Progress 09/01/02 to 08/31/06
Outputs
Infection of fish by the marine bacterium Vibrio anguillarum causes vibriosis, a lethal hemorrhagic septicemia. Regulation of two virulence activities, EmpA metalloprotease and hemolysin, were examined in two wild type strains of V. anguillarum (M93 and NB10). Our investigation of EmpA has revealed: 1) the transcriptional start site of empA (by 5' RACE) is 85 bp upstream of the translational start codon; 2) a sigma-S promoter consensus sequence at -10 and -35 upstream of the transcriptional start; 3) that empA expression is dependent upon rpoS and is only transcribed during stationary phase; 3) the presence of vanT, a luxR homologue, in both strains; 4) that mutations of vanT abolish empA expression in both strains; 5) that while strain M93Sm does not exhibit EmpA activity in the absence of fish gastrointestinal (GI) mucus, real-time-RT-qPCR demonstrated that both strains transcribe empA mRNA during stationary phase, even in the absence of GI mucus ; 6) that EmpA is
synthesized as a pre-pro-enzyme that is secreted as an inactive pro-EmpA, which is then processed to active EmpA; 7) strain M93Sm requires incubation in fish GI mucus for the final processing step of pro-EmpA to EmpA; and 8) that the promoter-lux box region of empA binds an unknown protein from cell extracts of M93Sm, but not from extracts of NB10. Our results clearly demonstrate that expression of the EmpA metalloprotease virulence factor is highly regulated from the transcriptional level to the post-translational level. This implies that proper regulation of this protease is essential to successful pathogenesis of host organisms. We also characterized hemolytic activity of V. anguillarum strains M93Sm and NB10. To date, we have demonstrated that both strains of V. anguillarum contain two gene clusters responsible for hemolytic activity. Hemolysin gene cluster #1 contains four genes: plp, vah1, llpA, and llpB. The plp gene acts to repress hemolytic activity, since plp mutants exhibit
2-3-fold increased hemolytic activity. Further, we demonstrated using real-time-RT-qPCR that plp mutants exhibit several fold higher rates of vah1 transcription and when the plp mutation was complemented, vah1 transcription was reduced to wild type levels. Additionally, null mutants of vah1 exhibited reduced virulence. We also found that only in a vah1 null mutant background were we able to select for a hemolysin-negative mutant. DNA surrounding the second site hemolysin mutation was cloned and sequenced to reveal hemolysin gene cluster #2. This cluster of genes is homologous to RTX genes and contains five genes, rtxACBDE. The rtxA gene encodes the RTX hemolysin/cytotoxin. The rtxC gene is an acylase. The rtxBDE genes encode three ABC-transporter genes; all three are necessary for export of the acylated RtxA protein. Finally, double mutants of vah1 and any rtx gene exhibit no hemolytic activity and very reduced virulence in Atlantic salmon. Our results show that hemolytic activity in
V. anguillarum is the result of the activities of two gene clusters containing at least 9 genes. The regulation of these gene clusters appears to be complex and involves at least one repressor gene.
Impacts
Vibriosis is one of the most destructive bacterial diseases of fish, causing significant economic loss to the aquaculture industry. This disease is caused by the bacterium Vibrio anguillarum. The overall goal of this investigation was to characterize the mechanisms that regulate the expression of two virulence genes, empA (encoding the metalloprotease) and vah1 (encoding the hemolysin). We have discovered during this research that the regulation of the EmpA metalloprotease is far more complex than we anticipated. Additionally, we also discovered that hemolytic activity is not encoded by only one gene, but requires the activities of at least nine genes arranged in two separate clusters. Determining the mechanisms for the regulation of virulence genes is essential to developing a more comprehensive understanding of V. anguillarum pathogenesis, specifically, and of bacterial pathogenesis in general. This knowledge will also reveal new means by which the disease process
can be interrupted and stopped. It is expected that this knowledge will permit the development of improved or new vaccines. Additionally, drugs that can target and disrupt specific processes required for pathogenesis may be developed for more effective treatments of bacterial infection.
