Progress 09/01/08 to 08/31/12
Outputs
OUTPUTS: Vibrio anguillarum is the causative agent of warm-water vibriosis in fish. The overall goal of this project has been to describe the molecular basis for virulence in this organism. The objectives for the grant period were to investigate the regulation and functions of the hemolysin and metalloprotease virulence factors of V. anguillarum. The transcriptional regulation of the three hemolysin genes, rtxA, plp, and vah1, was examined and analyzed. Additionally, the effects of each hemolysin on host cell killing and overall virulence were examined in both wild type and various hemolysin mutant strains of V. anguillarum. The mechanisms of hemolysin action were also examined. The regulation of the EmpA metalloprotease was examined and described with regard to post-translational regulation. The function of EmpA as a virulence factor was examined with regard to its role in suppressing the innate and adaptive immune systems of the host fish. Results of this investigation were disseminated by communicated to the target scientific community through publication of refereed journal articles, publication of abstracts and presentations at international, national, and regional scientific meetings, and by giving invited oral seminar presentations to investigators at other universities. The most significant dissemination activities were the publication of two refereed papers, an invited oral presentation and abstract at an international meeting in Spain, an invited presentation at another university, and several invited presentations at regional meetings. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Vibriosis, caused by Vibrio anguillarum, is one of the most destructive bacterial diseases of fish, causing large economic losses to the aquaculture industry. Our results have defined mechanisms of regulation for EmpA metalloprotease and the three hemolysins, RtxA, Plp, and Vah1. EmpA is regulated both transcriptionally and post-translationally. Previously, we described transcriptional regulation of EmpA and now describe post-translational regulation. Briefly, EmpA is initially secreted as pro-EmpA, which is processed to mature, active EmpA by cleavage of a C-terminal 10kDA peptide by a second protease, Epp. Like EmpA, Epp transcription is up-regulated by fish gastrointestinal mucus. Hemolysin gene expression is regulated by binding a repressor protein, HNS, to intergenic regions between the plp and vah1 genes and between the rtxHCA and rtxBDE genes. HNS binding overlaps the promoter regions of these genes. Binding of the anti-repressor protein, HlyU, disrupts HNS binding. HlyU binds to the center of the intergenic regions, but does not overlap with HNS binding sites. Thus balance between HNS and HlyU controls hemolysin gene expression. The roles of virulence proteins were examined. We showed that both EmpA and pro-EmpA degraded components (C3, C4, C5, and Factor B) of the fish complement pathway. It is interesting to note that pro-EmpA, which is not a general protease, targets complement components; however, EmpA is able to degrade both complement and many other proteins including casein, gelatin, and fish immunoglobins (IgT and IgM). Thus EmpA and pro-EmpA degrade components of the innate and adaptive immune systems of fish. The three hemolysins exhibit different activities. Plp is a phospholipase that specifically reacts with phosphatidylcholine, a major phospholipid of fish erythrocytes. Vah1 is a pore-forming hemolysin and induces excessive vacuolization in Atlantic salmon kidney cells. RtxA kills target cells primarily by inducing apoptosis. V. anguillarum mutants lacking RtxA are avirulent, while mutants lacking either Vah1 or Plp show little or no loss of virulence. However, all three hemolysin-cytotoxins preferentially target fish leukocytes, as purified leukocytes are killed with much lower doses of the toxins and the presence of any two hemolysins is enough to cause wild type levels of killing. Killing is only abolished when all three hemolysins are not present. Finally, we have begun to examine the effects of central metabolism on the expression of the hemolysin genes. While mutations in the TCA cycle will attenuate V. anguillarum, some TCA cycle mutations actually increase hemolysin gene expression. Results of these studies elucidate how virulence factors of V. anguillarum are regulated and function to evade and disarm the fish immune system. The results also suggest that we can generate specific mutants of V. anguillarum that are attenuated or avirulent that may have specific abilities to infect the fish host while retaining antigenic targets an immune response. Thus, we expect to be able to use this information to construct live vaccine strains of V. anguillarum.
