VIRULENCE FACTORS OF HORSE PATHOGEN STREPTOCOCCUS EQUI

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: ANIMAL HEALTH
• Reporting Frequency: Annual
• Accession No.: 0228223
• Project Start Date: Jan 1, 2012
• Project End Date: Dec 31, 2015
• Program Code: [(N/A)]- (N/A)

Recipient Organization
MONTANA STATE UNIVERSITY
(N/A)
BOZEMAN,MT 59717

Performing Department
Microbiology & Immunology

Non Technical Summary
The goal of this project is to identify virulence factors and protective antigens among the cell surface proteins of horse pathogen Streptococcus equi. This pathogen causes strangeles, one of the most common infectious diseases in horses. Most horses recovered from strangles have immunity against S. equi reinfection for at least 5 years. The immunity is primarily mediated by protective antibodies. Available strangle vaccines are not sufficiently protective or have safety issue. A safe and effective protein-based or subunit vaccine is desirable. Subunit strangle vaccine formulations consisting of known protective antigens are either insufficiently protective or need verification or field test. Our own testing found that three protective antigens cannot provide protection in horses. We hypothesize that an effective subunit vaccine needs to include more protective antigens. Thus, we propose to identify additional virulence factors and protective antigens among 13 uncharacterized cell surface proteins. In objective 1, we will identify cell wall surface protein genes that are critical for virulence of S. equi in a mouse model of intranasal infection. In objective 2, we will test whether the proteins encoded by identified virulence genes evoke protection against intranasal S. equi infection in mice. We expect to identify new protective antigens for the development of an effective and safe strangles subunit vaccine.

Animal Health Component

50%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	4010	1100	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1100 - Bacteriology;

Keywords

streptococcus equi

strangles

vaccine

virulence

protective antigens

cell wall-linked protein

Goals / Objectives
The goal of this project is to identify virulence factors and protective antigens among the cell surface proteins of horse pathogen Streptococcus equi. We will pursue two objectives to achieve the goal. First, we will identify new virulence factors among 13 uncharacterized cell wall proteins. Second, we will determine whether virulence factors identified in the first objective are protective antigens. We expect to identify new protective antigens that can be included in a vaccine formulation for the development of an effective and safe strangles subunit vaccine.

Project Methods
To achieve the first objective, we will first generate deletion mutants of S. equi for 13 genes that encode uncharacterized cell wall-linked proteins listed. Next, we will compare mutants with wild-type strain in intranasal infection of mice in which we will compare survival rates, throat colonization, and S. equi load in blood, lung and spleen. To achieve the second objective, we will clone genes of virulence factors identified in the first objective for overexpression of these genes in E. coli. The expressed recombinant proteins will be purified. Mice will be immunized intranasally with the purified proteins and challenged with intranasal S. equi infection.

Progress 01/01/12 to 12/31/15

Outputs
Target Audience:A seminar on evasion of innate immune response by Streptococcus equi is given to the faculty, students, and researchers at the Department of Aminal Sciences at Montana State University on October 29th, 2015. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided opportunities to train 1 graduate student, 5 undergraduate students, and 1 postdoctoral fellow. How have the results been disseminated to communities of interest?Part of the resultshave been presented in the 113th General meeting of American Society for Microbiologyin 2013,a seminar, and a publication for scientific community. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? To achieve the goal of this project to identify virulence factors and protective antigens among the cell surface proteins of horse pathogen Streptococcus equi, we pursued two objectives: 1) Identifying cell wall surface protein genes that are critical for virulence of S. equi and 2) testing whether identified virulence factors are protective antigens. In achieving objective 1, plasmids were constructed for inactivating target genes through recombination; 13 genes were successfully inactivated, including SEQ_0090, SEQ_0260, SEQ_0280, SEQ0555, SEQ_0633, SEQ_0646, SEQ_0855, SEQ_0904, SEQ_1116, SEQ_1259, SEQ_1278, SEQ_1606, SEQ_1817, and SEQ_2100 (The gene numbers are those used in the genome of S. equi subsp. equi strain 4047). Inactivation mutants of the SEQ_0855, SEQ_1278, SEQ_1817, and SEQ_2100 genes significantly increased the survival rates of infected mice compared with the parent strains. Thus, we identified 4 new virulence factors. In achieving objective 2; the DNA fragments of the SEQ_0855, SEQ_1278, SEQ_1817, and SEQ_2100 genes encoding mature proteins were cloned into plasmid vector pET-21d, and the SEQ_0855, SEQ_1278, SEQ_1817, and SEQ_2100 recombinant proteins were expressed and purified. The purified proteins were used to immunize mice in intranasal immunization using mouse IL-1alpha. The immunizations induced specific IgG antibody in blood. Immunized mice were challenged with S. equi. Although immunizations with these proteins increased survival rates of mice compared with adjuvant-treated mice, the protection was not significant. Thus, each of these proteins cannot provide significant protection against intranasal S. equi infection of mice, highlighting the difficulty to develop an efficacious vaccine to prevent strangles.

