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.     

Rv1698 of Mycobacterium tuberculosis represents a new class of channel-forming outer membrane proteins

Mycobacteria contain an outer membrane composed of mycolic acids and a large variety of other lipids. Its protective function is an essential virulence factor of Mycobacterium tuberculosis. Only OmpA, which has numerous homologs in Gram-negative bacteria, is known to form channels in the outer membrane of M. tuberculosis so far. Rv1698 was predicted to be an outer membrane protein of unknown function. Expression of rv1698 restored the sensitivity to ampicillin and chloramphenicol of a Mycobacterium smegmatis mutant lacking the main porin MspA. Uptake experiments showed that Rv1698 partially complemented the permeability defect of the M. smegmatis porin mutant for glucose. These results indicated that Rv1698 provides an unspecific pore that can partially substitute for MspA. Lipid bilayer experiments demonstrated that purified Rv1698 is an integral membrane protein that indeed produces channels. The main single channel conductance is 4.5 +/- 0.3 nanosiemens in 1 M KCl. Zero current potential measurements revealed a weak preference for cations. Whole cell digestion of recombinant M. smegmatis with proteinase K showed that Rv1698 is surface-accessible. Taken together, these experiments demonstrated that Rv1698 is a channel protein that is likely involved in transport processes across the outer membrane of M. tuberculosis. Rv1698 has single homologs of unknown functions in Corynebacterineae and thus represents the first member of a new class of channel proteins specific for mycolic acid-containing outer membranes.     

The Mycobacterium tuberculosis membrane protein Rv2560--biochemical and functional studies

The characterization of membrane proteins having no identified function in Mycobacterium tuberculosis is important for a better understanding of the biology of this pathogen. In this work, the biological activity of the Rv2560 protein was characterized and evaluated. Primers used in PCR and RT-PCR assays revealed that the gene encoding protein Rv2560 is present in M. tuberculosis complex strains, but transcribed in only some of them. Sera obtained from rabbits inoculated with polymer peptides from this protein recognized a 33 kDa band in the M. tuberculosis lysate and a membrane fraction corresponding to the predicted molecular mass (33.1 kDa) of this protein. Immunoelectron microscopy analysis found this protein on the mycobacterial membrane. Sixteen peptides covering its entire length were chemically synthesized and tested for their ability to bind to A549 and U937 cells. Peptide 11024 (121VVALSDRATTAYTNTSGVSS140) showed high specific binding to both cell types (dissociation constants of 380 and 800 nm, respectively, and positive receptor-ligand interaction cooperativity), whereas peptide 11033 (284LIGIPVAALIHVYTYRKLSGG304) displayed high binding activity to A549 cells only. Cross-linking assays showed the specific binding of peptide 11024 to a 54 kDa membrane protein on U937. Invasion inhibition assays, in the presence of shared high-activity binding peptide identified for U937 and A549 cells, presented maximum inhibition percentages of 50.53% and 58.27%, respectively. Our work highlights the relevance of the Rv2560 protein in the M. tuberculosis invasion process of monocytes and epithelial cells, and represents a fundamental step in the rational selection of new antigens to be included as components in a multiepitope, subunit-based, chemically synthesized, antituberculosis vaccine.     

Deoxyribonucleic acid replication time in Mycobacterium tuberculosis H37 Rv

The DNA increment method, designed for measuring the increment in the amount of DNA after inhibition of initiation of fresh rounds of replication initiation was employed to measure the rate of deoxyribonucleic acid (DNA) chain growth in Mycobacterium tuberculosis H37Rv growing in Youman and Karlson's medium at 37°C with a generation time of 24 h and also in relatively fast growing species like Mycobacterium smegmatis and Escherichia coli. From the results obtained, the time required for a DNA replication fork to traverse the chromosome from origin to terminus (C period) was calculated. The chain elongation rates of DNA of the three organisms was determined from the C period and the known genome sizes assuming that all these genomes have a single replication origin and bidirectional replication fork. The rate for M. tuberculosis was 3,200 nucleotides per min about 11 times slower than that of M. smegmatis and about 13–18 times slower than that of E. coli.Abbreviations DNA deoxyribonucleic acid - td delay in initiation - OD optical density - CAM chloramphenicol - RIF rifampicin     