Publications
- Mou, X. and Nelson, D.R. 2012. Effects of tricarboxylic acid cycle mutations on hemolysin gene expression in Vibrio anguillarum. 112th American Society for Microbiology General Meeting Abstracts. San Francisco, CA
- Mou, X., Spinard, E., Driscoll, M.V., and Nelson, D.R. 2012. HNS is a negative regulator of hemolysin/cytotoxin genes in Vibrio anguillarum. Boston Bacteriology Meeting Abstracts. Boston, MA
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Progress 09/01/10 to 08/31/11
Outputs
OUTPUTS: Conducting and analyzing experiments: Vibrio anguillarum is the causative agent of warm-water vibriosis. We have been investigating the molecular basis for virulence in this organism. For this reporting period we focused on the roles and regulation of the three hemolysin/cytotoxin genes and their encoded proteins. These genes are plp, vah1, and rtxA. We hypothesized that the RtxA cytotoxin (encoded by the rtxA gene) killed cells by either of two mechanisms: apoptosis or necrosis. Experiments were carried out to examine whether Atlantic salmon kidney (ASK) cells cultured in the presence of V. anguillarum cells induce caspase 3/7. ASK cells exposed to V. anguillarum cells containing an intact rtxA gene at moi values from 10-200 exhibited strong caspase 3/7 induction 4 h after initial exposure. No caspase activity was observed in rtxA-deficient strains of V. anguillarum. ASK cells treated with V. anguillarum strains lacking vah1 or plp showed no significant changes in caspase induction. These data suggest that V. anguillarum kills ASK cells primarily by apoptosis using the RtxA cytotoxin. Experiments were also conducted to examine whether culture supernatants from V. anguillarum cells could kill leukocytes isolated from rainbow trout head kidney and spleen. Freshly isolated leukocytes were mixed with V. anguillarum culture supernatants prepared from various hemolysin/cytotoxin mutant strains diluted to either 0.5x or 0.1x, incubated for 3 h, and then assessed for leukocyte killing. Observations revealed that each of the cytotoxins efficiently targeted leukocytes and only mutants lacking all three cytotoxins did not kill leukocytes. Experiments were conducted to examine the regulation of plp, vah1, and rtxA gene expression. It was found that plp and vah1 represent one gene cluster and are divergently transcribed, while rtxA is part of a 6-gene cluster on two divergently transcribed operons. The intergenic regions between plp and vah1 and between the various rtx genes were examined. It was found that an hlyU mutant of V. anguillarum produced reduced amounts of the three cytotoxins and that complementation of hlyU restored cytotoxin production. Gel shift experiments and DNase I protection assays revealed that HlyU protein binds in the middle of both intergenic regions to up-regulate cytotoxin production. Dissemination: The results of this investigation were disseminated to the scientific community by publication of our results in scientific journals and presentations at national meetings. Products: One student, Kenneth Gareau graduated with a M.S. degree in Cell and Molecular Biology PARTICIPANTS: Nelson, David R.: Project Director; directed entire project Sunyer, Oriol: co-PD; directed efforts examining effects of EmpA, pro-EmpA, Epp, Plp, Vah1, and RtxA on the immune system of fish Graduate Students: Gareau, Kenneth - Role: performed assays to characterize the cytotoxic activity of RtxA against Atlantic salmon kidney cells to determine whether RtxA induces apoptosis or necrosis Mou, Xiangyu - Role: worked on describing the regulation of the plp-vah1 and rtx gene clusters by hlyU and to characterize the activity of the Plp phospholipase Post-doctoral fellows: Jun Li and Yong-An Ahang Roles: Both performed complement hemolytic assays to test the capacity of bacterial culture supernatants (containing EmpA, pro-EmpA and/or Epp) to inhibit the activation of the rainbow trout complement system. In addition, they used the same bacterial culture supernatants to analyze their proteolytic activity against a number of complement proteins (C3, C4, C5, Bf) as well as trout immunoglobulins, including IgM and IgT. They also examined the effects of the hemolysins Plp, Vah1, and RtxA on rainbow trout leukocytes. TARGET AUDIENCES: The target audience is the scientific community, particularly those that are concerned with understanding the molecular basis for virulence and the development of vaccines and the aquaculture industry. Our efforts to deliver science-based knowledge have been to publish our results in peer-reviewed scientific journals and to present talks and abstracts at various meetings with members of both target audiences present. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
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 factors, the EmpA metalloprotease (encoded by empA) and hemolytic activity (encoded by three hemolysin genes, vah1, plp, and rtxA). We have now demonstrated that the three hemolysin genes are encoded by two gene clusters. One gene cluster includes plp and vah1. These two genes are divergently transcribed with an intergenic region between them containing RpoD-responsive promoters. The second gene cluster includes the six rtx genes rtxHCA and rtxBDE, which are divergently transcribed with an intergenic sequence containing RpoD-responsive promoters between them. We used degenerate PCR to identify a positive hemolysin regulatory gene, hlyU, from the unsequenced V. anguillarum genome. The hlyU gene of V. anguillarum encodes a 92-amino-acid protein and is highly homologous to other bacterial HlyU proteins. An hlyU mutant was constructed, which exhibited an ~5-fold decrease in hemolytic activity on sheep blood agar with no statistically significant decrease in cytotoxicity of the wild-type strain. Complementation of the hlyU mutation restored both hemolytic activity and cytotoxic activity. Both semiquantitative reverse transcription-PCR (RT-PCR) and quantitative real-time RT-PCR (qRT-PCR) were used to examine expression of the hemolysin genes under exponential and stationary-phase conditions in wild-type, hlyU mutant, and hlyU complemented strains. Compared to the wild-type strain, expression of rtx genes decreased in the hlyU mutant, while expression of vah1 and plp was not affected in the hlyU mutant. Complementation of the hlyU mutation restored expression of the rtx genes and increased vah1 and plp expression to levels higher than those in the wild type. The transcriptional start sites in both the vah1-plp and rtxH-rtxB genes' intergenic regions were determined using 5' random amplification of cDNA ends (5'-RACE), and the binding sites for purified HlyU were discovered using DNA gel mobility shift experiments and DNase protection assays.
