Mycobacterium tuberculosis PknK Substrate Profiling Reveals Essential Transcription Terminator Protein Rho and Two-Component Response Regulators PrrA and MtrA as Novel Targets for Phosphorylation.

The Mycobacterium tuberculosis protein kinase K regulates growth adaptation by facilitating mycobacterial survival in response to a variety of and stress conditions. Here, we further add that transcription is responsive to carbon and nitrogen starvation signals. The increased survival of an M.

Interplay of PhoP and DevR response regulators defines expression of the dormancy regulon in virulent .

The DevR response regulator of is an established regulator of the dormancy response in mycobacteria and can also be activated during aerobic growth conditions in avirulent strains, suggesting a complex regulatory system. Previously, we reported culture medium-specific aerobic induction of the DevR regulon genes in avirulent H37Ra that was absent in the virulent H37Rv strain. To understand the underlying basis of this differential response, we have investigated aerobic expression of the operon using H37Ra and H37Rv overexpression strains, designated as LIX48 and LIX50, respectively.

Vaccines: Conduits for Protective Antigens.

Vaccines afford a better and more cost-effective approach to combatting infectious diseases than continued reliance on antibiotics or antiviral or antiparasite drugs in the current era of increasing incidences of diseases caused by drug-resistant pathogens. Recombinant attenuated vaccines (RASVs) have been significantly improved to exhibit the same or better attributes than wild-type parental strains to colonize internal lymphoid tissues and persist there to serve as factories to continuously synthesize and deliver rAgs. Encoded by codon-optimized pathogen genes, Ags are selected to induce protective immunity to infection by that pathogen.

The Mycobacterium tuberculosis relBE toxin:antitoxin genes are stress-responsive modules that regulate growth through translation inhibition.

Toxin-antitoxin (TA) genes are ubiquitous among bacteria and are associated with persistence and dormancy. Following exposure to unfavorable environmental stimuli, several species (Escherichia coli, Staphylococcus aureus, Myxococcus xanthus) employ toxin proteins such as RelE and MazF to downregulate growth or initiate cell death. Mycobacterium tuberculosis possesses three Rel TA modules (Rel Mtb ): RelBE Mtb , RelFG Mtb and RelJK Mtb (Rv1246c-Rv1247c, Rv2865-Rv2866, and Rv3357-Rv3358, respectively), which inhibit mycobacterial growth when the toxin gene (relE, relG, relK) is expressed independently of the antitoxin gene (relB, relF, relJ).

Mycobacterium tuberculosis response regulators, DevR and NarL, interact in vivo and co-regulate gene expression during aerobic nitrate metabolism.

Mycobacterium tuberculosis genes Rv0844c/Rv0845 encoding the NarL response regulator and NarS histidine kinase are hypothesized to constitute a two-component system involved in the regulation of nitrate metabolism. However, there is no experimental evidence to support this. In this study, we established M.

Mycobacterium tuberculosis protein kinase K enables growth adaptation through translation control.

Mycobacterium tuberculosis serine/threonine protein kinases (STPKs) are responsible for orchestrating critical metabolic and physiological changes that dictate mycobacterial growth adaptation. Previously, we established that PknK participates in regulatory pathways that slow the growth of M. tuberculosis in a variety of in vitro stress environments and during persistent infection in mice.

Live attenuated Salmonella vaccines against Mycobacterium tuberculosis with antigen delivery via the type III secretion system.

Tuberculosis remains a global health threat, and there is dire need to develop a vaccine that is safe and efficacious and confers long-lasting protection. In this study, we constructed recombinant attenuated Salmonella vaccine (RASV) strains with plasmids expressing fusion proteins consisting of the 80 amino-terminal amino acids of the type 3 secretion system effector SopE of Salmonella and the Mycobacterium tuberculosis antigens early secreted antigenic target 6-kDa (ESAT-6) protein and culture filtrate protein 10 (CFP-10). We demonstrated that the SopE-mycobacterial antigen fusion proteins were translocated into the cytoplasm of INT-407 cells in cell culture assays.

Live attenuated Salmonella vaccines displaying regulated delayed lysis and delayed antigen synthesis to confer protection against Mycobacterium tuberculosis.

Live recombinant attenuated Salmonella vaccine (RASV) strains have great potential to induce protective immunity against Mycobacterium tuberculosis by delivering M. tuberculosis antigens. Recently, we reported that, in orally immunized mice, RASV strains delivering the M.

The prrAB two-component system is essential for Mycobacterium tuberculosis viability and is induced under nitrogen-limiting conditions.

The Mycobacterium tuberculosis prrA-prrB (Rv0903c-Rv0902c) two-component regulatory system is expressed during intracellular growth in human macrophages and is required for early intracellular multiplication in murine macrophages, suggesting its importance in establishing infection. To better understand the function of the prrA-prrB two-component system, we defined the transcriptional characteristics of the prrA and prrB genes during exponential and stationary growth and upon exposure to different environmental stresses and attempted to generate a prrA-prrB deletion mutant. The prrA and prrB genes constitute an operon and are cotranscribed during logarithmic growth, with transcriptional levels decreasing in stationary phase and during hypoxia.

Mycobacterium tuberculosis protein kinase K confers survival advantage during early infection in mice and regulates growth in culture and during persistent infection: implications for immune modulation.

