Thomas C. Zahrt

Mycobacterium tuberculosis signal transduction system required for persistent infections

It is estimated that nearly 2 billion people currently suffer from latent Mycobacterium tuberculosis infection. Although the key front-line antituberculosis drugs are effective in treating individuals with acute tuberculosis, these drugs are ineffective in eliminating M. tuberculosis during the persistent stages of latent infection. Consequently, therapeutics that directly target persistent bacilli are urgently needed. We have conducted a global analysis on a group of regulatory determinants that may play a role in M. tuberculosis virulence, and identified a two-component response regulator whose expression is required for entrance into and maintenance of persistent infection. Inactivation of this response regulator, Rv0981 (termed here mprA for mycobacterial persistence regulator), affected M. tuberculosis H37Rv growth in vivo in an organ- and infection stage-specific fashion. These results indicate that two-component systems are important for adaptation of the tubercle bacillus during stages of persistent infection.

An Essential Two-Component Signal Transduction System in Mycobacterium tuberculosis

The bacterial two-component signal transduction systems regulate adaptation processes and are likely to play a role in Mycobacterium tuberculosis physiology and pathogenesis. The previous initial characterization of an M. tuberculosis response regulator from one of these systems, mtrA-mtrB, suggested its transcriptional activation during infection of phagocytic cells. In this work, we further characterized the mtrA response regulator from M. tuberculosis H37Rv. Inactivation of mtrA on the chromosome of M. tuberculosis H37Rv was possible only in the presence of plasmid-borne functional mtrA, suggesting that this response regulator is essential for M. tuberculosis viability. In keeping with these findings, expression of mtrA in M. tuberculosis H37Rv was detectable during in vitro growth, as determined by S1 nuclease protection and primer extension analyses of mRNA levels and mapping of transcript 5 ends. The mtrA gene was expressed differently in virulent M. tuberculosis and the vaccine strain M. tuberculosis var. bovis BCG during infection of macrophages, as determined by monitoring of mtrA-gfp fusion activity. In M. bovis BCG, mtrA was induced upon entry into macrophages. In M. tuberculosis H37Rv, its expression was constitutive and unchanged upon infection of murine or human monocyte-derived macrophages. In conclusion, these results identify mtrA as an essential response regulator gene in M. tuberculosis which is differentially expressed in virulent and avirulent strains during growth in macrophages.

Construction and Characterization of a Highly Efficient Francisella Shuttle Plasmid

ABSTRACT Francisella tularensis is a facultative intracellular pathogen that infects a wide variety of mammals and causes tularemia in humans. It is recognized as a potential agent of bioterrorism due to its low infectious dose and multiple routes of transmission. To date, genetic manipulation in Francisella spp. has been limited due to the inefficiency of DNA transformation, the relative lack of useful selective markers, and the lack of stably replicating plasmids. Therefore, the goal of this study was to develop an enhanced shuttle plasmid that could be utilized for a variety of genetic procedures in both Francisella and Escherichia coli. A hybrid plasmid, pFNLTP1, was isolated that was transformed by electroporation at frequencies of >1 × 107 CFU μg of DNA−1 in F. tularensis LVS, Francisella novicida U112, and E. coli DH5α. Furthermore, this plasmid was stably maintained in F. tularensis LVS after passage in the absence of antibiotic selection in vitro and after 3 days of growth in J774A.1 macrophages. Importantly, F. tularensis LVS derivatives carrying pFNLTP1 were unaltered in their growth characteristics in laboratory medium and macrophages compared to wild-type LVS. We also constructed derivatives of pFNLTP1 containing expanded multiple cloning sites or temperature-sensitive mutations that failed to allow plasmid replication in F. tularensis LVS at the nonpermissive temperature. In addition, the utility of pFNLTP1 as a vehicle for gene expression, as well as complementation, was demonstrated. In summary, we describe construction of a Francisella shuttle plasmid that is transformed at high efficiency, is stably maintained, and does not alter the growth of Francisella in macrophages. This new tool should significantly enhance genetic manipulation and characterization of F. tularensis and other Francisella biotypes.