结核分枝杆菌蛋白Rv3425对细菌表型及毒力影响的研究

为探索蛋白Rv3425在结核分枝杆菌(Mycobacterium tuberculosis,M. tuberculosis)中的功能,本研究以耻垢分枝杆菌(Mycobacterium smegmatis,M. smegmatis)为模式菌株,构建重组了耻垢分枝杆菌Ms-Rv3425。分别将构建菌株(Ms-Rv3425)、野生株(Ms)及空载对照(Ms-Pact)接种于7H9-OADC培养基中37 ℃培养,观察Ms-Rv3425与Ms及Ms-Pact之间在生长速率、菌落形态、生物膜以及聚集度方面的差异。分别用低pH值以及含有十二烷基磺酸钠(sodium dodecyl sulfate,SDS)、氨苄西林、异烟肼及利福平的培养基进行培养,计算存活率以分析抗逆和抗药能力;用上述压力条件培养结核分枝杆菌标准株H37Ra,分析Rv3425内源表达量的变化;进行THP-1细胞感染和BALB/c小鼠攻毒实验分析菌株的毒性变化。结果显示,与Ms及Ms-Pact相比,Ms-Rv3425的菌落形态更为粗糙且隆起,成膜及聚集能力增强;在压力条件下,Ms-Rv3425表现出更高的抗逆和抗药能力,H37Ra中Rv3425的表达量也显著上调;胞内存活率及小鼠致死率更高,各脏器病理损伤更为严重。综上所述,过表达Rv3425能够改变耻垢分枝杆菌的表型,提高抗逆性、抗药性和毒力。深入探讨PPE家族蛋白Rv3425的功能,将为结核病的防治带来新的视角。     

Siderocalin inhibits the intracellular replication of Mycobacterium tuberculosis in macrophages

Siderocalin is a secreted protein that binds to siderophores to prevent bacterial iron acquisition. While it has been shown to inhibit the growth of Mycobacterium tuberculosis ( M.tb ) in extracellular cultures, its effect on this pathogen within macrophages is not clear. Here, we show that siderocalin expression is upregulated following M.tb infection of mouse macrophage cell lines and primary murine alveolar macrophages. Furthermore, siderocalin added exogenously as a recombinant protein or overexpressed in the RAW264.7 macrophage cell line inhibited the intracellular growth of the pathogen. A variant form of siderocalin, which is expressed only in the macrophage cytosol, inhibited intracellular M.tb growth as effectively as the normal, secreted form, an observation that provides mechanistic insight into how siderocalin might influence iron acquisition by the bacteria in the phagosome. Our findings are consistent with an important role for siderocalin in protection against M.tb infection and suggest that exogenously administered siderocalin may have therapeutic applications in tuberculosis.     

Peptides derived from Mycobacterium tuberculosis Rv2301 protein are involved in invasion to human epithelial cells and macrophages

The specific function of putative cut2 protein (or CFP25), encoded by the Rv2301 gene from Mycobacterium tuberculosis H37Rv, has not been identified yet. The aim of this study was to assess some of CFP25 characteristics and its possible biological role in Mycobacterium tuberculosis H37Rv invasion process to target cells. Molecular assays indicated that the gene encoding Rv2301 is present and transcribed in M. tuberculosis complex strains. The presence of Rv2301 protein over the bacilli surface was confirmed by Western blot and immunoelectron microscopy analyses, using goats sera inoculated with synthetic peptides derived from Rv2301 protein. Receptor–ligand binding assays with carcinomic human alveolar basal epithelial cells (A549) and macrophages derived from human histolytic lymphoma monocytes (U937) allowed us to identify five high activity binding peptides (HABPs) in both cell lines, and two additional HABPs only in A549 cells. U937 HABPs binding interactions were characterized by saturation assays, finding dissociation constants (K _d) within the nanomolar range and positive cooperativity (n _H?>?1). Inhibition assays were performed to assess the possible biological role of Rv2301 identified HABPs, finding that some of them were able to inhibit invasion at a 5?μM concentration, compared with the cytochalasin control. On the other hand, HABPs, and especially HABP 36507 located at the N-terminus of the protein, facilitated the internalization of fluorescent latex beads into A549 cells. These findings are of vital importance for the rational selection of Rv2301 HABPs, to be included as components of an antituberculosis vaccine.     

