Progress 10/01/14 to 09/30/19
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?7 undergraduate students, two professionals, and two technicians conducted research for this project. The project thus provide the students with the opportunity to gain research experience in gene cloning and deletion and gene expression and regulation. Two students were coauthors for published research papers. How have the results been disseminated to communities of interest?The results were presented at three international symposium and some of the results were published. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
The secovRS and semagA genes of Streptococcus equi were deleted, generating secovRS and semagA gene deletion mutants. The deletion mutants were compared with the parent strain in mouse model of intranasal S. equi infection, finding that these mutants were not attenuated in virulence. Then we compared the expression of the capsule synthase gene in the secovRS deletion mutant and wild-type strain and found that the secovRS deletion did not alter the expression of the capsule synthase gene. It is possible that the strain studied might have a mutation that compromised the function of SeCovRS to repress the capsule gene. To test this possibility, we sequenced the secovRS gene in 5 clinical isolates and did not find variation of the secovRS gene, ruling out the possibility. Similary, we compared the expression of the seM gene in wild-type strain and semagA deletion mutant and found no significant difference in seM gene expression.Our data suggest that, unlike in Group A Streptococcus, SeCovRS and SeMagA do not regulate major virulence factors in S. equi. We tested a mouse model of pulmonary infection using the signle and double deletion mutants of the streptolysin S and platelet-activating factor (PAF) acetylhydrolase see genes. We found that the single deletion mutants were not attenuated in virulence but the double deletion mutant was attenuated. Furthermore, we found that S. equi has the ability to invade the vascular system in this new mouse infection model.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2019
Citation:
1. Lei B, Minor D, Feng W, Jerome M, Quinn MT, Jutila MA, Liu M. 2019. Tissue Tropism in Streptococcal Infection: Wild-Type M1T1 Group A Streptococcus is Efficiently Cleared by Neutrophils Using an NADPH Oxidase-Dependent Mechanism in the Lung but not in the Skin. Infect Immun. 87: e00527-19.
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Progress 10/01/17 to 09/30/18
Outputs
Target Audience:
Nothing Reported
Changes/Problems:Since the results indicate that the deletion of secovRS and semga did not have significant effects on virulence and the new data show that S. equi can invade vascular system, we will search for virulence factors that contribute to S. equi invasion of the vascular system. What opportunities for training and professional development has the project provided?One undergraduate student and one graduate student participated in this project. They acquired experience in histological analyses of infected mouse tissues. How have the results been disseminated to communities of interest?Dr. Lei gave a keynote presentation at 2nd International Conference on Microbial Pathogenesis and Infectious Diseases, Vancouver, Canada: Molecular Mechanisms of Hypervirulent Group A Streptococcus to Evade Innate Immune Responses and to Invade the Vascular System in Mouse Model of Pulmonary Infection. What do you plan to do during the next reporting period to accomplish the goals?We will search for virulence factors that contribute to vascular invasion by S. equi.
Impacts
What was accomplished under these goals?
We compared the secovR and semga mutants with their parent strain in virulence in the mouse model of pulmonary infection. There were no differences in virulence among the strains. However, we found that S. equi invades the peribronchovascular interstitium in murine pulmonary infection. This phenomenon was similar to that in pulmonary infection of mice with a hypervirulent Group A Streptococcus (GAS) isolate. This GAS strain has a G1370T mutation in the sensor kinase covS gene of CovRS. Intratracheal inoculation of MGAS315 led to the lung infection that displayed extensive Gram staining at the alveolar ducts, alveoli, and peribronchovascular and perivascular interstitium. The correction of the covS mutation did not alter the infection at the alveolar ducts and alveoli but prevented GAS invasion of the peribronchovascular and perivascular interstitium. Furthermore, the covS mutation allowed MGAS315 to disrupt and degrade the smooth muscle and endothelial layers of the blood vessels, directly contributing to systemic dissemination. It is concluded that hypervirulent emm3 GAS covS mutants can invade the perivascular interstitium and directly attack the vascular system for systemic dissemination. Clinical S. equi show a similar phenotype with the hypervirulent GAS strain in peribronchovascular invasion, suggesting that S. equi may have the capacity to translocate the epithelial cells to enter the perivascular space where S. equi is drained to lymph nodes. The data also suggest that clinical S. equi isolates already have mutation in secovRS.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Lei B, Minor D, Feng W, Liu M. 2018. Hypervirulent Group A Streptococcus of Genotype emm3 Invades the Vascular System in Pulmonary Infection of Mice. Infect Immun 86: e00080-18.