MprAB Is a Stress-Responsive Two-Component System That Directly Regulates Expression of Sigma Factors SigB and SigE in Mycobacterium tuberculosis

The genetic mechanisms mediating the adaptation of Mycobacterium tuberculosis within the host are poorly understood. The best-characterized regulatory systems in this organism include sigma factors and two-component signal transduction systems. mprAB is a two-component system required by M. tuberculosis for growth in vivo during the persistent stage of infection. In this report, we demonstrate that MprAB is stress responsive and regulates the expression of numerous stress-responsive genes in M. tuberculosis. With DNA microarrays and quantitative real-time reverse transcription-PCR, genes regulated by MprA in M. tuberculosis that included two stress-responsive sigma factors were identified. Response regulator MprA bound to conserved motifs in the upstream regions of both sigB and sigE in vitro and regulated the in vivo expression of sigB and sigE in M. tuberculosis. In addition, mprA itself was induced following exposure to stress, establishing a direct role for this regulatory system in stress response pathways of M. tuberculosis. Induction of mprA and sigE by MprA in response to stress was mediated through the cognate sensor kinase MprB and required expression of the extracytoplasmic loop domain. These results provide the first evidence that recognition of and adaptation to specific stress in M. tuberculosis are mediated through activation of a two-component signal transduction system that directly regulates the expression of stress-responsive determinants.

Mycobacterial FurA is a negative regulator of catalase–peroxidase gene katG

In several bacteria, the catalase–peroxidase gene katG is under positive control by oxyR, a transcriptional regulator of the peroxide stress response. The Mycobacterium tuberculosis genome also contains sequences corresponding to oxyR, but this gene has been inactivated in the tubercle bacillus because of the presence of multiple mutations and deletions. Thus, M. tuberculosis katG and possibly other parts of the oxidative stress response in this organism are either not regulated or are controlled by a factor different from OxyR. The mycobacterial FurA is a homologue of the ferric uptake regulator Fur and is encoded by a gene located immediately upstream of katG. Here, we examine the possibility that FurA regulates katG expression. Inactivation of furA on the Mycobacterium smegmatis chromosome, a mycobacterial species that also lacks an oxyR homologue, resulted in derepression of katG, concomitant with increased resistance of the furA mutant to H2O2. In addition, M. smegmatis furA::Kmr was more sensitive to the front‐line antituberculosis agent isonicotinic acid hydrazide (INH) compared with the parental furA+ strain. The phenotypic manifestations were specific, as the mutant strain did not show altered sensitivity to organic peroxides, and both H2O2 and INH susceptibility profiles were complemented by the wild‐type furA+ gene. We conclude that FurA is a second regulator of oxidative stress response in mycobacteria and that it negatively controls katG. In species lacking a functional oxyR, such as M. tuberculosis and M. smegmatis, FurA appears to be a dominant regulator affecting mycobacterial physiology and intracellular survival.

Reactive nitrogen and oxygen intermediates and bacterial defenses: unusual adaptations in Mycobacterium tuberculosis.

The production of reactive oxygen and reactive nitrogen intermediates is an important host defense mechanism mediated in response to infection by bacterial pathogens. Not surprisingly, intracellular pathogens have evolved numerous defense strategies to protect themselves against the damaging effects of these agents. In enteric bacteria, exposure to oxidative or nitrosative stress induces expression of numerous pathways that allow the bacterium to resist the toxic effects of these compounds during growth in the host. In contrast, members of pathogenic mycobacterial species, including the frank human pathogens Mycobacterium tuberculosis and Mycobacterium leprae, are dysfunctional in aspects of the oxidative and nitrosative stress response, yet they remain able to establish and maintain productive acute and persistent infections in the host. This article reviews the current knowledge regarding reactive oxygen and nitrogen intermediates, and compares the adaptative mechanisms utilized by enteric organisms and mycobacterial species to resist the bactericidal and bacteriostatic effects resulting from exposure to these compounds.

Working toward the Future: Insights into Francisella tularensis Pathogenesis and Vaccine Development

SUMMARY Francisella tularensis is a facultative intracellular gram-negative pathogen and the etiological agent of the zoonotic disease tularemia. Recent advances in the field of Francisella genetics have led to a rapid increase in both the generation and subsequent characterization of mutant strains exhibiting altered growth and/or virulence characteristics within various model systems of infection. In this review, we summarize the major properties of several Francisella species, including F. tularensis and F. novicida, and provide an up-to-date synopsis of the genes necessary for pathogenesis by these organisms and the determinants that are currently being targeted for vaccine development.

Identification, Characterization and Immunogenicity of an O-Antigen Capsular Polysaccharide of Francisella tularensis

