Source: CORNELL UNIVERSITY submitted to
REGULATION OF SALMONELLA VIRULENCE BY INTESTINAL FATTY ACIDS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1002239
Grant No.
2014-67015-21697
Project No.
NYCVNYCV-478526
Proposal No.
2013-06805
Multistate No.
(N/A)
Program Code
A1241
Project Start Date
Jan 1, 2014
Project End Date
Dec 31, 2019
Grant Year
2014
Project Director
Altier, C.
Recipient Organization
CORNELL UNIVERSITY
(N/A)
ITHACA,NY 14853
Performing Department
PM & DS
Non Technical Summary
Salmonella is an important cause of foodborne illness in the United States. It is carried by many animal species and so can frequently contaminate meat, poultry and eggs. We hypothesize that the chemical environment of the animal intestine, generated by the bacteria that normally reside there, can affect the ability of Salmonella to cause disease. Specifically, we have found that fatty acids, by-products of intestinal bacteria, can inhibit the ability of Salmonella to penetrate animal tissues, a process called invasion. In this work, we will therefore identify the mechanism by which intestinal fatty acids prevent Salmonella invasion. We will then determine the importance of intestinal fatty acids to the control of Salmonella carriage in chickens. Finally, we will determine the importance of the intestinal environment to the prevention of infection by Salmonella. We expect to determine the means by which these chemical compounds affect Salmonella and thus to gain insight into possible means to prevent disease caused by this pathogen. We anticipate that the long-term impact of this work will be the development of improved means to prevent the colonization of production animals with Salmonella and thus an increase in the safety of our food supply.
Animal Health Component
0%
Research Effort Categories
Basic
75%
Applied
25%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
71240101040100%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins;

Subject Of Investigation
4010 - Bacteria;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
The long-range goal of our work is to understand how the environment of the intestinal tract, produced by the microbiota, affects Salmonella virulence. The central hypothesis is that the resident microbiota of the large intestine create an environment that is repressive for Salmonella virulence gene expression through the production of fatty acids, and that these fatty acids signal Salmonella by means of existing metabolic and regulatory pathways in the pathogen. Our first objective is to characterize the repressive effects of fatty acids on Salmonella virulence. Specifically, we aim to identify the molecular mechanism by which these fatty acids have their effects. We will further determine how dietary manipulation affects Salmonella virulence in animals using models that are relevant to human disease and animal production. Specifically, we aim to:1. Identify the mechanism by which intestinal fatty acids prevent Salmonella invasion.2. Determine the importance of intestinal fatty acids to the control of Salmonella carriage in chickens.3. Determine the importance of the intestinal environment to the prevention of infection by Salmonella in humans using an animal model of human disease.
Project Methods
Specific Aim 1: Identify the mechanism by which intestinal fatty acids prevent Salmonella invasion.The approach employed here will be to use a combination of biochemical and genetic means to characterize the physical interaction of fatty acids with HilD, a central regulator of Salmonella virulence. The experiments are designed to determine the means by which fatty acids repress Salmonella invasion genes through HilD. We will begin with a genetic approach, identifying essential elements of hilD, and then progress using biochemical methods to characterize modifications of HilD. These experiments will focus on propionate, with extension to other fatty acids.Specific Aim 2: Determine the importance of intestinal fatty acids to the control of Salmonella carriage in chickens.We will employ in this approach studies of in vivo Salmonella invasion gene expression using gfp fusions followed by a feeding trial using a commercially available microencapsulated n-butyric acid feed additive. Here we will investigate the in vivo control of Salmonella virulence in the environment of the chicken intestinal tract by a prebiotic dietary supplement. We will use gfp reporter fusions in combination with biochemical analyses to determine the effects of n-butyric acid on Salmonella carriage.Specific Aim 3: Determine the importance of the intestinal environment to the prevention of infection by Salmonella.As our approach, we will use a well studied animal model of septicemic salmonellosis, the conventional mouse, in combination with the antibiotic-treated mouse model, in which the intestinal microbiota and fatty acid composition are altered to produce an aberrant large intestinal disease. Here we will investigate the in vivo control of Salmonella virulence in the environment of the intestinal tract using the mouse as the experimental model. We will use gfp reporter fusions in combination with biochemical analyses to define the temporal and spatial variation in SPI-1 gene expression, determine what role fatty acids play in these expression changes, and find whether treatment with a dietary supplement known to exacerbate systemic salmonellosis does so by altering the expression of Salmonella invasion genes.