- Type:
Journal Articles
Status:
Published
Year Published:
2018
Citation:
Liu M., Lei B. 2018. Pathogenesis of hypervirulent Group A Streptococcus. Jpn J Med. 1:6:269-275
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Progress 10/01/16 to 09/30/17
Outputs
Target Audience:We gave two oral presentations at "A Haveneyer Foundation Workshop: Getting to Grips with Strangles and other Streptococcal Diseases", which was held on September 19-21, 2017 at Gallatin Gateway, Montana. The workshop was attended by 41 scientists. Changes/Problems:We had expected that SeCovRS and SeMga are the critical transcription regulators for the virulence factors of S. equi. However, the results so far indicate that their deletions had no significant effect on virulence and virulence gene expression. It is likely possible that the virulence genes of S. equi are regulated by other regulators. Thus, we will test other transcription regulators, especially other two component regulatory systems of S. equi for their role in virulence regulation. What opportunities for training and professional development has the project provided?Two undergraduate students and one technician participated in this project. They acaured experience in mouse model of bacterial infection and measurement of gene expression by real-time RT qPCR. How have the results been disseminated to communities of interest?We gavethe followingoral presentations at "A Haveneyer Foundation Workshop: Getting to Grips with Strangles and other Streptococcal Diseases", which was held on September 19-21, 2017 at Gallatin Gateway, Montana: Feng W., Liu M., Minor D., and Lei B. Mouse model for pulmonary infection with Streptococcus equi subspecies equi and Group A Streptococcus Lei B., Liu M., Xie G., and Zhu H. Evaluation of a tetravalent subunit strangle vaccine. What do you plan to do during the next reporting period to accomplish the goals?We plan to compare the secovR and semga mutants with their parent strain in virulence in the mouse model of pulmonary infection. If we still observe insignificant difference, we will test other transcription regulators for their role in the regulation of virulence factors including the capsule synthase HasA.
Impacts
What was accomplished under these goals?
In the second progress report for the period of 10/01/2015 to 09/30/2016, we reported unexpected results of the SecovRS mutant in terms of the effects of the CovR mutation on virulence and expression of the capsule synthase. We suspected that the strain we used might have mutation in the secovRS genes that led to the unexpected results. So we sequenced the secovRS genes of 5 clinical isolates and all had the same sequence. Thus, the unexpected results were unlikely due to a mutation of the secovRS genes. The data suggest that, unlike inGroup A Streptococcus,SecovRS may not regulatethe major virulence factors in S. equi. We compared the virulence of SeMga mutant with its parent strain using the mouse model ofintranasal infection. We did notobserve significant difference in virulence.Wealso test amouse model of pulmonary infection using the single and doubledeletion mutants of the streptolysin S andplatelet-activating factor (PAF) acetylhydrolasesee genes. We found that thesinglestreptolysin S or see deletion mutant was notattenuatedin virulence in this new model but the double mutant was attenuated. Thus, wedeveloped a newmouse infection model for S. equi thatshould be useful foridentification and evaluation virulence factors. This work was presented ina Haveneyer Foundation Workshop on Getting to Grips with Strangles and other streptococcal diseases.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2017
Citation:
Feng W, Minor D, Liu M, Lei B. 2017. Requirement and Synergistic Contribution of Platelet-Activating Factor Acetylhydrolase Sse and Streptolysin S to Inhibition of Neutrophil Recruitment and Systemic Infection by Hypervirulent emm3 Group A Streptococcus in Subcutaneous Infection of Mice. Infect Immun. 85: e00530-17.
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Progress 10/01/15 to 09/30/16
Outputs
Target Audience:The target audience includes: scientists in bacterial pathogenesis, strangle research and gene regulation and students in animal health. Changes/Problems:As described in the result, we need to figure out whether the S. equi strain had a natural SeCovRS mutation. However, this is not a major change in the approach. What opportunities for training and professional development has the project provided?Two undergraduate students, one professional, and one technician participated in this project. The project thus provide the students with the opportunity to gain research experience in gene cloning and deletion and gene expression and regulation. One student was a co-author of a recently accepted manuscript that will be published soon in Infection and Immunity. How have the results been disseminated to communities of interest?