Publications
- Denkin, S.M. and D.R. Nelson. 2004. Regulation of Vibrio anguillarum empA Metalloprotease Expression and Its Role in Virulence. Appl. Environ. Microbiol. 70:4193-4204
- Denkin, S.M., P. Sekaric, and D.R. Nelson. 2004. Gel shift analysis of the empA promoter region in Vibrio anguillarum. BMC Microbiology 4:42
- Rock J.L. and Nelson, D.R. 2004. Characterization and expression of the Vibrio anguillarum hemolysin genes. Abstr. 104th General Meeting Amer. Soc. Microbiol. B-38
- Rock, J.L. and Nelson, D.R. 2005. Identification and characterization of two hemolysin gene clusters in Vibrio anguillarum. Abstr. Vibrio 2005, pg. 52-53.
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Progress 01/01/05 to 12/31/05
Outputs
Vibrio anguillarum is a causative agent of vibriosis in fish. The zinc metalloprotease EmpA is a virulence factor in this bacterial pathogen of fish. Previous studies have shown that two strains of V. anguillarum regulate empA differently. Strain M93Sm exhibits protease activity only in the presence of fish gastrointestinal mucus, while protease activity is detected in NB10 culture supernatant under all stationary-phase conditions. We have used real-time reverse transcription-PCR to show that even in conditions where no protease activity is detected, empA transcription occurs. Western blot analysis revealed that EmpA is secreted as a 48-kDa proenzyme and that activation occurs extracellularly by the removal of a 10-kDa peptide. The presence of stable extracellular pro-EmpA in M93Sm culture supernatants suggests that activation of EmpA is not autolytic. Hemolytic activity has been suggested as a virulence factor in V. anguillarum infection by contributing to
hemorrhagic septicemia and diarrhea. In order to identify and characterize the hemolysin genes and examine the role of hemolytic activity in virulence, a mini-Tn10Kan mutagenesis clone bank of V. anguillarum was screened. While no hemolysin negative strains were observed, several mutants with 2-3-fold increased hemolytic activity were found. The region containing the insertion mutation was cloned, sequenced, and found to contain the V. anguillarum hemolysin (vah1) and two other open reading frames, a putative lactonizing lipase (llpA) and a putative phospholipase (plp). The mini-Tn10Kan was inserted into plp. Site-directed mutagenesis of each gene revealed that mutations in vah1 and llpA did not affect hemolytic activity, but insertions into plp caused a 2-3-fold increase in hemolysis. Double mutations in plp and either vah1 or llpA resulted in wild-type hemolytic activity. Complementation of plp restored hemolytic activity to wild type levels. Spectrophotometric determination of
hemolysin specific activity revealed that activity on a per cell basis peaked during the first 2 hours of growth in LB20. Real-time quantitative reverse transcriptase PCR used to quantitate transcription of the hemolysin genes plp and vah1 in V. anguillarum wild-type strains M93Sm and NB10 revealed that transcription of plp and vah1 peaked at 2 h of growth in LB20. Additionally, expression of vah1 measured in the plp mutant strain, JL01 during the first 2 h of growth was >8 times higher than in M93Sm. Mutations in plp and llpA did not affect virulence of V. anguillarum. The mutation in vah1 attenuated V. anguillarum virulence in fish. These data show that several genes are responsible for hemolytic activity in V. anguillarum. At least three genes (plp, llpA, and vah1) are responsible for one hemolytic activity. The data also suggest that plp acts as a negative-regulator of vah1 and llpA.