Progress 01/01/14 to 12/31/19

Outputs
Target Audience:The target audience of this work is scientists performing both basic and translational research in the area of bacterial pathogenesis and food safety. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?During the period of the award, this work has supported the training of six post-doctoral associates: Erick Bosire Maosa (Cornell University) Rimi Chowdhury (Cornell University) Colleen Eade (Cornell University) Chien-Che Hung (Cornell University) Michael Betteken (Cornell University) Lydia Bogomolnaya (Texas A&M University) Colleen Eade was awarded a NIFA National Needs Graduate Fellowship. Her training through this project contributed directly to the award of that fellowship, as it allowed her to gain the necessary experience and expertise to define her own project. How have the results been disseminated to communities of interest?Posters presented at national and international meetings: EM Bosire, CR Eade, L Bogomolnaya, H Andrews-Polymenis, and C Altier. Preventing Salmonella infection using drug-like inhibitors to repress invasion gene expression. ASM Microbe, Atlanta GA. June 7-11, 2018. CR Eade, JD Oliver, C Altier. Positive Feedback of hilD Achieves a Tunable Invasion Switch. ASM Microbe, Atlanta GA. June 7-11, 2018. Betteken MI, Reetz AE, Nugent SL, Hung CC, Pavinski Bitar PD, and Altier, C. Post-Translational Control of Salmonella Invasion is negatively regulated by HilE through the degradation of HilD mediated by Lon. ASM Microbe, Atlanta GA. June 7-11, 2018. EM Bosire, CR Eade, L Bogomolnaya, H Andrews-Polymenis, and C Altier. Targeting pathogen altruism to prevent bacterial infections in animals. 2017 USDA-NIFA Animal Health and Animal Well-Being Project Director Meeting, Chicago, IL. December 1, 2017. CR Eade, M Betteken, C-C Hung, S Nugent, and C Altier. A Tunable Switch: Controlling Salmonella Invasion of the Intestine. 2017 USDA NIFA Food Safety Project Directors' Meeting, Tampa FL. July 8, 2017. CR Eade, C-C Hung, S Nugent, and C Altier. Salmonella Invasion is Regulated through the Post-transcriptional Control of Transcriptional Activators. Gordon Research Conference: Microbial Toxins and Pathogenicity, Waterville Valley, NH. July 10-15, 2016. CR Eade, C-C. Hung, JS Gunn and C Altier. Bile Acids Repress Pathogenicity in Salmonella by Destabilizing the Invasion Regulator HilD. General Meeting of the American Society for Microbiology, New Orleans, LA. May 30 - June 2, 2015. CR Eade, C-C Hung, B Bullard, G Gonzalez-Escobedo, JS Gunn, C Altier. Regulation of Salmonella Virulence by the Intestinal Environment. USDA National Institute of Food and Agriculture Institute of Food Safety and Nutrition Project Director Meeting, Portland OR. July 24, 2015. Invited presentations: Controlling Salmonella Virulence using the Chemical Signals of the Intestine. Gettysburg College, Gettysburg PA. September 27 2018. Control of Salmonella invasion by chemical signals of the intestine. 5th ASM Conference on Salmonella, Potsdam, Germany. August 29-September 1, 2016. A tunable switch: Signals of the intestine modulate Salmonella virulence. Cornell University, Ithaca, NY, February 13, 2015. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. Identify the mechanism by which intestinal fatty acids prevent Salmonella invasion. We have found that fatty acids (and bile acids, another important component of the intestinal environment) work to reduce Salmonella virulence by controlling a central regulatory protein of Salmonella called HilD. Our initial findings have been reported (Eade et al., 2016). Importantly, we have recent evidence that chemical compounds related to fatty acids are extremely potent inhibitors of Salmonella virulence, functioning at sub-micromolar concentrations. This specific category of fatty acids, containing a 2-cis double bond, is extremely potent at reducing the expression of Salmonella virulence genes both in vitro and in vivo, using a mouse model. They function by directly inhibiting the HilD protein. We have more recently found that these same chemicals inhibit additional regulators of Salmonella virulence, RtsA and HilC, and also alter the expression of numerous genes of Salmonella, some of which are important for the survival of Salmonella within animal hosts. These chemicals might be employed as preventatives of salmonellosis in both humans and animals as medications or dietary supplements (see Aim 3 below). We are currently preparing a manuscript describing the effects of these compounds. 2. Determine the importance of intestinal fatty acids to the control of Salmonella carriage in chickens. We have developed a chick infection model for Salmonella virulence and have used this model to assess the importance of invasion to virulence in an animal host that carries Salmonella but does not exhibit clinical signs of disease. This system includes the use of fluorescent Salmonella strains that allow us to assess changes in virulence gene expression while they survive in the animal, a novel and useful tool. We have found that only a small fraction of Salmonella express virulence functions in chickens but that this population is required for chickens to carry Salmonella in their intestines. This is significant because carriage of this pathogen by poultry is an important source of human disease. The manuscript describing this was published by Infection and Immunity (Eade et al., 2018). 3. Determine the importance of the intestinal environment to the prevention of infection by Salmonella in humans using an animal model of human disease. This work is a follow-on to that of Aim 1. We first standardized the mouse model and developed the bacterial strains that we need (including the fluorescent strains being used in chickens). This allowed us to test various means to change the intestinal environment, including antibiotics and dietary supplements, and assess their effects on salmonellosis. We then tested compounds that inhibit Salmonella virulence gene expression for their effects in mice. Using a mouse colitis model of infection, administering a repressive chemical (2-cis-hexadecenoic acid) in drinking water reduced the expression of Salmonella invasion gene within the animal. This demonstrates that the administration of chemicals that repress Salmonella invasion in a water source is a practical means to alter the virulence of this pathogen.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Hung CC, Eade CR, Betteken MI, Pavinski Bitar PD, Handley EM, Nugent SL, Chowdhury R, Altier C. Salmonella invasion is controlled through the secondary structure of the hilD transcript. PLoS Pathog. 2019 Apr;15(4):e1007700. PubMed PMID: 31017982; PubMed Central PMCID: PMC6502421.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Miller, R.A., Betteken, M.I., Guo, X., Altier, C., Duhamel, G.E., and Wiedmann, M. (2018). The Typhoid Toxin Produced by the Nontyphoidal Salmonella enterica Serotype Javiana Is Required for Induction of a DNA Damage Response In Vitro and Systemic Spread In Vivo. mBio 9.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Sahler, J.M., Eade, C.R., Altier, C., and March, J.C. (2018). Salmonella Typhimurium increases functional PD-L1 synergistically with IFN gamma in intestinal epithelial cells via Salmonella Pathogenicity Island-2. Infection and immunity.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Eade, C. R., Bogomolnaya, L., Hung, C. C., Betteken, M. I., Adams, L. G., Andrews-Polymenis, H., and Altier, C. (2018) Salmonella Pathogenicity Island One is Expressed in the Chicken Intestine and Promotes Bacterial Proliferation. Infection and immunity
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Eade, C.R., Hung, C.C., Bullard, B., Gonzalez-Escobedo, G., Gunn, J.S., and Altier, C. (2016). Bile Acids Function Synergistically to Repress Invasion Gene Expression in Salmonella by Destabilizing the Invasion Regulator HilD. Infection and immunity.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Hung, C.C., Eade, C.R., and Altier, C. (2016). The protein acyltransferase Pat post-transcriptionally controls HilD to repress Salmonella invasion. Mol Microbiol 102, 121-136.