Mycobacterium tuberculosis serine/threonine protein kinases (STPKs) are key regulators of growth and metabolism; however, evidence for their roles in virulence is limited. In a preliminary screen based on comparative expression between strains H37Rv and H37Ra, six STPK genes, pknD, pknG, pknH, pknJ, pknK and pknL, showed higher expression in H37Rv. In the second screen, STPK expression was analysed in H37Rv-infected human macrophages.

DevR-mediated adaptive response in Mycobacterium tuberculosis H37Ra: links to asparagine metabolism.

The DevR transcriptional switch that defines the response of Mycobacterium tuberculosis to the lack of oxygen is now well established and likely helps the bacteria shift to a state of persistence. The M. tuberculosis two component signal transduction system (TCS), DevR-DevS, implicated in this transition to latency, is differentially expressed in H37Ra and H37Rv strains.

Three Mycobacterium tuberculosis Rel toxin-antitoxin modules inhibit mycobacterial growth and are expressed in infected human macrophages.

Mycobacterium tuberculosis protein pairs Rv1246c-Rv1247c, Rv2865-Rv2866, and Rv3357-Rv3358, here named RelBE, RelFG, and RelJK, respectively, were identified based on homology to the Escherichia coli RelBE toxin:antitoxin (TA) module. In this study, we have characterized each Rel protein pair and have established that they are functional TA modules. Overexpression of individual M.

The Mycobacterium tuberculosis TrcR response regulator represses transcription of the intracellularly expressed Rv1057 gene, encoding a seven-bladed beta-propeller.

The Mycobacterium tuberculosis TrcR response regulator binds and regulates its own promoter via an AT-rich sequence. Sequences within this AT-rich region determined to be important for TrcR binding were used to search the M. tuberculosis H37Rv genome to identify additional related TrcR binding sites.

Global expression analysis of two-component system regulator genes during Mycobacterium tuberculosis growth in human macrophages.

In the Mycobacterium tuberculosis H37Rv genome, there are 11 paired two-component regulatory system genes, two orphan histidine kinase genes, and six orphan response regulator genes. Expression of the 17 response regulator genes and the two orphan histidine kinase genes during growth of M. tuberculosis in human peripheral blood monocyte-derived macrophages has been analyzed by using cDNA mixtures prepared by the selective capture of transcribed sequences (SCOTS) technique.

Molecular genetics of Mycobacterium tuberculosis pathogenesis.

Tuberculosis (TB) has afflicted humankind throughout history. Approximately one third of the world's population is currently infected with Mycobacterium tuberculosis and nearly two million people die of TB annually. Although much has been learned about the structure of the tubercle bacillus, the epidemiology of TB, the physiological and immunological responses of the host to infection, and the physiology of M.

Mycobacterium avium genes expressed during growth in human macrophages detected by selective capture of transcribed sequences (SCOTS).

Selective capture of transcribed sequences (SCOTS) has been employed to identify 54 cDNA molecules that represent 46 genes that are expressed by Mycobacterium avium during growth in human macrophages. Some cDNA molecules correspond to genes that are apparently expressed 48 h after infection of macrophages, while others correspond to genes expressed 110 h after infection, and still others correspond to genes expressed throughout the course of infection in our model system. Genes expressed by M.

Expression, autoregulation, and DNA binding properties of the Mycobacterium tuberculosis TrcR response regulator.

The TrcRS two-component system of Mycobacterium tuberculosis is comprised of the TrcS histidine kinase and the TrcR response regulator, which is homologous to the OmpR class of DNA binding response regulators. Reverse transcription-PCRs with total RNA showed that the trcR and trcS two-component system genes are transcribed in broth-grown M. tuberculosis.

Identification of Mycobacterium tuberculosis RNAs synthesized in response to phagocytosis by human macrophages by selective capture of transcribed sequences (SCOTS).

A widely applicable, positive cDNA selection method was developed to identify RNAs synthesized by Mycobacterium tuberculosis in response to phagocytosis by cultured human primary macrophages. cDNAs for sigE and sigH (alternative sigma factors), aceA (isocitrate lyase), ponA (class I penicillin-binding protein), pks2 (polyketide synthase), uvrA (UvrABC endonuclease), and ctpV (putative cation transporter) were obtained from macrophage-grown bacteria. cDNAs for ORFs Rv3070, Rv3483c, Rv0903c (encoding a putative bacterial two-component transcriptional activator), and Rv0170 of the mce1 virulence operon also were obtained from phagocytized bacilli.

Cloning, sequencing, and expression of the mig gene of Mycobacterium avium, which codes for a secreted macrophage-induced protein.

Mycobacterium avium is an intracellular pathogen that has evolved to be a frequent cause of disseminated infection in immunocompromised patients. Although these bacilli are readily phagocytized, they are able to survive and even multiply within human macrophages. The process whereby mycobacteria circumvent the lytic functions of the macrophages is currently not well understood, but this is a key aspect in the pathogenicity of all pathogenic mycobacteria.

Immunological and functional characterization of Mycobacterium leprae protein antigens: an overview.

A major focus of leprosy research in the last 10 years has been the identification and characterization of antigens of Mycobacterium leprae that interact with antibodies and T cells of the host's immune response. Through the combined efforts of many different laboratories, a substantial number of protein antigens have been identified and characterized. In this MicroReview we present an updated list of M.

A Mycobacterium leprae gene encoding a fibronectin binding protein is used for efficient invasion of epithelial cells and Schwann cells.

Mycobacterium leprae, the causative agent of leprosy, is an obligate intracellular pathogen. M. leprae can infect a variety of cells in vivo, including epithelial cells, muscle cells, and Schwann cells, in addition to macrophages.