Vibrio salmonicida causes cold-water vibriosis in salmon populations around the world and causes financial damage to fisheries designed to farm these salmon. Very little is known about the physiology of how V. salmonicida causes disease and measures to contain vibriosis are restricted to either vaccinating individual fish against disease or administering antibiotics when an outbreak is detected. These procedures are costly and increase the risk for selection of antibiotic-resistant V. salmonicida strains. A recent reoccurrence of outbreaks in Norwegian fisheries provided incentive to better understand the virulence mechanisms of V. salmonicida. In this thesis, a proteomic approach was used to identify proteins that were differentially expressed when cells were grown in vitro under simulated virulence conditions (i.e. 5˚C and in the presence of exogenously supplied autoinducer 3-oxo-hexanoyl-homoserine lactone). Some examples of proteins with significantly altered expression that stood out at as homologs of potential virulence factors were: an exported serine protease DegQ, a multi-drug transporter HlyD, and an outer membrane protein OmpU. The proteomic approach allowed us to identify large numbers of proteins that are expressed by V. salmonicida, facilitating hypothesis-driven research in order to support possible roles for some of these proteins in virulence

A contributing factor in recurrent Listeria monocytogenes (L. monocytogenes) food contamination is that this bacterium produces biofilms on surfaces to persist in food-processing environments. Quorum sensing (QS) is a cell-to-cell communication system utilized by bacteria within biofilms to collaborate and adapt to environmental stresses. However, the details of how the QS-dependent network contributes to biofilm development of L. monocytogenes have yet to be well understood. By comparing the transfer rates of planktonic and biofilm (sessile) L. monocytogenes from stainless steel blades to bologna slices, we found that sessile bacteria had reduced transferability onto a single slice but caused the increase in the number of contaminated slices. This suggests that physiological adaptions derived during biofilm development affect bacterial dissemination. Given the contribution of proteins and environmental temperatures to the extracellular polymeric substances (EPS) synthesis and biofilm integrity, we evaluated the exoproteomes of biofilms formed at 25 and 37°C using 2D-gel electrophoresis and LC-MS/MS. We found exoproteases Lmo0186, Cwh, and Spl exclusively in biofilms formed at 25°C and their greater expression in the gene level at 25°C. By using the zymography and crystal-violet-staining assay with a protease inhibitor, we observed a greater proteolytic activity at lower temperatures and showed that the attenuated proteolytic activity of proteases is positively correlated with increased biofilmorming ability at 25°C. Considering the transcriptional role of QS systems during biofilm development, we investigated how the accessory gene regulator (Agr)-based and metabolite S-Adenosylmethionine (SAM)-involved QS systems modulate nutrient availability and EPS synthesis. The results revealed that the SAM signal interacts with the Agr QS at the transcriptional level during biofilm development, whereas SAM and Agr QS regulate distinct EPS synthesis pathways. Additionally, this interaction is dependent on bacterial life modes (planktonic and sessile). Overall, we conclude that L. monocytogenes manipulates the synthesis of EPS with the coregulation of metabolism and QS for biofilm formation and the production of exoproteases for biofilm dispersion. These precise regulations on EPS enable L. monocytogenes to prolong its survival and promote its dissemination in environments.

No description available.

No description available.

