Rv1268c protein peptide inhibiting Mycobacterium tuberculosis H37Rv entry to target cells

Tuberculosis (TB) remains one of the most worrying infectious diseases affecting public health around the world; 8.7 million new TB cases were reported in 2011. The search for an Mycobacterium tuberculosis H37Rv protein sequence which is functionally important in host-pathogen interaction has been proposed for developing a new vaccine which will allow efficient and safe control of the spread of this disease.The present study thus reports the results obtained for the Rv1268c protein described in the M. tuberculosis H37Rv genome as a hypothetical unknown, probably secreted, protein based on a highly robust, specific, sensitive and functional approach to the search for potential epitopes to be included in an anti-tuberculosis vaccine. Rv1268c presence was determined by immunoblotting after obtaining polyclonal sera against mycobacterial total sonicate or subcellular fractions. Such sera were used in electron immunomicroscopy (EIM) for confirming protein localisation on the M. tuberculosis envelop by recognising colloidal gold-labelled immunoglobulin. Screening assays revealed the presence of two sequences having high binding activity: one binding A549 alveolar epithelial cells (¹⁴¹TGMAALEQYLGSGHAVIVSI¹⁶⁰) and other binding U937 monocyte-derived macrophages (²¹AVALGLASPADAAAGTMYGD⁴⁰). Such sequences’ ability to inhibit mycobacterial entry during in vitro assays was analysed. The structure of synthetic peptides binding to target cells was also determined, bearing in mind the structure–function relationship. These results, together with those obtained for other proteins, have been involved in selecting peptides which might be included in a subunit-based anti-tuberculosis vaccine.     

Characterization of culture filtrate proteins Rv1197 and Rv1198 of ESAT-6 family from Mycobacterium tuberculosis H37Rv

BackgroundWe have characterized two immunogenic proteins, Rv1197 and Rv1198, of the Esx-5 system of the ESAT-6 family of Mycobacterium tuberculosis H37Rv.MethodsThe complex formation between Rv1197 and Rv1198 was characterized by biophysical techniques. The reactivity of serum from TB patients towards these proteins was characterized by ELISA. Lymphocyte proliferation and cytokine induction were followed in restimulated splenocytes from immunized mice by using MTT assay and CBA flowcytometry, respectively.ResultsRv1197 and Rv1198 strongly interact to form a heterodimeric complex under reducing conditions, which is characterized by a dissociation constant of 97 × 10^− 9 M and melting temperature, Tm, of 50.5 °C. Strong humoral responses to Rv1197, Rv1198, CFP-10 and MoaC1 (Rv3111) antigens were found in Indian patients with active pulmonary tuberculosis (n = 44), in comparison to non-infected healthy individuals (n = 20). The seroreactivity to Rv1198 was characterized by a sensitivity of 75% and specificity of 90%. In BALB/c mice, immunization with Rv1198-FIA induced a pro-inflammatory response with elevated levels of TNF and IL-6, along with low induction of IFN-γ, IL-2 and IL-10, but no induction of IL-4.ConclusionRv1197 and Rv1198 form a stable complex, which is regulated by the redox state of Rv1198. Rv1198 is immunogenic with highly specific seroreactivity towards TB patients' serum. Rv1198 elicits a pro-inflammatory recall response in immunized mice.General SignificanceThis study characterizes the interaction of Rv1197 and Rv1198, and establishes the immunogenic nature of Rv1198.     

Functional characterization of Mycobacterium tuberculosis Rv2969c membrane protein

Identifying Mycobacterium tuberculosis membrane proteins involved in binding to and invasion of host cells is important in designing subunit-based anti-tuberculosis vaccines. The Rv2969c gene sequence was identified by PCR in M. tuberculosis complex strains, being transcribed in M. tuberculosis H37Rv, M. tuberculosis H37Ra, and M. bovis BCG. Rabbits immunized with synthetic peptides from highly specific conserved regions of this protein produced antibodies recognizing 27 and 29 kDa bands in M. tuberculosis lysate, which is consistent with the molecular weight of the Rv2969c gene product in M. tuberculosis H37Rv. Immunoelectron microscopy revealed the protein was localized on the bacillus surface. Four and three specific high activity binding peptides (HABPs) to the A549 alveolar epithelial and U937 monocyte cell lines were found, respectively. Two of the HABPs found inhibited M. tuberculosis invasion of A549 cells, suggesting that these peptides might be good candidates to be included in a multiepitopic, subunit-based anti-tuberculosis vaccine.     

