isoniazid sensitivity and virulence by furA of Mycobacterium tuberculosis

Mycobacterium tuberculosis has two genes for ferric uptake regulator orthologues, one of which, furA, is situated immediately upstream of katG encoding catalase–peroxidase, a major virulence factor that also activates the prodrug isoniazid. This association suggested that furA might regulate katG and other genes involved in pathogenesis. Transcript mapping showed katG to be expressed from a strong promoter, with consensus −10 and −35 elements, preceding furA. No promoter activity was demonstrated downstream of the furA start codon, using different gene reporter systems, indicating that furA and katG are co‐transcribed from a common regulatory region. The respective roles of these two genes in the isoniazid susceptibility and virulence of M. tuberculosis were assessed by combinatorial complementation of a Δ(furA–katG) strain that is heavily attenuated in a mouse model of tuberculosis. In the absence of furA, katG was upregulated, cells became hypersensitive to isoniazid, and full virulence was restored, indicating that furA regulates the transcription of both genes. When furA alone was introduced into the Δ(furA–katG) mutant, survival in mouse lungs was moderately increased, suggesting that FurA could regulate genes, other than katG, that are involved in pathogenesis. These do not include the oxidative stress genes ahpC and sodA, or those for siderophore production.

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.

BacA, an ABC Transporter Involved in Maintenance of Chronic Murine Infections with Mycobacterium tuberculosis

BacA is an inner membrane protein associated with maintenance of chronic infections in several diverse host-pathogen interactions. To understand the function of the bacA gene in Mycobacterium tuberculosis (Rv1819c), we insertionally inactivated this gene and analyzed the resulting mutant for a variety of phenotypes. BacA deficiency in M. tuberculosis did not affect sensitivity to detergents, acidic pH, and zinc, indicating that there was no global compromise in membrane integrity, and a comprehensive evaluation of the major lipid constituents of the cell envelope failed to reveal any significant differences. Infection of mice with this mutant revealed no impact on establishment of infection but a profound effect on maintenance of extended chronic infection and ultimate outcome. As in alphaproteobacteria, deletion of BacA in M. tuberculosis led to increased bleomycin resistance, and heterologous expression of the M. tuberculosis BacA homolog in Escherichia coli conferred sensitivity to antimicrobial peptides. These results suggest a striking conservation of function for BacA-related proteins in transport of a critical molecule that determines the outcome of the host-pathogen interaction.

Mycobacterium tuberculosis in the post-genomic age

Since the publication of the complete genome sequence of Mycobacterium tuberculosis in 1998, there has been a marked intensification and diversification of activities in the field of tuberculosis research. Among the areas that have advanced spectacularly are comparative genomics, functional genomics-notably the study of the transcriptome and proteome - and cell envelope biogenesis, especially as it relates to the mechanism of action of antimycobacterial drugs.

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.

Preexposure to Live Brugia malayi Microfilariae Alters the Innate Response of Human Dendritic Cells to Mycobacterium tuberculosis

BACKGROUND Mycobacterium tuberculosis and helminth coinfection is highly prevalent, and the presence of helminths may modulate the Th1 response necessary for M. tuberculosis control. METHODS Elutriated human monocytes, differentiated into dendritic cells (DCs) and macrophages, were exposed in vitro to live microfilariae (mf). The influence that mf had on M. tuberculosis infectivity, expression of cell surface molecules, and production of cytokines was determined. RESULTS Compared with mf-unexposed, M. tuberculosis-infected cells, mf-exposed, M. tuberculosis-infected DCs had decreased expression of CD14, CD54, and human leukocyte antigen-DR, and mf-exposed, M. tuberculosis-infected macrophages had decreased expression of CD40. DCs that were mf exposed and M. tuberculosis infected produced more interleukin (IL)-1 beta than did mf-unexposed, M. tuberculosis-infected DCs. Also, mf-exposed, M. tuberculosis-infected DCs and macrophages expressed less IL-10 and interferon (IFN)- alpha than did mf-unexposed, M. tuberculosis-infected cells. When they were cultured with autologous CD4+ T cells, mf-exposed, M. tuberculosis-infected DCs were less capable of stimulating the production of IFN- gamma than were other DCs. Exposure of DCs to mf decreased the surface expression of DC-specific intercellular adhesion molecule-3 grabbing nonintegrin, a receptor required by M. tuberculosis for entry into DCs. CONCLUSIONS Exposure to mf reduces a key receptor on the DC surface, which perhaps renders these cells less susceptible to infection with M. tuberculosis. Exposure to mf changes the surface expression of adhesion and costimulatory molecules on DCs and macrophages and alters their expression of cytokines and chemokines in a way that renders them less capable of immunologic responses.

Reemergence and Amplification of Tuberculosis in the Canadian Arctic

BACKGROUND Between November 2011 and November 2012, a Canadian village of 933 persons had 50 culture-positive cases of tuberculosis, with 49 sharing the same genotype. METHODS We performed Illumina-based whole-genome sequencing on Mycobacterium tuberculosis isolates from this village, during and before the outbreak. Phylogenetic trees were generated using the maximum likelihood method. RESULTS Three distinct genotypes were identified. Strain I (n = 7) was isolated in 1991-1996. Strain II (n = 8) was isolated in 1996-2004. Strain III (n = 62) first appeared in 2007 and did not arise from strain I or II. Within strain III, there were 3 related but distinct clusters: IIIA, IIIB, and IIIC. Between 2007 and 2010, cluster IIIA predominated (11 of 22 vs 2 of 40; P < .001), whereas in 2011-2012 clusters IIIB (n = 18) and IIIC (n = 20) predominated over cluster IIIA (n = 11). Combined evolutionary and epidemiologic analysis of strain III cases revealed that the outbreak in 2011-2012 was the result of ≥6 temporally staggered events, spanning from 1 reactivation case to a point-source outbreak of 20 cases. CONCLUSIONS After the disappearance of 2 strains of M. tuberculosis in this village, its reemergence in 2007 was followed by an epidemiologic amplification, affecting >5% of the population.