"Characterization of SraB, a novel small RNA molecule, in the pathogenic bacterium Shigella dysenteriae"
CHARACTERIZATION OF SRAB, A NOVEL SMALL RNA MOLECULE, IN THE PATHOGENIC BACTERIUM SHIGELLA DYSENTERIAE _________________________________ A Thesis Presented to The Honors Tutorial College Ohio University _________________________________ In Partial Fulfillment Of the Requirements for Graduation From the Honors Tutorial College With the degree of Bachelor of Science in Biological Sciences _________________________________ By Eden Almasude June 2011 This thesis has been approved by The Honors Tutorial College and the Department of Biological Sciences __________________________ Dr. Erin R. Murphy Assistant Professor, Biomedical Sciences Thesis Advisor ___________________________ Dr. Soichi Tanda Associate Professor, Biological Sciences Honors Tutorial College, Director of Studies ___________________________ Dr. Jeremy Webster Dean, Honors Tutorial College 2 Table of Contents Acknowledgements 5 Abstract 6 List of Figures 8 List of Tables 9 Introduction 10 Shigella Cellular Pathogenesis 11 Small RNA (sRNA) Mediated Gene Regulation 13 The Bacterial sRNA SraB 15 Purpose of Study 17 Materials and Methods 18 Bacterial Strains and Growth 18 sraB Over-expression Plasmid 18 Real-Time PCR 18 Tissue Culture 20 Protein Isolation 20 Differential Gel Electrophoresis (DiGE) 21 Lysozyme Sensitivity Assay 22 Results 24 SraB Production over the Course of the Bacterial Growth Curve 24 Influence of Environmental Factors on the Production of SraB in S. dysenteriae 29 3 Impact of SraB Production on in vitro S. dysenteriae Virulence Analysis 33 Impact of SraB on Resistance of S. dysenteriae to Lysozyme 35 Differential Protein Expression in the presence of SraB Over-production 36 Discussion 41 Future Directions 44 References 46 4 Acknowledgements First and foremost, I must extend my thanks to Dr. Erin Murphy, my thesis advisor, for her teaching, guidance, and encouragement throughout this project. All the members of the Murphy Lab were also essential. Thanks to Jian Li for providing consultation regarding the Jiang et al. (2010) paper which was published in Chinese. I am grateful to the Honors Tutorial College, in particular Dean Jeremy Webster and Assistant Dean Jan Hodson, for providing me with continual support, funding, and opportunities. I was able to jump-start my research in the Murphy Lab due to a Summer Research Apprenticeship received through the Honors Tutorial College. I am also indebted to my director of studies, Dr. Soichi Tanda, for his mentorship and Drs. Lorie LaPierre and Donald Holzschu, who are responsible for initiating and nurturing my interest in gene regulation. The Provost’s Undergraduate Research Fund (PURF) provided funding for this project and I have been able to travel to conferences through the Honors Tutorial College Dean’s Discretionary Fund. Proteomic analysis was performed by the Ohio State University Campus Chemical Instrument Center. Finally, I would like to thank my partner, Stephen Pearson, for support and encouragement. 5 Abstract Shigella dysenteriae is a pathogenic bacterium that causes severe diarrheal disease in humans. Precise regulation of S. dysenteriae virulence-related gene expression is essential for the pathophysiology of infection within the human host. The contribution of small non-coding RNAs (sRNAs) in controlling Shigella species virulence-related gene expression has begun to emerge but nevertheless many sRNAs remain uncharacterized. This study aims to confirm the existence of the predicted S. dysenteriae sRNA SraB, to characterize the expression profile of sraB, to determine the role of SraB in controlling virulence-related gene expression, and to identify the proteins that are differentially produced as a result of sraB overexpression. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was used to determine whether the sRNA SraB is produced in S. dysenteriae. Semi-quantitative real-time PCR (qRT-PCR) was used to investigate the expression profile of sraB in both virulent and avirulent S. dysenteriae and to determine the effect of various environmental conditions on sraB expression. SraB is produced in S. dysenteriae and production varies between time points in the growth curve. sraB has a distinct expression profile in Escherichia coli as well as in virulent and avirulent S. dysenteriae. Although the presence of deoxycholic acid (DOC) and the absence of iron do not significantly influence sraB expression, sraB expression is significantly lower in bacteria cultured at 37°C compared to that measure in bacteria cultured at 30°C. SraB levels are also upregulated in the presence of lysozyme. 6 SraB over-production results in differential expression of 61 proteins. Many of the upregulated proteins are involved in the Krebs cycle and future studies should focus on determining the direct effects of SraB on S. dysenteriae metabolism. IpaA and IpaD, Shigella-specific proteins essential for invasion in the course of natural infection, were downregulated. This suggested that SraB inhibits S. dysenteriae virulence, a hypothesis which should be further investigated. The results of this study show that the role of SraB relates to virulence and survival under certain environmental conditions. Future research is aimed at determining additional environmental factors that affect SraB regulation, the direct mRNA targets of SraB, and the mechanism of action of SraB. 7 List of Figures Figure 1: Schematic of S. flexneri pathogenesis 13 Figure 2: RT-PCR confirmed sraB expression in S. dysenteriae 16 Figure 3: A) 2-Dimensional Gel Electrophoresis (DiGE) methodology (CCIC, 2011). B) Example 2-D gel using a wild-type S. dysenteriae whole protein sample. 22 Figure 4: qRT-PCR determined sraB expression across the bacterial growth curve in virulent S. dysenteriae. 25 Figure 5: sraB expression across the bacterial growth curve in virulent and avirulent S. dysenteriae. 26 Figure 6: sraB expression across the bacterial growth curve in virulent and avirulent S. dysenteriae and E. coli. 27 Figure 7: sraB expression in the presence and absence of deoxycholic acid (DOC). 30 Figure 8: sraB expression in the presence and absence of iron stress. 31 Figure 9: sraB expression at 30˚C and 37˚C. 32 Figure 10: sraB expression in the presence and absence of lysozyme. 