Mycobacterium tuberculosis Rv3034c regulates mTORC1 and PPAR‐γ dependant pexophagy mechanism to control redox levels in macrophages

Mycobacterium tuberculosis survives inside the macrophages by employing several host immune evasion strategies. Here, we reported a novel mechanism in which M. tuberculosis acetyltransferase, encoded by Rv3034c, induces peroxisome homeostasis to regulate host oxidative stress levels to facilitate intracellular mycobacterial infection. Presence of M. tuberculosis Rv3034c induces the expression of peroxisome biogenesis and proliferation factors such as Pex3, Pex5, Pex19, Pex11b, Fis‐1 and DLP‐1; while depletion of Rv3034c decreased the expression of these molecules, thereby selective degradation of peroxisomes via pexophagy. Further studies revealed that M. tuberculosis Rv3034c inhibit induction of pexophagy mechanism by down‐regulating the expression of pexophagy associated proteins (p‐AMPKα, p‐ULK‐1, Atg5, Atg7, Beclin‐1, LC3‐II, TFEB and Keap‐1) and adaptor molecules (NBR1 and p62). Inhibition was found to be dependent on the phosphorylation of mTORC1 and activation of peroxisome proliferator activated receptor‐γ. In order to maintain intracellular homeostasis during oxidative stress, M. tuberculosis Rv3034c was found to induce degradation of dysfunctional and damaged peroxisomes through activation of Pex14 in infected macrophages. In conclusion, this is the first report which demonstrated that M. tuberculosis acetyltransferase regulate peroxisome homeostasis in response to intracellular redox levels to favour mycobacterial infection in macrophage.     

A Novel Method of Identifying Mycobacterium tuberculosis Beijing Strains by Detecting SNPs in Rv0444c and Rv2629

A particular genotype of tuberculosis, named Beijing strain, is strongly associated with drug resistance and high virulence. Therefore, rapid prospective identification of Mycobacterium tuberculosis Beijing strains is very important for identifying and controlling tuberculosis of Beijing genotype. In the present study, we found that the co-mutation, A191C in Rv2629 and G243C in Rv0444c, is closely related to Beijing genotype. Gene Rv2629 and Rv0444c of 139 clinical isolates of M. tuberculosis were analyzed by PCR amplification and sequencing. Among 99 Beijing strains, 86 % (n = 85) isolates had the mutation G243C in Rv0444c and 92.93 % (n = 92) isolates had the mutation A191C in Rv2629. Among 40 non-Beijing isolates, only six isolates carried the mutation G243C in Rv0444c and eight isolates carried the mutation A191C in Rv2629. The co-mutation existed in 84.85 % (n = 84) of 99 clinical genome samples of W-Beijing strains and in only 12.5 % (n = 5) of the 40 non-Beijing strains, and the positive predictive value of 94.38 %, obtained in our experiment with a designed ratio of Beijing isolates, is similar to that in China at present. This result suggested that the detection method of the co-mutation, A191C in Rv2629 and G243C in Rv0444c, proposed in this study was a rapid, reliable, and sensitive one for identifying tuberculosis with Beijing genotype.     

Inaugural structure from the DUF3349 superfamily of proteins, Mycobacterium tuberculosis Rv0543c

The first structure for a member of the DUF3349 (PF11829) family of proteins, Rv0543c from Mycobacterium tuberculosis, has been determined using NMR-based methods and some of its biophysical properties characterized. Rv0543c is a 100 residue, 11.3 kDa protein that both size exclusion chromatography and NMR spectroscopy show to be a monomer in solution. The structure of the protein consists of a bundle of five α-helices, α1 (M1 – Y16), α2 (P21 – C33), α3 (S37 – G52), α4 (G58 – H65) and α5 (S72 – G87), held together by a largely conserved group of hydrophobic amino acid side chains. Heteronuclear steady-state {¹H}–¹⁵N NOE, T₁, and T₂ values are similar through-out the sequence indicating that the backbones of the five helices are in a single motional regime. The thermal stability of Rv0543c, characterized by circular dichroism spectroscopy, indicates that Rv0543c irreversibly unfolds upon heating with an estimated melting temperature of 62.5 °C. While the biological function of Rv0543c is still unknown, the presence of DUF3349 proteins predominately in Mycobacterium and Rhodococcus bacterial species suggests that Rv0543 may have a biological function unique to these bacteria, and consequently, may prove to be an attractive drug target to combat tuberculosis.     