Impacts
Vibriosis is one of the most destructive bacterial diseases of fish, causing significant economic loss to the aquaculture industry. This disease is caused by the bacterium Vibrio anguillarum. The overall goal of this investigation is to characterize the mechanisms that regulate the expression of two virulence genes, empA (encoding the metalloprotease) and vah1 (encoding the hemolysin). Determining the mechanisms for the regulation of virulence genes is essential to developing a more comprehensive understanding of V. anguillarum pathogenesis, specifically, and of bacterial pathogenesis in general. This knowledge will also reveal new means by which the disease process can be interrupted and stopped. It is expected that this knowledge will permit the development of improved or new vaccines. Additionally, drugs that can target and disrupt specific processes required for pathogenesis may be developed for more effective treatments of bacterial infection.
Publications
- Staroscik, A.M., S.M. Denkin, and D.R. Nelson. 2005. Regulation of the Vibrio anguillarum metalloprotease EmpA by post-translational modification. J. Bacteriol. 187:2257-2260
- Rock, J.L. and D.R. Nelson. 2006. Identification and characterization of a hemolysin gene cluster in Vibrio anguillarum. Infect. Immun. 74:xxx-xxx (in press)
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Progress 01/01/03 to 12/31/03
Outputs
We continue to examine the regulation of two virulence activities, the empA metalloprotease and hemolysis in two strains of V. anguillarum (M93Sm and NB10). To date we have: 1) determined the transcriptional start site of empA (by 5'-RACE) is 85 bp upstream of the translational start codon; 2) revealed a sigma-S promoter consensus sequence at -10 and -35 upstream of the transcriptional start; 3) confirmed the presence of vanT, a luxR homologue, in both strains of V. anguillarum; and 4) shown that mutations of vanT abolish empA expression in both strains. These results confirm that empA expression requires stationary-phase conditions and is dependent upon the presence of transcriptional regulator VanT. We have now demonstrated by real-time RT-PCR that M93Sm cells grown to stationary phase in LB20 express empA at 50% the level measured when grown in intestinal mucus (NSSM). However, LB20-grown cells exhibit very low levels of EmpA activity. Western-blot analysis
revealed that NSSM-grown cells have both mature EmpA (MW36 kDa) and a secreted pro-EmpA (MW44kDa), while LB20-grown cells produce only pro-EmpA and an unprocessed cytoplasmic full-length pre-pro-EmpA (MW76kDa). Following mini-Tn10 mutagenesis, an EmpA-negative mutant of M93Sm was selected (MSD-1). DNA sequence analysis revealed that the mutated gene was not empA, but another protease. Western-blot analysis of NSSM-grown cells revealed that in MSD1, the amount of pro-EmpA is increased while the amount of mature EmpA is decreased compared to NSSM-grown M93Sm cells. These results suggest that we have identified a protease responsible for processing pro-EmpA into an active mature form. Previously, we identified by mini-Tn10 mutagenesis a region that contains three genes (vah1, plp, llp) that together may define hemolysis activity. Mutations in plp result in a 2-3-fold increase in hemolytic activity. Mutations in either vah1 or llp restore hemolytic activity to wild-type levels in a plp
mutant. Further, complementation of the plp mutation reduces hemolytic activity to wild-type levels. These data demonstrate that plp is a negative regulator of hemolytic activity. A second round of mini-Tn10 mutagenesis carried out in a vah1 mutant resulted in a hemolysis-negative mutant. Sequence analysis of the mutated DNA revealed homologues of RTX-hemolysin genes (rtxA-C), with the mutation in rtxB. Additionally, real-time RT-PCR analysis of gene expression strongly suggests that hemolytic activity is controlled post-transcriptionally. Our findings show that hemolytic activity in V. anguillarum M93Sm is the result of two hemolysin operons.
Impacts
Vibriosis is one of the most destructive bacterial diseases of fish, causing significant economic loss to the aquaculture industry. This disease is caused by the bacterium Vibrio anguillarum. The overall goal of this investigation is to characterize the mechanisms that regulate the expression of two virulence genes, empA (encoding the metalloprotease) and vah1 (encoding the hemolysin). Determining the mechanisms for the regulation of virulence genes is essential to developing a more comprehensive understanding of V. anguillarum pathogenesis, specifically, and of bacterial pathogenesis in general. This knowledge will also reveal new means by which the disease process can be interrupted and stopped. It is expected that this knowledge will permit the development of improved or new vaccines. Additionally, drugs that can target and disrupt specific processes required for pathogenesis may be developed for more effective treatments of bacterial infection.