33 Figure 11: A) Visual and B) quantitative plaquing ability of S. dysenteriae in the presence and absence of sraB overexpression. 35 Figure 12: S. dysenteriae resistance to lysozyme in the presence and absence of SraB over-production 36 Figure 13: 2-D gel with proteins from S. dysenteriae in the presence and absence of SraB over-production 37 8 List of Tables Table 1: Bacterial strains and plasmids. 18 Table 2: Average relative expression of sraB across strains as determined by qRT-PCR 28 Table 3: Differential protein expression in the presence and absence of SraB over-production. 39 9 Introduction Shigella dysenteriae is a pathogenic bacterium that causes severe diarrheal disease in humans. This acute intestinal infection, shigellosis, accounts for 5-15% of worldwide diarrheal episodes. There are over 165 million cases of shigellosis annually that result in over 1.1 million deaths out of 1.8 million total deaths from diarrheal disease. 99% of all diarrheal cases, including Shigella species infections, take place in economically poor countries (Schroeder and Hilbi, 2008). Poor quality drinking water, malnutrition, and lack of access to medical care are all contributing factors to the high incidence of diarrheal disease in these countries. At least 60% of shigellosis mortality is in children under 5, who are more susceptible to the dehydration and other complications that can result from diarrheal disease (Initiative for Vaccine Research, 2009). Among these complications are peritonitis, septicemia, and Hemolytic Uremic Syndrome (HUS) (Initiation for Vaccine Research, 2009). In recent decades shigellosis has been especially problematic in regions with political disturbances or natural disasters, including Latin America, sub-Saharan Africa, and South-East Asia. For example, in a single region of the present day Democratic Republic of the Congo 20,000 refugees of the 1994 Rwandan genocide died from an extensively-drug-resistant strain of S. dysenteriae (Initiative for Vaccine Research, 2009). Diarrheal disease, including shigellosis, is also linked to poverty. According to World Health Statistics, up to 30% of mortality in children under 5 is caused by diarrheal disease, and nearly all of these deaths occur in economically poor countries (World Health Organization, 2010). Water sanitation and malnutrition, 10 factors highly associated with diarrheal morbidity and mortality, are also strongly linked to poverty. Although diarrheal disease, including shigellosis, can be treated effectively with antibiotics and/or oral rehydration (Schroeder and Hilbi, 2008), economically poor countries have low densities of physicians, nurses, and midwives to provide the required treatment (World Health Organization, 2010). However, even when healthcare is accessible, antibiotic resistance of Shigella species is rapidly rising. Despite research into a Shigella vaccine, success of experimental vaccines has yet to be achieved in clinical trials (Chompook et al., 2006; Jennison and Verma, 2004). Several attempts have been made to create a vaccine against type 1 S. dysenteriae bacteria, which cause the most severe diarrheal cases. Multi-drug- resistant strains of Shigella species have also become increasingly frequent and concerning. In three studies at least 90% of Shigella isolates were found to be resistant to at least two antibiotics; in one study, 73-95% of isolates were resistant to four antibiotics (von Seidlein et al., 2006; Agtini et al., 2005; Chompook et al., 2006). In light of the emerging antibiotic resistance in Shigella species and continual difficulty in developing a successful Shigella vaccine, a greater knowledge of virulence-related gene regulation proves particularly urgent as these studies may lead to novel control strategies. Cellular Pathogenesis Shigellosis is characterized by symptoms of diarrhea containing blood and mucus, abdominal cramping, and fever (Schroeder and Hilbi, 2008). The infection is 11 transmitted via the fecal-oral route through the ingestion of food or water contaminated with Shigella bacteria. Shigellosis is highly infectious; as few as 10 bacteria can cause an active diarrheal case. Shigella spp. pathogenesis has been most thoroughly studied in S. flexneri, so this species will be used to demonstrate the mechanism of infection. S. flexneri crosses the intestinal epithelium via uptake by microfold (M) cells; M cells sample particles in the large intestine for delivery to the macrophages in the mucosal tissue for immune response (Schroeder and Hilbi, 2008; Jennison and Verma, 2004). The bacteria are then able to induce macrophage apoptosis, thus freeing Shigella organisms to the basolateral colonic epithelium. Via actin rearrangement the bacteria induce uptake into epithelial cells and lyse the vesicles into which they were taken up. In doing so, they are capable of replicating in epithelial cells. Finally, the bacterium spreads from the infected eukaryotic cell to neighboring cells by polymerizing a polar actin tail. These processes are shown in Figure 1 (adapted from Schroeder and Hilbi, 2008). As a result of Shigella spp. infection, tissue damage, uninhibited ion and fluid release, and electrolyte imbalance result (Schroeder and Hilbi, 2008). Shigella toxins are partly to blame for these consequences. Shigella enterotoxin (ShET) 1 and 2 activate cellular fluid secretion into the intestine while the cytotoxic Shiga toxin, produced by type 1 S. dysenteriae, can lead to colonic, renal, and central nervous system (CNS) lesions (Schroeder and Hilbi, 2008). 