FILAMENT FORMATION AND THE PATHOGENESIS OF SALMONELLOSIS

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0222768
• Project Start Date: Oct 1, 2010
• Project End Date: Sep 30, 2013
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218

Performing Department
Bacteriology

Non Technical Summary
Salmonella causes an estimated 2-4 million cases of human gastroenteritis per year in the United States. Although most cases resolve in 5-7 days, the very young, elderly, and immunocompromised are susceptible to more severe infections that result in an estimated 500 deaths per year. It has been estimated that 95% of these cases result from consumption of contaminated food. Salmonella encounters myriad stresses in the pre-harvest and processing environments. Desiccation and temperature stress are a few of the commonly encountered stresses. In response, Salmonella triggers stress protection systems that render cells more tolerant to the inducing stress as well as cross protection to other stresses. As a consequence, there is greater survival and prolonged persistence of the organism in the environment or food. One of the long-term goals of our research is to define the role of stress-responses in the dissemination, fitness, and pathogenesis of salmonellae. One interesting response to stress that we and other groups have observed in salmonellae is the formation of filaments (elongated cells without septation) that can be greater than 200 microns long. The underlying processes and purpose of filament formation are unknown, but we have observed filament formation in response to several stresses including; desiccation, temperature, and ultraviolet light. This common response to different stresses points to a central system of importance to persistence and dissemination of this important human pathogen. Key questions center on whether filamentous salmonellae have enhanced survival properties and are infectious. Of equal significance is the formation of septa once encountering favorable conditions that results in a sudden burst in Salmonella numbers. Thus, filaments can affect tests to enumerate Salmonella in a food, which could influence retrospective assessments of the infectious dose and risk assessments. Likewise, the occurrence of filamentous Salmonella in the processing environment may influence the effectiveness of processing parameters. It is critical to understand under what conditions filaments are formed, whether filament formation affects the survival of Salmonella under other stress conditions, and if in turn these changes alter the virulence properties of the Salmonella cells. Our proposed research will provide insights into these important food safety questions.

Animal Health Component

20%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
712	1199	1100	30%
712	1219	1100	30%
712	1499	1100	40%

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

Subject Of Investigation
1499 - Vegetables, general/other; 1199 - Deciduous and small fruits, general/other; 1219 - Edible tree nuts, general/other;

Field Of Science
1100 - Bacteriology;

Keywords

salmonella

stress

filamentation

virulence

Goals / Objectives
The goal of this project is to understand the physiology of stress-induced filamentation by Salmonella and to determine the role filaments play in acid tolerance and pathogenesis. This will be accomplished by the following specific objectives: 1. Assess the ability of different stresses to induce filament formation 2. Compare macromolecular synthesis between control cells and filaments 3. Determine the mechanism of enhanced acid tolerance in filaments 4. Determine the virulence of filaments in mice. Outputs: Results generated from this project will be presented at relevant national conferences such as annual meetings of the American Society for Microbiology, International Association for Food Protection, and Institute of Food Technology. In addition, as all three co-PIs are core members of the Food Research Institute (FRI), University of Wisconsin-Madison, the results will be presented at FRI annual meetings. Manuscripts describing the research will be submitted to relevant journals for publication.

Project Methods
We will use two Salmonella enterica serovar Typhimurium strains, M-09-0001A-1 isolated from a peanut butter outbreak, and LT-2, a less robust isolate. We have shown that S. Typhimurium can form filaments over 100 microns long when grown under low water activity (aw) conditions, using NaCl as the humectant. Other stresses commonly encountered in the pre-harvest and food processing environment will be evaluated for their ability to induce filamentation. These include ultraviolet radiation, oxidative stress (e.g. bleach and oxidative sanitizers), and acid. DNA damage occurs after exposure to uv radiation, which induces the SOS response, ultimately leading to filament formation. The mechanism of filamentation under other stress conditions is unknown. We will determine whether DNA damage occurs and could be a cause of filamentation under these stresses. Ultraviolet treatment of bacteria results in inhibition of DNA synthesis but not RNA and protein, while DNA continues to be synthesized in cold-shock induced filamentous E. coli and is inhibited only slightly before inhibition of RNA and protein synthesis. We will monitor DNA, RNA, and protein synthesis in Salmonella grown under stress and non-stress conditions to determine if perturbation of any macromolecular synthesis can be related to filament formation. Cross protection is an important result of stress response. Our preliminary data showed that low aw induced Salmonella filaments are more tolerant to low pH than control cells. We will explore the mechanism of the enhanced acid tolerance in filaments by determining the expression levels of acid tolerance related genes such as gad, dps, and rpoS, enzyme activity levels of decarboxylase, urease, ATPase, and potential differences in membrane components such as cyclic fatty acids and outer membrane proteins OmpC and OmpF. We have shown that S. Enteritidis filaments are as virulent if not more in mice. Enhanced acid tolerance will also contribute to increased virulence. To test this, we will inoculate mice with and without pretreatment with bicarbonate with control and filamentous S. Typhimurium. Results generated will be presented at relevant conferences where the target audiences consist of members of academia, food industry, and food regulatory agencies. Knowledge gained on growth, survival, and virulence of Salmonella under stressful conditions will be used to make informed recommendations on interventions practices and risk assessments of Salmonella contamination of foods.