Publications
- Denkin, S.M., Sekaric, P., and Nelson, D. R. 2003. Regulation of empA Metalloprotease in Vibrio anguillarum Strains M93Sm and NB10. Abstr. 103rd General Meeting Amer. Soc. Microbiol. B-163.
- Denkin, S.M. and Nelson, D.R. 2004. Regulation of Vibrio anguillarum empA metalloprotease expression and its role in virulence. Appl. Environ. Microbiol. In press
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Progress 01/01/02 to 12/31/02
Outputs
We have begun to examine the regulation of two virulence genes, the empA metalloprotease and the hemolysin encoded by vah1, in two strains of V. anguillarum (M93Sm and NB10). The transcriptional start site of empA was determined by a combination of RT-PCR and sequencing of 5'-RACE (Rapid Amplification of cDNA ends) products. The start site was 85 bp upstream of the translational start codon. Further, examination of DNA sequence at -10 and -35 upstream of the transcriptional start revealed a sigma-S promoter consensus sequence. Additionally, regulation of empA expression by binding of a transcriptional regulator to the putative lux box in the promoter was examined by gel shift DNA mobility assays. V. anguillarum NB10 was found to have a protein that binds to the lux box under all conditions, in contrast strain M93Sm does not produce a binding protein. The presence of the luxR homologue, vanT, in both strains of V. anguillarum was confirmed. Knockout mutations of vanT
were made in both strains, which abolished empA expression, but did not affect protein binding to the lux box in NB10. These results confirm previous findings that empA expression requires stationary-phase conditions and is dependent upon the presence of transcriptional regulator VanT. The results also suggest that VanT does not directly regulate empA expression by binding to the promoter region, but that VanT acts indirectly through other proteins to regulate empA expression. Two mutants of V. anguillarum M93 that exhibit 2-4-fold increased hemolysin activity were created by mini-Tn10kan mutagenesis, isolated, and the DNA containing the insertion cloned. Restriction mapping revealed that the two clones are distinct and represent two different regions of DNA. DNA sequencing and analysis of the two clones has begun. Initial sequence analysis of one of the clones (pJLO1.3) using BLAST has revealed that the mini-Tn10 insertion is in a region of DNA that contains a several genes that
together may define hemolysin activity in V. anguillarum. These genes include a phospholipase hemolysin, a thermolabile hemolysin, a lactonizing lipase, and a second lipase. Additionally, the genes contained in this region are highly homologous in sequence and arrangement to similar regions in V. cholerae, V. vulnificus, and V. parahaemolyticus. It is hypothesized that the vah1 gene is in an unsequenced portion of the DNA and is adjacent to the thermostable hemolysin. These results suggest that vah1 is part of an operon containing several genes that regulate and are responsible for lipase/ hemolysin activity.
Impacts
Vibriosis is one of the most destructive bacterial diseases of fish, causing significant economic loss to the aquaculture industry. This disease is caused by the bacterium Vibrio anguillarum. The overall goal of this investigation is to characterize the mechanisms that regulate the expression of two virulence genes, empA (encoding the metalloprotease) and vah1 (encoding the hemolysin). Determining the mechanisms for the regulation of virulence genes is essential to developing a more comprehensive understanding of V. anguillarum pathogenesis, specifically, and of bacterial pathogenesis in general. This knowledge will also reveal new means by which the disease process can be interrupted and stopped. It is expected that this knowledge will permit the development of improved or new vaccines. Additionally, drugs that can target and disrupt specific processes required for pathogenesis may be developed for more effective treatments of bacterial infection.
Publications
- No publications reported this period
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