Mechanistic analysis of Mycobacterium tuberculosis Rv1347c, a lysine Nepsilon-acyltransferase involved in mycobactin biosynthesis

Mycobactin acylation plays a crucial role in the ability of Mycobacterium tuberculosis to acquire intracellular iron during infection. M. tuberculosis Rv1347c, the lysine N^ε-acyltransferase responsible for mycobactin acylation, represents a valid target for the development of novel anti-tubercular agents. Here we investigate the substrate specificity of Rv1347c, evaluate its kinetic mechanism and probe the contributions of active-site residues to catalysis. Our results confirm that Rv1347c demonstrates a preference for longer acyl-chains and suggest that mycobactin acylation occurs subsequent to mycobactin core assembly. Steady-state bisubstrate kinetics and dead-end inhibitor studies support a random sequential kinetic mechanism. Analysis of the pH dependence of k_cat/K_m revealed the presence of two groups that must be deprotonated for efficient catalysis. Mutagenesis of His¹³⁰ and Asp¹⁶⁸ indicated that both residues are critical for acyltransferase activity and suggests that His¹³⁰ is responsible for general base activation of the ε-amino group of lysine.     

The role of ABC efflux pump,Rv1456c-Rv1457c-Rv1458c,from <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> clinical isolates in China

Recently the ATP-binding cassette (ABC) efflux pumps have been proved to be a major component of drug resistance in Mycobacterium tuberculosis. The objective of this study was to investigate the expression profiles of Rv1456c-Rv1457c-Rv1458c efflux system in clinical isolates of M. tuberculosis and its involvement in drug-resistance mechanisms. Significantly increased mRNA expression of Rv1456c, Rv1457c, and Rv1458c appeared among the clinical isolates (P < 0.05), which are resistant to at least one of the four first-line drugs including rifampin, isoniazid, streptomycin, and ethambutol. In addition, overexpression of this efflux system was more frequently found in multidrug-resistant and extensively drug-resistant M. tuberculosis strains. Therefore, Rv1456c-Rv1457c-Rv1458c efflux pumps may play an important role in drug resistance of treatment of M. tuberculosis. Further investigation of this gene may lead to the development of countermeasures against M. tuberculosis drug resistance.     

Structural and Biochemical Insight into the Mechanism of Rv2837c from Mycobacterium tuberculosis as a c-di-NMP Phosphodiesterase

The intracellular infections of Mycobacterium tuberculosis, which is the causative agent of tuberculosis, are regulated by many cyclic dinucleotide signaling. Rv2837c from M. tuberculosis is a soluble, stand-alone DHH-DHHA1 domain phosphodiesterase that down-regulates c-di-AMP through catalytic degradation and plays an important role in M. tuberculosis infections. Here, we report the crystal structure of Rv2837c (2.0 Å), and its complex with hydrolysis intermediate 5′-pApA (2.35 Å). Our structures indicate that both DHH and DHHA1 domains are essential for c-di-AMP degradation. Further structural analysis shows that Rv2837c does not distinguish adenine from guanine, which explains why Rv2837c hydrolyzes all linear dinucleotides with almost the same efficiency. We observed that Rv2837c degraded other c-di-NMPs at a lower rate than it did on c-di-AMP. Nevertheless, our data also showed that Rv2837c significantly decreases concentrations of both c-di-AMP and c-di-GMP in vivo. Our results suggest that beside its major role in c-di-AMP degradation Rv2837c could also regulate c-di-GMP signaling pathways in bacterial cell.     

Chemical shift assignments for Rv0577, a putative glyoxylase associated with virulence from Mycobacterium tuberculosis

Approximately one-third of mankind has been exposed to Mycobacterium tuberculosis, the etiological agent responsible for tuberculosis (TB). As part of an effort to develop a new generation of anti-TB agents, the chemical shifts for the 261-residue, virulence-associated protein Rv0577 from M. tuberculosis has been extensively assigned.     

