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Salmonella Attachment and Invasion in Poultry Intestinal Epithelia Cells: Determination of the Responsible Molecular Mechanisms and Methods for Prevention


Our hypothesis is that Salmonella expresses undefined genes, in addition to or in response to the absence of Type III Secretion System genes (T3SS), which facilitate active attachment and infection of gut epithelial cells in poultry. <P>The first research objective of this work is to isolate and identify these undefined genes that are expressed by Salmonella during the events of attachment and infection of epithelia cells of the small intestine from chickens. Identification of these undefined genes is vital to further understanding of the mechanisms which allow Salmonella to establish colonization of the gut in chickens. By supplementing our findings with known genes that are expressed during infection (i.e. Type III Secretion System genes), we will be able to construct a complete genetic profile for the genes expressed by Salmonella during attachment and infection of the gut. <P>The second research objective is to analyze this genetic database to determine critical genes necessary for attachment and infection that can act as suitable targets for pre-harvest mitigation methods, such as vaccines. By establishing which gene functions to inhibit, we can develop innovative, effective pre-harvest methods that interrupt initial attachment and infection of gut epithelial cells by Salmonella, thus reducing the bacteria load on the chickens entering the processing plant.

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Non-Technical Summary:<br/>
New Food Safety Inspection Service (FSIS) performance standards have reduced the number of allowable Salmonella positive broiler carcasses from 12 previously, to 5 out of 51. This increased regulatory pressure imposes greater challenges upon the poultry industry to reduce pathogen incidence, at all levels of production and processing, in order to decrease the presence of the bacteria at the FSIS regulatory point: exit of the immersion chill tank. Existing pre- and post- harvest control measures have shown varying degrees of success to reduce and control Salmonella in broiler flocks, including various vaccination regimens, on-farm environmental hygiene measures, as well as in-plant antimicrobial programs. However, a particular need exists to develop innovative, precise pre-harvest methods to significantly reduce the prevalence of Salmonella in the field, which translates to a reduced pathogen load on the birds upon entering the processing plant. One obstacle to meeting this need is a lack of knowledge in completely understanding all genetic aspects involved with Salmonella attachment and infection in broilers. One of the virulence systems found in Salmonella to be directly involved in infection of host cells, is the Type III Secretion System (T3SS). Found in many Gram-negative pathogenic bacteria, T3SS is a needle-like structure that physically connects the cytoplasm of bacterial and host cells, permitting direct translocation of bacterial effector proteins into the cytosol of host cells to alter the function of host cells in favor of disease progression. T3SS is found in two locations within the genome of Salmonella, Salmonella Pathogenicity Island 1 and 2 (SPI-1 and SPI-2), and these islands play several roles in different organs within the host. In vitro studies have characterized the role of these pathogenicity islands in Salmonella by comparing inactive-T3SS strains (inactivated by point deletions and whole gene deletions) with parent strains. Results show that by altering different strains of wild-type Salmonella to make them T3SS-deficient, the then mutated pathogen shows a decreased ability to colonize the gut of chickens when compared to the parent strain. However, these mutant Salmonella, with their reduced ability to invade gut epithelial cells, have been recovered from the cecum and spleen after oral administration to chickens, suggesting undefined genetic elements are enabling the bacteria to actively attach and enter host cells. This study is unique in that specific gene-deletions within Salmonella are designed to occur in such a way that undefined genes involved in host/pathogen interaction are primarily analyzed using a proteomic approach. This research combines our time-dependent infection assay with established proteomic analytical methods to isolate and identify undefined genes which enable Salmonella to attach and infect gut epithelial cells in poultry. Since intestinal epithelial cells are the first site of contact between Salmonella and host, this research is fundamental in developing innovative, effective pre-harvest strategies to controlling this pathogen within broilers.
