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p1 {margin: 0.0px 0.0px 0.0px 0.0px; font: 12.0px ‘Times New Roman’; -webkit-text-stroke: #000000}p.p2 {margin: 0.0px 0.

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0px ‘Times New Roman’; -webkit-text-stroke: #000000; min-height: 15.0px}span.s1 {font-kerning: none}span.s2 {text-decoration: underline ; font-kerning: none}span.Apple-tab-span {white-space:pre}Listeria monocytogenes is a gram positive bacterium that causes many of food borne diseases in humans and animals (Bakker et al, 2010).

This rod shaped bacterium was first isolated in 1924 by E.G.D Murray from the blood of laboratory animals. Murray was not able to categorize this pathogen to a bacterial genus at the time.

He decided to name this newly discovered bacteria Bacterium monocytogenes. In 1940, researcher Harvey Pirie renamed the bacteria Listeria monocytogenes in honor of the British surgeon, Joseph Lister. This bacteria was not recognized as a pathogen until the listeriosis epidemic in 1949. This epidemic occurred in Germany and mostly effected newborns (Hof, 2003).   Listeria monocytogenes has a high incidence rate in meat, poultry, seafood, and dairy products (Oliver et al, 2010). According to the CDC, Listeria outbreaks in the early 1990’s were linked to deli meats and hot dogs. Today, Listeria outbreaks have been traced back to cheeses, celery, sprouts, and cantaloupe.

Listeria monocytogenes can lead to mild or severe infections (Kazmierczak et al, 2003). One disease in particular that this bacteria causes is Listeriosis. It is fairly common for Listeria monocytogenes to make its way into a host. The adult immune system will attack the pathogen and after a few days, Listeriae will be excreted by the body in the feces (Hof, 2003). Rarely does L.

monocytogenes cause listeriosis in healthy individuals, but the consumption can lead to mortality in those with compromised immune systems (Oliver et al, 2010). Individuals that are most at risk are the elderly, pregnant women, and infants. Infections can lead to fever, muscle aches, diarrhea, meningitis or encephalitis. This infection can also spread to the nervous system (Disson, 2012). These symptoms can include stiff neck, loss of balance, confusion, and convulsions. Pregnant women have an increase in risk of acquiring listeriosis compared to the normal population.

This pathogen can spread from the mother to the fetus through the placenta. Although the mother will only experience mild  symptoms. These symptoms usually present themselves like the flu, although this could be detrimental for the fetus (Hof, 2003). Listeriosis in pregnant women can lead to stillbirth, premature delivery, or neonatal meningitis (Dowd et al, 2011). Listeriosis causes about 10 percent of deaths related to food borne illnesses in the United States each year (Palmer et al. 2011).  Listeria monocytogenes strains come from different lineages which affects the bacteria’s ability to be transmitted to the host. This bacterium belongs to two distinct lineages: I and II.

Lineage I has higher pathogenic potential and is also associated with human cases of listeriosis. Its pathogenic potential is shown by its ability to spread to neighboring cells in the host. Linage II is more common in food isolates.  (Oliver et al, 2010). Listeria monocytogenes is able to break down and digest decayed organic matter. This process makes Listeria monocytogenes saprotrophic.

This bacteria is able to convert from a saprotroph into an intracellular pathogen  (Palmer et al. 2011). This occurs through regulatory factors that allows for the survival of the bacteria in different environments (Kazmierczak et al, 2003).

  Listeria monocytogenes has the ability to adapt to different environmental conditions which allows it the ability to successfully be transferred from food to its host. This bacteria can survive in a wide range of environments. Listeria monocytogenes is able to grow in temperatures that range from 0 to 45°C and in environments of pH ranging from 4.4-9.4 (Oliver et al, 2010). This pathogen has to overcome many barriers in the intestinal system to have an effect on the host. The largest barrier that this organism encounters is the bile released from the liver.

L. monocytogenes has the ability to be bile resistant which is essential for its survival and colonization in the human gallbladder.  Bile is an aqueous solution that is mostly composed of bile acids, cholesterol, phospholipids, and biliverdin.

It is synthesized in the hepatocyte of the liver and stored in the gallbladder. When food in consumed, the gallbladder is stimulated to contract and bile is secreted from the gallbladder into the small intestine. Its main function is to act as a detergent to emulsify and solubilizes lipids for fat digestion (Begley et al, 2006). Bile has many properties which allows it to cause damage to DNA, RNA, and macromolecules (Dowd et al, 2011). Listeria monocytogenes is able to be pathogenic due to its ability to resist the damaging properties of bile.  Sigma B has been shown to be the main contributor for ability of  Listeria monocytogenes to survive under environmental stress conditions (Kazmierczak et al, 2003). This stress responsive sigma factor has been conserved throughout the gram-positive bacterial genera (Palmer et al. 2011).

It can be found in bacteria such as Staphylococcus aureus Bacillus anthracis, and Bacillus licheniformis. These Gram-positive bacteria are able to survive in stressful environments are oxidative, acidic, and high energy (Oliver et al, 2010). Sigma factors bind to the core RNA polymerase during certain environmental conditions. This binding regulates the region transcription, causing the bacteria to respond to environmental signals (Palmer et al.