Deep N-terminomics of Mycobacterium tuberculosis H37Rv extensively correct annotated encoding genes

Mycobacterium tuberculosis (MTB) is a severe causing agent of tuberculosis (TB). Although H37Rv, the type strain of M. tuberculosis was sequenced in 1998, annotation errors of encoding genes have been frequently reported in hundreds of papers. This phenomenon is particularly severe at the 5′ end of the genes. Here, we applied a TMPP [(N-Succinimidyloxycarbonylmethyl) tris (2,4,6-trimethoxyphenyl) phosphonium bromide] labeling combined with StageTip separating strategy on M. tuberculosis H37Rv to characterize the N-terminal start sites of its annotated encoding genes. Totally, 1047 proteins were identified with 2058 TMPP labeled N-terminal peptides from all the 2625 mass spectrometer (MS) sequenced proteins. Comparative genomics analysis allowed the re-annotation of 43 proteins' N-termini in H37Rv and 762 proteins in Mycobacteriaceae. All revised N-termini start sites were distributed in 5’-UTR of annotated genes due to over-annotation of previous N-terminal initiation codon, especially the ATG. In addition, we identified and verified a novel gene Rv1078A in +3 frame different from the annotated gene Rv1078 in +2 frame. Altogether, our findings contribute to the better understanding of N-terminal of H37Rv and other species from Mycobacteriaceae that can assist future studies on biological study.     

Intranasal immunization with Mycobacterium tuberculosis Rv3615c induces sustained adaptive CD4+ T‐cell and antibody responses in the respiratory tract

Sustained adaptive immunity to pathogens provides effective protection against infections, and effector cells located at the site of infection ensure rapid response to the challenge. Both are essential for the success of vaccine development. To explore new vaccination approach against Mycobacterium tuberculosis (M.tb) infection, we have shown that Rv3615c, identified as ESX‐1 substrate protein C of M.tb but not expressed in BCG, induced a dominant Th1‐type response of CD4⁺ T cells from patients with tuberculosis pleurisy, which suggests a potential candidate for vaccine development. But subcutaneous immunization with Rv3615c induced modest T‐cell responses systemically, and showed suboptimal protection against virulent M.tb challenge at the site of infection. Here, we use a mouse model to demonstrate that intranasal immunization with Rv3615c induces sustained capability of adaptive CD4⁺ T‐ and B‐cell responses in lung parenchyma and airway. Rv3615c contains a dominant epitope of mouse CD4⁺ T cells, Rv3615c_41‐50, and elicits CD4⁺ T‐cell response with an effector–memory phenotype and multi‐Th1‐type cytokine coexpressions. Since T cells resident at mucosal tissue are potent at control of infection at early stage, our data show that intranasal immunization with Rv3615c promotes a sustained regional immunity to M.tb, and suggests a potency in control of M.tb infection. Our study warranties a further investigation of Rv3615c as a candidate for development of effective vaccination against M.tb infection.     

Aptamer inhibits <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> (H37Rv) invasion of macrophage

There is an urgent need to develop new anti-tuberculosis drugs due to the rising tendency in tuberculosis (TB) around the world. It is known that Mycobacterium tuberculosis (M. tuberculosis) generally infects mammalian host via aerosol route. The pathogenic process has been fully studied that it can initially invade alveolar macrophage, then established stable residence within those phagocytic cells, suggesting that one of the possible ways to prevent this pathogen is to inhibit its invasion and growth in the macrophage. Aptamers from SELEX (Systematic Evolution of Ligands by Exponential Enrichment) have been used to rival virulent M. tuberculosis (H37Rv) in our previous work, and the materials to which aptamers bound were proved to be some outer membrane proteins of H37Rv. In the present study, the interaction between M. tuberculosis and macrophage in the presence of aptamers was investigated in more details. The results suggested that the selective aptamers significantly inhibited H37Rv invasion of macrophage in vitro, and the effect correspond to the binding affinity of these aptamers to H37Rv. The values of equilibrium dissociation constant (Kd) was calculated by flow cytometry, all in the nanomolar range, showed much higher affinity to H37Rv than M. bovis Bacillus Guerin (BCG). Moreover, the aptamer-treated H37Rv can stimulate IFN-γ, IL-15 and IL-17 secretion of macrophages compared with H37Rv (no treated). In summary, our data indicated that the NK2 aptamer not only acted as anti-tuberculosis agent by inhibiting virulent M. tuberculosis (H37Rv) invasion of macrophage, but also might be used as molecular probe for exploring the interaction between the outer membrane of M. tuberculosis and macrophage.     