Progress 01/01/18 to 12/31/18

Outputs
Target Audience:The target audience of this work is scientists performing both basic and translational research in the area of bacterial pathogenesis and food safety. Changes/Problems:A problem with this project has been the great success of the trainees who have conducted it. All three post-docs trained at Cornell have gone on to permanent positions. That is, of course, the goal of their training. The time required to recruit talented trainees has, however, made it difficult to complete the project to date. A new post-doc, Rimi Chowdhury, joined the lab on 15 January, having experience with Salmonella pathogenesis. We anticipate that she and another post-doc, with the help of a laboratory technician, will complete the project. What opportunities for training and professional development has the project provided?During the reporting period, this work has supported the training of two post-doctoral associates: Michael Betteken, PhD (Cornell University) Lydia Bogomolnaya PhD (Texas A&M University) How have the results been disseminated to communities of interest?Posters presented at national and international meetings: EM Bosire, CR Eade, L Bogomolnaya, H Andrews-Polymenis, and C Altier. Preventing Salmonella infection using drug-like inhibitors to repress invasion gene expression. ASM Microbe, Atlanta GA. June 7-11, 2018. CR Eade, JD Oliver, C Altier. Positive Feedback of hilD Achieves a Tunable Invasion Switch. ASM Microbe, Atlanta GA. June 7-11, 2018. Betteken MI, Reetz AE, Nugent SL, Hung CC, Pavinski Bitar PD, and Altier, C. Post-Translational Control of Salmonella Invasion is negatively regulated by HilE through the degradation of HilD mediated by Lon. ASM Microbe, Atlanta GA. June 7-11, 2018. Invited presentations: Controlling Salmonella Virulence using the Chemical Signals of the Intestine. Gettysburg College, Gettysburg PA. September 27 2018. What do you plan to do during the next reporting period to accomplish the goals?Aim 1. Identify the mechanism by which intestinal fatty acids prevent Salmonella invasion. The final portion of this aim will be to define the biochemical interactions of the Salmonella regulatory protein HilD with the chemicals that repress invasion. We have established that repressive chemicals reduce HilD stability. We have purified HilD and can establish the direct interactions using protein-ligand binding techniques Aim 2. Determine the importance of intestinal fatty acids to the control of Salmonella carriage in chickens. We will test our most promising inhibitory chemicals in chickens for their ability to reduce intestinal carriage. We will also use this animal model to assess directly, using fluorescent Salmonella strains, the effects of chemicals on virulence gene expression in the animal. Aim 3. Determine the importance of the intestinal environment to the prevention of infection by Salmonella in humans using an animal model of human disease. We will continue the work described in the progress report above, testing chemicals for their ability to reduce human disease, using a mouse model. Our goal is to identify ways to reduce the expression of Salmonella virulence genes in vivo, and thus reduce its potential as a human pathogen.