12 Figure 1: Schematic of S. flexneri pathogenesis. Bacteria are transcytosed through M cells where they find macrophages. S. flexneri induces macrophage apoptotsis, freeing bacteria into the basolateral side of the lumen. They invade epithelial cells via actin rearrangement and spread between cells in the epithelial layer. sRNA-Mediated Gene Regulation The genome of S. dysenteriae has been sequenced and several genes encoding putative regulatory small RNA molecules (sRNAs) have been identified. sRNAs can be identified by a variety of computational approaches, including machine learning (i.e. a bioinformatics tool using algorithms that allow a computer to ‘evolve’ from data sets to learn pattern recognition), base-composition statistics, comparative genomics, and searches for unknown transcripts of an appropriate size or with certain transcription signals (Hershberg et al., 2003; Pichon and Felden, 2008). Bacterial sRNAs are RNA molecules that do not typically code for a protein but instead 13 function to regulate the expression of specific target genes in the bacterium. The first bacterial regulatory RNA was discovered in 1981, when it was found that RNA I blocks the replication of a plasmid by base pairing with another RNA that needs to be cleaved in order to allow replication to proceed (Waters and Storz, 2009). Since then, the field has shown to be extremely promising and bacterial regulatory RNAs have been found to affect nearly every aspect of cell physiology including metabolism, survival, and virulence (Waters and Storz, 2009; Romby et al., 2006). In recent years, the identification and characterization of novel bacterial sRNAs has exploded, but the functions and/or mechanisms of action of many of these sRNAs have not yet been discovered (Wassarman, 2002). Bacterial sRNA molecules have been shown to function by a variety of mechanisms, many of which require a physical interaction with the RNA-binding protein Hfq (Zhang et al., 2003). For example, the Escherichia coli sRNA OxyS functions by binding to the ribosomal binding sequences of its target mRNA molecules, thus preventing translation (Altuvia et al., 1998). OxyS is also an example of an sRNA that requires Hfq for proper function (Wassarman, 2002). Another E. coli sRNA, CsrB, prevents the function of specific proteins through binding and sequestration (Babitzke and Romeo, 2007). By doing so, CsrB regulates carbon metabolism in the presence of specific growth or environmental conditions. sRNAs can also enhance expression, with one mechanism of activation being the anti- antisense model. E. coli RyhB works through this mechanism, using direct base- pairing to increase translation (Fröhlich and Vogel, 2009). 14 RyhB is one of the most well-characterized sRNAs in S. dysenteriae. RyhB acts to suppress virulence through negative regulation of virB, a transcriptional activator (Murphy and Payne, 2007). RyhB is differentially produced in the presence of iron. Iron-responsive regulation of RyhB production is achieved by Fur, a transcriptional repressor that acts globally in response to iron. Like many bacterial sRNAs, RyhB uses an Hfq-based interaction to regulation the expression of several of its mRNA targets. While it is known that sRNAs are essential for functional virulence mechanisms and that the expression of these sRNAs is often regulated by environmental conditions, this area is relatively uninvestigated (Romby et al., 2006). The Bacterial sRNA SraB SraB was first identified as a putative sRNA and its existence and expression was confirmed in Escherichia coli in 2001 using Northern blot analysis (Argaman et al.). SraB production was shown to be substantial only during stationary phase under normal growth conditions. Heat shock, cold shock, and growth in minimal media did not lead to the production of detectable sraB levels. SraB is also present and expressed in S. dysenteriae (A. Edon unpublished data, Figure 2). Preliminary experiments showed that SraB production may be influenced by certain environmental conditions and that SraB overproduction may have some effect on virulence (A. Edon unpublished data). Although those experiments were not repeated and no solid conclusions could be drawn at the time, they suggested that SraB may be a promising target to investigate further. The 15 functions of SraB and its mechanism of action remained unknown in both E. coli, the bacterium it was first identified in, and S. dysenteriae, the bacterium of interest for this study. Figure 2: RT-PCR was performed using RNA isolated from S. dysenteriae. Lane1: sraB product (170bps); lane 2: sraB negative control; lane 3: spf product; lane 4: spf négative control. (Spf was a predicted sRNA which does not exist in S. dysenteriae (A. Edon, unpublished data). A recent publication by Jiang et al. (2010) suggested that at least one of the roles of SraB in Salmonella enteridis is to promote survival, specifically resistance to lysozyme. Lysozyme is an enzyme possessing the ability to damage the bacterial cell wall leading to the death of the organism. It is present in large quantities in egg white, where S. enteridis is commonly found. Experiments demonstrated that S. enteridis lacking sraB survives poorly in the presence of lysozyme as compared to wild type S. enteridis. Jiang et al. (2010) concluded that sraB plays a major role in the ability of S. enteridis to survive in the presence of egg lysozyme. The sraB sequence is highly conserved among strains of E. coli and Shigella species with 99-100% identity. sraB is more dissimilar in Salmonella strains, with a similarity of 82% (Altschul et al., 1997). 16 Purpose of Study Although there is an appreciation in the literature of the importance of sRNAs in controlling bacterial gene expression, little is known about the role of sRNAs in regulating genes in S. dysenteriae or, more specifically, the role of SraB. The characterization of sRNAs, such as SraB, in S. dysenteriae will provide important insights into how the bacterium regulates the expression of genes required for survival and pathogenesis. Understanding how a pathogen survives and causes disease in the human host is a clear step towards developing therapies that will prevent or limit the survival and pathogenesis of the organism and thus lessen the morbidity and mortality associated with infection. This study aims to determine when SraB is produced and to elucidate the regulatory functions of SraB and the role of SraB in controlling the ability of S. dysenteriae to cause disease in the human host. 17 Materials and Methods Bacterial Strains and Growth: Bacterial strains and plasmids used are shown in Table 1. Bacterial colonies were grown on tryptic soy broth agar (TSBA) plates containing 0.01% (wt/vol) Congo Red at 37˚C. Table 1: Bacterial Strains and Plasmids Description Source E. coli Strains DH5α Life Technologies S. dysenteriae Strains O4576S1-G Spontaneous Strr mutant of S. Payne clinical strain O4576S1-GW O4576S1-G minus a portion S. Payne of the virulence plasmid Plasmids pQE2 Expression vector Qiagen pSraB sraB in pQE2 A. Edon & E. Murphy sRNA Over-expression Plasmid: sraB was cloned into a plasmid (pSraB) under control of an IPTG-inducible promoter. A similar plasmid (pQE2) lacking the sraB sequence was used as a negative control. Both plasmids contained an ampicillin- resistant cassette allowing for selection of bacteria containing the plasmid by the addition of ampicillin to the growth media. Real-Time PCR Quantitative Real-Time Polymerase Chain Reaction (PCR) was used to determine SraB levels across the bacterial growth curve and to determine the effects of 18 the presence of deoxycholic acid (DOC), iron availability, and environmental temperature on SraB production levels. For the growth curve analysis, optical density (OD) was measured hours 2 through 8 and 24 hours into the growth assay. At each time point 2mL of bacterial culture were removed and mixed with 500µL StayRNA (95% ethanol, 5% phenol at pH 4.5) prior to total RNA isolation. 