2011). Sigma B has been shown to regulate the transcription of more than 150 genes directly and indirectly (Oliver et al, 2010). These genes involve a wide range of functions but more specifically contributing to the virulence and survival of the bacteria in the host (Palmer et al. 2011). Some of the virulence genes include prfA, bsh, inlA, and inlB (Oliver et al, 2010). Previous studies have shown that deletion of the gene bsh (bile salt hydrolase) in Listeria monocytogenes causes the destruction of the pathogen (Dowd et al, 2011).

Sigma B has also been shown to contribute to the formation of biofilm (Kazmierczak et al, 2003). Biofilm is a group of bacterial cells that are embedded in a matrix. Bacteria that form biofilms tend to resist antibiotics (Bjarnsholt, 2013). This may enhance the survival of Listeria monocytogenes in stressful environmental conditions (Kazmierczak et al, 2003)Previous research  Previous studies led us design our experiment for the contribution of sigma B to adaptability of Listeria monocytogene in the stressful environment of the gallbladder. Garner et al. performed the study, Sigma B Contributes to Listeria monocytogenes Gastrointestinal Infection but Not to Systemic Spread in the Guinea Pig Infection Model. Mechanisms of  Listeria monocytogenes were known but studies were limited on how L.

monocytogenes was able to survive during the preiavasion and gastrointestinal stage of Listeriosis. They believed that the limitations were due to the lack of information regarding the study of food-borne listeriosis (Garner et al, 2005). In this study, guinea pigs were used to imitate Listeria monocytogenes and its virulence characteristics when humans are infected. They stated that previous studies used murine models but that did not appropriately model the gastrointestinal infection process of humans.

This was because humans and mice have different E-cadherin amino acid sequences. Listeria monocytogenes InlA will not effectively interact with E-cadherin in the murine model. This causes mice to have limited susceptibility of listeriosis. In humans, E-cadherin interacts with InlA, an 800 amino acid protein, the bacteria is able to enter the epithelial cells of the host (Garner et al, 2005). This study gave insight on how to properly conduct an animal model experiment for listeriosis.   The study, The Listeria monocytogenes prfAP2 promoter is regulated by sigma B in a growth phase dependent manner by Schweb et al, also influenced the focus on sigma factor B in our proposed study. Sigma B was shown to be stress responsive in the stationary phase of sigma factor.

The sigma factor was conserved in other Gram-positive bacteria. Sigma B was also shown to contribute to the transcriptional regulation of sar in S. aureus. The locus of sar controlled the expression o other loci. These regulatory elements were shown to control the synthesis of different extracellular and cell surface proteins which contributed to the pathogenies of the bacterium S. aureus.

They believed that sigma B was the regulator of not only the virulence of S. aureus, but also the virulence of Listeria monocytogenes. It was stated that Listeria monocytogenes prfA had three promotors that contributed to its pathogenesis. These promotors included prfAP1, prfAP2, and plcA. In this study, they were able to determine that prfAP2 promotor was directly regulated by sigma B (Schwab, 2005).  The influence of the stressful environment on Listeria monocytogenes was shown in the study, Identification of Components of the Sigma B Regulon in Listeria monocytogenes That Contribute to Acid and Salt Tolerance (Abram et al, 2008). This study was able to determine that differences in sigma B was not detected in the exponential growth phase when Listeria monocytogenes was cultured in BHI medium.

This suggested that cells cultured in this medium did not experience stress during growth. They were able to show that normal Listeria monocytogenes cells grew slowly in medium that contained NaCl. When these cells were mutated causing a lack in sigma B, there was rapid growth in medium with NaCl. Osmolarity and pH also played a role in the survival of cells with a normal or mutant sigma B.

This study was also able to show that cells with a sigma B mutant had a different Gram staining that the control cells. This suggested that sigma B played a role in the maintenance of the cell wall (Abram et al, 2008). Aim/Hypothesis The aim of this study is to show that Sigma B contributes to the adaptability if Listeria monocytogenes to tolerate the stressful environment of bile. To understand this mechanism, the identification of genes that regulate sigma B must be shown. Next, the environmental stress conditions will be tested to determine if there is a change in the expression of Sigma B. Lastly, Sigma B will be down regulated to determine if this will in fact influence the survival of Listera monocytogenes. The goal is to understand the mechanisms behind this pathogen and to develop protocols that can be used to reduce the disease process.

Experimental DesignMicroarray To identify Sigma B dependent genes in L. monocytogenes, microarray will be used. For the microarray protocol, RNA will be extracted for analysis. The bacteria will be cultured in BHI broth. This medium is ideal for the cultivation of pathogenic microorganisms. The cells will then be collected and RNAProtect bacterial agent will be added to stabilize the mRNA.

Next, the cells will be lysed with lysozyme and sonication then centrifuged. The pellets will be stored at -80°C. RNA will then be isolated and purified using the RNeasy kit. The total RNA will be eluted and incubated with RNasin to inhibit RNases.