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

Outputs
Target Audience: Results from this study will be useful to industry, government, and academic food safety and public health professionals as well as students in this area of study. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The student supported by this project (A.J. Stasic) presented results from these studies at monthly lab meetings and at the Kenneth Raper Symposium at the University of Wisconsin-Madison. How have the results been disseminated to communities of interest? The results from this study have been disseminated to communities and target audiences at scientific meetings and publications. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Salmonella and many foodborne pathogens can survive for long periods in low-water activity foods, which have been implicated with increased frequency as vehicles of foodborne outbreaks. Salmonella develops filaments (elongated cells without septation) when exposed to osmotic stress induced by the addition of a humectant such as NaCl to the culture medium. When the stress is removed, the filaments septate and divide into multiple normal-sized cells, resulting in a rapid increase in viable counts. The formation of filaments by foodborne pathogens is significant to food safety because the detection and estimations of pathogen numbers may be compromised. The mechanism(s) of filamentation is not understood. It is unknown if the formation of filaments is a survival strategy or a consequence of stress. A major goal of this project is to characterize and further understand the mechanisms of filament formation and their significance to pathogen persistence. We showed that filamentation occurred with multiple Salmonella serotypes and strains, although there were differences in the degree of filamentation. The filaments were not more tolerant of low- or high-temperature stresses than non-filamented cells. However, there was greater survival of filaments in 10% bile salts, during pH 2 acid challenge, and under desiccation on stainless steel surfaces. Filamentation was accompanied by an increase in biomass (measured by optical density or wet weight) but not viable count. When the stress is removed, the filaments septate and divide into multiple normal-sized cells, resulting in a rapid increase in viable counts. The mechanism(s) of filamentation is not understood. When bacterial cells are exposed to ultraviolet radiation, DNA damage induces the SOS response, which ultimately leads to filament formation. However, this mechanism is not involved in filamentation induced by osmotic stress as we found no evidence of DNA damage in the Salmonella filaments. Filaments and control cells had similar ratios of proteins and DNA relative to their biomass, indicating that protein and DNA synthesis was not impaired in the filaments. To investigate what genes might be involved in filamentation, we determined the osmotic stress and desiccation survival of E. coli O157:H7 and its isogenic mutants in several stress response genes, dps, recA, and rpoS. These mutants were generated for a separate study. As E. coli is a closely related to Salmonella, we used them as a model to screen for potential gene candidates for further investigation in Salmonella. The rpoS mutant was consistently most sensitive to osmotic and desiccation stress, while survival of the dps mutant was less than that of the control at 37C but not at 30C when exposed to medium containing 12% NaCl. When dried on filter paper or in cow feces, survival of the control and the recA and dps isogenic mutants was similar. We further investigated bile salt tolerance and showed that heterogeneity existed among cells in a Salmonella population in their ability to grow in 10% bile salts. Isolates that were tolerant of > 15% bile salts were obtained, while the remainder of the population was sensitive to 8%. The tolerant phenotype was stable and was maintained even after multiple generations of subculturing in the absence of bile salts. These bile salt tolerant strains were not more resistant to other stresses including acid pH and detergent exposure; however, they were able to develop filaments at a higher rate than the parent and their bile salt sensitive counterparts. Preliminary results showed no differences in membrane protein or lipid profiles between bile salt tolerant and sensitive clones; however, RNA-Seq has identified a phage-encoded gene(s) expressed in the bile salt tolerant strains that is absent or expressed at significantly lower quantities in the bile salt sensitive strains. The bile salt tolerant and sensitive strains were clonal with identical biochemical, membrane protein and lipids, and genomic restriction enzyme digestion profiles.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2011 Citation: Stasic, A.J., A.C.L. Wong, and C.W. Kaspar. 2011. Survival of Escherichia coli O157:H7 in low water activity. Kenneth Raper Symposium, Madison, WI.
• Type: Conference Papers and Presentations Status: Published Year Published: 2012 Citation: Stasic, A.J., A.C.L. Wong, and C.W. Kaspar. 2011. Osmotic and desiccation tolerance in Escherichia coli O157:H7 requires rpoS. Kenneth Raper Symposium, Madison, WI.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Stasic, A.J., A.C.L. Wong, and C.W. Kaspar. 2011. Osmotic and desiccation tolerance in Escherichia coli O157:H7 requires rpoS. Current Microbiology 65:660-665.
• Type: Journal Articles Status: Other Year Published: 2013 Citation: Stasic, A.J., E. Stanton, A.C.L. Wong, and C.W. Kaspar. 2013. Phage-mediated population heterogeneity in Salmonella enterica. Manuscript in preparation

