Descriptive proteomic analysis shows protein variability between closely related clinical isolates of Mycobacterium tuberculosis.
ABSTRACT: The use of isobaric tags such as iTRAQ allows the relative and absolute quantification of hundreds of proteins in a single experiment for up to eight different samples. More classical techniques such as 2-DE can offer a complimentary approach for the analysis of complex protein samples. In this study, the proteomes of secreted and cytosolic proteins of genetically closely related strains of Mycobacterium tuberculosis were analyzed. Analysis of 2-D gels afforded 28 spots with variations in protein abundance between strains. These were identified by MS/MS. Meanwhile, a rigorous statistical analysis of iTRAQ data allowed the identification and quantification of 101 and 137 proteins in the secreted and cytosolic fractions, respectively. Interestingly, several differences in protein levels were observed between the closely related strains BE, C28 and H6. Seven proteins related to cell wall and cell processes were more abundant in BE, while enzymes related to metabolic pathways (GltA2, SucC, Gnd1, Eno) presented lower levels in the BE strain. Proteins involved in iron and sulfur acquisition (BfrB, ViuB, TB15.3 and SseC2) were more abundant in C28 and H6. In general, iTRAQ afforded rapid identification of fine differences between protein levels such as those presented between closely related strains. This provides a platform from which the relevance of these differences can be assessed further using complimentary proteomic and biological modeling methods.
Project description:BACKGROUND: Enterohaemorrhagic E. coli (EHEC) can cause severe disease such as bloody diarrhoea and haemolytic uraemic syndrome in humans. Besides production of Shiga toxins, the presence of LEE (eae-gene) and non-LEE (nle) encoded effector genes harboured on O-islands OI-122, OI-71 and OI-57 is associated with EHEC virulence and their frequency in outbreaks. Genes encoded by the EHEC-plasmid are putative virulence markers of EHEC. EHEC-plasmids, LEE and non-LEE effector genes have also been detected in some strains of enteropathogenic E. coli (EPEC). The objective of this study was to analyze the relationship between EHEC and EPEC for virulence genes encoded by genomic O-islands and by the EHEC-plasmids. RESULTS: Nle genes ent/espL2, nleB and nleE (OI-122), nleA, nleF and nleH1-2 (OI-71), nleG5-2 and nleG6-2 (OI-57), espK (CP-933N) and the EHEC-plasmid encoded genes ehxA, espP, etpD and katP were searched in 73 typical and in 235 atypical enteropathogenic E. coli (EPEC) strains. Typical and atypical EPEC each fall into two clusters. Cluster 1 typical (n = 46) and atypical (n = 129) EPEC strains were characterized by the presence of OI-122 encoded genes and grouped together with 64 investigated EHEC strains. Cluster 2 typical (n = 27) and atypical (n = 106) strains grouped together with 52 LEE-negative, Shiga toxin-producing E. coli (STEC) and with 21 apathogenic E. coli strains. Typical EPEC Cluster 1 strains belonged to serotypes frequently involved in severe illness and outbreaks in children (O111:H2, O114:H2, O55:H6, O127:H6 and O142:H6). Atypical EPEC Cluster 1 strains were characterized by serotypes related to EHEC (O26:H11, O55:H7, O145:H28, O103:H2 and O103:H25). CONCLUSION: The OI-122 encoded nleB gene was found to be most closely associated with Cluster 1 strains and may serve as a diagnostic tool for the identification of virulent EHEC and EPEC seropathotypes. OI-71 encoded genes nleA, nleF and nleH1-2 are less associated with Cluster 1 strains. EHEC-plasmid, OI-57 and CP-933 associated genes showed only weak similarities with virulent Cluster 1 EHEC and EPEC strains.
