THE MECHANISM MEDITATING LONG TERM COLONIZATION OF PIGS BY SALMONELLA ENTERICA

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0233479
• Project Start Date: Apr 1, 2013
• Project End Date: Sep 30, 2014
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108

Performing Department
Veterinary Biomedical Sciences

Non Technical Summary
Salmonella is a common cause of food borne diarrhea. Most infections are directly or indirectly linked to animals. One such animal, the pig, is frequent carrier of Salmonella and fecal contamination of meats or produce are a likely source of infection. Our research is targeted toward understanding how Salmonella can infect food animals for long periods of time during their growth but not cause disease. This is important because infected aniamls appear to be healthy. We identified a mechanism whereby Salmonella can switch between two forms; one of which is infectious and the other is not. We believe that the mechanism responsible for this switching is responsible for its ability to be kept in pigs. The goal of this project is to create mutants that can't switch between these two forms. We will use modern techniques in molecular biology to identify the mechanism controlling this switching with the long term goal to identify a means to block this switching and reduce food borne illnesses cause by Salmonella.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	3510	1100	10%
311	4010	1100	10%
712	3510	1100	10%
712	4010	1100	70%

Knowledge Area
311 - Animal Diseases; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
3510 - Swine, live animal; 4010 - Bacteria;

Field Of Science
1100 - Bacteriology;

Keywords

salmonella

food safety

mutations

phase variation

transposon

molecular biology

virulence

colonization

carriers

Goals / Objectives
The rationale for the proposed research is that pigs become colonized early in life by S. enterica and become life long carriers of this bacterial food borne pathogen. Carrier animals remain asymptomatic but the stresses of shipping, feed withdrawal, and lairage prior to slaughter increase fecal shedding of this microbe and leads to contamination of food products that then can cause disease in humans. We identified a process of phenotypic phase variation that allows S. enterica to exist in a virulent phenotype or a non-virulent phenotype. Our data shows that cells can shift from one phenotype to the other. Using the technique RNA-Seq we were able to determine all of the genes differentially expressed in each phenotype. We hypothesize that the carrier state is mediated by modulation of the fraction of cells in a population that are in each phenotype. Thus, there are sufficient numbers to grow in the pig, but insufficient to cause disease. The proposal was developed to test this hypothesis. The most direct way to test this hypothesis is to first create appropriate mutations that lock cells in each phenotype.

Project Methods
The most direct way to test the hypothesis that phenotypic phase variation controls the ability of S. Typhimurium to cause persistent asymptomatic infections of pigs is to create mutants that are locked in each phenotypic phase. We developed a two color screen to aid in the selection of these mutants. LacZ and PhoA gene fusions will be made with two different genes that are in the repertoire of genes expressed during the virulent phenotype : namely SipC and ompF. We will then mutagenize the S. Typhimurium with an antibiotic resistance transposon and using the two color screen identify mutants that lose both gene activities. These mutants will be characterized by DNA sequencing and phenotype analysis to identified bone fide phase locked off mutants. The mutant locus will then be transduced to cells in the virulent phenotype creating a phase locked on mutant. The mechanism of phase variation will then be inferred based on the location of the mutations and the genes they are associated with.

Progress 04/01/13 to 09/30/14

Outputs
Target Audience: Nothing Reported Changes/Problems: The experimental approach did not yield the desired mutants. What opportunities for training and professional development has the project provided? Two undergraduate students were employed in this project. The project did give them an experience with basic research and as a result, one of the students has settled in on some career goals. 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? This is the project final report.

Impacts
What was accomplished under these goals? The goal of this project was to create mutants in Salmonella enterica serovar Typhimurium that were phase locked in either the virulent or avirulent phenotype using directed transposon mutagenesis. The approach remains plausible but in the time frame of the project the desired mutants were not selected.

Publications

Progress 04/01/13 to 09/30/13

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? This project started employing the research technician in the laboratory. However, shortly after the initiation of the project she resigned to pursue a Ph.D. degree in Ireland. Currently, two undergraduates have been employed to continue this work. The project has given the two students new opportunities for hands on research including media preparation, gene cloning, transposon mutagenesis, and bacteriophage mediated transduction. The students are in the process of learning these new skills. 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? Once the omp::phoA fusion is verified both fusions (sipB::lacZ and omp::phoA) will be moved into the virulent phenotype of S. enterica strain 798 by P22 transduction. This new strain will be mutagenized using a transposon and mutants that knockout the virulence phenotype identified. The location of the transposon insertion will be identified by DNA sequencing. In the second specific aim, we will move the mutation caused by the transposon (or use more classical mutagenesis techniques) to knockout the virulent or non-virulent phenotypes. These mutants will represent locked on or locked off mutants and they will be tested for virulence first in mice and then in pigs.

Impacts
What was accomplished under these goals? The work performed was specific to the first specific aim of the project: the selection of phase locked off mutants. To date we have cloned the two indicator genes from Salmonella enterica (sipB and ompF) individually into the cloning vector pBR322. These constructs were verified by PCR amplification followed by electrophoretic separation and by DNA sequencing. The second step was to place unique fusions at the 3’prime ends of the two cloned genes (lacZ for sipB and phoA for ompF). The sipB::lacZ fusion has been constructed and verified by PCR. Potential ompF::phoA fusions have been made and these are currently being screened to identify the correct fusions. Once the omp::phoA fusion is verified both fusions will be moved into The virulent phenotype of S. enterica strain 798 by P22 transduction. We have prepared the P22 lysates, tittered them, and this reagent is ready for this step.

Publications