Crystal structure of the conserved hypothetical protein Rv1155 from Mycobacterium tuberculosis

With the aim of elucidating the biological function of hypothetical proteins unique amongst the Actynomyces sub-group of bacteria, we have solved the crystal structure of the conserved hypothetical protein Rv1155 from Mycobacterium tuberculosis at 1.8 A resolution. Rv1155 is a homodimer both in the crystal structure and in solution and folds into two separate domains consisting of a six-stranded anti-parallel beta-barrel fold flanked by two alpha-helices and a helix-turn-helix domain. Both domains contribute to the formation of two deep clefts at the dimer interface. The overall fold of Rv1155 strikingly resembles that of flavin mononucleotide-binding protein and pyridoxamine 5'-phosphate oxydase, but the architecture of the putative binding pocket is markedly different, consistent with the lack of color of Rv1155 and its inability to bind FMN. Rv1155 thus appears to belong to a group of proteins with stringent conservation of the binding cleft, having evolved towards a new binding function.     

Solution structure of the conserved hypothetical protein Rv2302 from Mycobacterium tuberculosis

The Mycobacterium tuberculosis protein Rv2302 (80 residues; molecular mass of 8.6 kDa) has been characterized using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. While the biochemical function of Rv2302 is still unknown, recent microarray analyses show that Rv2302 is upregulated in response to starvation and overexpression of heat shock proteins and, consequently, may play a role in the biochemical processes associated with these events. Rv2302 is a monomer in solution as shown by size exclusion chromatography and NMR spectroscopy. CD spectroscopy suggests that Rv2302 partially unfolds upon heating and that this unfolding is reversible. Using NMR-based methods, the solution structure of Rv2302 was determined. The protein contains a five-strand, antiparallel beta-sheet core with one C-terminal alpha-helix (A61 to A75) nestled against its side. Hydrophobic interactions between residues in the alpha-helix and beta-strands 3 and 4 hold the alpha-helix near the beta-sheet core. The electrostatic potential on the solvent-accessible surface is primarily negative with the exception of a positive arginine pocket composed of residues R18, R70, and R74. Steady-state {(1)H}-(15)N heteronuclear nuclear Overhauser effects indicate that the protein's core is rigid on the picosecond timescale. The absence of amide cross-peaks for residues G13 to H19 in the (1)H-(15)N heteronuclear single quantum correlation spectrum suggests that this region, a loop between beta-strands 1 and 2, undergoes motion on the millisecond to microsecond timescale. Dali searches using the structure closest to the average structure do not identify any high similarities to any other known protein structure, suggesting that the structure of Rv2302 may represent a novel protein fold.     

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.     

