Josephine E. Clark-Curtiss

Mycobacterium avium Genes Expressed during Growth in Human Macrophages Detected by Selective Capture of Transcribed Sequences (SCOTS)

ABSTRACT 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. avium during growth in macrophages include genes encoding enzymes of several biosynthetic pathways (pyrimidines, mycobactin, and polyketides); genes that encode enzymes involved in intermediary metabolism, energy metabolism (tricarboxylic acid cycle, glyoxalate shunt), and nitrogen metabolism; and genes that encode regulatory proteins. A number of genes of unknown function were also identified, including genes that code for proteins similar to members of the PPE family of proteins of Mycobacterium tuberculosis and proteins similar to those encoded by the M. tuberculosis mce genes, which have been previously associated with mycobacterial virulence. The SCOTS technique, followed by enrichment for cDNA molecules that are up-regulated or are uniquely expressed by M. avium during growth in human macrophages (compared to growth in laboratory broth culture), allows recovery and identification of a greater diversity of cDNA molecules than does subtractive hybridization between cDNA mixtures from macrophage-grown and broth-grown M. avium. Data are presented demonstrating the reproducibility of recovery of a subset of cDNA molecules from cDNA mixtures purified by SCOTS on several different occasions. These results further demonstrate the beneficial utility of the SCOTS technique for identifying genes whose products are needed for successful survival and growth by an organism in a specific environment.

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. tuberculosis rel toxin genes relE, relG, and relK induced growth arrest in Mycobacterium smegmatis; a phenotype that was completely reversible by expression of their cognate antitoxin genes, relB, relF, and relJ, respectively. We also provide evidence that RelB and RelE interact directly, both in vitro and in vivo. Analysis of the genetic organization and regulation established that relBE, relFG, and relJK form bicistronic operons that are cotranscribed and autoregulated, in a manner unlike typical TA modules. RelB and RelF act as transcriptional activators, inducing expression of their respective promoters. However, RelBE, RelFG, and RelJK (together) repress expression to basal levels of activity, while RelJ represses promoter activity altogether. Finally, we have determined that all six rel genes are expressed in broth-grown M. tuberculosis, whereas relE, relF, and relK are expressed during infection of human macrophages. This is the first demonstration of M. tuberculosis expressing TA modules in broth culture and during infection of human macrophages.

Molecular and immunological analysis of a fibronectin‐binding protein antigen secreted by Mycobacterium leprae

By screening a Mycobacterium lepraeλgt11 genomic DNA library with leprosy‐patient sera we have previously identified 50 recombinant clones that expressed novel M. leprae antigens (Sathish et al., 1990). In this study, we show by DNA sequencing and immunoblot analysis that three of these clones express a M. leprae homologue of the fibronectin‐binding antigen 85 complex of mycobacteria. The complete gene was characterized and it encodes a 327‐amino‐acid polypeptide, consisting of a consensus signal sequence of 38 amino acids followed by a mature protein of 289 amino acids. This is the first sequence of a member of the M. leprae antigen 85 complex, and Southern blotting analysis indicated the presence of multiple genes of the 85 complex in the genome of M. leprae. The amino acid sequence displays 75–85% sequence identity with components of the antigen 85 complex from M. tuberculosis, M. bovis BCG and M. kansasii. Furthermore, antibodies to the antigen 85 complex of M. tuberculosis and M. bovis BCG reacted with two fusion proteins containing the amino acid regions 55–266 and 265–327 of the M. leprae protein. The M. leprae 30/31 kDa protein induces strong humoral and cellular responses, as judged by Western blot analysis with patient sera and proliferation of T cells derived from healthy individuals and leprosy patients. Amino acid regions 55–266 and 265–327 both were shown to bind to fibronectin, indicating the presence of at least two fibronectin‐binding sites on the M. leprae protein. These data indicate that this 30/31 kDa protein is not only important in the immune response against M. leprae, but may also have a biological role in the interaction of this bacillus with the human host.