Identification of two Mycobacterium tuberculosis H37Rv ORFs involved in resistance to killing by human macrophages

Background  

The ability of Mycobacterium tuberculosis to survive and replicate in macrophages is crucial for the mycobacterium's ability to infect the host and cause tuberculosis. To identify Mycobacterium tuberculosis genes involved in survival in macrophages, a library of non-pathogenic Mycobacterium smegmatis bacteria, each carrying an individual integrated cosmid containing M. tuberculosis H37Rv genomic DNA, was passed through THP-1 human macrophages three times.     

Mycobacterium tuberculosis Rv3651 is a triple sensor‐domain protein

The genome of the human pathogen Mycobacterium tuberculosis (Mtb) encodes ～4,400 proteins, but one third of them have unknown functions. We solved the crystal structure of Rv3651, a hypothetical protein with no discernible similarity to proteins with known function. Rv3651 has a three‐domain architecture that combines one cG MP‐specific phosphodiesterases, a denylyl cyclases and F hlA (GAF) domain and two P er‐A RNT‐S im (PAS) domains. GAF and PAS domains are sensor domains that are typically linked to signaling effector molecules. Unlike these sensor‐effector proteins, Rv3651 is an unusual sensor domain‐only protein with highly divergent sequence. The structure suggests that Rv3651 integrates multiple different signals and serves as a scaffold to facilitate signal transfer.     

Analysis of intracellular expressed proteins of Mycobacterium tuberculosis clinical isolates

Background

Tuberculosis (TB) is the most threatening infectious disease globally. Although progress has been made to reduce global incidence of TB, emergence of multidrug resistant (MDR) TB threatens to undermine these advances. To combat the disease, novel intervention strategies effective against drug resistant and sensitive subpopulations of M. tuberculosis are urgently required as adducts in the present treatment regimen. Using THP-1 cells we have analyzed and compared the global protein expression profile of broth-cultured and intraphagosomally grown drug resistant and sensitive M.tuberculosis clinical isolates.

Results

On comparing the two dimensional (2-DE) gels, many proteins were found to be upregulated/expressed during intracellular state which were identified by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). Four proteins (adenosylhomocysteinase, aspartate carbomyltransferase, putatitive thiosulfate sulfurtransferase and universal stress protein) were present in both intracellular MDR and sensitive isolates and three of these belonged to intermediary metabolism and respiration category. Two proteins (alanine dehydrogenase and adenosine kinase) of intracellular MDR isolate and two (glucose-6-phosphate isomerase and ATP synthase epsilon chain) of intracellular sensitive isolate belonged to intermediary metabolism and respiration category. One protein (Peroxidase/Catalase) of intracellular MDR and three (HSPX, 14 kDa antigen and 10 kDa chaperonin) of sensitive isolate belonged to virulence, detoxification and adaptation category. ESAT-6 of intracellular MDR belonged to cell wall and cell processes category. Two proteins (Antigen 85-C and Antigen 85-A) of intracellular sensitive isolate were involved in lipid metabolism while probable peptidyl-prolyl cis-trans isomerase A was involved in information pathways. Four (Rv0635, Rv1827, Rv0036c and Rv2032) of intracellular MDR and two proteins (Rv2896c and Rv2558c) of sensitive isolate were hypothetical proteins which were functionally characterized using bioinformatic tools. Bioinformatic findings revealed that the proteins encoded by Rv0036, Rv2032c, Rv0635, Rv1827 and Rv2896c genes are involved in cellular metabolism and help in intracellular survival.

Conclusions

Mass spectrometry and bioinformatic analysis of both MDR and sensitive isolates of M. tuberculosis during intraphagosomal growth showed that majority of commonly upregulated/expressed proteins belonged to the cellular metabolism and respiration category. Inhibitors of the metabolic enzymes/intermediate can therefore serve as suitable drug targets against drug-resistant and sensitive subpopulations of M. tuberculosis.