Survival of Food-borne Pathogens

<p>NON-TECHNICAL SUMMARY:<br/> Listeria monocytogenes is a bacterium that is widely distributed in the environment; in foods, it is generally associated with dairy products and processed meats. In susceptible individuals, pregnant women, the very old, the very young and patients on immune system suppressants, it causes a disease called listeriosis. Although infection is relatively rare compared to Salmonellosis, listeriosis as more than 25 percent fatal. Consequently, LM the second largest killer due to food-borne illness, after Salmonella. A hallmark of LM's prevalence as a danger to the food supply is its ability to grow in high salt concentrations, and in the refrigerator. Thus a small amount of contamination can lead to an infections dose during refrigerated storage. LM is also found repeatedly in food-processing environments, despite arduous efforts at sanitization.
Moreover, persistent strains exist that are continually found in these environments.The discovery of LM in processed packages foods has led to major recalls, including the record 27.4 million pound recall of processed poultry meat by Pilgrim's Pride, in October of 2002. The impact of LM on society is therefore two fold: it poses a significant health risk for immunocompromised individuals, including pregnant women, and it can have a devastating economic impact on food processing companies or producers. In order to control LM, and protect both consumers and food companies, a better understanding of LM's peculiar ability to survive harsh environments and grow in the refrigerator. Our research has shown that transport proteins found in the cell membrane of LM enable its growth under salt stress and refrigeration. These proteins actively pump protective molecules found in milk and meat into
the cell, and the accumulation of these protectants allow LM to grow. These transporters only pump in the presence of the stress. So we are interested in the molecular mechanism of the pumps and how they are turned on by the stress. The membrane of LM cells is also unusual, and plays an important role in tolerance to cold. Our objectives are to determine the mechanism of pumping by the most important of the stress-related transporters, Gbu, and the mechanism by which it is turned on by cold. The results of this research will be used to find ways of preventing the growth of LM in the refrigerator and the recurrence of persistent strains in food-processing environments. We are also investigating the synthesis of the components of the membrane, with the goal of finding inhibitors that can be used in sanitizers.
<p>APPROACH: <br/>We found the phenomenon of cold-activated transport and traced the cold- and salt-hardiness of LM to 3 solute transporters. BetL, OpuC and Gbu. BetL causes influx of glycine betaine when the cell is stressed by high sodium chloride concentrations; OpuC causes influx of carnitine in response to either high osmotic strength or low temperature (refrigerator temps.); Gbu causes influx of glycine betaine in response to either high osmotic strength or low temperature. We identified Gbu as the most important of the 3 transporters. Gbu is composed of 3 kinds of protein, presumably with 2 copies of each. A pair of ATP-hydrolyzing proteins, GbuA, is on the inner surface of the membrane, 2 molecules of GbuB form transmembrane pore and 2 molecules of GbuC, which are tethered to the outer surface of the membrane bind the solute. We cloned and expressed genes for all 3
proteins in Escherichia coli and LM. We created mutants that lack GbuC, and one that lacks the attachment site for the membrane tether. In most bacteria, fluidity of the membrane is adjusted by varying the proportion of unsaturated fatty acids in the phospholipids portion of the membrane. The membrane of LM contains a large proportion of branched-chain fatty acids (C15 and C17, with iso- and anteiso-branches). Branching accomplishes the same reduction in phase transition temperature, but does not expose the membrane to oxidative damage. LM grown at lower temperatures has a higher proportion of shorter, anteiso-branched fatty acids. Membrane lipids show a broad melting curve between o and 20 degrees; Gbu is most active (in the absence of osmotic stress) when the membrane is about 80 percent gel. We will focus on 3 aspects of transporter structure and function. In one experiment we will
use our Gbu mutant strains to investigate the nature of the lipid tether. One of our constructs involves cloned wild-type gbuC contained on a plasmid harbored by a strain of M in which the chromosomal copy of gbuC has been deleted. Cloned gbuC contains a C-terminal hexahistidine tag foe easy purification. We will isolate the GbuC from this strain grown under different conditions, and determine the identity of the fatty acids of the diacylglycerol membrane tether, by using LC/MS. We will compare growth rate and glycine betaine transport rate of the wild type, gbuC deletion and a serine-substitutes GbuC to asses the requirement for GbuC and the lipid tether under conditions of activation by cold and by salt. 2) involves our hypothesis that thickness of the fluid portion of the cell membrane decreases at low temperature because longer-chain fatty acids have formed a gel phase and this
