Browse
Submit Data
Databases
API
Help

Dataset Information

21 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Cloning and characterization of a nonhemolytic phospholipase C gene from Burkholderia pseudomallei.

ABSTRACT: We cloned and characterized a phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC) gene from Burkholderia pseudomallei. DNA sequence analysis of the gene indicated an open reading frame coding for 700 amino acids with a 34-amino-acid signal peptide. When cleaved, this yields a secreted 73-kDa mature protein. The deduced amino acid sequence exhibited 48% similarity to that of a nonhemolytic PLC from Pseudomonas aeruginosa. The expressed PC-PLC was heat stable, nonhemolytic for sheep erythrocytes, and active between pH 2 and 8. Western blot analysis with sera from melioidosis patients indicated that they produced immunoglobulin M antibodies against this PC-PLC protein.

SUBMITTER: Korbsrisate S

PROVIDER: S-EPMC85747 | biostudies-literature | 1999 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Cloning and characterization of a nonhemolytic phospholipase C gene from Burkholderia pseudomallei.

Korbsrisate S S Suwanasai N N Leelaporn A A Ezaki T T Kawamura Y Y Sarasombath S S

Journal of clinical microbiology 19991101 11

We cloned and characterized a phosphatidylcholine-hydrolyzing phospholipase C (PC-PLC) gene from Burkholderia pseudomallei. DNA sequence analysis of the gene indicated an open reading frame coding for 700 amino acids with a 34-amino-acid signal peptide. When cleaved, this yields a secreted 73-kDa mature protein. The deduced amino acid sequence exhibited 48% similarity to that of a nonhemolytic PLC from Pseudomonas aeruginosa. The expressed PC-PLC was heat stable, nonhemolytic for sheep erythrocy ...[more]

PMID: 10523590

Similar Datasets

Cloning, expression, and characterization of a peptidoglycan hydrolase from the Burkholderia pseudomallei phage ST79.

Project description:The lytic phage ST79 of Burkholderia pseudomallei can lyse a broad range of its host including antibiotic resistant isolates from within using a set of proteins, holin, lysB, lysC and endolysin, a peptidoglycan (PG) hydrolase enzyme. The phage ST79 endolysin gene identified as peptidase M15A was cloned, expressed and purified to evaluate its potential to lyse pathogenic bacteria. The molecular size of the purified enzyme is approximately 18 kDa and the in silico study cited here indicated the presence of a zinc-binding domain predicted to be a member of the subfamily A of a metallopeptidase. Its activity, however, was reduced by the presence of Zn(2+). When Escherichia coli PG was used as a substrate and subjected to digestion for 5 min with 3 μg/ml of enzyme, the peptidase M15A showed 2 times higher in lysis efficiency when compared to the commercial lysozyme. The enzyme works in a broad alkaligenic pH range of 7.5-9.0 and temperatures from 25 to 42 °C. The enzyme was able to lyse 18 Gram-negative bacteria in which the outer membrane was permeabilized by chloroform treatment. Interestingly, it also lysed Enterococcus sp., but not other Gram-positive bacteria. In general, endolysin cannot lyse Gram-negative bacteria from outside, however, the cationic amphipathic C-terminal in some endolysins showed permeability to Gram-negative outer membranes. Genetically engineered ST79 peptidase M15A that showed a broad spectrum against Gram-negative bacterial PG or, in combination with an antibiotic the same way as combined drug methodology, could facilitate an effective treatment of severe or antibiotic resistant cases.

| S-EPMC5025407 | biostudies-literature

Cloning and expression of class A beta-lactamase gene blaA(BPS) in Burkholderia pseudomallei.

Project description:The beta-lactamase gene blaA(BPS) in Burkholderia pseudomallei was cloned and expressed in Escherichia coli. BPS-1 is a cephalosporinase with an isoelectric point of 7.7. Sequence analysis of BPS-1 revealed conserved motifs typical of class A beta-lactamases and a relationship to the PenA (in B. cepacia) and BlaI (in Yersinia enterocolitica) lineages.

| S-EPMC127091 | biostudies-literature

Functional characterization of Burkholderia pseudomallei trimeric autotransporters.

Project description:Burkholderia pseudomallei is a tier 1 select agent and the causative agent of melioidosis, a severe and often fatal disease with symptoms ranging from acute pneumonia and septic shock to a chronic infection characterized by abscess formation in the lungs, liver, and spleen. Autotransporters (ATs) are exoproteins belonging to the type V secretion system family, with many playing roles in pathogenesis. The genome of B. pseudomallei strain 1026b encodes nine putative trimeric AT proteins, of which only four have been described. Using a bioinformatic approach, we annotated putative domains within each trimeric AT protein, excluding the well-studied BimA protein, and found short repeated sequences unique to Burkholderia species, as well as an unexpectedly large proportion of ATs with extended signal peptide regions (ESPRs). To characterize the role of trimeric ATs in pathogenesis, we constructed disruption or deletion mutations in each of eight AT-encoding genes and evaluated the resulting strains for adherence to, invasion of, and plaque formation in A549 cells. The majority of the ATs (and/or the proteins encoded downstream) contributed to adherence to and efficient invasion of A549 cells. Using a BALB/c mouse model of infection, we determined the contributions of each AT to bacterial burdens in the lungs, liver, and spleen. At 48 h postinoculation, only one strain, Bp340::pDbpaC, demonstrated a defect in dissemination and/or survival in the liver, indicating that BpaC is required for wild-type virulence in this model.

| S-EPMC3719557 | biostudies-literature

Functional redundancy of Burkholderia pseudomallei phospholipase C enzymes and their role in virulence.