Impacts
What was accomplished under these goals? Aim 1. Identify the mechanism by which intestinal fatty acids prevent Salmonella invasion. We have found that fatty acids (and bile acids, another important component of the intestinal environment) work to reduce Salmonella virulence by controlling a central regulatory protein of Salmonella called HilD. Our initial findings have been reported (Eade et al., 2016). Importantly, we have recent evidence that chemical compounds related to fatty acids are extremely potent inhibitors of Salmonella virulence, functioning at sub-micromolar concentrations. They function by directly inhibiting the HilD protein. These chemicals might be employed as preventatives of salmonellosis in both humans and animals (see Aim 3 below). We are currently preparing a manuscript describing the effects of these compounds. Aim 2. Determine the importance of intestinal fatty acids to the control of Salmonella carriage in chickens. We have developed a chick infection model for Salmonella virulence and are currently conducting experiments using this model. This includes the use of fluorescent Salmonella strains that allow us to assess changes in virulence gene expression while they survive in the animal, a novel and useful tool. We have found that only a small fraction of Salmonella express virulence functions in chickens but that this population is required for chickens to carry Salmonella in their intestines. This is significant because carriage of this pathogen by poultry is an important source of human disease. The manuscript describing this was published by Infection and Immunity (Eade et al., 2018). Aim 3. Determine the importance of the intestinal environment to the prevention of infection by Salmonella in humans using an animal model of human disease. This work is a follow-on to that of Aim 1. We have tested compounds that inhibit Salmonella virulence gene expression for their effects in mice. The results are promising: Using a mouse septicemic model of infection, administering a repressive chemical (2-cis-hexadecenoic acid) reduced the ability of Salmonella to infect tissues. This is an exciting outcome, but one that must be repeated and verified.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Eade, C. R., Bogomolnaya, L., Hung, C. C., Betteken, M. I., Adams, L. G., Andrews-Polymenis, H., and Altier, C. (2018) Salmonella Pathogenicity Island One is Expressed in the Chicken Intestine and Promotes Bacterial Proliferation. Infection and immunity
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Sahler, J.M., Eade, C.R., Altier, C., and March, J.C. (2018). Salmonella Typhimurium increases functional PD-L1 synergistically with IFN gamma in intestinal epithelial cells via Salmonella Pathogenicity Island-2. Infection and immunity.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Miller, R.A., Betteken, M.I., Guo, X., Altier, C., Duhamel, G.E., and Wiedmann, M. (2018). The Typhoid Toxin Produced by the Nontyphoidal Salmonella enterica Serotype Javiana Is Required for Induction of a DNA Damage Response In Vitro and Systemic Spread In Vivo. mBio 9.