Nothing Reported
What do you plan to do during the next reporting period to accomplish the goals?We will check the hasA expression in clinical S. equ isolates in our collection and sequence the SeCovRS genes to determine whether there was a natural SeCovRS mutation in the strain from which the SeCovR deletion mutant was obtained. Addressing this question is critical for the SeCovR part of the objectives. If the strain had a natural SeCovRS mutation, we will generate SeCovR mutant of a strain with wild-type SeCovRS genes if the strain and then perform transcriptome analysis. Otherwise, wewillperform transcriptome analysis for theSeCovR mutantwe have now and its parent strain. We will also compare the virulence of the SeMga deletion mutant with its parent strain in mouse model of intranasal infection.
Impacts
What was accomplished under these goals?
In the first year, we got putative inactivation mutants of the SeCovR gene of Streptococcus equi. In this yearwe have confirmed the SeCovR gene inactivation by DNA sequencing. We also obtained the SeMga inactivation mutant of Streptococcus equi. We compared the virulence of the SeCovR gene deletion mutant with its parent strain using mouse model of intranasal infection. We found that the deletion of the SeCovR gene did not enahnce virulence. The result was unexpected. To figure out the reason for the unexpected result, we compared the expression levels of the hasA gene in the SeCovR deletion mutant and the parent strain, the gene encoding synthase for production of the hyaluronic capsule. Both strains expressed the high levels of the hasA transcript. CovRS suppresses hasA expression, and CovR deletion enhances hasA expression. Our results suggest that the clinical isolate may have a natural CovRS mutation that enhanced hasA expression, a frequent phenomenon in Group A Streptococcus. We will need to determine whether the S. equi strain had a CovRS mutation prior to transcriptome analysis.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2016
Citation:
2. Feng W, Liu M, Chen DG, Yiu R, Fang FC, Lei B. 2016. Contemporary Pharyngeal and Invasive emm1 and Invasive emm12 Group A Streptococcus Isolates Exhibit Similar In Vivo Selection for CovRS Mutants in Mice. PLoS One 11:e0162742.
- Type:
Journal Articles
Status:
Awaiting Publication
Year Published:
2017
Citation:
1. Feng W, Minor D, Liu M, Li J, Ishaq SL, Yeoman C, Lei B. 2016. Null Mutations of Group A Streptococcus Orphan Kinase RocA: Selection in Mouse Infection and Comparison with CovS Mutations in Alteration of in vitro and in vivo Protease SpeB Expression and Virulence. Infect Immun doi:10.1128/IAI.00790-16
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Progress 10/01/14 to 09/30/15
Outputs
Target Audience:
Nothing Reported
Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?One undergraduate student, one technican, and a professional participated in this project. Their participation provided opportunity for them to gain experience and knowledge in microbiology and bacteriology. How have the results been disseminated to communities of interest?Some resultswill bepresented in a seminar on October 29th, 2015 for faculty, students, and researchers in the Department of Animal Sciences at Montana State University. What do you plan to do during the next reporting period to accomplish the goals?We will first confirm the inactivation of the SeCovR gene in putative mutants obtained and generate the SeMga deletion mutants. After the mutants are obtained, we will start to identify genes that are regulated by the SeCovRS and SeMga virulence regulators by comparing mutants with parent S. equi strain in transcriptome.
Impacts
What was accomplished under these goals?
In the first year of this project, we mainly focused on generation of Streptococcus equi mutants that are defective in the SeCovR and SeMga genes. We have contructed plasmids for the inactivation of the SeCovR and SeMga genes, and we have obtained putative inactivation mutants of the SeCOvR gene.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2015
Citation:
Liu G, Feng W, Li D, Liu M, Nelson DC, Lei B. 2015. The Mga Regulon but not Deoxyribonuclease Sda1 of Invasive M1T1 Group A Streptococcus Contributes to in vivo Selection of CovRS Mutations and Resistance to Innate Immune Killing Mechanisms. Infect Immun. 83:4293-4303
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