SummaryProtein tyrosine kinases and tyrosine phosphatases from several bacterial pathogens have been shown to act as virulence factors by modulating the phosphorylation and dephosphorylation of host proteins. The identification and characterization of two tyrosine phosphatases namely MptpA and MptpB from Mycobacterium tuberculosis has been reported earlier.MptpB is secreted by M. tuberculosis into extracellular mileu and exhibits a pH optimum of 5.6, similar to the pH of the lysosomal compartment of the cell. To determine the role of MptpB in the pathogenesis of M. tuberculosis , we constructed a mptpB mutant strain by homologous recombination and compared the ability of parent and the mutant strain to survive intracellularly. We show that disruption of the mptpB gene impairs the ability of the mutant strain to survive in activated macrophages and guinea pigs but not in resting macrophages suggesting the importance of its role in the host-pathogen interaction. Infection of guinea pigs with the mutant strain resulted in a 70-fold reduction in the bacillary load of spleens in infected animals as compared with the bacillary load in animals infected with the parental strain. Upon reintroduction of the mptpB gene into the mutant strain, the complemented strain was able to establish infection and survive in guinea pigs at rates comparable to the parental strain. These observations demonstrate a role of MptpB in the pathogenesis of M. tuberculosis .

The devR-devS two-component system of Mycobacterium tuberculosis was identified earlier and partially characterized in our laboratory. A devR: :kan mutant of M. tuberculosis was constructed by allelic exchange. The devR mutant strain showed reduced cell-tocell adherence in comparison to the parental strain in laboratory culture media. This phenotype was reversed on complementation with a wild-type copy of devR. The devR mutant and parental strains grew at equivalent rates within human monocytes either in the absence or in the presence of lymphocytic cells. The expression of DevR was not modulated upon entry of M. tuberculosis into human monocytes. However, guinea pigs infected with the mutant strain showed a significant decrease in gross lesions in lung, liver and spleen ; only mild pathological changes in liver and lung; and a nearly 3 log lower bacterial burden in spleen compared to guinea pigs infected with the parental strain. Our results suggest that DevR is required for virulence in guinea pigs but is not essential for entry, survival and multiplication of M. tuberculosis within human monocytes in vitro. The attenuation in virulence of the devR mutant in guinea pigs together with DevR-DevS being a bona fide signal transduction system indicates that DevR plays a critical and regulatory role in the adaptation and survival of M. tuberculosis within tissues.

We had recently reported that the mymA operon (Rv3083 to Rv3089) of Mycobacterium tuberculosis is regulated by AraC/XylS transcriptional regulator VirS (Rv3082c) and is important for the cell envelope of M. tuberculosis. In this study, we further show that a virS mutant (Mtb⌬virS) and a mymA mutant (Mtbmym::hyg) of M. tuberculosis exhibit reduced contents and altered composition of mycolic acids along with the accumulation of saturated C 24 and C 26 fatty acids compared to the parental strain. These mutants were markedly more susceptible to major antitubercular drugs at acidic pH and also showed increased sensitivity to detergent (sodium dodecyl sulfate) and to acidic stress than the parental strain. We show that disruption of virS and mymA genes impairs the ability of M. tuberculosis to survive in activated macrophages, but not in resting macrophages, suggesting the importance of the mymA operon in protecting the bacterium against harsher conditions. Infection of guinea pigs with Mtb⌬virS, Mtbmym::hyg, and the parental strain resulted in an ϳ800-fold-reduced bacillary load of the mutant strains compared with the parental strain in spleens, but not in the lungs, of animals at 20 weeks postinfection. Phenotypic traits were fully complemented upon reintroduction of the virS gene into Mtb⌬virS. These observations show the important role of the mymA operon in the pathogenesis of M. tuberculosis at later stages of the disease.Mycobacterium tuberculosis, the etiological agent of extremely serious human infections, is a highly successful intracellular pathogen because it can adapt itself to various hostile environments. The cell envelope of mycobacteria is known to play a major role in their virulence and resistance to hostile environments. Besides, interaction of the mycobacterial cell envelope with host cell receptors facilitates uptake of the bacterium and modulation of host immune responses (12). Mycobacterium tuberculosis has approximately 250 genes involved in its lipid metabolism, and the lipid contents of the pathogen contribute to 60% of the cell dry weight (10,12). A number of genes involved in the synthesis of essential components of the cell envelope for maintaining appropriate cell wall architecture of M. tuberculosis have been identified, and their requirement for the virulence of M. tuberculosis has been established, suggesting their importance as targets for the development of new antitubercular drugs (4,8,19,22,57). Genes responsible for the biosynthesis of oxygenated mycolic acids and cyclopropanated mycolic acids were shown to be important for the in vivo growth and persistence of M. tuberculosis (19,22,57). The gene cluster involved in the biosynthesis of major complex lipids phthiocerol and phenolphthiocerol dimycocerosates of M. tuberculosis have been shown to play an important role in its virulence (11). Induction of various genes involved in fatty acid metabolism in caseous granuloma (9) or human macrophages (20) or after exposure to sodium dodecyl sulfate (SDS) (37, 57) or acidic stress (21) su...

The gamma interferon (IFN-␥) enzyme-linked immunospot (ELISPOT) assay is used routinely to evaluate the potency of human immunodeficiency virus (HIV) vaccine candidates and other vaccine candidates. In orderto compare candidates and pool data from multiple trial laboratories, validated standardized methods must be applied across laboratories. Proficiency panels are a key part of a comprehensive quality assurance program to monitor inter-and intralaboratory performance, as well as assay performance, over time. Seven International AIDS Vaccine Initiative-sponsored trial sites participated in the proficiency panels described in this study. At each laboratory, two operators independently processed identical sample sets consisting of frozen peripheral blood mononuclear cell (PBMC) samples from different donors by using four blind stimuli. PBMC recovery and viability after overnight resting and the IFN-␥ ELISPOT assay performance were assessed. All sites demonstrated good performance in PBMC thawing and resting, with a median recovery of 78% and median viability of 95%. The laboratories were able to detect similar antigen-specific T-cell responses, ranging from 50 to >3,000 spot-forming cells per million PBMC. An approximate range of a half log in results from operators within or across sites was seen in comparisons of antigen-specific responses. Consistently low background responses were seen in all laboratories. The results of these proficiency panels demonstrate the ability of seven laboratories, located across three continents, to process PBMC samples and to rank volunteers with differential magnitudes of IFN-␥ ELISPOT responses. These findings also illustrate the ability to standardize the IFN-␥ ELISPOT assay across multiple laboratories when common training methods, reagents such as fetal calf serum, and standard operating procedures are adopted. These results are encouraging for laboratories that are using cell-based immunology assays to test HIV vaccines and other vaccines.Most human immunodeficiency virus (HIV) vaccines currently in development aim to induce cellular immune responses, since these have been shown previously to temporally correlate with the containment of virus in infected individuals and, more significantly, to be crucial in the suppression of virus in the rhesus macaque model (2,13,15,25). The ability to measure and quantitate cellular immune responses has been facilitated through the development of enzyme-linked immunospot (ELISPOT) and flow cytometry assays which determine the number of antigen-specific cells through surrogate markers of effector function, such as cytokine production or the degranulation of lytic granules (1,8,23,29), and are more quantitative and less labor-intensive than traditional assays that detect T-cell responses, such as 51 Cr release and lymphoproliferation assays (19). The gamma interferon (IFN-␥) ELISPOT assay is a primary assay employed to measure vaccine immunogenicity in HIV vaccine clinical trials, in addition to trials in the cancer, malaria, and tuberculosi...