shrinkage causes a change in the structure of GbuB, which effects activation. Experiments involve expression and purification of the Gbu proteins, and reconstitution into artificial membranes of known composition. Artificial membranes will be composed of phospholipids with different length fatty acids, which will adjust the thickness of the membrane. 3) will be to continue our search for inhibitors of branched-chain fatty acid synthesis. Such an inhibitor would strongly reduce the ability of LM to grow in the refrigerator.</p><p>
PROGRESS: <br/>2012/01 TO 2012/12 <br/>OUTPUTS: The gene gbuC encodes a solute binding protein of the Gbu transport system of the food-borne pathogen Listeria monocytogenes (LM). This transport system is critical to the survival of LM under chill or osmotic stress. We are investigating the compositions and function of the lipid tether that fastens GbuC to the membrane. The gene contains a signaling sequence for the secB pathway, which targets the protein to the exterior of the cell, and causes the attachment of a diacylglycerol to the sulfur of the N-terminal cysteine residue after proteolytic processing of the signal peptide. An LM strain harboring a plasmid encoding a hexahistidine-tagged GbuY, and strains with either a deletion or gbuY or a C1->S1 mutant of gbuY, which lacks the cysteine have been revived from storage after a brief period during which the project was not
staffed. The hexahistidine-tagged protein has been expressed, and we are now expressing the gene under conditions of high salt and in the cold (7 degrees C). The proteins are to be cleaved using trypsin, and the hydrolysate submitted to LC/MS to determine the molecular weights of the two fatty acids to determine if they are fixed, or if they chance along with the overall fatty acid composition of the membrane, which shofts from longer-chained, iso-branched fatty acids at room temperature, to C15:0 anteiso-branched fatty acids. A new graduate student has been added to the project, and a visiting scholar is scheduled to arrive in the fall to work on expression of gbuA and gbuB in E. coli, which will be used in separate studies of the structure and function of the Gbu system. PARTICIPANTS: Ms. Xinyi Song, a student from Jiangnan University in China, has joined the project to work on GbuC. A
visiting professor from Jiangnan University is scheduled to join us in the Fall for a sabbatical leave, during which he will express gbuA and gbuB in E. coli TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.<br/>
PROGRESS: <br/>2011/01/01 TO 2011/12/31 <br/>OUTPUTS: The project is currently un-staffed. Activity has been restricted to culture maintenance until personnel can be recruited. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.<br/>
PROGRESS: <br/>2010/01/01 TO 2010/12/31 <br/>OUTPUTS: The focus of the current stage of the project is to determine how the glycine betaine transport system Gbu is activated by chill. Our hypothesis is that transport is activated at low temperature because phospholipids containing longer-chain fatty acids crystallize and leave the protein in a membrane that is enriched in lower-melting lipids, which are two carbon atoms shorter. This narrowing of the membrane causes a further tilt of the transmembrane helices of the pore protein GbuB, which activates transport. Our approach is to clone and express the three proteins comprising the system, and to reconstitute them into artificial phospholipids bilayers of defined thickness. All genes have been cloned for expression in E. coli, and the solute-binding protein-encoding gene can be expressed in L. monocytogenes (because it is
processed differently in E. coli). And all have been expressed and purified. The project has stalled temporarily due to manpower shortage. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.<br/>
PROGRESS: <br/>2009/01/01 TO 2009/12/31 <br/>OUTPUTS: The mechanism of chill and osmotic tolerance of the food-borne pathogen Listeria monocytogenes is being investigated by examining the structure and function of a transport system that imports protective molecules and my analysis of the composition of the cell membrane. The genes encoding the most important stress-related transporter, Gbu, have been cloned and are being used in various substudies: the investigation of the function of the lipid tether that attaches the binding protein GbuC to the membrane, the mechanism of coupling between ATP hydrolysis and transport, the overall organization of the six polypeptides in the L. monocytogenes membrane, and the nature of the alterations in the membrane of mutants that are unable to synthesize branched-chain fatty acids that form a substantial but variable fraction of the
membrane in the wild type. Both the lipid experiments and the experiments involving GbuC are awaiting GC/MS analysis. Therefore, no outputs were completed. PARTICIPANTS: Patchanee Yasurin was a graduate student in the Food Biotechnology program at the Assumption University of Thailand (Bangkok). She pursued her PhD research under my direction at UC Davis under the auspices of an Agreement of Cooperation. She is now teaching at Assumption University. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.<br/>
