Turkeys.

2020-21 Rapid Ag: Characterization of Novel Bacterial Outer Membrane Proteins as Vaccine Targets to Reduce or Eliminate Salmonella Colonization in Turkeys

May 17, 2019

Principal Leader

Sinisa Vidovic

Department

Veterinary Biomedical Sciences Department

The Problem

Non-typhoidal Salmonella is one of the most common food-borne pathogens in the U.S. The annual cost associated with salmonellosis in this country is approximately $14.6 billion. According to the World Health Organization, poultry is significantly associated with salmonellosis. Infection of newly hatched birds by Salmonella often results in death. Older birds become asymptomatic carriers, harboring a large pathogen reservoir that may contaminate hatched birds and the human food chain. An effective Salmonella vaccine will prevent intestinal and cecal colonization, thereby reducing shedding of Salmonella. However, there is no effective vaccine or prophylaxis is available to prevent Salmonella colonization in birds. As a result, multistate outbreaks of salmonellosis involving poultry products continue to occur. In this proposal, we plan to create and evaluate two different types of Salmonella mutants that lack key proteins associated with adhesion, invasion, and replication. Our preliminary results demonstrate that these Salmonella mutants are not able to colonize and form biofilms on abiotic surfaces. We propose to determine the role of these proteins in Salmonella colonization of the avian host. By characterizing these novel targets in Salmonella, our findings will provide valuable information leading to the production of a novel, avian-specific live vaccine against this important food-borne pathogen.

Background

Minnesota has the fourth largest poultry exports in the U.S. These exports attained a value of $162 million in 2016. In addition to steady growth in exports, the poultry industry in Minnesota supports over 26,000 jobs. Non-typhoidal salmonellosis is a leading cause of foodborne gastroenteritis worldwide (2). This zoonotic pathogen colonizes avian hosts at an early age and can persist in birds for weeks or indefinitely (3). In most cases, birds colonized by Salmonella become asymptomatic carriers, constituting a reservoir of the pathogen that poses a significant biohazard to humans. Contaminated poultry products represent the primary transmission route of human salmonellosis (4, 5), which has profound commercial consequences for the poultry industry in terms of food recalls and loss of consumer confidence. Preventing colonization of poultry and subsequent contamination of poultry products by Salmonella has been challenging. Vaccination is a promising approach to prevent Salmonella colonization. In turkeys and chickens, an effective Salmonella pre-harvest prevention program should prevent intestinal and cecal colonization, which will reduce transmission and egg contamination by this zoonotic pathogen (6). The existing Salmonella vaccines in the US have not been effective in completely preventing human infections from poultry sources.

Bacterial mutants: Responses to the extracytoplasmic stresses that affect components of the cell envelope, including periplasmic and outer membrane proteins, play a critical role in the biology of Gram-negative bacteria (7). The alternative sigma factor E (RpoE) and the two-component regulator (CpxAR) are regulatory proteins that govern the bacterial response to various stressors that affect the synthesis, assembly, and homeostasis of lipopolysaccharides and outer-membrane porins (8). Recently, we investigated the role of these two regulators in aggregation, colonization and biofilm formation of S. Enteritidis and discovered that they play novel roles in colonization and biofilm formation of this pathogen in vitro. Comparing biofilms of the wild type and rpoE/cpxR mutant strain (double mutant), we determined two important characteristics. First, the rpoE/cpxR mutant was unable to form a mature biofilm, generating significantly less biomass compared to that of the wild type. Second, this deficiency lasted during the entire experiment (e.g. 78 h). It should be emphasized that many antimicrobial agents and/or mutations (9-11) may reduce biofilm formation but only for a short period of time; usually, after 24 hours, bacteria re-establish a mature biofilm. In our study, the rpoE/cpxR deletion had a profound and long-lasting effect on Salmonella colonization and biofilm formation, clearly underscoring their critical role in the overall fitness of Salmonella.

To gain an insight into the rpoE/cpxR mediated inhibition of Salmonella mature biofilm formation, we carried out global transcriptomics of the wild type and mutant strains. The global transcriptomics approach revealed that the rpoE/cpxR mutant is not able to express the major operons and genes involved in the sessile (i.e. biofilm) lifestyle of S. Enteritidis. Our studies on the iron transporter, IroN, indicated that this unique Salmonella OMP is under strong immune selective pressure in the mammalian and bird hosts, further demonstrating its importance in the adaptation of Salmonella to its hosts. The proposed investigation will determine the role of the extracytoplasmic regulators, RpoE and CpxR as well as the unique Salmonella iron transporter, IroN, in the process of Salmonella colonization and invasion of the avian host. Findings from the proposed study will make a significant contribution to the design and implementation of vaccines and other pre-harvest interventions to prevent Salmonella colonization of poultry and contamination of poultry products.

Objectives

Our long-term goal is to develop a live, attenuated vaccine against critical outer membrane proteins (OMPs), which can prevent Salmonella colonization of chickens and turkeys. The extra-cytoplasmic stress response regulators RpoE and CpxR and iron transport proteins are among these important OMPs. Recently, we discovered that RpoE and CpxR function in Salmonella colonization and biofilm formation as well. Moreover, we have found that the critical Salmonella iron transporter, IroN appears to be under strong host immune pressure, and may be an important factor in the adaptation of Salmonella to different hosts (1). Based on these exciting data, we hypothesize that RpoE and CpxR, play a crucial role in Salmonella adherence and colonization of an avian host, and IroN confers an important growth advantage to Salmonella during the process of avian colonization. We plan to test these hypotheses through the following two specific aims:

Specific Aim 1: We will determine the ability of a double mutation (rpoE/cpxR) on the adherence and colonization of Salmonella in avian cell lines.

Specific Aim 2: We will compare the ability of double (rpoE, cpxR) and triple (rpoE, cpxR, and iroN) mutants to establish themselves in the gut mucosa of turkey poults and invade these hosts.

References

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