Identification ofMycobacterium tuberculosisCounterimmune (cim) Mutants in Immunodeficient Mice by Differential Screening

Abstract

Tuberculosis (TB) is characterized by lifetime persistence of Mycobacterium tuberculosis. Despite the induction of a vigorous host immune response that curtails disease progression in the majority of cases, the organism is not eliminated. Subsequent immunosuppression can lead to reactivation after a prolonged period of clinical latency. Thus, while it is clear that protective immune mechanisms are engaged during M. tuberculosis infection, it also appears that the pathogen has evolved effective countermechanisms. Genetic studies with animal infection models and with patients have revealed a key role for the cytokine gamma interferon (IFN-␥) in resistance to TB. IFN-␥ activates a large number of antimicrobial pathways. Three of these IFN-␥-dependent mechanisms have been implicated in defense against M. tuberculosis: inducible nitric oxide synthase (iNOS), phagosome oxidase (phox), and the phagosome-associated GTPase LRG-47. In order to identify bacterial genes that provide protection against specific host immune pathways, we have developed the strategy of differential signature-tagged transposon mutagenesis. Using this approach we have identified three M. tuberculosis genes that are essential for progressive M. tuberculosis growth and rapid lethality in iNOS-deficient mice but not in IFN-␥-deficient mice. We propose that these genes are involved in pathways that allow M. tuberculosis to counter IFN-␥-dependent immune mechanisms other than iNOS.Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is capable of long-term survival in vivo despite a vigorous host immune response. Although 90 to 95% of infected individuals initially control bacterial replication and do not manifest clinical signs and symptoms, reactivation of latent TB infection (LTBI) can occur many years after infection (42). Human immunodeficiency virus infection has become the most important risk factor for reactivation of LTBI, increasing the risk of reactivation from 5 to 10% per lifetime to 5% per year (9). Immunosuppressive agents, such as Infliximab, an antitumor necrosis factor alpha antibody used to treat chronic inflammatory conditions, have also been associated with reactivation of LTBI (42). Approximately half of all active cases of pulmonary TB are thought to result from endogenous reactivation of LTBI (40).Exogenous reinfection of asymptomatic individuals also appears to be common in areas where TB is endemic, despite the presence of detectable antigen-specific immune responses established by the previous infection (6,17,46). Similarly, vaccination with live-attenuated Mycobacterium bovis BCG has yielded highly variable and generally poor efficacy in prevention of pulmonary TB in adults despite eliciting robust antigenspecific immune responses in the majority of vaccinated individuals (4).A key component of antimycobacterial immunity is provided by the cytokine gamma interferon (IFN-␥), as evidenced by infection of IFN-␥-deficient mice (12, 19) and IFN-␥R1-deficient mice (25) and by naturally occurring mutations in IFN-...

Spontaneous Phthiocerol Dimycocerosate-Deficient Variants of Mycobacterium tuberculosis Are Susceptible to Gamma Interferon-Mediated Immunity

“…M. tuberculosis strains from the McKinney lab were H37Rv (parental strain) and Tn5370 (Tn) mutagenized derivatives rv0072::Tn, rv0405::Tn, and rv2958c::Tn, described previously (12). M. tuberculosis strains from the Guilhot lab were H37Rv (parental strain), ⌬rv2957, ⌬rv2958c, ⌬rv2959c, and ⌬rv2962c, described previously (24,25).…”

“…Previously, we reported the results of a pilot signature-tagged mutagenesis screen to identify M. tuberculosis genes involved in countering the impact of IFN-␥-dependent immune mechanisms other than NOS2 (12). This was accomplished by parallel screening of Tn-induced mutants in intravenously infected gene knockout mice to identify mutants that were attenuated for growth and virulence in NOS2 Ϫ/Ϫ mice but unimpaired for growth and virulence in IFN-␥ Ϫ/Ϫ mice.…”

“…The Rv2962c, Rv2958c, and Rv2957 glycosyl transferases add rhamnose 3 rhamnose 3 fucose to p-hydroxyphenylalkanoic acid (25), and the Rv2959c methyltransferase O-methylates the C2 ring position of the proximal rhamnosyl residue (24). genetic screen that was designed to identify M. tuberculosis genes involved in countering IFN-␥-dependent host immune mechanisms other than NOS2 (12). Disruption of these "counterimmune" (cim) genes severely attenuated growth and virulence in NOS2 Ϫ/Ϫ mice but had little or no impact on bacterial growth and virulence in IFN-␥ Ϫ/Ϫ mice.…”

Mycobacterium tuberculosis persistence mutants identified by screening in isoniazid-treated mice

“…Mutant library construction, genomic DNA isolation, signature tag amplification, and Southern blot hybridizations were as described (13). Hybridization signal intensities of individual signature-tagged mutagenesis tags were measured using an Amersham Biosciences Typhoon 9400 Imager.…”

“…Historically, signature-tagged mutagenesis has been used to identify mutants with growth or survival defects in untreated animals (11)(12)(13). Our screening scheme was complicated by the fact that the entire population of pooled mutants was progressively reduced by INH therapy, which could lead to artifactual underrepresentation or overrepresentation of individual mutants.…”