PROGRESS: <br/>2008/01/01 TO 2008/12/31 <br/>OUTPUTS: L. monocytogenes is a serious food-borne pathogen that is notable in its resistance to environmental stresses, including chill and high osmolarity. It is the second largest cause of death from food-borne illness in the US. A key aspect of its ability to survive and grow at refrigeration temperatures is the unusual composition of its membrane, which contains highly saturated fatty acids, many of which are ranched. C15 and C17, both iso- and anteiso- branches are common. The membrane also contains transport proteins Gbu and OpuC that transport glycine betaine and carnitine, respectively, which contribute to both osmotic and chill tolerance and a third transporter, BetL, that transports glycine betaine in response to high salt concentration in the medium. The regulaton of the synthesis of the three transporters, and the
mechanism of activation of Gbu and OpuC by chill are all of interest in the effort to reduce food-borne illness due to L. monocytogenes. We have therefore created operon fusions in which the lacZ gene is inserted in the genome preceding a copy of the gene or first gene in the operon that encodes the transporters for studies on the regulation of the synthesis of each transporter at the genetic level. Gbu, the most important of the three transporters, has been cloned and expressed in a project that has recently expired. Additional studies of the L. monocytogenes membrane were also begun. In these studies, the membrane composition was altered by growth at low temperatre, which increases the proportion of C15 fatty acids and anteiso branches, In addition, cold-sesitive mutants exist that are deficient in branched-chain fatty acids. The growth and membrane composition of the membranes of
these mutants was studied in the presence and absence of likely alternative primers for unsaturated, atypically-branched or multiply-branched fatty acid synthesis were determined. Glycine betaine transport will be determined in these cultures. The membrane lipids of these mutants have been isolated for future reconstitution of the cloned GbuA,B and C proteins to determine how transport activity can be affected by differing membrane composition. The reconstitution experiments are a follow-up to our expired project. PARTICIPANTS: Gary M. Smith, PI, Professor, UC Davis. Originated all experiments. Patchanee Yasurin, graduate student, Assumption University of Thailand, visiting scholar at UC Davis. Constructed reporter-gene operon fusions, conducted initial quantitative growth experiments and fatty acid analysis of branched chain deficient mutants. Mun Hua Tan, Undergraduate studet, UC
Davis, conducted preliminary growth experiments on branched chain deficient mutants. Yinghua Xiao, Graduate student, Waginingen University, visiting scholar, UC Davis, conducted preliminary qualitative screening of potential fatty acid primers in branched chain deficient mutants. Raymond C. Valentine, Prof. Emer., UC Davis, consulted on membrane fluidity and fatty acid primers in L. monocytogenes. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.<br/>
PROGRESS: <br/>2007/01/01 TO 2007/12/31
<br/>OUTPUTS: When stressed by high salt concentration or refrigeration temperature L. monocytogenes (LM) cells accumulate the protective molecules glycine betaine and carnitine from the environment by the action of three transport systems, Gbu, OpuC and BetL. Regulation of synthesis is being studied using a promoterless lacZ gene inserted behind a copy of the promoter region of the genes encoding the three stress-related transporters. The new emphasis is to determine whether presence of the substrates glycine betaine, carnitine or other factors alter the expression, and if so, by what mechanism. We previously characterized the membrane lipids of LM and found them to contain highly saturated, often branched-chain fatty acids, largely C17:0anteiso at higher temperatures, shifting to C15:0anteiso at lower temperature. Wilkinson showed mutants unable to make branched chain fatty acids are cold
sensitive. We have provided alternate fatty acid primers to Wilkinson's mutants, and found that linoleic, oleic, vaccenic, isolaleric and t-butylactic acids reestablished chill tolerance to the level of the wild type. The properties and compositions of the membranes synthesized with these primers and under various stress conditions are being determined.
PARTICIPANTS: Pachanee Yasurin, Graduate Student, Biotechnology Program, The Assumption University of Thailand. Mun Hua Tan, Undergraduate Student, UC Davis. Yinghua Xiao, Graduate Student, Wagenengin University, The Netherlands
TARGET AUDIENCES: This work will be presented to microbiology and microbial food safety professionals via publication in professional journals and in food science literature.
PROJECT MODIFICATIONS: All changes involved changes in methodology to enhance the probability of success.