<p>Virulence in <em>Staphylococcus aureus </em>is mediated by the <em>accessory gene regulator </em>(agr) quorum sensing system. This regulatory system is activated by a secreted thiolactone peptide termed autoinducing peptide (AIP) and its receptor histidine kinase, AgrC. Interaction of extracellular AIP with a cognate AgrC receptor generates an intracellular signal that is transduced by conformational changes and phosphorylation events in a two-component sensor histidine kinase system. At the heart of the <em>agr</em> quorum-sensing cascade lies the two-component histidine kinase, AgrC, and the response regulator protein, AgrA. Interaction of AgrC and AgrA, and the resulting phosphotransfer event results in expression from the divergent promoters P2 and P3, inducing expression of the master quorum-sensing regulator RNAIII and upregulating the <em>agr</em> operon respectively. Signal transduction systems function as intracellular information-processing pathways that link sensation of external stimuli to specific adaptive processes. In <em>S. aureus</em> , these include the up-regulation of virulence factors and hemolysis production, biofilm formation, and colonization-based regulation of surface proteins and adhesion factors. As such, the interactions of these systems have become key targets in the design of small inhibitor compounds.</p> <p>Through the creation of a protein truncation series, we proposed the development of a small protein for the inhibition of key protein-protein interactions involved in <em>S. aureus</em> <em>agr</em> two-component signaling. Herein, we demonstrate the efficacy of these protein truncations as dominant negative inhibitors of AgrC:AgrA interactions, likely acting as a dominant phosphoacceptor in place of endogenous AgrA. We provide evidence of this function through <em>in vitro </em>hemolysis assays and phosphate-detection based gel electrophoresis.</p> / Master of Science (MSc)

Quorum sensing in Gram-negative bacteria is best understood in the bioluminescent marine microorganism, <i>Vibrio fischeri</i>. In <i>V. fischeri</i>, the luminescence or <i>lux</i> genes are regulated in a cell density-dependent manner by the activator LuxR in the presence of an acylated homoserine lactone autoinducer molecule (3-oxo-hexanoyl homoserine lactone). LuxR, which binds to the <i>lux</i> operon promoter at position -42.5, is thought to function as an ambidextrous activator making multiple contacts with RNA polymerase (RNAP). The specific role of the <font face = "symbol">a</font>CTD of RNAP in LuxR-dependent transcriptional activation of the <i>lux</i> operon promoter has been investigated. The effect of seventy alanine substitution variants of the <font face = "symbol">a</font> subunit was determined <i>in vivo</i> by measuring the rate of transcription of the <i>lux</i> operon via luciferase assays in recombinant <i>Escherichia coli</i>. The mutant RNAPs from strains exhibiting at least two fold increased or decreased activity in comparison to the wild-type were further examined by <i>in vitro</i> assays. Since full-length LuxR has not been purified to date, an autoinducer-independent N-terminal truncated form of LuxR, LuxR<font face = "symbol">D</font>N, was used for <i>in vitro</i> studies. Single-round transcription assays were performed using reconstituted mutant RNAPs in the presence of LuxR<font face = "symbol">D</font>N, and fourteen residues in the <font face = "symbol">a</font>CTD were identified as having negative effects on the rate of transcription from the <i>lux</i> operon promoter. Five of these fourteen residues were also involved in the mechanism of both LuxR and LuxR<font face = "symbol">D</font>N-dependent activation <i>in vivo</i> and were chosen for further analysis by DNA mobility shift assays. Results from these assays indicate that while the wild-type <font face = "symbol">a</font>CTD is capable of interacting with the <i>lux</i> DNA fragment tested, all five of the variant forms of the <font face = "symbol">a</font>CTD tested appear to be deficient in their ability to recognize and bind the DNA. These findings suggest that <font face = "symbol">a</font>CTD-DNA interactions may play a role in LuxR-dependent transcriptional activation of the <i>lux</i> operon during quorum sensing. / Master of Science