0.1% DOC was used to determine differential SraB levels in the presence of DOC. To induce iron stress, 30µM EDDHA was used and iron stress was confirmed by real-time analysis of shuA, a gene whose expression is known to be affected by iron stress. To look at SraB levels in the presence of lysozyme, bacteria were grown in 3mL LB with 50µg lysozyme for 5 hours. Following growth under the conditions being investigated, total RNA was isolated using RNeasy MiniKit (Qiagen) as per product directions. Reverse transcription of RNA to cDNA was performed using iScript cDNA synthesis kit (BIO- RAD) using 150µg RNA as template in each reaction. Expression of sraB is normalized to the expression level of a constitutively expressed gene (rrsA) in each sample so that the results are not influenced by RNA quality, variation between sample dilutions, or global changes in transcriptional activity under the given environmental conditions. All real-time experiments were performed in biological triplicate. The sequences of the forward and reverse real-time primers for sraB cDNA were 5’ GGTTGATGTTCGTTATCAGCACTG 3’ and 5’AGCCTGTGGTTGCCTTTTGC 3’, respectively (Integrated DNA Technologies). A primer concentration of 500 nM and 19 an annealing temperature of 58.6º C were used. Reactions were performed in a CFX96 real-time PCR system (BIO-RAD). Tissue Culture Henle cells were cultured in six-well polystyrene plates in Gibco minimum essential medium (MEM) (BioExpress) supplemented with 10% fetal bovine serum (BioExpress), 10% tryptose phosphate broth, 2mM glutamine, and 1X non-essential amino acids. The cells were incubated at 37ºC in an atmosphere with 5% CO2 until a confluent monolayer was formed (approximately 48 hours). The methodology for the plaque assay was similar to that of Oaks et al. (1985). A single S. dysenteriae colony was used to inoculate 3mL LB broth containing 50µg/mL ampicillin, to be grown overnight at 30˚C. This culture was then diluted 1:100 in LB containing 200µM IPTG, 50µg/mL ampicillin, and 0.1% DOC. These conditions promote production of SraB from the pSraB plasmid (IPTG) and production of S. dysenteriae virulence factors (DOC). The bacteria were grown at 37˚C to an OD of 0.4-0.7, corresponding to mid-logarithmic phase of growth. 1x103 cells/well, diluted in LB, were added to each tissue culture well in addition to 2mL MEM, 50µg/mL ampicillin, and 200µM IPTG. After 1 hour of incubation at 37˚C in 5% CO2, and overlay was added consisting of 2mL MEM, 200µg/mL gentamicin, and 0.3% glucose. The plates were then incubated for 72 hours before being washed with water and stained with Wright-Giemsa stain (EMD Chemicals) 20 Protein Isolation A single colony of wild-type S. dysenteriae carrying either pQE2 or pSraB was picked and grown in 3mL LB with 50µg/mL ampicillin overnight at 30˚C. This culture was then diluted 1:100 into 25mL LB with 50µg/mL ampicillin and 200µM IPTG and grown 24 hours before pelleting at 4˚C. The 24 hour time point was selected for protein analysis because sraB expression in virulent S. dysenteriae is the highest at this time point, suggesting that its effects may be the greatest at that time. The cells were re-suspended in a wash buffer (10mM Tris at pH 8.0, 5mM MgAc), pelleted at 12,000xg for 4 minutes at 4˚C, and supernatant removed. This process was repeated three times. The pellet was then re-suspended in 1mL cell lysis buffer (7M Urea, 30mM Tris, 4% CHAPS; pH 8.5) and left on ice for 10 minutes before freezing at -80˚C. Over several days, the cells were removed from -80˚C, thawed at 37˚C, and re-frozen at -80˚C 6 times. Protein concentration was determined using a Bradford assay. Protein samples, and thus results, were obtained in biological quadruplicate. Differential Gel Electrophoresis (DiGE) DiGE protein analysis and identification was performed by the Mass Spectrometry & Proteomics Facility of Ohio State University’s Campus Chemical Instrument Center. Intended to avoid gel-to-gel variance, protein samples from each condition, in the presence and absence of SraB over-production, are labeled with different fluorescent dyes (Cy1 and Cy2). The samples are then mixed together in equal amounts and run using gel electrophoresis, with the end result being that relative expression can be measured on a single gel (Figure 3). 21 A B Figure 3: A) Up to 3 protein extracts can be mixed after labeling with different fluorescent dyes. They are then mixed in equal amounts for 2- dimensional gel electrophoresis. Differential expression is measured by dye- specific excitation. (CCIC, 2011). B) An example of a 2-D gel run using a wild-type S. dysenteriae whole protein preparation. 22 Lysozyme Sensitivity Assay The methodology to test S. dysenteriae survival in the presence of lysozyme is similar to that of Jiang et al. (2010). A 3mL culture of bacteria was grown at 37˚C for 3 hours with 25µg/mL ampicillin before 200µM IPTG was added. The culture was allowed to grow 21 more hours. The culture was centrifuged and the supernatant removed. The pellet was brought up in 3mL 10mM Tris buffer (pH 7.0) with 25µg lysozyme, then incubated in a 37˚C shaker. At 0, 3, 5, 8 and 24 hours after dilution OD was measured. Survival was determined by comparing the number of surviving bacteria at the given time point to time point ‘0.’ Results were obtained in biological triplicate. 