Progress 01/01/17 to 12/31/17

Outputs
Target Audience:The target audience of this work is scientists performing both basic and translational research in the area of bacterial pathogenesis and food safety. Changes/Problems:A problem with this project has been the great success of the trainees who have conducted it. All three post-docs trained at Cornell have gone on to permanent positions. That is, of course, the goal of their training. The time required to recruit talented trainees has, however, made it difficult to complete the project to date. A new post-doc, Rimi Chowdhury, will join the lab on 15 January, having experience with Salmonella pathogenesis. We anticipate that she, with the help of a laboratory technician, will complete the project. What opportunities for training and professional development has the project provided?During the reporting period, this work has supported the training of two post-doctoral associates: Michael Betteken, PhD (Cornell University) Lydia Bogomolnaya (Texas A&M University) How have the results been disseminated to communities of interest?Posters presented at national and international meetings: EM Bosire, CR Eade, L Bogomolnaya, H Andrews-Polymenis, and C Altier. Preventing Salmonella infection using drug-like inhibitors to repress invasion gene expression. ASM Microbe, Atlanta GA. June 7-11, 2018. CR Eade, JD Oliver, C Altier. Positive Feedback of hilD Achieves a Tunable Invasion Switch. ASM Microbe, Atlanta GA. June 7-11, 2018. Betteken MI, Reetz AE, Nugent SL, Hung CC, Pavinski Bitar PD, and Altier, C. Post-Translational Control of Salmonella Invasion is negatively regulated by HilE through the degradation of HilD mediated by Lon. ASM Microbe, Atlanta GA. June 7-11, 2018. Invited presentations: Controlling Salmonella Virulence using the Chemical Signals of the Intestine. Gettysburg College, Gettysburg PA. September 27 2018. A Tunable Switch: Controlling Salmonella Invasion of the Intestine. Texas A&M University. April 10, 2017. A Tunable Switch: Controlling Salmonella Invasion of the Intestine. University of California at Santa Cruz. April 20, 2017. What do you plan to do during the next reporting period to accomplish the goals?Aim 1. Identify the mechanism by which intestinal fatty acids prevent Salmonella invasion. The final portion of this aim will be to define the biochemical interactions of the Salmonella regulatory protein HilD with the chemicals that repress invasion. We have established that repressive chemicals reduce HilD stability. We have purified HilD and can establish the direct interactions using protein-ligand binding techniques Aim 2. Determine the importance of intestinal fatty acids to the control of Salmonella carriage in chickens. We will test our most promising inhibitory chemicals in chickens for their ability to reduce intestinal carriage. We will also use this animal model to assess directly, using fluorescent Salmonella strains, the effects of chemicals on virulence gene expression in the animal. Aim 3. Determine the importance of the intestinal environment to the prevention of infection by Salmonella in humans using an animal model of human disease. We will continue the work described in the progress report above, testing chemicals for their ability to reduce human disease, using a mouse model. Our goal is to identify ways to reduce the expression of Salmonella virulence genes in vivo, and thus reduce its potential as a human pathogen.

Impacts
What was accomplished under these goals? Aim 1. Identify the mechanism by which intestinal fatty acids prevent Salmonella invasion. We have found that fatty acids (and bile acids, another important component of the intestinal environment) work to reduce Salmonella virulence by controlling a central regulatory protein of Salmonella called HilD. Our initial findings have been reported (Eade et al., 2016). Importantly, we have recent evidence that chemical compounds related to fatty acids are extremely potent inhibitors of Salmonella virulence, functioning at sub-micromolar concentrations. They function by directly inhibiting the HilD protein. These chemicals might be employed as preventatives of salmonellosis in both humans and animals (see Aim 3 below). We are currently preparing a manuscript describing the effects of these compounds. Aim 2. Determine the importance of intestinal fatty acids to the control of Salmonella carriage in chickens. We have developed a chick infection model for Salmonella virulence and are currently conducting experiments using this model. This includes the use of fluorescent Salmonella strains that allow us to assess changes in virulence gene expression while they survive in the animal, a novel and useful tool. We have found that only a small fraction of Salmonella express virulence functions in chickens but that this population is required for chickens to carry Salmonella in their intestines. This is significant because carriage of this pathogen by poultry is an important source of human disease. The manuscript describing this is currently in revision by Infection and Immunity (Eade et al., 2018). Aim 3. Determine the importance of the intestinal environment to the prevention of infection by Salmonella in humans using an animal model of human disease. This work is a follow-on to that of Aim 1. We have tested compounds that inhibit Salmonella virulence gene expression for their effects in mice. The results are promising: Using a mouse septicemic model of infection, administering a repressive chemical (2-cis-hexadecenoic acid) reduced the ability of Salmonella to infect tissues. This is an exciting outcome, but one that must be repeated and verified.