Project description:Phospholipase C (PLC) enzymes are key virulence factors in several pathogenic bacteria. Burkholderia pseudomallei, the causative agent of melioidosis, possesses at least three plc genes (plc1, plc2 and plc3). We found that in culture medium plc1 gene expression increased with increasing pH, whilst expression of the plc3 gene was pH (4.5 to 9.0) independent. Expression of the plc2 gene was not detected in culture medium. All three plc genes were expressed during macrophage infection by B. pseudomallei K96243. Comparing B. pseudomallei wild-type with plc mutants revealed that plc2, plc12 or plc123 mutants showed reduced intracellular survival in macrophages and reduced plaque formation in HeLa cells. However, plc1 or plc3 mutants showed no significant differences in plaque formation compared to wild-type bacteria. These findings suggest that Plc2, but not Plc1 or Plc3 are required for infection of host cells. In Galleria mellonella, plc1, plc2 or plc3 mutants were not attenuated compared to the wild-type strain, but multiple plc mutants showed reduced virulence. These findings indicate functional redundancy of the B. pseudomallei phospholipases in virulence.

| S-EPMC7648637 | biostudies-literature

Molecular characterization of putative virulence determinants in Burkholderia pseudomallei.

Project description:The Gram-negative saprophyte Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease which is endemic in Southeast Asia and northern Australia. This bacterium possesses many virulence factors which are thought to contribute to its survival and pathogenicity. Using a virulent clinical isolate of B. pseudomallei and an attenuated strain of the same B. pseudomallei isolate, 6 genes BPSL2033, BP1026B_I2784, BP1026B_I2780, BURPS1106A_A0094, BURPS1106A_1131, and BURPS1710A_1419 were identified earlier by PCR-based subtractive hybridization. These genes were extensively characterized at the molecular level, together with an additional gene BPSL3147 that had been identified by other investigators. Through a reverse genetic approach, single-gene knockout mutants were successfully constructed by using site-specific insertion mutagenesis and were confirmed by PCR. BPSL2033::Km and BURPS1710A_1419::Km mutants showed reduced rates of survival inside macrophage RAW 264.7 cells and also low levels of virulence in the nematode infection model. BPSL2033::Km demonstrated weak statistical significance (P = 0.049) at 8 hours after infection in macrophage infection study but this was not seen in BURPS1710A_1419::Km. Nevertheless, complemented strains of both genes were able to partially restore the gene defects in both in vitro and in vivo studies, thus suggesting that they individually play a minor role in the virulence of B. pseudomallei.

| S-EPMC4158159 | biostudies-other

Characterization of an autotransporter adhesin protein shared by Burkholderia mallei and Burkholderia pseudomallei.

Project description:BackgroundAutotransporters form a large family of outer membrane proteins specifying diverse biological traits of Gram-negative bacteria. In this study, we report the identification and characterization of a novel autotransporter gene product of Burkholderia mallei (locus tag BMA1027 in strain ATCC 23344).ResultsDatabase searches identified the gene in at least seven B. mallei isolates and the encoded proteins were found to be 84% identical. Inactivation of the gene encoding the autotransporter in the genome of strain ATCC 23344 substantially reduces adherence to monolayers of HEp-2 laryngeal cells and A549 type II pneumocytes, as well as to cultures of normal human bronchial epithelium (NHBE). Consistent with these findings, expression of the autotransporter on the surface of recombinant E. coli bacteria increases adherence to these cell types by 5-7 fold. The gene specifying the autotransporter was identified in the genome of 29 B. pseudomallei isolates and disruption of the gene in strain DD503 reduced adherence to NHBE cultures by 61%. Unlike B. mallei, the mutation did not impair binding of B. pseudomallei to A549 or HEp-2 cells. Analysis of sera from mice infected via the aerosol route with B. mallei and B. pseudomallei revealed that animals inoculated with as few as 10 organisms produce antibodies against the autotransporter, therefore indicating expression in vivo.ConclusionsOur data demonstrate that we have identified an autotransporter protein common to the pathogenic species B. mallei and B. pseudomallei which mediates adherence to respiratory epithelial cells and is expressed in vivo during the course of aerosol infection.

| S-EPMC4021183 | biostudies-literature

Characterization and analysis of the Burkholderia pseudomallei BsaN virulence regulon.

