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Lethal factor (LF), a zinc-dependent protease of high specificity produced by Bacillus anthracis, is the effector component of the binary toxin that causes death in anthrax. New therapeutics targeting the toxin are required to reduce systemic anthrax-related fatalities. In particular, new insights into the LF catalytic mechanism will be useful for the development of LF inhibitors. We evaluated the minimal length required for formation of bona fide LF substrates using substrate phage display. Phage-based selection yielded a substrate that is cleaved seven times more efficiently by LF than the peptide targeted in the protein kinase MKK6. Site-directed mutagenesis within the metal-binding site in the LF active center and within phage-selected substrates revealed a complex pattern of LF-substrate interactions. The elementary steps of LF-mediated proteolysis were resolved by the stopped-flow technique. Pre-steady-state kinetics of LF proteolysis followed a four-step mechanism as follows: initial substrate binding, rearrangement of the enzyme-substrate complex, a rate-limiting cleavage step, and product release. Examination of LF interactions with metal ions revealed an unexpected activation of the protease by Ca(2+) and Mn(2+). Based on the available structural and kinetic data, we propose a model for LF-substrate interaction. Resolution of the kinetic and structural parameters governing LF activity may be exploited to design new LF inhibitors.

We studied the effects of alpha1-acid glycoprotein preparations on the survival rate of BALB/c mice infected with the lethal dose of B. anthracis STI-1. Apart from native alpha1-acid glycoprotein from donor blood, we studied 3 glycoforms differing in the affinity for concanavalin A and structure of carbohydrate chains. The protective effect of alpha1-acid glycoprotein preparations did not depend on its dose and was observed 3 months after treatment (0.3 mg per mouse). The protective effect was revealed in mice receiving alpha1-acid glycoprotein preparations 2 h before infection and 24 h after inoculation of the bacterial culture. In the latter case the survival rate of animals was much higher compared to that observed in preventive administration of alpha1-acid glycoprotein. The protective effect practically did not depend on the time of treatment with glycoforms. Pretreatment with alpha1-acid glycoprotein preparations significantly decreased plasma interferon-gamma concentration. Administration of the test preparations 24 h after infection decreased the concentration of tumor necrosis factor-alpha.

Tuberculosis imposes a major burden of death and disease on the human population. Each year, tens of millions of people become infected with Mycobacterium tuberculosis , several millions develop clinical disease, and more than 2 million die of tuberculosis (1)(2). The successful treatment of tuberculosis depends heavily on timely diagnostics and selection of an adequate treatment strategy. Use of genotyping tools based on molecular techniques decreases the time necessary for the detection of drug resistance in M. tuberculosis from several weeks to a few days or even less, and a patient’s treatment regimen can be adjusted more rapidly to account for any detected drug resistance. Several mutations conferring resistance to the first-line drugs used for antituberculosis treatment have been described [for a review, see Ref (3)], making detection of these mutations by molecular biology techniques feasible. Rifampin and isoniazid belong to the first-line drugs used for treatment of tuberculosis (4). Resistance to rifampin, one of the most efficient antitubercular drugs, is conferred mainly by mutations in the rpoB gene. Ninety-six percent of rifampin-resistant cases are attributable to point mutations, insertions, or deletions located in an 81-nucleotide segment of the rpoB gene (5). Although resistance to isoniazid can be conferred by mutations in several different genes, mutations in katG represent the most frequent cause of the resistance (3)(6). Here we describe the allele-specific depletory PCR (ASD-PCR) assay, which was developed for the detection of mutations associated with M. tuberculosis resistance to first-line drugs, specifically isoniazid and rifampin. The accuracy and reproducibility of classic allele-specific PCR assays depend greatly on the careful selection of amplification conditions, primer design, and template concentration. Elongation through the mismatched duplex leads to accumulation of mutant template fully complementary to the initially mismatched primer, yielding the false-positive amplicon. Depending on the nature of …