Publications

  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Miller, R.A., Betteken, M.I., Guo, X., Altier, C., Duhamel, G.E., and Wiedmann, M. (2018). The Typhoid Toxin Produced by the Nontyphoidal Salmonella enterica Serotype Javiana Is Required for Induction of a DNA Damage Response In Vitro and Systemic Spread In Vivo. mBio 9.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Sahler, J.M., Eade, C.R., Altier, C., and March, J.C. (2018). Salmonella Typhimurium increases functional PD-L1 synergistically with IFN gamma in intestinal epithelial cells via Salmonella Pathogenicity Island-2. Infection and immunity.
  • Type: Journal Articles Status: Accepted Year Published: 2018 Citation: Eade, C.R., Bogomolnaya, L., Hung, C.C., Betteken, M.I.*, Adams, L.G., Andrews-Polymenis, H., Altier, C. Salmonella Pathogenicity Island One is Expressed in the Chicken Intestine and Promotes Bacterial Proliferation. Accepted for publication by Infection and Immunity.


Progress 01/01/16 to 12/31/16

Outputs
Target Audience:The target audience of this work is scientists performing both basic and translational research in the area of bacterial pathogenesis and food safety. Changes/Problems:The work has proceeded, but identifying and retaining capable scientists able to complete the studies remains challenging. What opportunities for training and professional development has the project provided?This work has supported the training of three post-doctoral associates in the laboratory. Colleen Eade, PhD Chien-Che Hung, DVM, PhD, DACVM Michael Betteken, PhD Lydia Bogomolnaya, PhD. How have the results been disseminated to communities of interest?Posters presented at national and international meetings: EM Bosire, CR Eade, L Bogomolnaya, H Andrews-Polymenis, and C Altier. Targeting pathogen altruism to prevent bacterial infections in animals. 2017 USDA-NIFA Animal Health and Animal Well-Being Project Director Meeting, Chicago, IL. December 1, 2017. CR Eade, M Betteken, C-C Hung, S Nugent, and C Altier. A Tunable Switch: Controlling Salmonella Invasion of the Intestine. 2017 USDA NIFA Food Safety Project Directors' Meeting, Tampa FL. July 8, 2017. CR Eade, C-C Hung, S Nugent, and C Altier. Salmonella Invasion is Regulated through the Post-transcriptional Control of Transcriptional Activators. Gordon Research Conference: Microbial Toxins and Pathogenicity, Waterville Valley, NH. July 10-15, 2016. CR Eade, C-C. Hung, JS Gunn and C Altier. Bile Acids Repress Pathogenicity in Salmonella by Destabilizing the Invasion Regulator HilD. General Meeting of the American Society for Microbiology, New Orleans, LA. May 30 - June 2, 2015. CR Eade, C-C Hung, B Bullard, G Gonzalez-Escobedo, JS Gunn, C Altier. Regulation of Salmonella Virulence by the Intestinal Environment. USDA National Institute of Food and Agriculture Institute of Food Safety and Nutrition Project Director Meeting, Portland OR. July 24, 2015. Invited presentations: Control of Salmonella invasion by chemical signals of the intestine. 5th ASM Conference on Salmonella, Potsdam, Germany. August 29-September 1, 2016. A tunable switch: Signals of the intestine modulate Salmonella virulence. Cornell University, Ithaca, NY, February 13, 2015. What do you plan to do during the next reporting period to accomplish the goals?Aim 1. Identify the mechanism by which intestinal fatty acids prevent Salmonella invasion. We have recent evidence that chemical compounds related to fatty acids are extremely potent inhibitors of Salmonella virulence. We will therefore next test the potency and efficacy of these chemicals. Such chemicals might be employed as preventatives of salmonellosis in both humans and animals Aim 2. Determine the importance of intestinal fatty acids to the control of Salmonella carriage in chickens. We will use the fluorescent Salmonella strains that allow us to assess changes in virulence gene expression while they survive in the animal as a means to assess virulence of the pathogen in vivo. We will therefore test the importance of invasion to Salmonella carriage in chickens. Aim 3. Determine the importance of the intestinal environment to the prevention of infection by Salmonella in humans using an animal model of human disease. We will continue the work described in the progress report above, testing chemicals for their ability to reduce human disease, using a mouse model. Our goal is to identify ways to reduce the expression of Salmonella virulence genes in vivo, and thus reduce its potential as a human pathogen.

