PE is a functional domain responsible for protein translocation and localization on mycobacterial cell wall (original) (raw)
Related papers
PLoS ONE, 2011
PE are peculiar exported mycobacterial proteins over-represented in pathogenic mycobacterial species. They are characterized by an N-terminal domain of about 110 amino acids (PE domain) which has been demonstrated to be responsible for their export and localization. In this paper, we characterize the PE domain of PE_PGRS33 (PE Rv1818c ), one of the best characterized PE proteins. We constructed several mutated proteins in which portions of the PE domain were deleted or subjected to defined mutations. These proteins were expressed in different mycobacterial species and their localization was characterized. We confirmed that the PE domain is essential for PE_PGRS33 surface localization, and demonstrated that a PE domain lacking its first 30 amino acids loses its function. However, single amino acid substitutions in two regions extremely well conserved within the N-terminal domain of all PE proteins had some effect on the stability of PE_PGRS33, but not on its localization. Using Mycobacterium marinum we could show that the type VII secretion system ESX-5 is essential for PE_PGRS33 export. Moreover, in M. marinum, but not in Mycobacterium bovis BCG and in Mycobacterium tuberculosis, the PE domain of PE_PGRS33 is processed and secreted into the culture medium when expressed in the absence of the PGRS domain. Finally, using chimeric proteins in which different portions of the PE Rv1818c domain were fused to the N-terminus of the green fluorescent protein, we could hypothesize that the first 30 amino acids of the PE domain contain a sequence that allows protein translocation. Citation: Cascioferro A, Daleke MH, Ventura M, Donà V, Delogu G, et al. (2011) Functional Dissection of the PE Domain Responsible for Translocation of PE_PGRS33 across the Mycobacterial Cell Wall. PLoS ONE 6(11): e27713.
Functional characterization of Mycobacterium tuberculosis Rv2969c membrane protein
Biochemical and Biophysical Research Communications, 2008
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, subunitbased anti-tuberculosis vaccine.
Infection and immunity, 1989
Only a limited number of proteins from Mycobacterium tuberculosis have so far been shown to possess species-specific epitopes as defined by monoclonal antibodies. One such protein is protein antigen b (Pab) of molecular weight 38,000, which binds the monoclonal antibodies HYT 28, HAT 2, HBT 12, HGT 3, TB 71, and TB 72. The gene encoding this protein was isolated from a lambda gt11 M. tuberculosis DNA library. The nucleotide sequence of the recombinant mycobacterial insert was determined, and an open reading frame of 374 amino acids was identified. The amino acid sequence exhibited 30% homology to a phosphate-binding protein, PstS, from Escherichia coli. The pab gene was subcloned into pBR322 in conjunction with the lacZ gene, and deletions were obtained from the 3' end. The anti-Pab monoclonal antibodies were used to probe crude protein lysates of E. coli transformed with the deletion plasmids. The monoclonal antibodies showed two reactivity patterns; one group of antibodies wer...
Protein Science, 2008
The Rv2707 gene encoding a putative alanine-and leucine-rich protein was found to be present in all Mycobacterium tuberculosis complex strains (by PCR) and its transcription was shown by RT-PCR in all but M. bovis and M. microti. Antibodies raised against Rv2707 peptides specifically recognized the native protein by Western blot and were able to locate this protein on the M. tuberculosis membrane by immunoelectron microscopy. A549 and U937 cells lines were used in binding assays involving synthetic peptides covering the whole Rv2707 protein. High A549 cell-binding peptide 16083 ( 281 QEEWPAPATHAHRLGNWLKAY 300 ) was identified. Peptides 16072 ( 61 LFGPDTLPAIEKSALSTAHSY 80 ) and 16084 ( 301 RIGVGTTTYSSTAQHSAVAA 320 ) presented high specific binding to both A549 and U937 cells. Cross-linking assays revealed that peptide 16084 specifically bound to a 40-kDa and a 50-kDa U937 cell membrane protein. High activity binding peptides (HABPs) 16083 and 16084 were able to inhibit M. tuberculosis invasion of A549 cells. Our results suggest that these sequences could be part of the binding sites used by the bacillus for interacting with target cells, and thus represent good candidates to be tested in a future subunit-based, multiepitope, antituberculosis vaccine.