The Type III Secretion System (T3SS) genes must be inactivated in order to isolate and identify additional genes which play an active role in the colonization of the gut in poultry. Datsenko and Wanner have developed a method to inactivate Escherichia coli K-12 chromosomal genes using PCR products. This method has been successfully applied to inactivate genes within Salmonella enterica serovar Typhimurium. In this study T3SS apparatus genes, located within Salmonella pathogenicity islands 1 and 2 (SPI-1 and -2), are targeted for deletion which results in a gene-deletion strain of Salmonella with inactivated T3SS (?T3SS). Following literature review, it appears our study is the first in which portions of the pathogenicity islands will be deleted instead of whole deletions of the pathogenicity islands, leaving specific regulatory genes intact. These regulatory genes may have activating effects outside of the pathogenicity islands which previous studies failed to account for. To determine quality and quantity of bacteria proteins linked to attachment and infection of the gut in poultry, we will develop a time-dependent infection assay to identify proteins isolated from Salmonella at pre-determined time points of infection. The assay consists of individual tissue culture plates that are assigned to specific time points: prior to infection, 0 h infection, 0.5 h post-infection, 1 h post-infection, 1.5 post-infection, 2 h post-infection, 2.5 h post-infection, 3 h post-infection, 3.5 h post-infection, 4 h post-infection, and 24 h post-infection. Results from the tissue culture plates observed as a whole represent a time-line of infection. Small intestinal epithelia cells originially isolated from poultry by BioNutriTech are seeded to the bottom of each well and a solution of the gene-deletion strain of Salmonella (?T3SS) is added to all plates in parallel. At a time point, the epithelia cells are lysed with Triton X-100 and the bacteria are isolated from the cells. Following isolation, the cells are concentrated by centrifugation and the cell pellets are frozen for storage until time for protein prep. This approach collects snapshots of protein expression within Salmonella over the course of infection and comparison of the results detects changes in quality and quantity of its protein profile. To determine bacteria proteins linked to attachment and infection of the gut in poultry, a 2D-liquid chromatography (LC)-mass spectrometry (MS)-based proteomic approach is used to identify proteins isolated from Salmonella following our time-dependent infection assay. All samples are prepared and analyzed by following protocols. Tryptic pepties are separated over a gradient prior to their mass spectrometric analysis. Tandem mass spectrometry (MS/MS) is used to elucidate the chemical structures of the peptides. Raw spectrometry data is processed and matched to a database using Bioworks software. For proteins not in a database, de novo sequencing is performed based on automated MS/MS data acquisition. Our study represents the first systemic investigation of the bacterial proteins associated with gut colonization using a global proteomics approach.
2012/01 TO 2012/12<br/>
OUTPUTS: Genetic mutations were created within Salmonella enterica serovar Kentucky and serovar Typhimurium to delete genes that are specific to the expression of Type III Secretion System. The genetic mutations were created through the adaptation of a previously published method for creating chromosome deletions. The current protocol used in this analysis is specialized for Salmonella enterica and has been documented. The protocol and its results have been presented to the scientific community at American Society for Microbiology South Central Branch Annual Meeting (Starkville; Oct. 2012) and Conference of Research Workers in Animal Diseases (Chicago; Dec. 2012). Our method has also been disseminated to colleagues and graduate students within the College of Veterinary Medicine through scientific research presentations for practical use in their laboratories.
<br/>PARTICIPANTS: Kevin Howe, Sanaz Salehi, Dr. Attila Karsi, Dr. Mark Lawrence, Dr. Hart Bailey. Funds are being used to develop methods as part of dissertation research for Kevin Howe and Sanaz Salehi.
<br/>TARGET AUDIENCES: The results of research have been presented to the scientific community at American Society for Microbiology South Central Branch Annual Meeting (Starkville; Oct. 2012) and Conference of Research Workers in Animal Diseases (CRWAD; Chicago, IL; Dec. 2012). Kevin Howe received recognition for Outstanding graduate student presentation at American College of Veterinary Microbiologists, Molecular Section, entitled "Evaluation of Invasion by Nonpathogenic Salmonella enterica serovar Kentucky in Poultry Intestinal Epithelial Cells" (abstract no. 005) at CRWAD. Sanaz Salehi received recognition for Outstanding graduate student presentation at the Association for Veterinary Epidemiology and Preventive Medicine Award, Food and Environmental Safety division, entitled "The Role of Flagella in the Attachment of Salmonella enterica serovar Kentucky to Broiler Skin" (abstract no. 079) at CRWAD.
<br/>PROJECT MODIFICATIONS: The cell line originally proposed, B5 poultry intestinal epithelia cell line, was discontinued from the manufacturer. The current cell line used in this analysis is HTB-37 human intestinal Caco-2 cell line.
IMPACT: The results of our genetic mutations experiments provide evidence that our adaptation of a previously described method is successful and efficient to create deletion mutations within the chromosome of Salmonella enterica. These mutant strains will play a critical role in our invasion assay in determining the molecular components involved in attachment and invasion.

Bailey, R. Hartford
Mississippi State University
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