Publications
- Gareau, K.W. and Nelson, D.R. 2011. Vibrio anguillarum induces apoptosis in Atlantic salmon Kidney cells. 111th American Society for Microbiology General Meeting Abstracts. New Orleans, LA
- Li, L., Mou, X., and Nelson, D.R. 2011. HlyU is a positive regulator of hemolysin expression in Vibrio anguillarum. J. Bacteriol. 193:4779-4789.
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Progress 09/01/09 to 08/31/10
Outputs
OUTPUTS: Infection of fish by the bacterium Vibrio anguillarum causes warm-water vibriosis, a lethal hemorrhagic septicemia. We have been investigating the regulation of two virulence factors, EmpA metalloprotease and hemolysin, in V. anguillarum. The two hemolysin gene clusters previously identified in V. anguillarum, the vah1 cluster and the rtxACHBDE cluster, are responsible for the hemolytic and cytotoxic activities of V. anguillarum in fish. We used degenerate PCR to identify a positive hemolysin regulatory gene, hlyU, from the unsequenced V. anguillarum genome. The hlyU gene of V. anguillarum encodes a 92-amino acid protein and is highly homologous to other bacterial HlyU proteins. An hlyU mutant was constructed, which exhibited ~5-fold decrease in hemolytic activity on sheep blood agar and ~80% of the cytotoxicity of the wild type strain. Complementation of the hlyU mutation restored both hemolytic and cytotoxic activity. Both semi-quantitative RT-PCR and real time RT-qPCR were used to examine expression of the hemolysin genes under exponential and stationary phase conditions in wild type, hlyU mutant, and hlyU complemented strains. Compared to the wild type strain, expression of rtx genes deceased in the hlyU mutant while expression of vah1 and plp was not affected in the hlyU mutant. Complementation of the hlyU mutation restored expression of the rtx genes and increased vah1 and plp expression to levels higher than in the wild type. The transcriptional start sites in the intergenic regions of both vah1/plp and rtxH/rtxB genes were determined using 5'-RACE and the binding sites for purified HlyU was discovered using DNA gel mobility shift experiments and DNase protection assays. We also examined the effects of the secreted metalloprotease EmpA and the hemolytic cytotoxins RtxA and Vah1 upon the innate and adaptive immune systems of rainbow trout (Oncorhynchus mykiss) using culture supernatants from wild type, mutant, and complemented mutant strains of V. anguillarum. Supernatants containing EmpA and pro-EmpA (secreted precursor of EmpA) inhibited complement-induced lysis of rabbit erythrocytes, while supernatants from an EmpA mutant did not inhibit lysis. Degradation of specific complement components was tested by western blot analysis. EmpA and pro-EmpA degraded C3, C4, and C5, while EmpA and Epp (EmpA processing protease) degraded factor B. EmpA degraded IgM and IgT. The effects of the cytotoxins upon phagocytic cells were investigated. These toxins were extremely lethal to phagocytic cells. Our data show that V. anguillarum virulence factors incapacitate both the innate and adaptive immune systems of fish. Additionally, three graduate students (Xiangyu Mou, Maureen Varina, and Kenneth Gareau) were mentored by the PD and one (Maureen Varina) graduated with her doctorate. PARTICIPANTS: Nelson, David R.: Project Director; directed entire project Sunyer, Oriol: co-PD; directed efforts examining effects of EmpA, pro-EmpA, and Epp on the immune system of fish Graduate Students: Gareau, Kenneth - Role: performed assays to characterize the cytotoxic activity of RtxA against Atlantic salmon kidney cells to determine whether RtxA induces apoptosis or necrosis Mou, Xiangyu - Role: worked on describing the regulation of the vah1 and rtx gene clusters by hlyU and to characterize the activity of the Plp phospholipase Varina, Maureen; Graduate student; worked on EmpA and Epp metalloproteases to construct mutants to determine protein functions and roles in affecting the innate immune functions of the fish host by degrading complement proteins and immunoglobins Post-doctoral fellows: Jun Li and Yong-An Ahang Roles: Both performed complement hemolytic assays to test the capacity of bacterial culture supernatants (containing EmpA, pro-EmpA and/or Epp) to inhibit the activation of the rainbow trout complement system. In addition, they used the same bacterial culture supernatants to analyze their proteolytic activity against a number of complement proteins (C3, C4, C5, Bf) as well as trout immunoglobulins, including IgM and IgT. TARGET AUDIENCES: The target audience is the scientific community, particularly those that are concerned with understanding the molecular basis for virulence and the development of vaccines and the aquaculture industry. Our efforts to deliver science-based knowledge have been to publish our results in peer-reviewed scientific journals and to present talks and abstracts at various meetings with members of both target audiences present. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
