Successful bacterial pathogens manipulate crucial intracellular host processes as a virulence strategy. One particular potent mechanism utilized by bacterial phytopathogens is to inject virulence factors (effectors) directly into the host cell. While many effectors have been identified and shown to suppress plant immune responses, very few have well-characterized enzymatic activities or host targets. To overcome the challenges of functional analysis of effectors, I designed two heterologous screens to characterize effector proteins of the bacterial phytopathogen Pseudomonas syringae. Specifically, my objective was to identify those P. syringae effectors that target evolutionarily conserved host proteins or processes and to subsequently elucidate the molecular mechanisms of these effectors. The first heterologous screen that I performed was to utilize tandem-affinity-purification (TAP)-tagged effectors in human cells to identify potential interacting host proteins. The second heterologous screen iii utilized a high-throughput genomics approach in yeast, known as the pathogenic genetic array (PGA), to characterize P. syringae effectors. Using the first heterologous approach, I have identified HopZ1a as the first bacterial phytopathogen effector that binds tubulin. I have shown that HopZ1a is an acetyltransferase activated by the eukaryotic co-factor, phytic acid. In vitro, activated HopZ1a acetylates itself and tubulin. In Arabidopsis thaliana, activated HopZ1a causes microtubule destruction, disrupts the secretory pathway and suppresses cell wall-mediated defense. The acetyltransferase activity of HopZ1a is dependent on the conserved catalytic cysteine residue (C216) and a conserved lysine residue (K289). Using the second heterologous screen in yeast, I have shown that HopZ1a may target the mitogen-activated protein kinase (MAPK) signaling cascades. Together, my work has identified novel eukaryotic targets and elucidated the virulence functions of HopZ1a.

The pathogenicity of plant pathogen Pseudomonas syringae depends on the type III secretion system which translocates effector proteins into host cells. In response, plants have evolved resistance proteins to detect presence of specific effectors and activate defense responses. The constant host surveillance imposes a strong selective pressure on effector proteins to evolve rapidly in order to evade detection. The P. syringae HopZ1 effector has evolved into three allelic forms as a result of diversifying selection. In this thesis, I aimed to investigate how sequence divergence contributes to the distinct allelic specificities of HopZ1. Mutational analysis of HopZ1a identified three amino acid residues that were potentially involved in dampening host defense responses, and two HopZ1a mutants partially lost the ability to trigger defense responses yet did not lose their virulence functions. These results suggested that distinct host targets could be involved in the defense-eliciting activity and virulence function of HopZ1a.

The pathogenicity of plant pathogen Pseudomonas syringae depends on the type III secretion system which translocates effector proteins into host cells. In response, plants have evolved resistance proteins to detect presence of specific effectors and activate defense responses. The constant host surveillance imposes a strong selective pressure on effector proteins to evolve rapidly in order to evade detection. The P. syringae HopZ1 effector has evolved into three allelic forms as a result of diversifying selection. In this thesis, I aimed to investigate how sequence divergence contributes to the distinct allelic specificities of HopZ1. Mutational analysis of HopZ1a identified three amino acid residues that were potentially involved in dampening host defense responses, and two HopZ1a mutants partially lost the ability to trigger defense responses yet did not lose their virulence functions. These results suggested that distinct host targets could be involved in the defense-eliciting activity and virulence function of HopZ1a.

Enteropathogenic Escherichia coli (EPEC) is an enteric pathogen that causes potentially fatal infantile diarrhea. A type III secretion system is employed by EPEC to inject bacterial effector proteins directly into host intestinal epithelial cells. The multivalent chaperone, CesT, interacts with nine effectors and is a significant contributor to EPEC pathogenesis. A putative transcriptional promoter region was identified directly upstream of cesT. In silico analyses identified conserved elements that suggest the cesT promoter is recognized by ?70. Using transcriptional fusions to lux reporter genes I showed that the cesT promoter region is active under conditions known to induce virulence-gene expression. I conclude that the cesT promoter is active early during an in vitro assay, and regulated by different mechanisms than those affecting the Ptir operon promoter.