Turning self-destructing Salmonella into a universal DNA vaccine delivery platform

We previously developed a biological containment system using recombinant Salmonella Typhimurium strains that are attenuated yet capable of synthesizing protective antigens. The regulated delayed attenuation and programmed self-destructing features designed into these S. Typhimurium strains enable them to efficiently colonize host tissues and allow release of the bacterial cell contents after lysis. To turn such a recombinant attenuated Salmonella vaccine (RASV) strain into a universal DNA vaccine-delivery vehicle, our approach was to genetically modify RASV strains to display a hyperinvasive phenotype to maximize Salmonella host entry and host cell internalization, to enable Salmonella endosomal escape to release a DNA vaccine into the cytosol, and to decrease Salmonella-induced pyroptosis/apoptosis that allows the DNA vaccine time to traffic to the nucleus for efficient synthesis of encoded protective antigens. A DNA vaccine vector that encodes a domain that contributes to the arabinose-regulated lysis phenotype but has a eukaryotic promoter was constructed. The vector was then improved by insertion of multiple DNA nuclear-targeting sequences for efficient nuclear trafficking and gene expression, and by increasing nuclease resistance to protect the plasmid from host degradation. A DNA vaccine encoding influenza WSN virus HA antigen delivered by the RASV strain with the best genetic attributes induced complete protection to mice against a lethal influenza virus challenge. Adoption of these technological improvements will revolutionize means for effective delivery of DNA vaccines to stimulate mucosal, systemic, and cellular protective immunities, and lead to a paradigm shift in cost-effective control and prevention of a diversity of diseases.

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. Analysis of the trcR and trcS genes using various SCOTS (selective capture of transcribed sequences) probes also confirmed that these genes are expressed in broth-grown cultures and after 18 h of M. tuberculosis growth in cultured human primary macrophages. To determine if the TrcR response regulator is autoregulated, a trcR-lacZ fusion plasmid and a TrcR expression plasmid were cotransformed into Escherichia coli. Upon induction of the TrcR protein, there was a >500-fold increase in beta-galactosidase activity from the trcR-lacZ fusion, indicating that TrcR is involved in transcriptional autoactivation. Gel mobility shift assays with the trcR promoter and TrcR established that the response regulator was autoregulating via direct binding. By use of a delimiting series of overlapping trcR PCR fragments in gel mobility shift assays with TrcR, an AT-rich region of the trcR promoter was shown to be essential for TrcR binding. Additionally, this AT-rich sequence was protected by TrcR in DNase I protection assays. To further analyze the role of the AT-rich region in TrcR autoregulation, the trcR promoter was mutated and analyzed in lacZ transcriptional fusions in the presence of TrcR. Alteration of the AT-rich sequence in the trcR promoter resulted in the loss of trcR transcriptional activation in the presence of TrcR. This report indicates that the M. tuberculosis TrcR response regulator activates its own expression by interacting with the AT-rich sequence of the trcR promoter.

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. Interestingly, significant expression of pknK was detected only at 18 h post-infection, suggesting its involvement in early infection events. We have investigated the roles of PknK in vitro and in vivo. PknK levels were induced under stationary phase and deletion of pknK resulted in increased resistance of the mutant to acidic pH, hypoxia, oxidative and stationary-phase stresses in vitro. These results, together with the increased survival of the ΔpknK strain during persistent infection in mice, reveal a role for PknK in adaptive mechanisms that slow the growth of mycobacteria. A novel finding of this study was the inhibition of growth of ΔpknK strain during acute infection in mice that correlated with the significant upregulation of tumour necrosis factor as well as the simultaneous downregulation of interleukin-12p40, interferon-γ and induced nitric oxide synthase transcripts. Finally, we provide evidence for the localization of PknK during infection and discuss its implications in pathogenesis.