<br/>PROGRESS: <br/>2006/01/01 TO 2006/12/31 <br/>When stressed by high salt concentration or refrigeration temperature L. monocytogenes (LM) cells accumulate the protective molecules glycine betaine and carnitine from the environment by the action of three transport systems, Gbu, OpuC and BetL. Regulation of synthesis is being studied using a promoterless lacZ gene inserted behind a copy of the promoter region of the genes encoding the three stress-related transporters. Our fusion for the opuC operon proved to be faulty, and we generated a new mutant. All three systems are expressed to some extent during balanced growth without added salt stress. Expression is enhanced by the addition of NaCl. Enhancement of expression by chill is more difficult to determine because all processes are slower. The new emphasis is to determine whether presence of the substrates glycine betaine or
carnitine alter the expression, and if so, by what mechanism. We previously characterized the membrane lipids of LM and found them to contain highly saturated, often branched-chain fatty acids, largely C17:0anteiso at higher temperatures, shifting to C15:0anteiso at lower temperature. Wilkinson showed mutants unable to make branched chain fatty acids are cold sensitive. We have provided alternate fatty acid primers to Wilkinson's mutants, and found that some of them conferred cold tolerance. We are now analyzing the lipid composition in these cells.<br/>
PROGRESS: <br/>2005/01/01 TO 2005/12/31When stressed by high salt concentration or low (e.g., refrigeration) temperature L. monocytogenes cells accumulate the protective molecules glycine betaine and carnitine from the environment by the action of three transport systems, Gbu, OpuC and BetL. The activities of the transport systems are regulated by the presence of the stress. Previous work in which genes encoding the transport proteins were deleted showed the relative importance of each transport system and the conditions under which it functions. Besides regulation of transport activity at the biochemical level, it is likely that L. monocytogenes regulates the amount of each transport system by controlling synthesis of the proteins at the genetic level. Regulation of synthesis is being studied using a promoterless lacZ gene inserted behind a copy of the promoter region
of the gbu gene, and similar constructs involving two other stress-related transporters, BetL and OpuC. We grow the cells in the asence of stress and the absence of glycine betaine and carnitine, and test the expression of the reporter gene when the cells are subjected to salt stress, salt stress not involving sodium ion, sugar-mediated osmotic stress and chill stress. We are also testing whether the presence or absence of substrates glycine betaine and carnitine affect expression. Assays using the chromogenic substrate OMPG proved not to be sufficiently sensitive, and we have begun using the fluorogenic substrate MUG. Preliminary results indicate that the transporters are at least partially constitutive, and are present even in rich media unsupplemented with salt.<br/>
PROGRESS: <br/>2005/01/01 TO 2005/12/31 <br/>When stressed by high salt concentration or low (e.g., refrigeration) temperature L. monocytogenes cells accumulate the protective molecules glycine betaine and carnitine from the environment by the action of three transport systems, Gbu, OpuC and BetL. The activities of the transport systems are regulated by the presence of the stress. Previous work in which genes encoding the transport proteins were deleted showed the relative importance of each transport system and the conditions under which it functions. Besides regulation of transport activity at the biochemical level, it is likely that L. monocytogenes regulates the amount of each transport system by controlling synthesis of the proteins at the genetic level. Regulation of synthesis is being studied using a promoterless lacZ gene inserted behind a copy of the promoter region
of the gbu gene, and similar constructs involving two other stress-related transporters, BetL and OpuC. We grow the cells in the asence of stress and the absence of glycine betaine and carnitine, and test the expression of the reporter gene when the cells are subjected to salt stress, salt stress not involving sodium ion, sugar-mediated osmotic stress and chill stress. We are also testing whether the presence or absence of substrates glycine betaine and carnitine affect expression. Assays using the chromogenic substrate OMPG proved not to be sufficiently sensitive, and we have begun using the fluorogenic substrate MUG. Preliminary results indicate that the transporters are at least partially constitutive, and are present even in rich media unsupplemented with salt.
<br/>PROGRESS: <br/>2004/01/01 TO 2004/12/31 <br/>Our objectives were to measure expression of the three compatible solute transporters (Gbu, BetL and OpuC) of Listeria monocytogenes using reporter gene fusions, express green fluorescent protein (GFP)-labeled proteins of the Gbu transport system, and purify histidine-tagged GbuA, GbuB and GbuC. The expression of Gbu and BetL was measured in response to salt stress, osmotic stress (sucrose) and chill stress in the presence and absence of glycine betaine and carnitine. The OpuC label proved to be faulty and we are currently preparing a new strain. E. coli strains harboring plasmids containing genes encoding hexahistidine-tagged GbuA, GbuB and GbuC under the control of a Lac promoter have been prepared and the proteins are being purified using zinc affinity chromatography. The gene encoding GFP has been cloned into a plasmid
suitable for our purposes.
<br/>PROGRESS: <br/>2003/01/01 TO 2003/12/31 <br/>LISTERIA MONOCYTOGENES is a foodborne pathogen that survives under hyperosmotic stress and grows at refrigeration temperatures. Solute transport systems aid in the stress-hardiness by importing the protective molecules glycine betaine and carnitine, which are found in the food. Deletion experiments showed that there are three separate transport systems that are responsible for stress-activated transport of these solutes; they are encoded by the gbu and opuC operons and the betL gene. Gbu and BetL are primarily glycine betaine transporters and OpuC is a carnitine transporter, although Gbu and OpuC show some transport of the other solute. These two transporters are activated by chill or by hyperosmotic stress, whereas BetL is activated by hyperosmotic stress. Operon fusions of a reporter gene show that expression of gbu and betL is
enhanced under stress; the fusion mutant involving opuC is defective and it is being re-designed.</p>