Shigellae are pathogenic bacteria that cause the disease shigellosis. Two methods for studying secreted effectors encoded by this pathogen’s virulence plasmid are described. First, protein microarrays were used to identify substrates of an E3 ubiquitin ligase called IpaH7.8. Second, a deletion collection containing mutants for every gene on the virulence plasmid was used in two screens: one to identify mutants that elicit atypical levels of Interleukin-8 (IL-8) from U937 cells, and one to identify mutants that bind the dye Congo red abnormally. Although protein microarrays were an ineffective tool, the deletion collection proved valuable. Most mutants were less effective at sequestering Congo red than wild-type S. flexneri, although this ability was enhanced in several mutants. Four mutants, ?ospB, ?orf186, ?mxiH and ?mxiK, elicited higher levels of IL-8 from U937 cells than wild type S. flexneri. These results validate the use of the deletion collection as a tool for studying bacterial pathogenesis.

Successful pathogens like Pseudomonas syringae translocate type III effector proteins (T3SE) into host cells. Plant hosts react by specifically recognizing these effectors via R proteins that trigger defense responses. The T3SE family HopZ1 has evolved into three allelic forms as a result of diversifying selection. In this thesis, I investigated how virulence and immune-eliciting functions are determined in HopZ1a and HopZ1b in Arabidopsis. Mutational analysis of HopZ1a identified ten residues important for immune elicitation and at least three are involved in virulence functions. These results suggest that distinct key amino acid residues in HopZ1a mediate the two activities. The closely related HopZ1b T3SE elicits an inconsistent immune response in Arabidopsis. We found that HopZ1b-triggered immune response involves a TIR-type R protein and plastid-derived SA. Together, these results highlight an uncharacterized ETI response to the HopZ1 family of T3SEs.

La bactérie phytopathogène du sol, Ralstonia solanacearum cause la maladie du flétrissement bactérien sur un grand nombre de plante hôtes. Un des déterminants clefs de son pouvoir pathogène est le système de sécrétion de type III. Celui-ci permet à la bactérie d'injecter des protéines directement dans les cellules de l'hôte. Dans ma thèse je me suis attaché à décrire et analyser finement la contribution au pouvoir pathogène de la bactérie de certains de ces substrats de l'appareil de sécrétion de type III (RipAX2, RipH2, RipH3 et RipG). Des expériences de double-hybride nous ont permis d'identifier des protéines des plantes hôtes pouvant être ciblées par ces effecteurs de type III. Dans une autre partie de mon travail j'ai contribué à l'étude de RipAX2 qui est induit spécifiquement la résistance dans des lignées d'aubergines porteur d'un locus de résistance. J'ai également travaillé sur l'identification des cellules de plantes soumises à l'injection de type III dans une interaction compatible. Pour cela j'ai utilisé la plante hôte Medicago truncatula, en exprimant dans certaines lignées cellulaires un effecteurs (RipG7) pour lequel nous avions démontré que son expression constitutive das M. truncatula pouvait restaurer l'infection de ces plantes par un mutant bactérien dans ce même effecteur. Enfin, j'ai aussi contribué à l'analyse structure-fonction fine de l'effecteur RipG7 dans sa fonction de contribution au pouvoir pathogène de R. solanacearum sur la plante hôte M. truncatula. Ce travail nous a permis d'identifier les acides aminés de RipG7 qui sont sous sélection positive, et parmi ceux-là, ceux qui contribuent directement à la fonction de RipG7 sur M. truncatula. / The soil-borne pathogen Ralstonia solanacearum causes bacterial wilt in a broad range of plants. The type III secretion system (T3SS) and its associated type III effectors (T3Es) are the main virulence determinants of R. solanacearum. In my PhD study, to understand the mode of action of several "core" effectors (RipH2, RipH3, RipG7, RipG6) from R. solanacearum in host cells. We performed yeast two-hybrid screening of plant cDNA library to identify their protein targets. Besides, we also collaborated on the identification and characterization of a specific type III effector RipAX2 which is an avirulence factor that triggers a hypersensitive response in specific Eggplant lines. To understand which plant root cells are actually subjected to type III injection during the compatible interaction, I have generated transiently transformed Medicago truncatula lines (hairy root), expressing a host specificity and core T3E RipG7 in different root cell layers. When the transformed plant expressing RipG7 under 35S promoter, the plant can be infected and colonized by the ripG7 single mutant strain. The study could be refined by using specific root cell layer promoter to identify the root cell layers that are key players in this interaction. We also worked on the characterization of the structure-function of RipG7 from R. solanacearum. Our work revealed the genetic and functional variation of RipG7. Furthermore, positive selection study and mutagenesis analysis enabled us to identify essential functional residues which likely to have been differentially selected during the host-pathogen co-evolution. The potential plant targets of RipG7 were also studies further in our study by differential yeast two-hybrid.