Project description:Extraintestinal pathogenic Escherichia coli (ExPEC) strains cause a large spectrum of infections. The majority of ExPEC strains are closely related to the B2 or the D phylogenetic group. The aim of our study was to develop a protein-based vaccine against these ExPEC strains. To this end, we identified ExPEC-specific genomic regions, using a comparative genome analysis, between the nonpathogenic E. coli strain K-12 MG1655 and ExPEC strains C5 (meningitis isolate) and CFT073 (urinary tract infection isolate). The analysis of these genomic regions allowed the selection of 40 open reading frames, which are conserved among B2/D clinical isolates and encode proteins with putative outer membrane localization. These genes were cloned, and recombinant proteins were purified and assessed as vaccine candidates. After immunization of BALB/c mice, five proteins induced a significant protective immunity against a lethal challenge with a clinical E. coli strain of the B2 group. In passive immunization assays, antigen-specific antibodies afforded protection to naive mice against a lethal challenge. Three of these antigens were related to iron acquisition metabolism, an important virulence factor of the ExPEC, and two corresponded to new, uncharacterized proteins. Due to the large number of genetic differences that exists between commensal and pathogenic strains of E. coli, our results demonstrate that it is possible to identify targets that elicit protective immune responses specific to those strains. The five protective antigens could constitute the basis for a preventive subunit vaccine against diseases caused by ExPEC strains.
Project description:In June 2013, the first human infection by avian influenza A(H6N1) virus was reported in Taiwan. This incident raised the concern for possible human epidemics and pandemics from H6 viruses. In this study, we performed structural and functional investigation on the hemagglutinin (HA) proteins of the human-infecting A/Taiwan/2/2013(H6N1) (TW H6) virus and an avian A/chicken/Guangdong/S1311/2010(H6N6) (GD H6) virus that transmitted efficiently in guinea pigs. Our results revealed that in the presence of HA1 Q226, the triad of HA1 S137, E190 and G228 in GD H6 HA allows the binding to both avian- and human-like receptors with a slight preference for avian receptors. Its conservation among the majority of H6 HAs provides an explanation for the broader host range of this subtype. Furthermore, the triad of N137, V190 and S228 in TW H6 HA may alleviate the requirement for a hydrophobic residue at HA1 226 of H2 and H3 HAs when binding to human-like receptors. Consequently, TW H6 HA has a slight preference for human receptors, thus may represent an intermediate towards a complete human adaptation. Importantly, the triad observed in TW H6 HA is detected in 74% H6 viruses isolated from Taiwan in the past 14 years, suggesting an elevated threat of H6 viruses from this region to human health. The novel roles of the triad at HA1 137, 190 and 228 of H6 HA in binding to receptors revealed here may also be used by other HA subtypes to achieve human adaptation, which needs to be further tested in laboratory and closely monitored in field surveillance.
Project description:Proteolysis is a major protein posttranslational modification that, by altering protein structure, affects protein function and, by truncating the protein sequence, alters peptide signatures of proteins analyzed by proteomics. To identify such modified and shortened protease-generated neo-N-termini on a proteome-wide basis, we developed a whole protein isobaric tag for relative and absolute quantitation (iTRAQ) labeling method that simultaneously labels and blocks all primary amines including protein N- termini and lysine side chains. Blocking lysines limits trypsin cleavage to arginine, which effectively elongates the proteolytically truncated peptides for improved MS/MS analysis and peptide identification. Incorporating iTRAQ whole protein labeling with terminal amine isotopic labeling of substrates (iTRAQ-TAILS) to enrich the N-terminome by negative selection of the blocked mature original N-termini and neo-N-termini has many advantages. It enables simultaneous characterization of the natural N-termini of proteins, their N-terminal modifications, and proteolysis product and cleavage site identification. Furthermore, iTRAQ-TAILS also enables multiplex N-terminomics analysis of up to eight samples and allows for quantification in MS2 mode, thus preventing an increase in spectral complexity and extending proteome coverage by signal amplification of low abundance proteins. We compared the substrate degradomes of two closely related matrix metalloproteinases, MMP-2 (gelatinase A) and MMP-9 (gelatinase B), in fibroblast secreted proteins. Among 3,152 unique N-terminal peptides identified corresponding to 1,054 proteins, we detected 201 cleavage products for MMP-2 and unexpectedly only 19 for the homologous MMP-9 under identical conditions. Novel substrates identified and biochemically validated include insulin-like growth factor binding protein-4, complement C1r component A, galectin-1, dickkopf-related protein-3, and thrombospondin-2. Hence, N-terminomics analyses using iTRAQ-TAILS links gelatinases with new mechanisms of action in angiogenesis and reveals unpredicted restrictions in substrate repertoires for these two very similar proteases.