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 factors, the EmpA metalloprotease (encoded by empA) and hemolytic activity (encoded by three hemolysin genes, vah1, plp, and rtxA). We discovered that the hemolysin RtxA (encoded by rtxA) is the major hemolysin in V. anguillarum. Our most recent findings demonstrate that the RtxA is a member of the MARTX (multifunctional autoprocessing repeat-in-toxin) family of toxins. Specifically, we demonstrated that RtxA contains several domains with specific activities. These include a cysteine (CPD) protease domain, a cell-rounding domain (CRD), and the A, B, and C repeat regions. Our data demonstrate that the CPD is responsible for the autoproteolytic cleavage of RtxA. CPD activity is not required for toxicity or hemolysis, as an in-frame deletion of the CPD did not affect the toxicity of RtxA. In contrast, the CPD is required for toxicity but not for secretion, as deletion of this domain resulted a non-toxic, non-hemolytic secreted protein. Our data raise the possibility that this CRD-deleted variant of RtxA may be a candidate for vaccine development against V. anguillarum. Further, we have now demonstrated that both hemolysin gene clusters, vah1 (which includes vah1 and plp) and rtx, are positively regulated by the HlyU protein (encoded by hlyU). We have also demonstrated that the EmpA protease, the pro-EmpA precursor, and Epp protease (EmpA processing protease) are able to degrade components of the fish innate and adaptive immune responses, including complement compenents and the immunoglobulins IgM and IgT. These results demonstrate that there are specific mechanisms by which V. anguillarum is able to evade and degrade the host immune responses which enable this organism to establish and maintain an infection.
Publications
- Nelson, D.R., Varina, M., Li, J., and Sunyer, J.O. 2010. Effects of secreted protease and hemolysins of Vibrio anguillarum upon innate and adaptive immune systems of rainbow trout. Abstracts Ninth International Congress on the Biology of Fish.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: Infection of fish by the bacterium Vibrio anguillarum causes warm-water vibriosis, a lethal hemorrhagic septicemia. We have been investigating the regulation of two virulence factors, EmpA metalloprotease and hemolysin, in V. anguillarum. We have previously shown that hemolytic activity by V. anguillarum is required for virulence and is mediated by two gene clusters, the vah1 (four genes) and rtx (six genes) clusters. V. anguillarum RtxA is a member of the multifunctional autoprocessing RTX (MARTX) protein family and causes fish cell rounding and death. We demonstrated that the 440 kDa full-length RtxA toxin was processed into multiple products, including a 130 kDa C-terminal portion, by immunoblot analysis using anti-RtxA C-repeat antibody. A conserved domain, homologous to the cysteine protease domain (CPD) in V. cholerae RtxA, was identified in the RtxA of V. anguillarum and shown to have cysteine protease activity. In vitro characterization of purified recombinant CPD peptide revealed that CPD activity is activated by Atlantic salmon kidney (ASK) cell lysate, but not by GTP. The cleavage site for CPD was identified between Lys3289 and Ala3290 by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF), and suggested that the 130 kDa carboxyl portion of RtxA is released into bacterial culture supernatant after cleavage. Additionally, a mutant with the entire C-terminal region after the CPD cleavage site deleted (in a vah1 background) completely lost hemolytic and cytotoxic activity, indicating that the C-terminal region behind the CPD cleavage site was important for RtxA toxin function. Further, an in-frame deletion of CPD did not affect either hemolytic or cytotoxic activity of RtxA. Interestingly, in-frame deletion of a 515 amino acid region between the CPD and the C-repeat regions resulted in the loss of both hemolytic activity and ASK cell rounding. This region was termed the cell-rounding domain (CRD). Western blot analysis revealed that RtxA lacking CRD was secreted by V. anguillarum cells. These