23 Results SraB Production in the Course of the Bacterial Growth Curve The first step of my analysis was to investigate the relative expression of sraB at various points throughout the bacterial growth curve in virulent and avirulent S. dysenteriae as well as in E. coli. This is the first step in developing an expression profile of sraB and can provide clues as to its regulation and function(s). SraB production in virulent S. dysenteriae was shown to be substantial throughout the bacterial growth curve (Figure 4) although highest expression was during early death phase (24hrs after dilution) and lowest expression was at late stationary phase (8hrs after dilution) though these differences were not statistically significant. These results were unexpected as they differ from those previously obtained by Argaman et al. (2001) in E. coli, whose genomic content is highly similar to S. dysenteriae but which lacks the Shigella virulence plasmid (Yang et al., 2005). 24 100.00 2 Relative amount of SraB 10.00 1.5 OD 600 1.00 1 0.10 0.5 0.01 0 3 5 8 24 Time points (hrs) Figure 4: The bars show relative SraB levels at growth time points 3, 5, 8, and 24 hours are expressed relative to the expression at 3 hours. The secondary vertical axis shows growth of virulent S. dysenteriae measured in optical density (OD) at the given time point, represented by the line. Due to the differing results obtained previously in E. coli as compared to S. dysenteriae and the different techniques used, I next analyzed sraB expression in K12 E. coli cells, the same strain used by Argaman et al. (2001). The differences in SraB levels between E. coli and S. dysenteriae are only significant at the 24 hour time point (Figure 5). The northern blot results of Argaman et al. (2001) showed that SraB was only detectable in stationary phase and its levels were highest in late stationary phase but in my qRT-PCR results I saw tangible SraB production in all phases of bacterial growth. This observed difference may be explained by the fact that my studies use a 25 more sensitive technique to measure SraB levels as compared to that used by Argaman et al. (2001). Within E. coli, the differences in SraB levels between certain time points are statistically significant: between early logarithmic phase (3hrs) and death phase (24hrs) (P = 0.0136), between late logarithmic phase (5hrs) and death phase (P = 0.0001), and between stationary phase (8hrs) and death phase (P = 0.0001). These statistical differences are not highlighted in Figure 5. 100 2.5 Relative amount of SraB 2 10 1.5 E. coli * OD 600 1 * 1 virulent 0.1 * 0.5 0.01 0 3 5 8 24 Time points (hrs) Figure 5: Relative sraB expression levels in E. coli and virulent S. dysenteriae (-G) at growth time points 3, 5, 8, and 24 hours expressed relative to the expression at 3 hours. p < 0.05 (*) Given that the main difference between S. dysenteriae and E. coli is the presence of the Shigella-specific virulence plasmid, the significant difference between SraB levels in E. coli as compared to S. dysenteriae 24 hours after dilution suggests 26 that the S. dysenteriae virulence plasmid may be influencing sraB expression in some way. In order to determine whether this is the case or if another difference between the bacterial species is causing the differential expression of sraB I looked at SraB levels in an avirulent strain of S. dysenteriae. This strain of S. dysenteriae is identical to virulent S. dysenteriae except that a portion of the virulence plasmid is missing. A difference in SraB levels between virulent and avirulent S. dysenteriae would provide strong evidence that the S. dysenteriae virulence plasmid does affect sraB expression by an unknown mechanism. E. coli 100 2 virulent avirulent Relative amount of SraB 10 1.5 1 OD 600 1 1 2 0.5 0.1 1 2 0 0.01 -0.5 3 5 8 24 Time points (hrs) Figure 6: Relative sraB expression levels in E. coli, virulent S. dysenteriae, and avirulent S. dysenteriae at growth time points 3, 5, 8, and 24 hours expressed relative to the expression at 3 hours. Differences between bars with the same numbers are statistically significant (p< 0.05) 27 Table 2: Average relative expression of sraB determined by qRT-PCR Strain 3hr 5hr 8hr 24hr Virulent S. dysenteriae 0.52 ± 0.44 0.33 ± 0.31 0.088 ± 0.13 0.43 ± 0.20 Avirulent S. dysenteriae 0.70 ± 0.34 0.22 ± 0.052 0.045 ± 0.00055 0.12 ± 0.067 E. coli 0.54 ± 0.27 0.29 ± 0.027 0.26 ± 0.023 0.024 ± 0.007 P valuea P = 0.68 P = 0.067 P valueb P = 0.073 P = 0.026* P valuec P = 0.0011* P = 0.055 a P values are the results of non-paired Student’s t-test between virulent and avirulent S. dysenteriae expression levels. b P values are the results between virulent S. dysenteriae and E. coli. c P values are the results between avirulent S. dysenteriae and E. coli. The results from comparing sraB expression levels in E. coli as well as in both virulent and avirulent S. dysenteriae are intriguing (Figure 6). While I expected that sraB expression in avirulent S. dysenteriae would be similar to expression in E. coli because the genetic content is nearly identical, I found that this was not the case. Across all strains, sraB expression remains high and consistent at the 3 hour and 5 hour time points. At 8 hours after dilution, however, expression levels in avirulent S. dysenteriae are the most different from E. coli, results which contradicted my hypothesis, and this difference is statistically significant (0.045 ± 0.0006 and 0.26 ± 0.022 in avirulent S. dysenteriae and E. coli, respectively, P = 0.0011). At 24 hours 28 after dilution there is only a significant difference, as mentioned, between expression levels in E. coli as compared to virulent S. dysenteriae (0.024 ± 0.007 and 0.43 ± 0.20, respectively, P = 0.026). Between time points in avirulent S. dysenteriae, there was a statistically significant difference between SraB levels at stationary phase (8hrs) and death phase (24hrs) (P = 0.0449). Influence of Environmental Factors on the Production of SraB in S. dysenteriae I next examined SraB production in response to growth in four specific environmental conditions. Many bacteria regulate the expression of specific genes in response to environmental signals such as temperature and the presence of certain nutrients. In S. dysenteriae, it is known that the expression of many virulence- associated genes is highly regulated in response to several environmental conditions including, but not limited to, temperature, iron availability, and the presence of bile acids such as deoxycholic acid (DOC). The effects of each of these environmental conditions on the expression of virulence-associated genes are mediated via a variety of regulatory molecules including, in some cases, small RNAs (Murphy and Payne, 2007; Schroeder and Hilbi, 2008). DOC is a bile acid found in the intestine, where S. dysenteriae invades its host. Production of several virulence-related factors in S. dysenteriae is regulated in response to DOC. I found that sraB expression was slightly increased in the presence of DOC although the response was not statistically significant (Figure 7, 1.83 ± 0.62 29 and 1.19 ± 0.19 for plus and minus DOC, respectively, P = 0.16). 