Posttranslational modification of proteins with farnesyl and geranylgeranyl isoprenoids is a widespread phenomenon in eukaryotic organisms. Isoprenylation is conferred by three protein prenyltransferases: farnesyl transferase (FTase), geranylgeranyl transferase type-I (GGTase-I), and Rab geranylgeranyltransferase (RabGGTase). Inhibitors of these enzymes have emerged as promising therapeutic compounds for treatment of cancer, viral and parasite originated diseases, as well as osteoporosis. However, no generic nonradioactive protein prenyltransferase assay has been reported to date, complicating identification of enzyme-specific inhibitors. We have addressed this issue by developing two fluorescent analogues of farnesyl and geranylgeranyl pyrophosphates {3,7-dimethyl-8-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-octa-2,6-diene-1}pyrophosphate (NBD-GPP) and {3,7,11-trimethyl-12-(7-nitro-benzo[1,2,5]oxadiazo-4-ylamino)-dodeca-2,6,10-trien-1} pyrophosphate (NBD-FPP), respectively. We demonstrate that these compounds can serve as efficient lipid donors for prenyltransferases. Using these fluorescent lipids, we have developed two simple (SDS-PAGE and bead-based) in vitro prenylation assays applicable to all prenyltransferases. Using the SDS-PAGE assay, we found that, in contrast to previous reports, the tyrosine phosphatase PRL-3 may possibly be a dual substrate for both FTase and GGTase-I. The on-bead prenylation assay was used to identify prenyltransferase inhibitors that displayed nanomolar affinity for RabGGTase and FTase. Detailed analysis of the two inhibitors revealed a complex inhibition mechanism in which their association with the peptide binding site of the enzyme reduces the enzyme's affinity for lipid and peptide substrates without competing directly with their binding. Finally, we demonstrate that the developed fluorescent isoprenoids can directly and efficiently penetrate into mammalian cells and be incorporated in vivo into small GTPases.

Mutations in the btk gene encoding Bruton’s tyrosine kinase cause X-linked immune deficiency, with impaired B lymphocyte function as the major phenotype. Earlier, we demonstrated that CBA/N-xid mice, unlike the wild-type CBA mice, were not protected by bacillus Calmette-Guérin (BCG) vaccination against tuberculosis infection. Because IFN-γ–producing T cells and activated macrophages are key elements of antituberculosis protection, it remained unclear how the mutation predominantly affecting B cell functions interferes with responses along the T cell–macrophage axis. In this study, we show that B cell deficiency leads to an abnormally rapid neutrophil migration toward the site of external stimulus. Using adoptive cell transfers and B cell genetic knockout, we demonstrate a previously unappreciated capacity of B cells to downregulate neutrophil motility. In our system, an advanced capture of BCG by neutrophils instead of macrophages leads to a significant decrease in numbers of IFN-γ–producing T cells and impairs BCG performance in X-linked immune-deficient mice. The defect is readily compensated for by the in vivo neutrophil depletion.

We previously demonstrated that mice of the I/St strain are extremely susceptible to Mycobacterium tuberculosis, as well as to the taxonomically distant intracellular bacteria Chlamydia pneumoniae and Salmonella enterica. To broaden our knowledge about the control of susceptibility to intracellular pathogens, we studied the infection caused by Mycobacterium avium virulent strain 724 in a panel of inbred mouse strains and found that I/St mice are resistant to M. avium. By comparing I/St mice with B6 mice, we demonstrated that (i) B6 mice are much more susceptible to infection caused by M. avium in terms of bacterial multiplication in the lung tissue and severity of lung pathology; (ii) in B6 mice but not in I/St mice infection leads to prolonged leukocyte infiltration of the lung tissue, development of necrotic lung granulomata, and lethality; and (iii) the unfavorable infectious course in B6 mice is accompanied by elevated production of gamma interferon, tumor necrosis factor alpha, and especially interleukin-12 in the lungs. Importantly, M. avium-resistant I/St mice carry a functional r allele of the Slc11a1 (formerly Nramp1) gene, while B6 mice have the Slc11a1 s genotype. Segregation genetic analysis of (I/St × B6) F2 hybrids demonstrated that susceptibility or resistance to infection caused by M. avium largely depended upon the Slc11a1 genotype and that other genetic traits had a relatively weak influence. This close-to-monogenic pattern differs sharply from the host control of many other intracellular bacterial infections, for which the involvement of numerous quantitative trait loci has been ubiquitously observed.