Project description:Burkholderia pseudomallei is a facultative intracellular pathogen and the causative agent of melioidosis. A conserved type III secretion system (T3SS3) and type VI secretion system (T6SS1) are critical for intracellular survival and growth. The T3SS3 and T6SS1 genes are coordinately and hierarchically regulated by a TetR-type regulator, BspR. A central transcriptional regulator of the BspR regulatory cascade, BsaN, activates a subset of T3SS3 and T6SS1 loci.To elucidate the scope of the BsaN regulon, we used RNAseq analysis to compare the transcriptomes of wild-type B. pseudomallei KHW and a bsaN deletion mutant. The 60 genes positively-regulated by BsaN include those that we had previously identified in addition to a polyketide biosynthesis locus and genes involved in amino acid biosynthesis. BsaN was also found to repress the transcription of 51 genes including flagellar motility loci and those encoding components of the T3SS3 apparatus. Using a promoter-lacZ fusion assay in E. coli, we show that BsaN together with the chaperone BicA directly control the expression of the T3SS3 translocon, effector and associated regulatory genes that are organized into at least five operons (BPSS1516-BPSS1552). Using a mutagenesis approach, a consensus regulatory motif in the promoter regions of BsaN-regulated genes was shown to be essential for transcriptional activation.BsaN/BicA functions as a central regulator of key virulence clusters in B. pseudomallei within a more extensive network of genetic regulation. We propose that BsaN/BicA controls a gene expression program that facilitates the adaption and intracellular survival of the pathogen within eukaryotic hosts.

| S-EPMC4236580 | biostudies-literature

Characterization of BPSS1521 (bprD), a regulator of Burkholderia pseudomallei virulence gene expression in the mouse model.

Project description:The Gram-negative saprophytic bacterium Burkholderia pseudomallei is the causative agent of melioidosis, a severe infectious disease of both humans and animals. Severity of the disease is thought to be dependent on both the health status of the host, including diabetes mellitus and kidney disease, and bacterial-derived factors. To identify the bacterial factors important during an acute infection, gene expression profiles in the spleen, lung, and liver of BALB/c (Th2 prototype) and C57BL/6 mice (Th1 prototype) were determined using DNA microarrays. This analysis identified BPSS1521 (bprD), a predicted transcriptional regulator located in the type III secretion system (T3SS-3) operon, to be up regulated, specifically in C57BL/6 mice. BALB/c mice infected with a bprD mutant showed a shorter time to death and increased inflammation, as determined by histopathological analysis and enumeration of bacteria in the spleen. Elevated numbers of multinucleated giant cells (MNGCs), which is the hallmark of melioidosis, were detected in both the wild-type and the bprD mutants; a similar elevation occurs in melioidosis patients. One striking observation was the increased expression of BPSS1520 (bprC), located downstream of bprD, in the bprD mutant. BprC is a regulator of T6SS-1 that is required for the virulence of B. pseudomallei in murine infection models. Deletion of bprD led to the overexpression of bprC and a decreased time to death. bprD expression was elevated in C57BL/6--as compared to BALB/c--mice, suggesting a role for BprD in the natural resistance of C57BL/6 mice to B. pseudomallei. Ultimately, this analysis using mice with different immune backgrounds may enhance our understanding of the outcomes of infection in a variety of models.

| S-EPMC4128674 | biostudies-literature

The Identification and Differentiation between Burkholderia mallei and Burkholderia pseudomallei Using One Gene Pyrosequencing.

Project description:The etiologic agents for melioidosis and glanders, Burkholderia mallei and Burkholderia pseudomallei respectively, are genetically similar making identification and differentiation from other Burkholderia species and each other challenging. We used pyrosequencing to determine the presence or absence of an insertion sequence IS407A within the flagellin P (fliP) gene and to exploit the difference in orientation of this gene in the two species. Oligonucleotide primers were designed to selectively target the IS407A-fliP interface in B. mallei and the fliP gene specifically at the insertion point in B. pseudomallei. We then examined DNA from ten B. mallei, ten B. pseudomallei, 14 B. cepacia, eight other Burkholderia spp., and 17 other bacteria. Resultant pyrograms encompassed the target sequence that contained either the fliP gene with the IS407A interruption or the fully intact fliP gene with 100% sensitivity and 100% specificity. These pyrosequencing assays based upon a single gene enable investigators to reliably identify the two species. The information obtained by these assays provides more knowledge of the genomic reduction that created the new species B. mallei from B. pseudomallei and may point to new targets that can be exploited in the future.

| S-EPMC4897507 | biostudies-literature

Methylome Characterization of Burkholderia pseudomallei Strain 982 at Single-Base Resolution.

Project description:Burkholderia pseudomallei is the etiological agent of melioidosis, which has been studied by transcriptome and secretome analyses. However, little is known about the methylome of this pathogen. Here, we present the complete genome and methylome of melioidosis-causing B. pseudomallei strain 982.

| S-EPMC6813387 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data