Sleep disturbance and behavioural difficulties are both prevalent problems in the intellectual and developmental disability population and can have a significant impact on quality of life for the individual and their family. This thesis investigated sleep, behaviour and circadian rhythm functioning in children with intellectual and developmental disabilities, and is presented in three sections. The first two papers have been prepared in accordance with the author guidelines of the journals proposed for submission, excluding tables and figures for ease of reading. The first paper is a systematic review of the literature examining the relationship between sleep disturbance and challenging behaviour in children with intellectual and developmental disabilities. 15 studies were included in the review and overall there were consistent findings of an association between the presence of sleep disruption and increased behavioural difficulties. A causal relationship could not be inferred due to the cross-sectional methodology of studies. Other factors, such as parental wellbeing, child level of intellectual disability and comorbidity of physical health conditions, need to be considered to understand the complexity of this relationship. Children with the neurodevelopmental disorder mucopolysaccharidosis type III (MPS III or Sanfilippo syndrome) present with high rates of sleep disturbance and challenging behaviour. The second paper investigates circadian rhythm functioning and activity levels in children with MPS III, compared to typically developing controls. Objective measurement of circadian rhythm and activity levels was obtained through actigraphic recording for 7-10 days. Children with MPS III had increased fragmentation of circadian rhythm, less stability of rhythm in relation to external cues and a differential pattern of activity across the day compared to controls. Overall, results were indicative of a disruption of circadian rhythm function in children with MPS III. The implications for clinical practice and future research are discussed. The third paper provides a critical appraisal of the overall research process, including further consideration of the strengths and limitations, implications for clinical practice, wider context of the research and personal reflections. An account of the project that was originally proposed with the MPS III population is also presented, alongside reflections on its termination.

Indiana University-Purdue University Indianapolis (IUPUI) / The Gram-negative bacterium Pseudomonas aeruginosa causes numerous acute and chronic opportunistic infections in humans. One of its most formidable weapons is a type III secretion system (T3SS), a multi-protein molecular syringe that injects powerful toxins directly into host cells. The toxins lead to cell dysfunction and, ultimately, cell death. Identification of regulatory pathways that control T3SS gene expression may lead to the discovery of novel therapeutics to treat P. aeruginosa infections. In a previous study, it was found that expression of the magnesium transporter gene mgtE inhibits T3SS gene transcription. MgtE-dependent inhibition appeared to interfere with the synthesis or function of the master T3SS transcriptional activator ExsA, although the exact mechanism was unclear. In this work, we demonstrate that mgtE expression acts through the GacAS two-component system to activate transcription of the small regulatory RNAs RsmY and RsmZ. This event ultimately leads to inhibition of exsA translation. Moreover, our data reveal that MgtE acts solely through this pathway to regulate T3SS gene transcription. Our study reveals an important mechanism that may allow P. aeruginosa to fine-tune T3SS activity in response to certain environmental stimuli. In addition, a previous study has shown that the P. aeruginosa gene algR abrogates mgtE mediated regulation of cytotoxicity. AlgR has pleiotropic effects in P. aeruginosa, including regulation of synthesis of the exopolysaccharide alginate. In the second part of my thesis, I show that algR and mgtE genetically crosstalk to inhibit ExsA driven T3SS gene transcription. This genetic interaction between algR and mgtE seems to be specifically directed towards regulation of T3SS gene expression rather than having an indiscriminate effect on multiple virulence attributes in P. aeruginosa. Additionally, we have further demonstrated that AlgR inhibits mgtE transcription. These studies suggest the presence of a T3SS inhibitor that is inhibited by both AlgR and MgtE. Future work will involve transcriptomic and proteomic analysis to identify such an inhibitor. Taken together, this study provides important insight into the molecular mechanisms of mgtE expression and function in P. aeruginosa. We have established that mgtE has pleiotropic effects on cytotoxicity in P. aeruginosa. Thus, given the role that cytotoxicity regulation plays in shaping P. aeruginosa pathogenesis and associated clinical outcomes, mgtE might be an interesting drug target, though extensive future studies are required to validate this proposition. Nevertheless, this research, provides clues for identification of novel therapeutic targets in P. aeruginosa. Hence this work, in the long run, serve to ameliorate the morbidity and mortality in patients infected with P. aeruginosa.

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