A phenol-chloroform extract will be used for purification. The integrity of the RNA will be assessed by gel electrophoresis. Microarray will then be performed on the extracted RNA to identify the mRNA for sigma B (Oliver et al, 2010).      Guinea pig listeriosis model. Guinea pig listeriosis infection model will be used to assess the virulence of Listera monocytogenes and sigma B. Animal protocol will be followed during this experiment. Three week old male guinea pigs will be used for the listeriosis model. The guinea pigs will be housed individually and will be provided a standard chow diet and water ad libitum.

They will be acclimated for 7 days prior to intragastric inoculation (Oliver et al, 2010). The bacteria will be grown in BHI broth. The cells will be cultured and centrifuged and treated with PBS containing 15% glycerol. The cells will be spiral plated on a petri dish and incubate for 24 hours to determine cell viability. The guinea pigs will then be intragastric inoculated with the viable Listera monocytogenes cells (Oliver et al, 2010).

  Listera monocytogenes will be administered by gavage. The stomach of the guinea pigs will be neutralized with calcium carbonate containing PBS before bacteria is given. Guinea pigs will euthanized by CO2 72 hours being administered Listera monocytogenes. Before euthanization, the guinea pigs will be weighed.  The small intestines and liver will be removed and treated with PBS and gentamicin.

These organs will also be weighed before testing. The small intestines and liver will be homogenized separately. The homogenates will be diluted with PBS and  plated on Oxford medium. Listera monocytogenes colonies will be measured. The levels of recovery for Listera monocytogenes in the liver and small intestines will be measured for the virulence. RT-PCR will be used to measure the expression of sigma B mRNA in the control guinea pigs and the guinea pigs with listeriosis (Oliver et al, 2010).     Statistical analysis will be performed using Statistical Analysis Software (SAS)Sigma B gene down regulation. A common strategy for understanding the biological network of a factor is to disrupt the gene and monitor the consequences (Palmer et al.

2011). In this study, mutations in the sigma B gene will be created for for L. monocytogenes.

This mutation will down regulate the sigma B gene. This will be achieved by creating antisense RNA which was first discovered in 1963 by Singer et al. In their study they were able to show that polyuridylic acid could be blocked by using antisense polyuridylic acid. Antisense RNA by can be used for gene silencing in bacteria. This noncoding RNA binds to complementary mRNA sequences which induces gene down regulation.

This occurs because antisense RNA is able to inhibit translation of a protein or degrade the proteins mRNA through base pairing. The target sites for the antisense RNA are located in the mRNA translation initiation region. This will affect the ribosome binding that will cause a change in translation (Park et al, 2014).Mutated sigma B L. monocytogenes survival.  Once the sigma B gene is down regulated, the survival of Listeria monocytogenes will be tested under normal conditions.

Previous studies have shown that a mutation in Listeria monocytogenes 10403S causing a silencing of sigma B caused the reduction in virulence intragastricaly of guinea pigs and also reduced its ability to invade human intestinal epithelial cells (Oliver et al, 2010). In the  Bile stress survival assay. Listeria monocytogenes cells will be exposed to bile conditions. After exposure, the expression of sigma B genes will be analyzed. Bile conditions can be replicated by using powdered bile. Oxgall or bile extract porcine will be used. Porcine bile is considered to be the most similar to human bile.

Listeria monocytogenes cells will be cultured in BHI. Normal cells and mutated cells will be treated with the bile extract. Survival of the Listeria monocytogenes cells will be monitored.

  Predicted results For the microarray procedure, different sigma B dependent genes should be shown. This procedure should allow use to identify 140-160 genes. One important gene that would be studied  is bsh. Bile salt hydrolase (bsh) is typically not seen in bacteria that does not have bile in their natural environment, but Listeria monocytogenes is on bacteria that is an exception (Begley et al, 2006).  The guinea pig model should show properly recreate the human listeriosis infection. Guinea pigs inoculated with normal Listeria monocytogenes should be more susceptible to  listeriosis than the guinea pigs inoculated with mutated Listeria monocytogenes without sigma B.

  This should be due to the inability of sigma B to transcribe bile salt hydrolase (bsh). This should ultimately cause Listeria monocytogenes to be destroyed by the stress of the bile in the gallbladder.  Sigma B should be down regulated by using antisense RNA. Down regulation should be checked by performing western blot analysis. The predicted results should be that the down regulated sigma B and the control sigma B should be tested in normal conditions and in bile conditions. Listeria monocytogenes should show an increase in transcription in the high stress environmental condition of the bile. Mutated sigma B should decrease the survival rate of Listeria monocytogenes.

    Alternative results The microarray procedure should show different sigma B dependent genes. If the gene Bsh is not identified, then other sigma B dependent genes can be tested to determine which gene is able to detoxify bile. If the guinea pig model does not work efficiently, clinical trails on humans with listeriosis can be used. Although, sigma B mutants can not be used for clinical trial, fecal samples can still be tested to determine the normal expression of sigma B mRNA in individuals infected with Listeria monocytogenes. 

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