10 Relative amount of SraB plus DOC 1 minus DOC 0.1 Figure 7: Relative SraB expression levels in S. dysenteriae grown in either the presence or absence of DOC. Expression values are expressed relative to that measure in the minus DOC condition. The same basic analysis was used to compare the expression of sraB in the presence and absence of iron stress (Figure 8). Iron stress is known to affect the expression of bacterial sRNAs, including the S. dysenteriae sRNA RyhB. Although SraB levels may be slightly higher in the presence of iron, the difference is not significant (2.14 ± 0.34 and 1.525 ± 0.50 for iron rich and iron limited, respectively, P = 0.40). 30 10 Relative amount of SraB iron rich 1 iron limited 0.1 Figure 8: Relative SraB levels in S. dysenteriae grown in an iron rich or iron limited environment. Expression values are expressed relative to that measure in the iron limited condition. SraB levels were compared at 30˚C to that at 37˚C. 37˚C is the temperature of the human body, and thus is the temperature at which S. dysenteriae infects the human host. As a result, it is also the temperature at which Shigella-specific virulence factors are maximally expressed (Turner and Dorman, 2007). Real-time analysis showed that SraB levels are different between the two temperatures and that that difference is statistically significant (Figure 9). SraB levels are lower at 37°C as compared to 30°C (0.63 ± 0.02 and 0.97 ± 0.04, respectively, P = 0.00016). Because 37°C is the temperature at which virulence factors are maximally expressed (Turner and Dorman, 2007), this result shows that it is possible that SraB represses virulence in some way. This hypothesis required further experimental analysis to determine the effect of SraB on virulence, which is 31 explored in the following section. 10 Relative amount of SraB * 30˚C 1 37˚C 0.1 Figure 9: Relative sraB expression levels in S. dysenteriae grown at 30°C and 37°C. Expression values are expressed relative to that measure in the 30°C condition. p < 0.05 (*). The results of Jiang et al. (2010), the only researchers to elucidate possible function(s) of SraB, justify the experiments involving lysozyme in this study. While their analyses were conducted in S. enteridis, which encounters lysozyme in the environment of the egg. Protection against lysozyme is relevant for S. dysenteriae because lysozyme is present in mucus and encountered by S. dysenteriae in the human host. The results of Jiang et al. (2010) suggested that sraB expression levels may be up-regulated by the presence of lysozyme. This was found to be the case (Figure 10), and the up-regulation of SraB levels in the presence of lysozyme is statistically significant (0.7 ± 0.26 and 1.4 ± 0.34 for minus and plus lysozyme, respectively, P = 0.04). 32 10 Relative amount of SraB * minus lysozyme 1 plus lysozyme 0.1 Figure 10: Relative sraB expression levels in S. dysenteriae grown in the presence and absence of lysozyme. Expression values are relative to that measure in the minus lysozyme conditions. p < 0.05 (*). Impact of SraB Production on in vitro S. dysenteriae Virulence I used established in vitro tissue culture based experiments to examine the effect of SraB over-production on S. dysenteriae virulence (Oaks et al., 1985). Because S. dysenteriae is an intracellular obligate human pathogen, in vitro tissue culture analysis is the best way to determine the role of a given gene product on virulence. Using a plaque assay I measured the ability of S. dysenteriae to invade eukaryotic cells, grow within the eukaryotic cells, and spread from one eukaryotic cell to the next in both the presence and absence of SraB over-production. When the bacteria can successfully complete these three processes, they are able to kill eukaryotic cells, producing clearings (i.e. plaques) in a contiguous monolayer of 33 eukaryotic cells. Over-production of SraB was achieved using the expression plasmid already constructed (pSraB) (Edon, unpublished data). The control plasmid, lacking the sraB gene sequence, is denoted pQE. If the over-production of SraB negatively impacts any of the essential steps of pathogenesis listed above, then I would expect to see that bacteria over-producing SraB would not be able to form plaques as effectively as the bacteria not over-producing SraB (this strain provide a control for the experiment). S. dysenteriae virulence, as measured in vitro by established tissue culture assays, is not significantly affected by SraB production, although there are fewer plaques in the presence of SraB over-production (Figure 11). There was no significant difference in the number of plaques produced by bacteria over-producing SraB as compared to those with normal SraB levels (71.3 ± 18.7 versus 92.3 ± 25.9; P = 0.32). 34 A B 120 pQE 100 80 pQE 60 pSraB 40 pSraB 20 0 Figure 11: (A) Clearings in a stained monolayer of Henle cells can be clearly seen in the control (pQE) and the S. dysenteriae strain overexpressing SraB (pSraB). (B) The number of plaques were quantified for each strain and no significant difference was found. Impact of SraB on Resistance of S. dysenteriae to Lysozyme Although previous researchers found that SraB increased resistance of Salmonella enteridis to lysozyme at stationary and early death phases (Jiang et al., 2010), I looked at survival at all stages in the bacterial growth curve to obtain more complete data (Figure 12). At 3, 5, and 8 hours after initially encountering lysozyme, S. dysenteriae over-producing SraB have higher survival rates than the control. At 24 hours, however, this result is reversed and the bacteria over-producing SraB have lower survival in the presence of lysozyme. This experiment needs to be repeated for statistical analysis. 