Project description:A novel Streptomyces, strain MUSC 149(T) was isolated from mangrove soil. A polyphasic approach was used to study the taxonomy of MUSC 149(T), which shows a range of phylogenetic and chemotaxonomic properties consistent with those of the members of the genus Streptomyces. The diamino acid of the cell wall peptidoglycan was LL-diaminopimelic acid. The predominant menaquinones were identified as MK9(H8) and MK9(H6). Phylogenetic analysis indicated that closely related strains include Streptomyces rhizophilus NBRC 108885(T) (99.2% sequence similarity), S. gramineus NBRC 107863(T) (98.7%) and S. graminisoli NBRC 108883(T) (98.5%). The DNA-DNA relatedness values between MUSC 149(T) and closely related type strains ranged from 12.4 ± 3.3% to 27.3 ± 1.9%. The DNA G + C content was determined to be 72.7 mol%. The extract of MUSC 149(T) exhibited strong antioxidant activity and chemical analysis reported identification of an antioxidant agent, Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro-. These data showed that metabolites of MUSC 149(T) shall be useful as preventive agent against free-radical associated diseases. Based on the polyphasic study of MUSC 149(T), the strain merits assignment to a novel species, for which the name S. mangrovisoli sp. nov. is proposed. The type strain is MUSC 149(T) (=MCCC 1K00699(T)=DSM 100438(T)).
Project description:Bats are considered important animal reservoirs for many viruses pathogenic to humans. An approach based on viral metagenomics was used to study gut specimens from 78 insectivorous bats in Yunnan Province, China. Seventy-four reads were found to be related to group A rotavirus (RVA). Further reverse transcription-PCR screening and viral isolation on cell cultures confirmed the presence of a novel RVA strain, named RVA/Bat-tc/MSLH14/2012/G3P, in 1 (6%) of 16 lesser horseshoe bats. Full genomic sequencing analyses showed that MSLH14 possessed the genotype constellation G3-P-I8-R3-C3-M3-A9-N3-T3-E3-H6, which is akin to human and animal rotaviruses believed to be of feline/canine origin. Phylogenetic analysis indicated that VP7 was most closely related to bovine RVA strains from India, whereas VP4 was most closely related to an unusual human RVA strain, CMH222, with animal characteristics isolated in Thailand. The remaining gene segments were only distantly related to a range of animal RVA strains, most of which are believed to be related to feline/canine RVAs. Experimental infection showed that bat RVA strain MSLH14 was highly pathogenic to suckling mice, causing 100% mortality when they were inoculated orally with a titer as low as 5 × 10² 50% tissue culture infective doses. As this virus is not closely related to any known RVA strain, it is tempting to speculate that it is a true bat RVA strain rather than a virus transmitted between species. However, further screening of bat populations, preferably juvenile animals, will be crucial in determining whether or not this virus is widely distributed in the bat population.
Project description:<i>Alexandrium minutum</i> and <i>Alexandrium tamutum</i> are two closely related harmful algal bloom (HAB)-causing species with different toxicity. Using isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics and two-dimensional differential gel electrophoresis (2D-DIGE), a comprehensive characterization of the proteomes of <i>A. minutum</i> and <i>A. tamutum</i> was performed to identify the cellular and molecular underpinnings for the dissimilarity between these two species. A total of 1436 proteins and 420 protein spots were identified using iTRAQ-based proteomics and 2D-DIGE, respectively. Both methods revealed little difference (10-12%) between the proteomes of <i>A. minutum</i> and <i>A. tamutum</i>, highlighting that these organisms follow similar cellular and biological processes at the exponential stage. Toxin biosynthetic enzymes were present in both organisms. However, the gonyautoxin-producing <i>A. minutum</i> showed higher levels of osmotic growth proteins, Zn-dependent alcohol dehydrogenase and type-I polyketide synthase compared to the non-toxic <i>A. tamutum</i>. Further, <i>A. tamutum</i> had increased S-adenosylmethionine transferase that may potentially have a negative feedback mechanism to toxin biosynthesis. The complementary proteomics approach provided insights into the biochemistry of these two closely related HAB-causing organisms. The identified proteins are potential biomarkers for organismal toxicity and could be explored for environmental monitoring.