Impacts
What was accomplished under these goals? Aim 1. Identify the mechanism by which intestinal fatty acids prevent Salmonella invasion. We have found that fatty acids (and bile acids, another important component of the intestinal environment) work to reduce Salmonella virulence by controlling a central regulatory protein of Salmonella called HilD. Our findings have been reported (Eade et al., 2016). This demonstrates that signals produced by host animals in the intestinal tract can alter the virulence of Salmonella using a sensitive genetic switch. Aim 2. Determine the importance of intestinal fatty acids to the control of Salmonella carriage in chickens. We have developed a chick infection model for Salmonella virulence and are currently conducting experiments using this model. This includes the use of fluorescent Salmonella strains that allow us to assess changes in virulence gene expression while they survive in the animal, a novel and useful tool. We anticipate that these fluorescent strains will allow us to track the virulence of Salmonella in the animal host, a novel approach to the study of pathogenesis. Aim 3. Determine the importance of the intestinal environment to the prevention of infection by Salmonella in humans using an animal model of human disease. We are currently testing compounds that inhibit Salmonella virulence gene expression for their effects in mice. We have established the model, which requires that mice be provided with chemicals in drinking water, and will next examine the effects of specific chemicals on virulence.

Publications

  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Eade, C.R., Hung, C.C., Bullard, B., Gonzalez-Escobedo, G., Gunn, J.S., and Altier, C. (2016). Bile Acids Function Synergistically to Repress Invasion Gene Expression in Salmonella by Destabilizing the Invasion Regulator HilD. Infection and immunity.
  • Type: Journal Articles Status: Published Year Published: 2016 Citation: Hung, C.C., Eade, C.R., and Altier, C. (2016). The protein acyltransferase Pat post-transcriptionally controls HilD to repress Salmonella invasion. Mol Microbiol 102, 121-136.


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

Outputs
Target Audience:The target audience of this work is scientists performing both basic and translational research in the area of bacterial pathogenesis and food safety. Changes/Problems:There have been no major changes to the proposal. The construction of Salmonella strains required for the work took longer than expected, but we now have what we need. Our findings in chickens have been surprising, and likely will alter the direction of that part of the work. It was anticipated previously (and reported in the literature), that the ability of Salmonella to invade the intestinal epithelium was important for its carriage in this species. We are finding the opposite, using carefully constructed animal experiments. We do not consider this at all a problem, as it will allow us to better understand the pathogenesis of this disease, but it likely will lead to changes in our experimental plans. What opportunities for training and professional development has the project provided?This work has supported the training of two post-doctoral associates in the laboratory. Importantly, one of them, Dr. Colleen Eade, was recently awarded a NIFA National Needs Graduate Fellowship, which we anticipate will begin this year. Her training through this project contributed directly to the award of that fellowship, as it allowed her to gain the necessary experience and expertise to define her own project. 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? 1. Identify the mechanism by which intestinal fatty acids prevent Salmonella invasion. As described above, we are now focusing on the genetic and biochemical control of the virulence regulator HilD. We have conducted a genetic screen that is defining portions of the gene and protein required for its control. We have also identified protein acylation as a means by which this control is exerted. We will continue to investigate the molecular methods of control, focusing upon means that might lead to intervention strategies. 2. Determine the importance of intestinal fatty acids to the control of Salmonella carriage in chickens. We anticipate that chicken experiments conducted this year will elucidate the importance of Salmonella virulence functions to survival and carriage of the organism in this important agricultural species. 3. Determine the importance of the intestinal environment to the prevention of infection by Salmonella in humans using an animal model of human disease. This year, we will employ the mouse model that we have developed. We should be able to determine the effects of various treatments, including antibiotics and dietary supplements, on the virulence of Salmonella in this animal model. Our goal is to identify ways to reduce the expression of Salmonella virulence genes in vivo, and thus reduce its potential as a human pathogen.

Impacts
What was accomplished under these goals? 1. Identify the mechanism by which intestinal fatty acids prevent Salmonella invasion. We have found that fatty acids (and bile acids, another important component of the intestinal environment) work to reduce Salmonella virulence by controlling a central regulatory protein of Salmonella called HilD. The importance of this finding is that allows us to focus intervention methods for Salmonella control on this central regulator. We have also found that a number of chemical compounds similar to fatty acids have this same repressive effect on HilD and thus Salmonella virulence. Some of these are commercially available medications or dietary supplements. Thus, they might be employed as preventatives of salmonellosis. We anticipate reporting these results in publications during this coming year (with two publications already in review). 2. Determine the importance of intestinal fatty acids to the control of Salmonella carriage in chickens. We have developed a chick infection model for Salmonella virulence and are currently conducting experiments using this model. This includes the use of fluorescent Salmonella strains that allow us to assess changes in virulence gene expression while they survive in the animal, a novel and useful tool. Our preliminary results are surprising: It appears that Salmonella genes known to be required for virulence in humans, livestock and laboratory animals are not needed in chickens. In fact, expression of these virulence determinants incur a fitness cost to the pathogen that make it less viable in the host. These findings, if confirmed, would shift the way that we think about Salmonella in chickens, suggesting that it behaves more like a commensal organism than a pathogen. They also raise important questions about the ecology of Salmonella infection in chicken flocks. For example, if traditional virulence characteristics are in fact detrimental to the organism in chickens, are these characteristics lost from Salmonella that infect this species. We anticipate at least one publication on this subject in the coming year. 3. Determine the importance of the intestinal environment to the prevention of infection by Salmonella in humans using an animal model of human disease. This work is a follow-on to that of Aim 1. Thus far, we have standardized the mouse model and have developed the bacterial strains that we need (including the fluorescent strains being used in chickens). This allows us to test various means to change the intestinal environment, including antibiotics and dietary supplements, and assess their effects on salmonellosis. That work will be conducted this year.