35 1.20 1.00 0.80 0hrs 3hrs OD 600 0.60 5hrs 0.40 8hrs 24hrs 0.20 0.00 pQE1 pSraB1 Figure 12: Representative experiments of S. dysenteriae survival in the presence of lysozyme and either the presence or absence of SraB over-production. Survival at 3, 5, 8, and 24 hours is expressed as percent survival as compared to the start, 0 hrs. Differential Protein Expression in the Presence of SraB Over-production Proteomic analysis allows the identification of specific proteins whose expression is up- or down-regulated by SraB over-production. By comparing the total protein levels of bacteria over-producing SraB to a control, differential expression is seen and these proteins can be identified. The determination of differential protein expression is key in the characterization of SraB in S. dysenteriae as it will identify the specific targets of SraB, both direct and indirect. The differentially produced proteins will provide insight into the functions of SraB and its role in S. dysenteriae physiology. 36 2-Dimensional Gel Electrophoresis (DiGE) analysis showed that 61 proteins were differentially expressed in the presence of SraB over-production as compared to physiological SraB levels. Both significant up- and down-regulation were seen, with a P value of less than 0.05 (Figure 13, Table 3). Many of the upregulated proteins are involved in the tricarboxylic acid cycle, or Krebs cycle. Two outer membrane proteins are also upregulated and may help to account for lysozyme resistance. Of particular interest are invasion-related proteins, among them IpaA and IpaD, both of which are downregulated. This suggests that SraB over-production decreases the ability of S. dysenteriae to invade human epithelial cells and cause an active infection. Figure 13: 2D gel with total protein samples from S. dysenteriae in the presence and absence of SraB over- production. Protein spots showing significantly differential expression are circled in red and were removed for identification. 37 Table 3: Differential protein expression in the presence and absence of SraB over-production. The average ratio is the expression value of the protein in the presence of SraB over-production as compared to the absence of SraB over- production. The T-test shows statistical significance. Upregulated Proteins Av. Accession T-test Ratio number Protein bifunctional aconitate hydratase 2/2- 0.02 1.91 gi|16128111 methylisocitrate dehydratase 0.0014 2.26 gi|82775995 dihydrolipoamide succinyltransferase 0.0021 3.07 gi|300918382 succinate dehydrogenase, flavoprotein subunit 0.044 1.76 gi|82775552 serine endoprotease 0.0046 2.73 gi|16129570 fumarate hydratase (fumarase A), aerobic Class I fumarase A = fumarate hydratase class I; aerobic 0.016 1.68 gi|15802026 isozyme 0.038 1.61 gi|82778780 phosphoenolpyruvate carboxykinase 0.0095 1.45 gi|15799800 dihydrolipoamide dehydrogenase 0.042 1.19 gi|309784443 ATP synthase F1, beta subunit 0.0055 2.38 gi|82775995 dihydrolipoamide succinyltransferase 38 Upregulated Proteins Av. Accession T-test Ratio Number Protein 0.0079 1.48 gi|15803300 phosphopyruvate hydratase 0.024 1.89 gi|15800432 succinyl-CoA synthetase subunit beta 0.0079 2.49 gi|50513683 Chain A, Fatty Acid Transporter Fadl 0.0068 1.35 gi|82779098 L-lactate dehydrogenase glutamine ABC transporter, glutamine-binding periplasmic 0.0044 1.81 gi|194433178 protein 0.044 1.93 gi|226907 malate dehydrogenase 0.015 1.69 gi|82778271 L-asparaginase II 0.0095 2.94 gi|82775997 succinyl-CoA synthetase subunit alpha histidine ABC transporter, periplasmic histidine-binding 0.042 1.45 gi|194436225 protein 0.0052 3.07 gi|82775992 succinate dehydrogenase iron-sulfur subunit 0.0092 2.08 gi|82777721 lysine-, arginine-, ornithine-binding periplasmic protein 0.0044 1.97 gi|300918382 succinate dehydrogenase, flavoprotein subunit 0.038 1.87 gi|15800428 succinate dehydrogenase iron-sulfur subunit glutamine ABC transporter, periplasmic glutamine-binding 0.021 1.76 gi|261341396 protein 0.024 1.39 gi|146983 outer membrane protein II 0.014 1.51 gi|157159269 outer membrane protein A 39 Downregulated Proteins Av. Accession T-test Ratio Number Protein 0.05 -1.61 gi|1421648 Chain A, Chaperonin Groel 0.04 -1.22 gi|30063872 uracil phosphoribosyltransferase 0.044 -1.17 gi|2914323 Chain A, Enoyl Reductase 0.037 -1.25 gi|82779536 aromatic amino acid aminotransferase 0.028 -1.49 gi|82779098 L-lactate dehydrogenase 0.0044 -1.93 gi|82777069 6-phosphofructokinase 2 0.039 -1.3 gi|15800465 aldose 1-epimerase 0.014 -1.6 gi|145263 fumarate reductase flavoprotein subunit Major phosphate-irrepressible acid phosphatase; 0.0065 -1.64 gi|130122 Short=HPAP; Flags: Precursor 0.05 -2.41 gi|82524569 IpaA 0.0095 -2.23 gi|82524570 IpaD 0.044 -1.39 gi|13449093 invasion protein 40 Discussion Expression analysis (qRT-PCR) showed that sraB expression levels are uniquely regulated between virulent and avirulent S. dysenteriae as well as in E. coli at late stationary and early death phases. Although the mechanisms of sraB transcriptional regulation remain primarily unknown, this result suggests that the factors encoded on the S. dysenteriae virulence plasmid could play a role in the regulation of sraB expression levels. sraB expression among other enteric pathogens must be further investigated to determine whether unique regulation is present; sraB expression levels across the bacterial growth curve may be species- and/or strain- specific. Differential SraB production may reflect the fact that different pathogens, even those that are closely related, cause unique infections and thus experience unique environmental stresses throughout the course of infection within the human host. Further expression analysis (qRT-PCR) revealed that sraB expression levels are not significantly affected by the presence of DOC or iron stress during the late logarithmic/early stationary phase of bacterial growth. Temperature does significantly affect sraB expression; growing virulent S. dysenteriae at 30˚C up-regulates the production of SraB as compared to that at 37˚C. Because 37˚C is the temperature of the human host and the temperature at which virulence factors are maximally expressed, this result suggested