Role of the DinB Homologs Rv1537 and Rv3056 in Mycobacterium tuberculosis

The environment encountered by Mycobacterium tuberculosis during infection is genotoxic. Most bacteria tolerate DNA damage by engaging specialized DNA polymerases that catalyze translesion synthesis (TLS) across sites of damage. M. tuberculosis possesses two putative members of the DinB class of Y-family DNA polymerases, DinB1 (Rv1537) and DinB2 (Rv3056); however, their role in damage tolerance, mutagenesis, and survival is unknown. Here, both dinB1 and dinB2 are shown to be expressed in vitro in a growth phase-dependent manner, with dinB2 levels 12- to 40-fold higher than those of dinB1. Yeast two-hybrid analyses revealed that DinB1, but not DinB2, interacts with the β-clamp, consistent with its canonical C-terminal β-binding motif. However, knockout of dinB1, dinB2, or both had no effect on the susceptibility of M. tuberculosis to compounds that form N²-dG adducts and alkylating agents. Similarly, deletion of these genes individually or in combination did not affect the rate of spontaneous mutation to rifampin resistance or the spectrum of resistance-conferring rpoB mutations and had no impact on growth or survival in human or mouse macrophages or in mice. Moreover, neither gene conferred a mutator phenotype when expressed ectopically in Mycobacterium smegmatis. The lack of the effect of altering the complements or expression levels of dinB1 and/or dinB2 under conditions predicted to be phenotypically revealing suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.The emergence and global spread of multi- and extensively drug-resistant strains of Mycobacterium tuberculosis have further complicated the already daunting challenge of controlling tuberculosis (TB) (15). The mechanisms that underlie the evolution of drug resistance in M. tuberculosis by chromosomal mutagenesis and their association with the conditions that tubercle bacilli encounter during the course of infection are poorly understood (6). It has been postulated that hypoxia, low pH, nutrient deprivation, and nitrosative and oxidative stress impose environmental and host immune-mediated DNA-damaging insults on infecting bacilli (64). In addition, the observed importance of excision repair pathways for the growth and survival of M. tuberculosis in murine models of infection (13, 55) and the upregulation of M. tuberculosis genes involved in DNA repair and modification in pulmonary TB in humans provide compelling evidence that the in vivo environment is DNA damaging (51).Damage tolerance constitutes an integral component of an organism''s response to genotoxic stress, preventing collapse of the replication fork at persisting, replication-blocking lesions through the engagement of specialized DNA polymerases that are able to catalyze translesion synthesis (TLS) across the sites of damage (19, 21, 60). Most TLS polymerases belong to the Y family, which comprises a wide range of structurally related proteins present in bacteria, archaea, and eukaryotes (44). Of these, the DinB subfamily of Y family polymerases, whose founder member is Escherichia coli Pol IV (63), is conserved among all domains of life (44). The association of Y family polymerases with inducible mutagenesis has implicated these enzymes in the adaptation of bacteria to environmental stress (17, 20, 39, 54, 58, 59, 66). Their key properties are exemplified in E. coli Pol IV: the polymerase catalyzes efficient and accurate TLS across certain N²-dG adducts (27, 28, 34, 40, 45, 67) and has been implicated in the tolerance of alkylation damage (4); furthermore, overexpression of Pol IV significantly increases mutation rates in E. coli (reviewed in references 21 and 26), and dinB is the only SOS-regulated gene required at induced levels for stress-induced mutagenesis in this organism (20). Furthermore, overproduction of E. coli Pol IV inhibits replication fork progression through replacement of the replicative polymerase to form an alternate replisome in which Pol IV modulates the rate of unwinding of the DnaB helicase (25) and also reduces colony-forming ability (61).The M. tuberculosis genome encodes two Y family polymerase homologs belonging to the DinB subfamily, designated herein as DinB1 (DinX, encoded by Rv1537) and DinB2 (DinP, encoded by Rv3056), as well as a third, distantly related homolog encoded by Rv3394c (see Fig. S1 in the supplemental material) (9). On the basis of sequence similarity with their counterparts from E. coli (63) and Pseudomonas aeruginosa (54), including the complete conservation of key acidic residues essential for catalysis, DinB1 and DinB2 may be functional DNA polymerases (see Fig. S1). In contrast, Rv3394c lacks these residues and as such is unlikely to have polymerase activity (see Fig. S1). Unlike most Y family polymerase-encoding genes investigated with other bacteria (17, 26, 54, 58), dinB1 and dinB2 expression in M. tuberculosis is not dependent on RecA, the SOS response, or the presence of DNA damage (5, 7, 52). That these genes are regulated by other mechanisms and so may serve distinct roles in DNA metabolism in M. tuberculosis is suggested by the observation that dinB1 is differentially expressed in pulmonary TB (51) and is a member of the SigH regulon (30), whereas expression of dinB2 is induced following exposure to novobiocin (5).In this study, we adopted a genetic approach to investigate the function of dinB1 and dinB2 in M. tuberculosis. Mutants with altered complements or expression levels of dinB1 and/or dinB2 were analyzed in vitro and in vivo under conditions predicted to be phenotypically revealing based on DinB function established with other model organisms. The lack of discernible phenotypes in any of the assays employed suggests that the DinB homologs from M. tuberculosis do not behave like their counterparts from other organisms.     

Investigating a putative transcriptional regulatory protein encoded by Rv1719 gene of Mycobacterium tuberculosis

The Protein Journal - Mycobacterium tuberculosis, the causative agent of tuberculosis, demonstrates immense plasticity with which it adapts to a highly dynamic and hostile host environment. This is...     