Publications


    Progress 01/01/14 to 12/31/14

    Outputs
    Target Audience: At this point in the project, the target audience has been other scientists, through presentations. Changes/Problems: The construction of the fluorescent strains needed to complete this work has been more challenging than anticipated. This has slowed the work somewhat, but we now have strains that we think will be sufficient to our needs. What opportunities for training and professional development has the project provided? This work is being done primarily by two Post-doctoral Associates. Both spend considerable time with the PI discussing the project, planning experiments and interpreting results. Both present their most recent findings during a weekly laboratory meeting. Both have also recently presented their work at a Cornell conference on Infection and Pathobiology. One of them has applied for a NIFA-AFRI Post-doctoral Fellowship, under the direction of the PI, the construction of which involved training in project planning and proposal construction. How have the results been disseminated to communities of interest? Thus far, results have been disseminated to the scientific community through oral presentations. Both the PI and post-docs have had presentations (oral and as posters) at meetings. What do you plan to do during the next reporting period to accomplish the goals? 1. Identify the mechanism by which intestinal fatty acids preventSalmonellainvasion. We plan to continue the line of investigation demonstrating the importance of protein acylation to Salmonella invasion. We will investigate the importance of deacylase inhibitors as preventative agents. This will be accomplished first in vitro, and if successful, using mice and/or chickens as models of infection. We will also continue to elucidate the molecule mechanisms of invasion control. We are working now on a number of genetic screens, as proposed in the original grant, to identify the genes required for invasion, as a means to identify new targets of intervention. 2. Determine the importance of intestinal fatty acids to the control ofSalmonellacarriage in chickens. We will continue experiments in chickens that we have recently begun. Our goal will be to determine where and when in the intestinal tract Salmonella invasion occurs, and the importance of invasion to carriage in this species. We plan after that to embark on studies in which we alter the intestinal environment through feeding changes, and assess Salmonella carriage. 3. Determine the importance of the intestinal environment to the prevention of infection bySalmonellain humans using an animal model of human disease. We will begin preliminary experiments using this animal model to determine, as we are for chickens, the location of Salmonella invasion and its importance to carriage and virulence.

    Impacts
    What was accomplished under these goals? Progress on our goals: 1. Identify the mechanism by which intestinal fatty acids preventSalmonellainvasion. We have determined that Salmonella invasion is inhibited by a mechanism that involves protein acylation. Additionally, we have found that chemicals that inhibit protein deacylation in eukaryotic organisms also inhibit Salmonella invasion. This is important, because a number of these chemicals exist as FDA-approved drugs. Thus, they present an attractive means to prevent Salmonella infection and carriage in animals. A manuscript on this subject will be submitted soon. 2. Determine the importance of intestinal fatty acids to the control ofSalmonellacarriage in chickens. We are performing our first experiments on chickens as this is written. These experiments required the construction of a number of specialized Salmonella strains. We created these strains to contain a constitutive fluorescent fusion of one color, and a second fusion linked to an invasion gene, such that it is expressed only when invasion genes are. This approach will allow us to detect Salmonella derived from the intestines of chickens, and to determine the extent of invasion gene expression in vivo, which to this point has not been known. Our initial experiments will test our recovery and analysis of bacteria, and will be used to determine whether the invasion gene regulator known to be controlled by fatty acids, hilD, is essential for Salmonella carriage in chickens. 3. Determine the importance of the intestinal environment to the prevention of infection bySalmonellain humans using an animal model of human disease. This work will be done using the mouse model. Thus far, we have created the bacterial strains necessary for the work (similar to those used for chickens). We will begin the mouse experiments in the coming months.

    Publications