that SraB may play a role in survival and/or virulence in the environmental conditions of the human host. 41 The observation that SraB production is up-regulated in the presence of lysozyme, which exists in mucus and is encountered by S. dysenteriae during the course of natural infection, provided further evidence that at least one function of SraB relates to survival within the host. Because lysozyme affects the bacterial cell wall, this result also suggests that the mRNA targets of SraB may relate to the cell wall or cell membranes. Together, temperature results suggest that generally, SraB levels are downregulated in the human body while SraB increase when S. dysenteriae reach the gut and are exposed to lysozyme. This suggests that SraB levels are tightly regulated and that exposure to lysozyme is an important factor signaling S. dysenteriae to upregulate SraB. Initial experiments showed that SraB over-production may confer resistance to lysozyme up to 8 hours after exposure. Although these experiments must be repeated for statistical analysis, the hypothesis that SraB increases resistance to lysozyme is supported by the upregulation of outer membrane proteins under SraB over- production. Plaque assays demonstrated that S. dysenteriae overexpressing sraB do not show significantly differential plaquing ability as compared to a control strain. It is important to note that not all aspects of virulence can be measured via a plaque assay, although it is the best means of assessing virulence in S. dysenteriae. A plaque assay result would only be affected by a differential ability to enter, replicate within, and spread between Henle cells, all processes essential for virulence in the human host. It is important to note that there are other aspects of S. dysenteriae pathophysiology that 42 cannot be assessed via a plaque assay, e.g. bacterial survival under physiological conditions. Differential protein production between the presence and absence of SraB over-production shows that SraB does have an active role in regulating, directly or indirectly, the expression of specific proteins. Proteomic analysis showed that SraB over-production leads to the downregulation of well-characterized proteins, e.g. IpaA and IpaD, which are known to be essential for invasion and thus virulence. Looking at Figure 11 we can see that although there is not a significant difference in plaquing ability, there are fewer plaques formed by S. dysenteriae over-producing SraB. It is possible that the lack of statistical significance between the number of plaques is due to the large error bars, or standard deviations. The downregulation of IpaA and IpaD by SraB is consistent with the result that sraB expression is downregulated at 37˚C. At 37˚C virulence factors, including IpaA and IpaD, are upregulated (Venkatesan et al., 1988). SraB, which downregulates IpaA and IpaD, is downregulated at that temperature, potentially to avoid interference with virulence. The upregulation of many metabolism-related proteins under SraB over- production provides strong evidence that the role of SraB is key to proper metabolic processes. It is possible that the role of SraB in metabolism may account for the differential expression levels of sraB between virulent and avirulent S. dysenteriae; an avirulent strain of S. dysenteriae has been found to have an altered ability to metabolize succinate, fumarate, and malate using the Krebs cycle (Kim and Corwin, 1973). Virulent S. dysenteriae were able to metabolize succinate, fumarate, and 43 malate faster than avirulent bacteria. This correlates well with my finding that SraB levels are higher in virulent S. dysenteriae. If metabolic enzymes are upregulated by SraB and SraB levels are higher in virulent S. dysenteriae, then it is logical that virulent bacteria are more able and efficient at metabolism. Future Directions In order to further investigate differential SraB levels across bacterial species and strains, an expression profile for SraB should be developed for other enteric pathogens including Salmonella spp. and enteropathogenic E. coli. The mRNA targets of SraB should be investigated in different bacterial species in order to identify the specific role of SraB in these species, as it may be different from the role played in virulent S. dysenteriae. Although some environmental factors that influence SraB levels have been identified, the effects of acid and fever temperature have yet to be determined. S. dysenteriae must encounter the stomach during its stay in the human host and the acidic environment there provides a strong environmental stress which the bacteria must survive. The upregulation of a glutamine transporter provides evidence that SraB may have a role in acid resistance because one system of acid resistance in enteropathogens is glutamine-dependent (Castanie-Cornet et al., 1999). Because temperature-dependent regulation of SraB production is seen, SraB levels should also be analyzed at 40˚C, corresponding to human fever temperature. Shigella infections cause fever and this is another environmental stress that S. dysenteriae bacteria must 44 encounter. Growth of a bacterial culture in minimal media provides another stress which may increase the levels of an sRNA, such as SraB, which appears to affect survival. Based on the large number of metabolic enzymes which were differentially expressed in the presence of SraB over-production, growth in minimal media may very likely increase SraB production levels. Based on the downregulation of invasion proteins under SraB over-production, invasion assays should be done to confirm that SraB over-production inhibits invasion. If this is the case, SraB plays an important role in regulating virulence in S. dysenteriae. Understanding the specific mechanisms by which SraB accomplishes this may be essential for an complete understanding of S. dysenteriae virulence. Finally, now that the proteins that are differentially expressed under SraB over-production have been identified, the direct targets of SraB must be identified. All of the proteins found to be differentially expressed under SraB over-production must be investigated further. Direct targets can be predicted by looking at complementarity of SraB to the mRNA transcript of a given protein. 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