Heat shock protein (HSP) are a family of proteins that serve as the main protein folding chaperones. They also participate in protein assembly, export, turn-over and regulation. Seven genes are known to be responsible for the encoding of HSP ( clpB , dnaK , groES , grpE , htpG , htpX and ibpB) . HSP are known to be expressed in both stressful and non- stressful situations. Stressful conditions include temperature UV light, and wound healing or tissue remodeling. When exposed to high temperature, the proteins has a tendency to become highly expressed inorder as a form of protection. Currently little information exists on the differences in expression of heat shock proteins in different organisms such as Escherichia coli and Salmonella enteritidis when placed under the same stress conditions.  Escherichia coli and Salmonella enterica are important foodborne pathogens capable of forming single-species biofilms or coexisting in multispecies biofilm communities. When both of these organisms are placed in similar temperature conditions different form their respective normal temperature conditions, the expression of their heat shock proteins will tend to change. E.coli and Salmonella both grow at different maximum temperature conditions. E. coli is known to have a lethal temperature of 49?C whiles Salmonella can survive up to a temperature of 60?C. The differences in the expression of heat shock proteins helps to provide a higher survival rate in one more than the other. Understanding the differences in the different expression of heat sock proteins between these two organisms explains what causes the differences in the survival rate. To solve this problem analysis of the expression of heat shock proteins present in Escherichia coli vs Salmonella enteritidis will be conducted. E. coli will be allowed to grow at 4, 37 and 40°C whiles Salmonella enteritidis will be allowed to grow in 4, 37, 40 and 60 °C. Knowing that

Further analysis will also be done to analyze the membrane proteins of these organisms by knocking out the genes that produce this protein under the above temperature conditions. The  genes dnaK, dnaJ, and grpE responsible for their corresponding heat shock proteins have been found to be present in both  Escherichia coli vs Salmonella enteritidis (Liberek et al 1991). DnaJ and GrpE heat shock proteins jointly stimulate ATPase activity of DnaK. ATPases  are a class of enzymes know to catalyze the decomposition of ATP into ADP and a free phosphate ion

Some such enzymes are integral membrane proteins. The innermost layer of these organism are known to regulate the passage of metabolites into and out of the cytoplasm,ATPases , also called transmembrane proteins, move solutes across the membrane. But what happens to these membrane proteins when placed on heat stress in the absence of their heat shock proteins. It can be hypothesize that there will be significant changes in the expression of heat shock proteins as well as the membrane proteins present in Escherichia coli vs Salmonella enteritidis.

Aim 1 will successfully determine and compare the expression of the heat shock proteins in both bacterial species grown at different temperatures.

Aim 2 will successfully knockout genes responsible for the production of HSP under different temperature conditions to analyze the changes in the membrane proteome

Background and significance

Escherichia coli commonly known as E. coli is from the genus Escherichia. It falls under the categories of a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium. Salmonella enteritidis on the other hand is from the genus Salmonella. It is a Gram-negative bacteria with a rod-shaped liked structure, flagellated, and facultative anaerobic. Escherichia coli and Salmonella, are known to not only be the two of the most common pathogens also known to cause bacterial foodborne illnesses such as diarrhea in the United States (Mead et al., 1999). Through direct or indirect contamination of food, humans tend to get infected with such illnesses. They can also circulate through fecal–oral route of transmission (Britton et al, 2004). over the years both bacteria have become increasingly important in the food industry considering that in worst cases it can be life –threating

Aside from the fact that both bacteria have been found to grow at 37 degrees Celsius which is also the temperature of human beings, they have also been found to survive in temperatures above or lower to this optimal temperature due to the presence of heat shock proteins (HSP). HSPs are main protein folding chaperones, and they are also a kind of heat stress protein, that have a tendency to become activated for protection when organisms are exposed to stress conditions not limited to just high temperature. 

Figure 1. This figure showcases two main functions of heat shock proteins with respect to protein folding and protection in stress conditions. The top part of the picture represents a new polypeptide chains (proteins) being produced by ribosome within the cell, heat shock proteins help in the modification of the right  folding of polypeptide chain into functional protein. Presence of heat shock protein (purple) assures that the new protein will assume its functional three-dimensional configuration. The Bottom part of the figure shows how heat shock proteins also assist in refolding of denatured proteins in the presence of heat stress. Figure from Mehat et al, 2005

 

A study investigated the survival of heat-shocked and non-heat-shocked Escherichia coli and Salmonella enterica when co-composting dairy manure and vegetable wastes in a field setting. It was detected that both E. coli and Salmonella survived a maximum of 9days in the summer compared to the winter where they survived 60days (Shepherd et al., 2010). The results indicated that composting dairy manure with vegetable wastes allowed survival of pathogens in the heap at low temperature.