Quorum sensing is utilized by many different proteobacteria, including the two studied for this dissertation work, Vibrio parahaemolyticus and Pantoea stewartii subsp. stewartii. V. parahaemolyticus causes acute gastroenteritis in people who eat contaminated raw or undercooked shellfish. It is found in warmer marine waters and in rare cases, causes systemic infections when bacteria enter the body through open wounds. P. stewartii, on the other hand, is a phytopathogen that causes Stewart's wilt in maize. It is found in soil or the mid-gut of the corn flea beetle, its insect vector. Both V. parahaemolyticus and P. stewartii utilize quorum sensing to control their pathogenicity. Quorum sensing enables coordinate gene expression across a bacterial population. The V. parahaemolyticus quorum-sensing system utilizes the master regulator OpaR, which is homologous to the V. harveyii LuxRVh and the P. stewartii system contains EsaR which is homologous to the V. fischeri LuxRVf regulator. While the two systems differ in the molecular details of their mechanistic control, they are both forms of cell density dependent regulation that are either directly or indirectly controlled by small signaling molecules. Three different signaling molecules are found in V. parahaemolyticus, and only one signal is used in P. stewartii. The focus of this dissertation has been on understanding the downstream targets of OpaR and EsaR in their respective quorum-sensing systems. Prior to this work, it was known that when OpaR is not present or is nonfunctional V. parahaemolyticus changes from an opaque to a translucent colony morphology phenotype and the cells also become swarm proficient and more pathogenic. The complete genome of the V. parahaemolyticus BB22OP strain was assembled and annotated (Chapter 2). RNA-Seq was then used to analyze the transcriptomes of OpaR-active and OpaR-deficient strains of V. parahaemolyticus and identify genes that were regulated via quorum sensing (Chapter 3). Similarly, P. stewartii was also analyzed using RNA-Seq to identify genes controlled by EsaR in the transcriptome that had not been detected through prior proteomic studies. The initial RNA-Seq work confirmed the control of some previously identified direct targets of EsaR and newly identified ten other genes also directly controlled by EsaR (Chapter 4). Two direct targets of EsaR, rcsA and lrhA, became the focus of additional studies to further define the hierarchy of gene control downstream of the quorum-sensing regulator EsaR. RcsA controls capsule production, while LrhA controls motility and adhesion in P. stewartii. The regulons of rcsA and lrhA were defined by RNA-Seq, which also revealed multi-level control of rcsA gene expression (Chapter 5). Tight coordinated and temporal control of virulence factors is important for successful disease progression by pathogens. This dissertation work aims to enable a better understanding of the quorum-sensing hierarchy of genetic control in V. parahaemolyticus and P. stewartii. / Ph. D.

Pantoea stewartii subsp. stewartii is a bacterium that causes Stewart’s wilt disease in corn plants. The bacteria are transmitted to the plants via an insect vector, the corn flea beetle Chaetocnema pulicaria. Once in the plant, the bacteria migrate to the xylem and grow to high cell densities, forming a biofilm by secreting excess capsular exopolysaccharide, which blocks water transport and causes wilting. The timing of virulence factor synthesis is regulated by the cell-density dependent quorum sensing (QS) system. Such temporal regulation is crucial in establishing infection and is orchestrated by the QS-dependent transcriptional regulator EsaR. EsaR represses expression of capsular exopolysaccharide at low cell densities. At high cell densities, an acylated homoserine lactone (AHL) molecule produced during growth by the cognate AHL-synthase EsaI accumulates. The AHL binds to and inactivates EsaR, causing derepression of capsule production. EsaR is a member of the LuxR family of QS-dependent transcriptional factors. Most LuxR homologs are unstable and/or insoluble in the absence of AHL which has hindered structural studies. Chapter Two describes the changes in the structure of EsaR due to binding of AHL ligand as determined through biochemical methods. EsaR was found to be stable and retain its multimeric state in the absence or presence of AHL, but intra- and inter-domain changes occurred that affect its DNA-binding capacity. Apart from repressing expression of capsule at low cell-densities, EsaR represses its own expression and activates production of a small RNA, EsaS, with unknown function. In Chapter Three a proteomic approach was used to identify an additional 30 QS-controlled proteins. Genes encoding three of these proteins are directly regulated by EsaR and the EsaR binding sites in the respective promoters were defined. In Chapter Four, a high-throughput RNA-Seq method identified even more genes in the QS regulon that the proteomic approach overlooked. RNA-Seq analysis of rRNA-depleted RNA from two strains of P. stewartii was used as a screen to help identify 11 promoters, subsequently shown to be directly regulated by EsaR in vitro. Most of the genes controlled by QS grouped into three major physiological responses, capsule & cell wall production, surface motility & adhesion and stress response. In Chapter Five, the role of two QS regulated genes, dkgA (encoding 2, 5-diketo-D-gluconate) and lrhA (encoding a repressor of chemotaxis, adhesion and motility), in plant virulence were examined. These studies have better characterized the QS regulator EsaR and its interaction with the AHL ligand, and shown that QS has a more global response in P. stewartii than previously recognized. Further characterization of the genes identified in this study could facilitate identification of factors crucial in plant pathogenesis or insect-vector symbiosis and aid in the development of molecular-based approaches for possible disease intervention. / Ph. D.