Peptides, 2011
The identification and characterization of hypothetical membrane proteins from Mycobacterium tuberculosis have led to a better understanding of the mechanisms used by this pathogen to invade and survive inside host cells. This study assessed the presence, transcription, localization and possible biological activity of the conserved hypothetical protein Rv0180c from M. tuberculosis. Bioinformatics analyses indicated that Rv0180c contains a signal peptide, six possible transmembrane helices and a Plasmodium Export Element (PEXEL)-like motif. PCR analyses showed the presence of the Rv0180c gene in strains from the M. tuberculosis complex; but transcription was not detected in Mycobacterium microti. Sera against synthetic peptides of Rv0180c recognized two protein bands in M. tuberculosis H37Rv sonicate: a ∼48-kDa band close to the predicted molecular mass of Rv0180c (47.6 kDa), and a 63-kDa band probably caused by protein modifications. Moreover, the same sera located the protein on the surface of M. tuberculosis H37Rv bacilli by immunoelectron microscopy. Twenty-three synthetic peptides spanning the entire length of Rv0180c were tested for their ability to bind to U937 and A549 cells, finding nine high-activity binding peptides (HABPs) specific for both cell types, two HABPs specific for A549 cells (namely 31032 and 31044) and two HABPs specific for U937 cells (namely 31025 and 31041). HABPs inhibited invasion of M. tuberculosis H37Rv into A549 or U937 cells by significant percentages and facilitated internalization of latex beads in A549 cells. The Rv0180c HABPs herein reported could be preliminary candidates to be assessed as components of a multiepitope, chemically synthesized, subunit-based vaccine against tuberculosis.
The trafficking of the Mycobacterium tuberculosis PE and PPE proteins
2007
The expansion of the Mycobacterium tuberculosis PE and PPE gene families seems to be linked to that of the immunologically-important ESAT-6 (esx) gene clusters secretion system, as the ancestral members of these families are found only within the ESAT-6 gene cluster regions. These ancestral members are also the only copies in the earlier mycobacteria like M. smegmatis. The later duplications of the PE and PPE families belonging to the PGRS and MPTR subgroups, have been implicated in virulence and are only found within the genomes of the pathogenic mycobacteria closely related to the M. tuberculosis complex. The aim of this study was to compare the subcellular localization of the later duplications of the PE and PPE gene families belonging to the PGRS and MPTR subgroups with that of the ancestral PE and PPE proteins found in M. smegmatis and to investigate whether the ESX secretion apparatus is involved in the trafficking of these proteins. The PE (Rv3872) and PPE (Rv3873) genes from M. smegmatis were PCR amplified with a C-terminal HA tag using M. smegmatis genomic DNA as template. Two PPE-MPTR genes, Rv0442c and Rv0878c, and one PE_PGRS gene, Rv2615c, were also PCR amplified with a C-terminal HA tag using M. tuberculosis genomic DNA as template. All genes were cloned into the mycobacterial expression vector p19Kpro. Expression and localization was investigated using SDS-PAGE and Western blotting. The PE and PPE genes expressed in M. smegmatis were found to be present within the cell wall, membrane, and cytosol fractions, but not in the culture filtrate, indicating no secretion. The PPE-MPTR and PE_PGRS genes expressed in M. smegmatis, were also found to be present within the cell wall, membrane and cytosol fractions, but not in the culture filtrate, indicating that they are also not secreted. We hypothesize that their secretion is dependent on ESAT-6 gene cluster region 5, which is absent from the genome of M. smegmatis. Ancestral PE and PPE proteins are secreted efficiently in M. tuberculosis. The ESAT-6 gene cluster Region 3 and Region 4 of M. smegmatis were knocked out, and these knockout mutants could be used in future studies to investigate if the ESAT-6 gene cluster region 1 is involved in the secretion of the ancestral and recent PE and PPE proteins.