IL-11 is multifunctional cytokine whose physiological role in the lungs during pulmonary tuberculosis (TB) is poorly understood. Here, using in vivo administration of specific antibodies against IL-11, we demonstrate for the first time that blocking IL-11 diminishes histopathology and neutrophilic infiltration of the lung tissue in TB-infected genetically susceptible mice. Antibody treatment decreased the pulmonary levels of IL-11 and other key inflammatory cytokines not belonging to the Th1 axis, and down-regulated IL-11 mRNA expression. This suggests the existence of a positive feedback loop at the transcriptional level, which is further supported by up-regulation of IL-11 mRNA expression in the presence of rIL-11 in in vitro cultures of lung cells. These findings imply a pathogenic role for IL-11 during the early phase of Mycobacterium tuberculosis-triggered disease in a genetically susceptible host.

Tuberculosis (TB) is currently the leading cause of death among bacterial infectious diseases. The spectrum of disease manifestations depends on both host immune responses and the ability of Mycobacterium tuberculosis to resist it. Small non-coding RNAs are known to regulate gene expression; however, their functional role in the relationship of M. tuberculosis with the host is poorly understood. Here, we investigated the effect of small non-coding sRNAs MTS1338 and MTS0997 on M. tuberculosis properties by creating knockout strains. We also assessed the effect of small non-coding RNAs on the survival of wild type and mutant mycobacteria in primary cultures of human alveolar macrophages and the virulence of these strains in a mouse infection model. Wild-type and mutants survived differentially in human alveolar macrophages. Infection of I/St mice with KO M. tuberculosis H37RV strains provided beneficial effects onto major TB phenotypes. We observed attenuated tuberculosis-specific inflammatory responses, including reduced cellular infiltration and decreased granuloma formation in the lungs. Infections caused by KO strains were characterized by significantly lower inflammation of mouse lung tissue and increased survival time of infected animals. Thus, the deletion of MTS0997 and MTS1338 lead to a significant decrease in the virulence of M. tuberculosis.

Earlier we demonstrated that blocking of interleukin 11 (IL-11) by systemic administration of anti-IL-11 antibodies attenuates severity of Mycobacterium tuberculosis infection in mice. The substitution W147A in the IL-11 molecule creates the form of cytokine capable to disrupt gp130/IL11R signaling complex formation, thus serving as a high-affinity specific antagonist of IL-11-mediated signaling. We hypothesized that this mutant form of IL-11 may serve as an effective tool for inhibition of native IL-11 activity in vivo. We established the recombinant W147A mutant form of IL-11 in an optimized Escherichia coli expression system and administered it as the aerosol in the lungs of M. tuberculosis-susceptible I/St mice infected with M. tuberculosis Our results show that this therapeutic approach markedly inhibits tuberculous inflammation in lungs, increases the survival time of infected animals, and decreases expression of key inflammatory factors at the RNA and protein levels. These findings are a step toward clinical evaluation of the anti-IL-11 therapy for tuberculosis.

It is supposed that alpha,gamma-diketo acids (DKAs) inhibit the activity of hepatitis C virus RNA-dependent RNA polymerase (RdRP HCV) via chelation of catalytic magnesium ions in the active center of the enzyme. However, DKAs display noncompetitive mode of inhibition with respect to NTP substrate, which contradicts the proposed mechanism. We have examined the NTP substrate entry channel and the active site of RdRP HCV for their possible interaction with DKAs. The substitutions R48A, K51A, and R222A greatly facilitated RdRP inhibition by DKAs and simultaneously increased K(m) values for UTP substrate. Interestingly, C223A was the only one of a number of substitutions that decreased K(m)(UTP) but facilitated the inhibitory action of DKAs. The findings allowed us to model an enzyme-inhibitor complex. According to the proposed model, DKAs introduce an additional Mg2+ ion into the active site of the enzyme at a stage of phosphodiester bond formation, which results in displacement of the NTP substrate triphosphate moiety to a catalytically inactive binding mode. This mechanism, in contrast to the currently adopted one, explains the noncompetitive mode of inhibition.

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