The mechanism of gene regulation used by Gram-negative bacteria during quorum sensing is well understood in the bioluminescent marine bacterium Vibrio fischeri. The cell-density dependent activation of the luminescence (lux) genes of V. fischeri relies on the formation of a complex between the autoinducer molecule, N-(3-oxohexanoyl) homoserine lactone, and the autoinducer-dependent transcriptional activator LuxR. LuxR, a 250 amino acid polypeptide, binds to a site known as the lux box centered at position -42.5 relative to the luxI transcriptional start site. During transcriptional activation of the lux operon, LuxR is thought to function as an ambidextrous activator capable of making multiple contacts with RNA polymerase (RNAP). The specific role of region 4 of the Escherichia coli sigma 70 subunit of RNAP in LuxR-dependent transcriptional activation of the luxI promoter has been investigated. Rich in basic amino acids, this conserved portion of sigma 70 is likely to be surface-exposed and available to interact with transcription factors bound near the -35 element. The effect of 16 single and 2 triple alanine substitution variants of sigma 70 between amino acid residues 590 and 613, was determined in vivo by measuring the rate of transcription from a luxI-lacZ translational fusion via b-galactosidase assays in recombinant E. coli. In vitro work was performed with LuxRDN, the autoinducer-independent C-terminal domain (amino acids 157 to 250) of LuxR because purified, full length LuxR is unavailable. Single-round transcription assays were performed in the presence of LuxRDN and 19 variant RNAPs, one of which contained a C-terminally truncated sigma 70 subunit devoid of region 4. Results indicate that region 4 is essential for LuxRDN-dependent luxI transcription with two specific amino acid residues, E591 and K597, having negative effects on the rate of LuxRDN-dependent luxI transcription in vivo and in vitro. None of the residues tested were identified as having any effect on LuxR-dependent luxI transcription in vivo. These findings suggest that region 4.2 is most likely to be in close proximity to LuxR when bound to the luxI promoter. However, unlike the situation found for other ambidextrous activators, no single residue within region 4.2 of sigma 70 may be critical by itself for LuxR-dependent during transcriptional activation. / Master of Science

Yes / N-acylhomoserine lactone (AHL)-based quorum sensing (QS) is important for the regulation of proteobacterial virulence determinants. Thus, the inhibition of AHL synthases offers non-antibiotics-based therapeutic potentials against QS-mediated bacterial infections. In this work, functional AHL synthases of Pseudomonas aeruginosa LasI and RhlI were heterologously expressed in an AHL-negative Escherichia coli followed by assessments on their AHLs production using AHL biosensors and high resolution liquid chromatography–mass spectrometry (LCMS). These AHL-producing E. coli served as tools for screening AHL synthase inhibitors. Based on a campaign of screening synthetic molecules and natural products using our approach, three strongest inhibitors namely are salicylic acid, tannic acid and trans-cinnamaldehyde have been identified. LCMS analysis further confirmed tannic acid and trans-cinnemaldehyde efficiently inhibited AHL production by RhlI. We further demonstrated the application of trans-cinnemaldehyde inhibiting Rhl QS system regulated pyocyanin production in P. aeruginosa up to 42.06%. Molecular docking analysis suggested that trans-cinnemaldehyde binds to the LasI and EsaI with known structures mainly interacting with their substrate binding sites. Our data suggested a new class of QS-inhibiting agents from natural products targeting AHL synthase and provided a potential approach for facilitating the discovery of anti-QS signal synthesis as basis of novel anti-infective approach. / University of Malaya High Impact Research (HIR) Grant (UM-MOHE HIR Grant UM.C/625/1/HIR/MOHE/CHAN/14/1, no. H-50001-A000027) given to K.G.C. and National Natural Science Foundation of China (no. 81260481) given to H.W.