Effect of recombinant Rv1009 protein on promoting the growth of Mycobacterium tuberculosis

Aims: To determine whether resuscitation-promoting factor (RPF) from Mycobacterium tuberculosis can promote mycobacterial growth and shorten culture time. Method and Results: We cloned, expressed and purified an RPF from M. tuberculosis, Rv1009 protein and subsequently studied the biological activity of the recombinant Rv1009 (rRv1009) in liquid and on solid media. Our results indicate that the molecular weight of rRv1009 protein expressed in Escherichia coli BL21 was approximately 39 kDa. At picomolar and micromolar concentrations, rRv1009 protein could increase the optical density of freeze-dried Mycobacterium bovis BCG three to fivefold in Middlebrook 7H9 medium, stimulate the growth of viable mycobacteria on solid medium, and shorten positive growth detection time of a small number of M. tuberculosis in BACTEC 960 medium. Conclusions: The rRv1009 could promote proliferation of mycobacteria. It may be useful for culture of mycobacteria presented in clinical samples. Significance and Impact of the Study: rRv1009 protein can be used as a growth-promoting reagent of mycobacteria in the medium to shorten the time of culture.     

Structural characterization of the multidomain regulatory protein Rv1364c from Mycobacterium tuberculosis

The open reading frame rv1364c of Mycobacterium tuberculosis, which regulates the stress-dependent σ factor, σ(F), has been analyzed structurally and functionally. Rv1364c contains domains with sequence similarity to the RsbP/RsbW/RsbV regulatory system of the stress-response σ factor of Bacillus subtilis. Rv1364c contains, sequentially, a PAS domain (which shows sequence similarity to the PAS domain of the B. subtilis RsbP protein), an active phosphatase domain, a kinase (anti-σ(F) like) domain and?a C-terminal anti-σ(F) antagonist like domain. The crystal structures of two PAS domain constructs (at 2.3 and 1.6??) and a phosphatase/kinase dual domain construct (at 2.6??) are described. The PAS domain is shown to bind palmitic acid but to have 100 times greater affinity for palmitoleic acid. The full-length protein can exist in solution as both monomer and dimer. We speculate that a switch between monomer and dimer, possibly resulting from fatty acid binding,?affects the accessibility of the serine of the C-terminal, anti-σ(F) antagonist domain for dephosphorylation by the phosphatase domain thus indirectly altering the availability of σ(F).     

Meningococcal outer membrane protein NhhA triggers apoptosis in macrophages

Phagocytotic cells play a fundamental role in the defense against bacterial pathogens. One mechanism whereby bacteria evade phagocytosis is to produce factors that trigger apoptosis. Here we identify for the first time a meningococcal protein capable of inducing macrophage apoptosis. The conserved meningococcal outer membrane protein NhhA (Neisseria hia/hsf homologue A, also known as Hsf) mediates bacterial adhesion and interacts with extracellular matrix components heparan sulphate and laminin. Meningococci lacking NhhA fail to colonise nasal mucosa in a mouse model of meningococcal disease. We found that exposure of macrophages to NhhA resulted in a highly increased rate of apoptosis that proceeded through caspase activation. Exposure of macrophages to NhhA also led to iNOS induction and nitric oxide production. However, neither nitric oxide production nor TNF-α signaling was found to be a prerequisite for NhhA-induced apoptosis. Macrophages exposed to wildtype NhhA-expressing meningococci were also found to undergo apoptosis whereas NhhA-deficient meningococci had a markedly decreased capacity to induce macrophage apoptosis. These data provide new insights on the role of NhhA in meningococcal disease. NhhA-induced macrophage apoptosis could be a mechanism whereby meningococci evade immunoregulatory and phagocytotic actions of macrophages.     