Project description:An unusual feature of lipid A from plant endosymbionts of the Rhizobiaceae family is the presence of a 27-hydroxyoctacosanoic acid (C28) moiety. An enzyme that incorporates this acyl chain is present in extracts of Rhizobium leguminosarum, Rhizobium etli, and Sinorhizobium meliloti but not Escherichia coli. The enzyme transfers 27-hydroxyoctacosanate from a specialized acyl carrier protein (AcpXL) to the precursor Kdo2 ((3-deoxy-d-manno-octulosonic acid)2)-lipid IV(A). We now report the identification of five hybrid cosmids that direct the overexpression of this activity by screening approximately 4000 lysates of individual colonies of an R. leguminosarum 3841 genomic DNA library in the host strain S. meliloti 1021. In these heterologous constructs, both the C28 acyltransferase and C28-AcpXL are overproduced. Sequencing of a 9-kb insert from cosmid pSSB-1, which is also present in the other cosmids, shows that acpXL and the lipid A acyltransferase gene (lpxXL) are close to each other but not contiguous. Nine other open reading frames around lpxXL were also sequenced. Four of them encode orthologues of fatty acid and/or polyketide biosynthetic enzymes. AcpXL purified from S. meliloti expressing pSSB-1 is fully acylated, mainly with 27-hydroxyoctacosanoate. Expression of lpxXL in E. coli behind a T7 promoter results in overproduction in vitro of the expected R. leguminosarum acyltransferase, which is C28-AcpXL-dependent and utilizes (3-deoxy-d-manno-octulosonic acid)2-lipid IV(A) as the acceptor. These findings confirm that lpxXL is the structural gene for the C28 acyltransferase. LpxXL is distantly related to the lauroyltransferase (LpxL) of E. coli lipid A biosynthesis, but highly significant LpxXL orthologues are present in Agrobacterium tumefaciens, Brucella melitensis, and all sequenced strains of Rhizobium, consistent with the occurrence of long secondary acyl chains in the lipid A molecules of these organisms.
Project description:Intimins are outer membrane proteins expressed by enteric bacterial pathogens capable of inducing intestinal attachment-and-effacement lesions. A eukaryotic cell-binding domain is located within a 280-amino-acid (Int280) carboxy terminus of intimin polypeptides. Polyclonal antiserum was raised against Int280 from enteropathogenic Escherichia coli (EPEC) serotypes O127:H6 and O114:H2 (anti-Int280-H6 and anti-Int280-H2, respectively), and Western blot analysis was used to explore the immunological relationship between the intimin polypeptides expressed by different clinical EPEC and enterohemorrhagic E. coli (EHEC) isolates, a rabbit diarrheagenic E. coli strain (RDEC-1), and Citrobacter rodentium. Anti-Int280-H6 serum reacted strongly with some EPEC serotypes, whereas anti-Int280-H2 serum reacted strongly with strains belonging to different EPEC and EHEC serotypes, RDEC-1, and C. rodentium. These observations were confirmed by using purified Int280 in an enzyme-linked immunosorbent assay and by immunogold and immunofluorescence labelling of whole bacterial cells. Some bacterial strains were recognized poorly by either antiserum (e.g., EPEC O86:H34 and EHEC O157:H7). By using PCR primers designed on the basis of the intimin-encoding eae gene sequences of serotype O127:H6, O114:H2, and O86:H34 EPEC and serotype O157:H7 EHEC, we could distinguish between different eae gene derivatives. Accordingly, the different intimin types were designated alpha, beta, delta, and gamma, respectively.
Project description:Until 2001, H6N1 influenza viruses in the Hong Kong bird markets were represented by a single stable A/teal/Hong Kong/W312/97-like lineage. Beginning in 2001, despite a reduction in overall prevalence, an increase was observed in the number of H6 viruses isolated from chickens and other hosts. To assess any changes in H6 viruses, we characterized 18 H6 viruses isolated in the Hong Kong bird markets from 2001 to 2003. Experimental data showed that the 2003 H6 viruses had similar infectivity for chickens as did A/teal/HK/W312/97, and they were unable to transmit. Although all hemagglutinin genes were closely related to A/teal/HK/W312/97, 7 isolates were reassortant viruses containing similar gene segments of co-circulating H9N2 or H5N1 viruses. The receptor specificity was different from that of A/teal/Hong Kong/W312/97. Interestingly, similar observations have been documented in H9N2 viruses in Hong Kong. This evolution strongly suggests that some change in the ecology of influenza in the region selected for these changes. Taken together, these findings suggest that the H6 influenza viruses isolated in the Hong Kong markets are not well adapted to chickens and that the likely continued source of these viruses are other "minor" poultry species in which they are undergoing genetic and biologic evolution.