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

Outputs
OUTPUTS: We investigated the physiology of osmotic stress-induced filamentation by Salmonella and the potential mechanism(s) of tolerance to osmotic stress. Portions of our results were presented at the Kenneth Raper Symposium and the Food Research Institute annual meeting at the University of Wisconsin-Madison. A paper was published in Current Microbiology. PARTICIPANTS: Amy C. Lee Wong, PI; Charles W. Kaspar, co-PI; Charles J. Czuprynski, co-PI; Andrew J. Stasic, graduate student. TARGET AUDIENCES: Target audiences include researchers, regulators, and other personnel in industry, government, and academia interested in and concerned about food safety. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
Salmonella and many foodborne pathogens can survive for long periods in low-water activity foods, which have been implicated with increased frequency as vehicles of foodborne outbreaks. Salmonella develops filaments (elongated cells without septation) when exposed to osmotic stress induced by the addition of a humectant such as NaCl to the culture medium. When the stress is removed, the filaments septate and divide into multiple normal-sized cells, resulting in a rapid increase in viable counts. The mechanism(s) of filamentation is not understood. It is unknown if the formation of filaments is a survival strategy or a consequence of stress. We showed that filamentation occurred with multiple Salmonella serotypes and strains, although there were differences in the degree of filamentation. The filaments were not more tolerant of low- or high-temperature stresses than non-filamented cells. However, there was greater survival of filaments in 10% bile salts, during pH 2 acid challenge, and under desiccation on stainless steel surfaces. We further investigated bile salt tolerance and showed that heterogeneity existed among cells in a Salmonella population in their ability to grow in 10% bile salts. Isolates that were tolerant of > 15% bile salts were obtained, while the remainder of the population was sensitive to 8%. The tolerant phenotype was stable and was maintained even after multiple generations of subculturing in the absence of bile salts. These bile salt tolerant clones were not more resistant to other stresses including acid pH and detergent exposure; however, they were able to develop filaments at a higher rate than the parent and their bile salt sensitive counterparts. Preliminary results showed no differences in membrane protein or lipid profiles between bile salt tolerant and sensitive clones. Work is continuing to determine what genotypic or other changes may account for the bile salt tolerant phenotype.To investigate what genes might be involved in filamentation, we determined the osmotic stress and desiccation survival of E. coli O157:H7 and its isogenic mutants in several stress response genes, dps, recA, and rpoS. These mutants were generated for a separate study. As E. coli is a closely related to Salmonella, we used it as a model to screen for potential gene candidates for further investigation in Salmonella. The rpoS mutant was consistently most sensitive to osmotic and desiccation stress. We examined a Salmonella strain whose RpoS expression was repressed, and found that it filamented at a lower NaCl concentration than other strains with intact RpoS expression. This suggested that RpoS is not directly required for filamentation. We have generated rpoS mutants in Salmonella, which will be used for further experiments. The formation of filaments by foodborne pathogens is significant to food safety because the detection and estimations of pathogen numbers may be compromised. Understanding the mechanism(s) of formation and survival of filaments will help develop mitigation strategies to reduce these pathogens on food and the food processing environment.