Membrane proteins have been discovered to have great importance in the functioning of cells. Such functions include ion transport, transport of nutrients, communication, links to extracellular matrix, receptors for viruses, and connections for the cytoskeleton (Vandeventer P., et al. 2011). Membrane proteins also have importance with regards to diseases such as diabetes, hypertension, depression, arthritis, cancer, and many other common diseases which is the reason they are targeted by over 75% of pharmaceuticals in use today. The membrane fatty acid composition of Salmonella Enteritidis was studied under heat treatment 54, 56, 58, and 60 °C (Yang et al, 2014).A decrease in the ratio of unsaturated to saturated fatty acids was observed as the growth temperature increased when compared to the control cells grown at 37 °C But the question still remains, do HPSs have anything to do with the structure of the membrane proteins when placed in stress conditions.

A study by Kusukawa  et al, was conducted to investigate the physiological roles of sigma 32 and heat shock proteins, when  isolated and at different temperatures. Their results demonstrated that the heat shock protein GroE levels increased and correlated with a maximum temperature of 40degrees.but not up to 42. This basically just gives a confirmation on the survival rate of e.coil under heat stress. However what the further discovered as that Dnak was hyper produced and this was able to survive up to a temperature of 42 degrees Celsius but at low levels of production. Less than 10%.

Cold storage and heat treatment are the most widely used intervention techniques to inhibit or inactivate foodborne pathogens during food storage and processing (Davidson et al, 2013). Identifying the changes in the expression of HSP in these commonly foodborne pathogens under temperature stress conditions brings light to how these organisms survive the temperature abuse and become more virulent and have greater resistance to subsequent exposure to heat condition . After analyzing studies form different papers, there is confirmation that these two organisms definitely grow at different temperature. Some have shown that certain genes enable them to survive at a higher temperature. Knocking out some of the genes related to heat shock proteins will bring about the understanding of what makes one survive longer than the other. It could be that due to the higher content of a certain gene in one compared to the other, or it could be that one has a specific gene that the other lacks. Difference in general will be seen because these bacteria need to find a way to survive, and what makes survive with respect to the heat shock proteins will be critically looked at.

Methods

Specific Aim 1 Determine and compare the expression of the heat shock proteins in both bacteria to be grown at different temperatures and one control and a recovery time point created determine.

It is important to know to what extent to which Salmonella enteritidis and Escherichia coli can survive under different temperatures. One of the ways that these organisms are able to survive such stress conditions are through the expressions of heat shock proteins (HSPs). Understanding the expression of the HSPs in different temperature conditions provides a better understanding of how to modify preventive measures in order to prevent illnesses generated by these organisms.

Both bacterial species will be grown in different temperature conditions ranging from 4, 37, 40 and 60°C. E. coli is known to grow at an optimal temperature of 49°C, so it will then be allowed to grow at the temperatures, 4, 37 and 40 °C.

 Cultures of both Salmonella enteritidis and Escherichia coli can be obtained from ATCC or the American Type Culture Collection (Helena et al, 2016).The ATCC is an accredited non-profit organization that collects, stores, and distributes standard reference microorganisms. These cultures will be extracted from petri dishes and inoculated into Trypticase soy broth (TSB) for liquid cultures and kept in the above temperature conditions for growth in replicates of 3 for both. TSB grows many types of bacteria including Salmonella enteritidis and Escherichia coli. . In order to standardize the extraction point, a growth curve for each temperature will be found. This will be done by measuring turbidity levels over time. The turbidity will be measured using an OD reader and will be measured every 4 hours (Driessen et al 1996) .Proteins will be extracted at the early late phase. At the phase, the cells have grown for a descent amount to generate a good quantity of proteins. Cell will then be spun down using a centrifuge at max speed for 15mins. Pellets will be re-suspended using lysis buffer at 1:1 to 1:4 dilutions, and lysed using the sonication protocol. An alternative for this method will be formed will be resuspended using ammonium bicarbonate and lysed with glass beads for 2.5mins.(Horth et al, 2003). Sonication has been used before and a lot of background knowledge has been developed however they are time where it has proven to be unsuccessful.  