data demonstrate that CRD is required for the hemolytic and cytotoxic activities of the RtxA toxin. The effects of the EmpA protease, pro-EmpA, and the Epp protease upon complement components of fish (Rainbow trout) were tested by examining inhibition of complement-mediated lysis of rabbit erythrocytes. V. anguillarum culture supernatants containing EmpA (wild-type) or pro-EmpA (epp mutant) inhibited complement activity by 75-80% compared to control samples without culture supernatant, while supernatants from empA mutants that contained neither EmpA nor pro-EmpA, but did contain Epp, exhibited no inhibition of complement activity. When the same culture supernatants were incubated in the presence of specific complement components (i.e. C3, C4, C5, and factor B) it was found that both EmpA and pro-EmpA degraded C3, C4, and C5. Both EmpA and Epp degraded factor B. Only EmpA degraded the immunoglobins IgM and IgT. Additionally, three graduate students (Ling Li, Maureen Varina, and Kenneth Gareau) were mentored by the PD and one (Ling Li) graduated with his doctorate. PARTICIPANTS: Nelson, David R.: Project Director; directed entire project Sunyer, Oriol: co-PD; directed efforts examining effects of EmpA, pro-EmpA, and Epp on the immune system of fish Graduate Students: Gareau, Kenneth - Role: performed assays to characterize the cytotoxic activity of RtxA against Atlantic salmon kidney cells to determine whether RtxA induces apoptosis or necrosis Li, Ling - Role: worked on characterizing RtxA structure and domain function and to characterize the activity of the Plp phospholipase Varina, Maureen; Graduate student; worked on EmpA and Epp metalloproteases to construct mutants to determine protein functions and roles in affecting the innate immune functions of the fish host by degrading complement proteins and immunoglobins Post-doctoral fellows: Jun Li and Yong-An Ahang Roles: Both performed complement hemolytic assays to test the capacity of bacterial culture supernatants (containing EmpA, pro-EmpA and/or Epp) to inhibit the activation of the rainbow trout complement system. In addition, they used the same bacterial culture supernatants to analyze their proteolytic activity against a number of complement proteins (C3, C4, C5, Bf) as well as trout immunoglobulins, including IgM and IgT. TARGET AUDIENCES: The target audience is the scientific community, particularly those that are concerned with understanding the molecular basis for virulence and the development of vaccines and the aquaculture industry. Our efforts to deliver science-based knowledge have been to publish our results in peer-reviewed scientific journals and to present talks and abstracts at various meetings with members of both target audiences present. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
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 factors, the EmpA metalloprotease (encoded by empA) and hemolytic activity (encoded by two hemolysin genes, vah1 and rtxA). We discovered that the hemolysin RtxA is the major hemolysin in V. anguillarum. Our most recent findings demonstrate that the RtxA is a member of the MARTX (multifunctional autoprocessing repeat-in-toxin) family of toxins. Specifically, we demonstrated that RtxA contains several domains with specific activities. These include a cysteine (CPD) protease domain, a cell-rounding domain (CRD), and the A, B, and C repeat regions. Our data demonstrate that the CPD is responsible for the autoproteolytic cleavage of RtxA. CPD activity is not required for toxicity or hemolysis, as an in-frame deletion of the CPD did not affect the toxicity of RtxA. In contrast, the CPD is required for toxicity but not for secretion, as deletion of this domain resulted a non-toxic, non-hemolytic secreted protein. Our data raise the possibility that this CRD-deleted variant of RtxA may be a candidate for vaccine development against V. anguillarum. We have also demonstrated that the EmpA protease, the pro-EmpA precursor, and Epp protease (EmpA processing protease) are able to degrade components of the fish innate and adaptive immune responses. This demonstrates that there are specific mechanisms by which V. anguillarum is able to evade the host immune response during infection.
Publications
- Li, L. and Nelson, D.R. 2009. The cell rounding domain (CRD) of RtxA is essential for hemolytic and cytotoxic activities of Vibrio anguillarum. FASEB Summer Research Conferences. Microbial Pathogenesis: Mechanisms of Infectious Disease. Abstract Number 20
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