Mycobacterium tuberculosis Rv0899 defines a family of membrane proteins widespread in nitrogen-fixing bacteria

The Mycobacterium tuberculosis membrane protein Rv0899 confers adaptation of the bacterium to acidic environments. Due to strong sequence homology of its C-terminus to bacterial OmpA-like domains, Rv0899 has been proposed to constitute an outer membrane porin of M. tuberculosis. However, OmpA-like domains are widespread in a wide variety of bacterial proteins with different functions. Furthermore, the three-dimensional structure of Rv0899 does not contain a transmembrane β-barrel, and recent evidence demonstrates that it does not have porin activity. Instead, the rv0899 gene is part of an operon (rv0899-rv0901) that is required for fast ammonia secretion, pH neutralization, and growth of M. tuberculosis in acidic environments. The mechanism whereby these functions are accomplished is not known. To gain further functional insights, a targeted search of the genomic databases was performed for proteins with sequence similarity beyond the OmpA-like C-terminus. The results presented here, show that Rv0899-like proteins are widespread in bacteria with functions in nitrogen metabolism, adaptation to nutrient poor environments, and/or establishing symbiosis with the host organism, and appear to form a protein family. These findings suggest that M. tuberculosis Rv0899 may also assist similar processes and lend further support to its role in ammonia secretion and M. tuberculosis adaptation to the host environment.     

Improving basic and membrane protein MS detection of the culture filtrate proteins from Mycobacterium tuberculosis H37Rv by biomimetic affinity prefractionation

The culture filtrate proteins (CFPs) from Mycobacterium tuberculosis have been shown to induce protective immune responses in human and animal models, making them a promising source of candidate targets for tuberculosis drugs, vaccines, and diagnostics. The constituents of the M. tuberculosis CFP proteome are complex and vary with growth conditions. To effectively profile CFPs, gel‐based prefractionation is usually performed before MS analysis. In this study, we describe a novel prefractionation approach by which the proteome is divided into seven partially overlapping fractions by biomimetic affinity chromatography (BiAC) using a six‐column cascade. The LC‐MS/MS analysis of individual fractions identified a total of 541 CFPs, including 61 first‐time identifications. Notably, ～1/3 (20/61) of these novel CFPs are membrane proteins, among which nine proteins have 2–14 transmembrane domains. In addition, ～1/4 (14/61) of the CFPs are basic proteins with pI values greater than 9.0. Our data demonstrate that biomimetic affinity chromatography prefractionation markedly improves protein detection by LC‐MS/MS, and the coverage of basic and hydrophobic proteins in particular is remarkably increased.     

Mycobacterium tuberculosis Rv2536 protein implicated in specific binding to human cell lines

The gene encoding the Mycobacterium tuberculosis Rv2536 protein is present in the Mycobacterium tuberculosis complex (as assayed by PCR) and transcribed (as determined by RT-PCR) in M. tuberculosis H37Rv, M. tuberculosis H37Ra, M. bovis BCG, and M. africanum strains. Rabbits immunized with synthetic polymer peptides from this protein produced antibodies specifically recognizing a 25-kDa band in mycobacterial sonicate. U937 and A549 cells were used in binding assays involving 20-amino-acid-long synthetic peptides covering the whole Rv2536 protein sequence. Peptide 11207 (161DVFSAVRADDSPTGEMQVAQY180) presented high specific binding to both types of cells; the binding was saturable and presented nanomolar affinity constants. Cross-linking assays revealed that this peptide specifically binds to 50 kDa U937 cell membrane and 45 kDa A549 cell membrane proteins.     

Characterization and site-directed mutagenesis of the putative novel acyl carrier protein Rv0033 and Rv1344 from Mycobacterium tuberculosis

Mycolic acids are generated in Mycobacterium tuberculosis as a result of the interaction of two fatty acid biosynthetic systems: type I fatty acid synthase (FAS) and type II fatty acid synthase. Acyl carrier protein (ACP) is a small, acidic protein in type II FAS systems. It plays a central role in mycolic acid biosynthesis by transferring the acyl groups from one enzyme to another for the completion of the fatty acid synthesis cycle. The nature of the proper recognition between ACPs and its many interactive proteins is not understood. Here, we report the over-expression, purification, and characterization of two putative ACPs: Rv0033 and Rv1344 in M. tuberculosis. In order to study the role of the conserved residues and the conformation of whole protein, some site-directed mutations of recombinant Acp1344 were made and the 3D structure of Acp1344 was modeled.