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. Despite the transcriptional differences, PrrA protein levels remained relatively stable throughout growth and in hypoxia. Under conditions of nitrogen limitation, prrAB transcription was induced, while acidic pH stress and carbon starvation did not significantly alter transcript levels. Deletion of the prrAB operon on the chromosome of M. tuberculosis H37Rv occurred only in the presence of an episomal copy of the prrAB genes, indicating that this two-component system is essential for viability. Characterization of the prrAB locus in M. tuberculosis Mt21D3, a previously described prrA transposon mutant, revealed that this strain is not a true prrA knockout mutant. Rather, Tn5367 transposon insertion into the prrA promoter only decreased prrA and prrB transcription and PrrA levels in Mt21D3 compared to those in the parental Mt103 clinical strain. These data provide the first report describing the essentiality of the M. tuberculosis prrAB two-component system and reveal insights into its potential role in mycobacterial growth and metabolism.

Live Attenuated Salmonella Vaccines against Mycobacterium tuberculosis with Antigen Delivery via the Type III Secretion System

ABSTRACT 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. Oral immunization of mice with RASV strains synthesizing SopE–ESAT-6–CFP-10 fusion proteins resulted in significant protection of the mice against aerosol challenge with M. tuberculosis H37Rv that was similar to the protection afforded by immunization with Mycobacterium bovis bacillus Calmette-Guérin (BCG) administered subcutaneously. In addition, oral immunization with the RASV strains specifying these mycobacterial antigens elicited production of significant antibody titers to ESAT-6 and production of ESAT-6- or CFP-10-specific gamma interferon (IFN-γ)-secreting and tumor necrosis factor alpha (TNF-α)-secreting splenocytes.

The Mycobacterium tuberculosis TrcR Response Regulator Represses Transcription of the Intracellularly Expressed Rv1057 Gene, Encoding a Seven-Bladed β-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. A similar AT-rich sequence was identified within the intergenic region located upstream of the Rv1057 gene. In the present work, we demonstrate that TrcR binds to a 69-bp AT-rich sequence within the Rv1057 intergenic region and generates specific contacts on the same side of the DNA helix. An M. tuberculosis trcRS deletion mutant, designated STS10, was constructed and used to determine that TrcR functions as a repressor of Rv1057 expression. Additionally, identification of the Rv1057 transcriptional start site suggests that a SigE-regulated promoter also mediates control of Rv1057 expression. Using selective capture of transcribed sequences (SCOTS) analysis as an evaluation of intracellular expression, Rv1057 was shown to be expressed during early M. tuberculosis growth in human macrophages, and the Rv1057 expression profile correlated with a gene that would be repressed by TrcR. Based on structural predictions, motif analyses, and molecular modeling, Rv1057 consists of a series of antiparallel beta-strands which adopt a beta-propeller fold, and it was determined to be the only seven-bladed beta-propeller encoded in the M. tuberculosis genome. These results provide evidence of TrcR response regulator repression of the Rv1057 beta-propeller gene that is expressed during growth of M. tuberculosis within human macrophages.

Microbial gene expression elucidated by selective capture of transcribed sequences (SCOTS)

Publisher Summary This chapter describes selective capture of transcribed sequences (SCOTS), a technique that combines total ribonucleic acid (RNA) isolation, followed by complementary deoxyribonucleic acid (cDNA) synthesis, hybridization with biotinylated genomic deoxyribonucleic acid (DNA), polymerase chain reaction (PCR) amplification, cloning, and Southern blot hybridization, all of which are commonly used techniques in molecular biology. SCOTS could be used to analyze gene expression of a pathogen present at different sites (i.e., different organs or tissues) within the host. SCOTS could also be used to compare gene expression between mutant and wild-type strains grown under a specific condition. Analysis of microbe-host interactions by SCOTS is a powerful tool for the global analysis of microbial gene expression inside the natural host. Moreover, any potential candidate antigen can be tested for its expression when bacteria are growing in the host cell. Understanding the metabolic pathways used by bacteria growing in host cells could suggest potential targets for development of new antibiotics. The information obtained by SCOTS can lead to identification of specific proteins produced at high levels within antigen-presenting cells, providing candidates for development of effective vaccines against bacterial infections.