Figure 1 This figure showcases the alternative method using glass beads as a method of lysing cells open (Yeong,.et al 2010)

 

The clear supernatant will then be subjected to protein concentration determination following the Bradford method (Bradford 1976). This is needed in order to not overload 2D gels. IPG strips will be rehydrated passively in solubilization solution (7 M urea, 2 M thiourea, 4 % CHAPS, 65 mM DTT, 0.8 % pH 3–10 ampholytes) containing 1.0 mg protein samples and focused using a Protean IEF Cell with total product time × voltage 80,000 Vh for each strip at 20 °C( Yanyu et al,2013) . DTT breaks the disulfide bonds in the protein. The IEF strips will then be further reduced with 1%w/v DTT and alkylated with 2.5 %w/v iodoacetamide in equilibration buffer (6 M urea, 25 %w/v glycerol, 2%SDS, and 0.375 mM TrisHCl, pH 8.8) for 15 min( Yanyu et al,2013). The equilibrated strips will further undergo SDS-PAGE analysis. the 2-DE gels will then be stained with Coomassie Brilliant Blue protocol. Protein spots of interest will be identified and cut out from the 2D and digested in accordance to the trypsin digestion protocol described by( Kim et al. 2004). It basically hydrolyzes certain peptide bonds.  Spots of interest will be cut out with alcohol rinsed scalpel and put into a microcentrifuge tube. 200ul of 50%methanol will then added and left to incubate for whiles shaking vigorously.50Mm of ammonium bicarbonate /50% acetonitrile will be further added and incubated for 30mins whiles also shaking vigorously. On removal of the acetonitrile, 0.02mg/ml of trypsin will then be added and incubated overnight at 37degrees. The digest solution will be transferred into a new tube and stored in the -80 freezer. The digested proteins will be analyzed using the MALDI on a mass spectrometry. MASCOT will then be used for identification using the peptide mass finger printing search

It is expected that there will be a large identification of heat shock proteins or proteins that have a correlation with HSP in both organisms compared between their controls and between the two species. The difference in temperature within each specie compared to their control will explain the how the HSP changes with temperature and the species will also be compared along with their respect max temperature conditions to provide a conclusive comparison

Specific Aim 2 successfully knockout genes responsible for the production of HSP under different temperature conditions to analyze the changes in the membrane proteins

Membrane proteins controls everything that goes in and out of the cells of organisms. Under stress conditions, the HSPs of the both Salmonella enteritidis and Escherichia coli will be become activated in order to serve as a form of protection for the cell. It is hypothesized that changes will be seen in the membrane protein when certain genes responsible for the heat stresses in these two species are knocked out.

Salmonella enteritidis and Escherichia coli both have dnaK, dnaJ, and grpE (Liberek et al 1991). These genes are known to express heat shock proteins under heat stress. Knocking out of these genes will be conducted using the site directed mutagenesis protocol which uses a PCR

Procedure (Zheng et al, 2004). Desired mutations will be introduced into a gene by replacing the sequence of the normal gene with that of the mutated gene thereby knocking out these specific genes. The cultures after the knock out will then be inoculated and left to grow in the different temperatures and spun down using the same procedure as in specific aim 1.

Protiens will be extracted at the early late phase same as in specific aim 1. Pellets will be re-suspended using lysis buffer at 1:1 to 1:4 dilutions, and lysed using the sonication protocol. An alternative for this method will be formed will be resuspended using ammonium bicarbonate and lysed with glass beads for 2.5mins.(Horth et al, 2003). Sonication has been used before and a lot of background knowledge has been developed however they are time where it has proven to be unsuccessful. 

 This supernatant will then undergo the Bradford assay protocol. A desired volume of the supernatant will undergo acetone precipitation in a 5:1 ratio of cold acetone to water for a minimum of 2 hours. The samples will further undergo the second part of shotgun proteomics which includes re-suspending in buffer (1% deoxycholate, 50mM TEAB PH 8.5), incubating in DTT and IAA before finally undergoing trypsin digestion( Proteomics lab, 2017). The digested proteins will be analyzed using the MALDI on a mass spectrometry. MASCOT will then be used for identification using the peptide mass finger printing search.

Results should not only indicate differences in the membrane protein within the two species when compared to their control but also differences should be seen between the two species. Salmonella enteritidis has a higher resistance to temperature, results from this should indicate if there are other genes other than the ones they both have in common which enables it to have a higher chance of survival. The results will also showcase how the membrane proteins functions when the genes related to the heat stress are knocked out. HSP are known to help contain the fluidity of membranes when under heat stress, results will provide a conclusion on how these membrane proteins function in the absence of their savior (Heat shock protein)