Publications

• Stasic, A.J., Wong, A. C. L. and Kaspar, C.W. 2012. Osmotic and desiccation tolerance in Escherichia coli O157:H7 requires rpoS. Curr. Microbiol. 65:660-665.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: We investigated the physiology of osmotic stress-induced filamentation by Salmonella and the potential mechanism(s) of tolerance to osmotic stress. We determined whether DNA damage occurred in filaments and compared the levels of DNA and total and outer membrane proteins in filaments and non-filamented cells. The potential involvement of several stress response genes in osmotic stress survival was also examined. Portions of our results were presented at the Department of Bacteriology annual retreat and the Kenneth Raper Symposium at the University of Wisconsin-Madison. PARTICIPANTS: Amy Wong, PI; Charles Kaspar, co-PI; Charles Czuprynski, co-PI; Andrew Stasic, graduate student TARGET AUDIENCES: Target audiences include consumers and personnel in industry, government and academia interested in and concerned about food safety. Portions of this work have been presented at the Department of Bacteriology annual retreat and the Kenneth Raper Symposium at the University of Wisconsin-Madison PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Salmonella develops filaments (elongated cells without septation) when exposed to osmotic stress induced by the addition of a humectant such as NaCl to the culture medium. Filamentation is accompanied by an increase in biomass (measured by optical density or wet weight) but not viable count. When the stress is removed, the filaments septate and divide into multiple normal-sized cells, resulting in a rapid increase in viable counts. The mechanism(s) of filamentation is not understood. When bacterial cells are exposed to ultraviolet radiation, DNA damage induces the SOS response, which ultimately leads to filament formation. However, this mechanism is not involved in filamentation induced by osmotic stress as we found no evidence of DNA damage in the Salmonella filaments. Filaments and control cells had similar ratios of proteins and DNA relative to their biomass, indicating that protein and DNA synthesis was not impaired in the filaments. However, there were differences in relative amounts of several membrane proteins. Notably, the level of penicillin binding protein 2 (PBP2) was greater in filaments than control cells. PBP2 is involved in cell elongation. In addition, the level of OmpX, which has been shown to play a role in stress survival in some organisms, was increased. To investigate what genes might be involved in filamentation, we determined the osmotic stress and desiccation survival of E. coli O157:H7 and its isogenic mutants in several stress response genes, dps, recA, and rpoS. These mutants were generated for a separate study. As E. coli is a closely related to Salmonella, we used them as a model to screen for potential gene candidates for further investigation in Salmonella. The rpoS mutant was consistently most sensitive to osmotic and desiccation stress, while survival of the dps mutant was less than that of the control at 37C but not at 30C when exposed to medium containing 12% NaCl. When dried on filter paper or in cow feces, survival of the control and the recA and dps isogenic mutants was similar. The formation of filaments by foodborne pathogens is significant to food safety because the detection and estimations of pathogen numbers may be compromised. Understanding the mechanism(s) of formation and survival of filaments will help develop mitigation strategies to reduce these pathogens on food and the food processing environment.

Publications

• No publications reported this period

Progress 10/01/10 to 12/31/10

Outputs
OUTPUTS: We initiated this project to understand the physiology of stress-induced filamentation by Salmonella and to determine the role of filaments in acid tolerance and pathogenesis. PARTICIPANTS: Amy Wong, PI; Charles Czuprynski, co-PI; Charles Kaspar, co-PI; Andrew Stasic, graduate student TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
We showed that there are strain differences in the ability of Salmonella to tolerate environmental stresses and to develop filamentous cells under stress. We selected two Salmonella enterica serovar Typhimurium strains that differ in these abilities and have started to investigate the physiological and genetic basis for these differences. The formation of filaments by foodborne pathogens is significant to food safety because detection and estimations of pathogen numbers may be compromised. Understanding the formation, survival, and virulence of Salmonella filaments will help develop mitigation strategies to reduce these pathogens on food and in the food processing environment.

Publications

• No publications reported this period