Capsular polysaccharides are important factors in bacterial pathogenesis and have been the target of a number of successful vaccines. Francisella tularensis has been considered to express a capsular antigen but none has been isolated or characterized. We have developed a monoclonal antibody, 11B7, which recognizes the capsular polysaccharide of F. tularensis migrating on Western blot as a diffuse band between 100 kDa and 250 kDa. The capsule stains poorly on SDS-PAGE with silver stain but can be visualized using ProQ Emerald glycoprotein stain. The capsule appears to be highly conserved among strains of F. tularensis as antibody 11B7 bound to the capsule of 14 of 14 F. tularensis type A and B strains on Western blot. The capsular material can be isolated essentially free of LPS, is phenol and proteinase K resistant, ethanol precipitable and does not dissociate in sodium dodecyl sulfate. Immunoelectron microscopy with colloidal gold demonstrates 11B7 circumferentially staining the surface of F. tularensis which is typical of a polysaccharide capsule. Mass spectrometry, compositional analysis and NMR indicate that the capsule is composed of a polymer of the tetrasaccharide repeat, 4)-α-D-GalNAcAN-(1->4)-α-D-GalNAcAN-(1->3)-β-D-QuiNAc-(1->2)-β-D-Qui4NFm-(1-, which is identical to the previously described F. tularensis O-antigen subunit. This indicates that the F. tularensis capsule can be classified as an O-antigen capsular polysaccharide. Our studies indicate that F. tularensis O-antigen glycosyltransferase mutants do not make a capsule. An F. tularensis acyltransferase and an O-antigen polymerase mutant had no evidence of an O-antigen but expressed a capsular antigen. Passive immunization of BALB/c mice with 75 µg of 11B7 protected against a 150 fold lethal challenge of F. tularensis LVS. Active immunization of BALB/c mice with 10 µg of capsule showed a similar level of protection. These studies demonstrate that F. tularensis produces an O-antigen capsule that may be the basis of a future vaccine.

Identification of Francisella tularensis Himar1-Based Transposon Mutants Defective for Replication in Macrophages

ABSTRACT Francisella tularensis, the etiologic agent of tularemia in humans, is a potential biological threat due to its low infectious dose and multiple routes of entry. F. tularensis replicates within several cell types, eventually causing cell death by inducing apoptosis. In this study, a modified Himar1 transposon (HimarFT) was used to mutagenize F. tularensis LVS. Approximately 7,000 Kmr clones were screened using J774A.1 macrophages for reduction in cytopathogenicity based on retention of the cell monolayer. A total of 441 candidates with significant host cell retention compared to the parent were identified following screening in a high-throughput format. Retesting at a defined multiplicity of infection followed by in vitro growth analyses resulted in identification of approximately 70 candidates representing 26 unique loci involved in macrophage replication and/or cytotoxicity. Mutants carrying insertions in seven hypothetical genes were screened in a mouse model of infection, and all strains tested appeared to be attenuated, which validated the initial in vitro results obtained with cultured macrophages. Complementation and reverse transcription-PCR experiments suggested that the expression of genes adjacent to the HimarFT insertion may be affected depending on the orientation of the constitutive groEL promoter region used to ensure transcription of the selective marker in the transposon. A hypothetical gene, FTL_0706, postulated to be important for lipopolysaccharide biosynthesis, was confirmed to be a gene involved in O-antigen expression in F. tularensis LVS and Schu S4. These and other studies demonstrate that therapeutic targets, vaccine candidates, or virulence-related genes may be discovered utilizing classical genetic approaches in Francisella.

Silencing of Oxidative Stress Response in Mycobacterium tuberculosis: Expression Patterns of ahpC in Virulent and Avirulent Strains and Effect of ahpC Inactivation

ABSTRACT Intracellular pathogens such as Mycobacterium tuberculosis are able to survive in the face of antimicrobial products generated by the host cell in response to infection. The product of the alkyl hydroperoxide reductase gene (ahpC) of M. tuberculosis is thought to be involved in protecting the organism against both oxidative and nitrosative stress encountered within the infected macrophage. Here we report that, contrary to expectations, ahpC expression in virulent strains of M. tuberculosis and Mycobacterium bovis grown in vitro is repressed, often below the level of detection, whereas expression in the avirulent vaccine strainM. bovis BCG is constitutively high. The repression of the ahpC gene of the virulent strains is independent of the naturally occurring lesions of central regulatoroxyR. Using a green fluorescence protein vector (gfp)-ahpC reporter construct we present data showing that repression of ahpC of virulentM. tuberculosis also occurred during growth inside macrophages, whereas derepression in BCG was again seen under identical conditions. Inactivation of ahpC on the chromosome ofM. tuberculosis by homologous recombination had no effect on its growth during acute infection in mice and did not affect in vitro sensitivity to H2O2. However, consistent with AhpC function in detoxifying organic peroxides, sensitivity to cumene hydroperoxide exposure was increased in theahpC::Kmr mutant strain. The preservation of a functional ahpC gene in M. tuberculosis in spite of its repression under normal growth conditions suggests that, while AhpC does not play a significant role in establishing infection, it is likely to be important under certain, as yet undefined conditions. This is supported by the observation that repression of ahpC expression in vitro was lifted under conditions of static growth.