Identification of outer membrane proteins of Mycobacterium tuberculosis
Tuberculosis, 2008
The cell wall of mycobacteria contains an unusual outer membrane of extremely low permeability. While Escherichia coli uses more than 60 proteins to functionalize its outer membrane, only two mycobacterial outer membrane proteins (OMPs) are known. The porin MspA of Mycobacterium smegmatis provided the proof of principle that integral mycobacterial OMPs share the β-barrel structure, the absence of hydrophobic α-helices and the presence of a signal peptide with OMPs of gram-negative bacteria. These properties were exploited in a multi-step bioinformatic approach to predict OMPs of M. tuberculosis. A secondary structure analysis was performed for 587 proteins of M. tuberculosis predicted to be exported. Scores were calculated for the β-strand content and the amphiphilicity of the β-strands. Reference OMPs of gram-negative bacteria defined threshold values for these parameters that were met by 144 proteins of unknown function of M. tuberculosis. Two of them were verified as OMPs of unknown functions by a novel two-step experimental approach. Rv1698 and Rv1973 were detected only in the total membrane fraction of M. bovis BCG in Western blot experiments, while proteinase K digestion of whole cells showed the surface accessibility of these proteins. These findings established that Rv1698 and Rv1973 are indeed localized in the outer membrane and tripled the number of known OMPs of M. tuberculosis. Significantly, these results provide evidence for the usefulness of the bioinformatic approach to predict mycobacterial OMPs and indicate that M. tuberculosis likely has many OMPs with β-barrel structure. Our findings pave the way to identify the set of proteins which functionalize the outer membrane of M. tuberculosis.
The Internet Journal of Health, 2008
Mycobacterium tuberculosis is a facultative intracellular pathogen that has evolved the ability to survive and multiply within human macrophages. These bacteria comprise of significant proteins, which were involve in the pathogenesis and regulation of cell activity. The insilico prediction of protein subcellular localization has been extensively studied for prokaryotic, virus and eukaryotic organisms. But, in the case of Mycobacterium, proteins are often involved in extensive interactions at various subcellular localizations in cell. Total thirty-nine putative proteins of M. tuberculosis were predicted for four locations viz cytoplasmic, integral membrane, secretory and protein attached to membrane by Lipid anchor in the subcellular localization. Such predictions provide a method to annotate Mycobacterium proteomes with subcellular localization information rapidly. They have widespread applications in function of proteins in the host cell and in designing the tuberculosis drugs.
Structure and function of a 40,000-molecular-weight protein antigen of Mycobacterium tuberculosis
Infection and Immunity, 1992
A gene encoding a protein antigen from Mycobacterium tuberculosis with a molecular weight of 40,000 has been sequenced. On the basis of sequence homology and functional analyses, we demonstrated that the protein is an L-alanine dehydrogenase (EC 1.4.1.1). The enzyme was demonstrated in M. tuberculosis and Mycobacterium marinum but not in Mycobacterium bovis BCG. The enzyme may play a role in cell wall synthesis because L-alanine is an important constituent of the peptidoglycan layer. Although no consensus signal sequence was identified, we found evidence which suggests that the enzyme is secreted across the cell membrane. The enzyme was characterized and purified by chromatography, thus enabling further studies of its role in virulence and interaction with the immune system of M. tuberculosis-infected individuals.
Biochemistry, 2010
The membrane protein, Rv0899 (OmpATb), from M. tuberculosis, has been proposed to act as an outer membrane porin and to contribute to the bacterium's adaptation to the acidic environment of the phagosome during infection. The gene is restricted to pathogenic mycobacteria and, thus, is an attractive candidate for the development of anti-TB chemotherapy. The 326-residue protein contains three domains: an N-terminal domain (residues 1-72) which includes a sequence of 20 hydrophobic amino acids that is required for membrane translocation; a central B domain (residues 73-200) with homology to the conserved putative lipid-binding BON (bacterial OsmY and nodulation) superfamily; and a C domain (residues 201-326) with homology to the OmpA-C-like superfamily of periplasmic peptidoglycan-binding sequences, found in several types of bacterial membrane proteins, including in the C-terminus of the E. coli outer membrane protein OmpA. We have characterized the structure and dynamics of the B and C domains, and have determined the three-dimensional structure of the B domain. Rv0899 does not form a transmembrane β-barrel. Residues 73 to 326 form a mixed α/β globular structure, encompassing two independently folded modules corresponding to the B and C domains connected by a flexible linker. The B domain folds with three parallel/ antiparallel α-helices packed against six parallel/antiparallel β-strands which form a flat β-sheet. The core is hydrophobic while the exterior is polar and predominantly acidic. The structure of a BON homology domain is revealed here for the first time. In light of this unexpected structure, it is hard to reconcile an outer membrane porin activity with the central domain of the protein. The structure of the B domain and the overall architecture of the protein suggest alternative modes of membrane association.