Tuberculosis ward of a prison in Russia.Molecular characterization of drug-resistant isolates.Isolates were collected from all tuberculosis patients occurring in the prison over a 1-year period.Of 130 patients studied, 17 patients produced pan-susceptible isolates and 113 produced isolates resistant to at least one drug, including 85 multidrug-resistant isolates. Mutations at katG315 occurred in 98% of isoniazid-resistant isolates. Mutations in rpoB were found in 89% of rifampicin-resistant isolates. Mutations in pncA occurred in 13% of the 75 isolates tested. By spoligotyping, members of the Beijing (55 isolates) and LAM (31 isolates) families were identified. By IS6110 genotyping, two groups (34 and 55 isolates) of related isolates were found, including three clusters (10, 12, and 16 isolates) with identical patterns. In a study of samples collected 3 months apart from 28 patients, four patients produced isolates containing a mixture of strains and five patients produced specimens containing distinctly different isolates. Isolates of nine patients acquired additional drug resistance.Three families of strains accounted for much of the drug-resistant tuberculosis in this population. Multiple resistance, acquisition of resistance, and infection with two or more strains as well as reinfection were observed.

We studied the effects of alpha1-acid glycoprotein on tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) production and lymphocyte response to phytohemagglutinin in cultured peripheral blood mononuclear leukocytes from 6 healthy donors. We observed 2 opposite responses to alpha1-acid glycoprotein: first, stimulation of TNF-alpha and IL-10 production and inhibition of lymphocyte proliferation, and second, suppression of cytokine production and stimulation of lymphocyte proliferation. In cell cultures isolated from 4 of 6 donors, the TNF-alpha/IL-10 ratio remained unchanged after addition of native alpha1-acid glycoprotein, but some fractions isolated by chromatography on concanavalin A-Sepharose changed this parameter. These changes were most pronounced after treatment with fraction C enriched with molecules with incomplete (biantennary) carbohydrate chains. The mechanisms of alpha1-acid glycoprotein-induced effects on peripheral blood mononuclear leukocytes are discussed.

State Research Center for Applied Microbiology, Russian Research Institute of Phthisiopulmonology (Ministry of Health, Moscow).To analyze drug-resistant clinical isolates of Mycobacterium tuberculosis obtained from patients referred to the institute from different parts of Russia, and to study the mechanisms of their rifampicin resistance.Fifty clinical isolates of M. tuberculosis were analysed. Polymerase chain reaction (PCR) and sequencing were used to study the mechanisms of rifampicin resistance in 25 isolates.Among cultures isolated from 50 patients, drug resistance was detected in 33. Most of the isolates were resistant to rifampicin (25 isolates), isoniazid (14 isolates), and streptomycin (seven isolates). Only 6% of the isolates were resistant to one drug, while 14% were resistant to two, 32% to three, 40% to four, and 8% to five drugs. Susceptible isolates were derived from 17 patients. The following point mutations and deletions in the rpoB locus, responsible for high level rifampicin resistance (more than 50 microg/ml in egg-based medium), were detected: G--A/395 (Arg--Gln), C--T/232 (His--Tyr), C--T/221 (Ser--Leu), G--T/202 (Asp--Tyr), GA--TT/202-203 (Asp--Phe), deltaATGGACCAG/199-207 (Met, Asp, Gin), A--T/91 (Met--Leu), TG--CC/227-228 (Leu--Ser), GAG--AGT/349-350-351 (Gln--Ser), deltaGGG/354(Gly).A number of previously unrecognised genetic modifications in the rpoB region were found in rifampicin-resistant strains isolated from patients from different parts of Russia.

Tuberculosis remains a major public health concern in Russia and worldwide. Given the great geographical, ethnic, and socio-economic heterogeneities between Russian regions, epidemiological data cannot be generalized from a regional to a country-wide level. We present data on the epidemiology of tuberculosis in Central Russia. We report a high level of resistance to major antitubercular drugs in both new and previously treated patients in the region. The level of drug resistance in new cases was almost twice as high as the estimated average national level. The Mycobacterium tuberculosis strains that circulated in the region were predominantly represented by LAM-RUS and Beijing genotypes. These two lineages were strongly associated with drug resistance and clustering. Using molecular epidemiology techniques, we showed a high interpenetration by M. tuberculosis strains between the prison and civilian populations. A limited number of identical strains were responsible for the majority of drug-resistant tuberculosis cases in both settings.