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
• Type: Journal Articles Status: Published Year Published: 2015 Citation: 2. Stetzner ZW, Li D, Feng W, Liu M, Liu G, Wiley J, Lei B. 2015. Serotype M3 and M28 Group A Streptococci Have Distinct Capacities to Evade Neutrophil and TNF-? Responses and to Invade Soft Tissues. PLoS One 10:e0129417.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: 3. Liu M, Feng W, Zhu H, Lei B. 2015. A Neutralizing Monoclonal IgG1 Antibody of Platelet-Activating Factor Acetylhydrolase SsE Protects Mice against Lethal Subcutaneous Group A Streptococcus Infection. Infect Immun. 83:2796-2805

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: We reached the research community by attending two meetings: The 114th General Meeting of American Society for Microbiology held on May 18-21, 2014 in Boston and The international Symposium on Microbiology in the Omics Era held on June 27-30, 2014 in Chongqing, China. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One posterdoctoral fellow (Dr. Guanghui Liu) and two undergraduate students (Emily Jackson and Robert Patrick Mullin) performed the gene cloning and generation of S. equi mutants. Research associate Wenchao Feng performed animal experiments. 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 clone the left 4 genes of the five viruence factor, express and purify recombinant proteins of the 5 virulence factors. Finally, we will immunize mice with the recombinant proteins and challenge the immunized mice in intranasal infection to find out whether these virulence factors are protective antigens or not.

Impacts
What was accomplished under these goals? We have finished the first objective. We generated 13 S. equi mutants that each had one of 13 target genes inactivated. These mutants were compared with parent strain in virulence using a mouse model of intranasal S. equi infecction. We found that five mutants were significantly attenuated. For the second objective, we have cloned one virulence gene for preparation of recombinant protein.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Li J, Liu G, Feng W, Zhou Y, Liu M, Wiley JA, Lei B. 2014. Neutrophils select hypervirulent CovRS mutants of M1T1 group A Streptococcus during subcutaneous infection of mice. Infect. Immun. 82:1579-1590.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Zhu H, Li D, Liu M, Copie V, Lei B. 2014. Non-heme-binding domains and segments of the Staphylococcus aureus IsdB protein critically contribute to the kinetics and equilibrium of heme acquisition from methemoglobin. PLoS One 6:e100744.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: We attended the 113th General Meeting of American Society for Microbiology in Denver to present a poster. We also published a paper in Infection and Immunity that characterized a virulence factor of Streptococcus equi and its homologue in Group A Streptococcus. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One undergraduate student, Emily Jackson, got training in molecular biology skills by making DNA constructs for gene inactivation. Dr. Guanghui Liu, a postdoctoral fellow, received training in studying the pathogenesis of S. equi using mouse model of infection. 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 continue the mouse infection study to compare S. equi mutants with the parent strain in virulence to identify virulence factors. We will start to clone the genes of identified virulence factors for protein expression and purify recombinant proteins.

Impacts
What was accomplished under these goals? The research activities in 2013 were directed at the first objective. We cloned the DNA fragments of 3 target genes forinactivation of the target genes. We successfully obtained Streptococcus equimutants for two of the three target genes. So far we have generated mutants for 12 of 13 target genes. We compared virulence of 4 mutants with the parent strain using a mouse model of intranasal S. equi infection, and one of the mutant was significantly attenuated in virulence.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Liu G, Liu M, Xie G, Lei B. 2013. Characterization of Streptococcal Platelet-Activating Factor Acetylhydrolase Variants That are Involved in Innate Immune Evasion. Infect. Immun. 81:3128-3138.

Progress 01/01/12 to 12/31/12

Outputs
OUTPUTS: The goal of this project is to identify virulence factors and protective antigens among uncharacterized cell wall-lined proteins of Streptococcus equi. We mainly focused on generating S. equi mutants. PARTICIPANTS: Wenchao Feng, a technician, is working on this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We proposed to generate deletion mutants of S. equi for 13 genes that encode uncharacterized cell wall-linked proteins. In the past year, we have generated mutants for 10 of the 13 genes. We will generate the mutants for the remaining 3 genes. Once we finish the construction of the mutants, we will move on to the next stage to the intranasal infection of mice to evaluate the virulence of these mutants and the parent strains.

Publications

• No publications reported this period