Thesis (Ph. D.)--University of Oregon, 2006. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 122- 131). Also available for download via the World Wide Web; free to University of Oregon users.

Orientador: Cristiano Gallina Moreira / Banca: Ana Marisa Fusco Almeida / Banca: Samar Freschi de Barros / Resumo: Salmonella Typhimurium é um patógeno causador de gastroenterite em humanos e outros mamíferos, e sua habilidade de invadir as células epiteliais e replicar-se dentro de macrófagos a torna um importante modelo de estudo de mecanismos de virulência e interação patógeno-hospedeiro. O estudo desta interação baseia-se na sinalização química via hormônios do hospedeiro e do patógeno, e durante a investigação destes processos foi detectada uma nova proteína denominada VisP, relacionada a funções celulares diversas como manutenção de membrana, metabolismo, virulência bacteriana e resposta ao estresse. A motilidade via flagelos, importante na colonização e exploração de novos nichos, é um dos importantes mecanismos de patogenicidade bacteriana durante o processo de infecção. O objetivo deste trabalho foi investigar a motilidade mediada por flagelos e seu funcionamento em S. Typhimurium, bem como a participação de VisP durante o processo. Para isso, foram feitos ensaios de motilidade, análise da expressão gênica de genes alvos importantes para a patogênese, expressão da flagelina FliC e microscopia em cada um dos mutantes isogênicos comparados à cepa selvagem. Foi evidenciado que a retirada do gene visP afeta a motilidade de S. Typhimurium, bem como promove um desequilíbrio na homeostase da membrana celular, gerando um aumento exacerbado de flagelina através de um mecanismo ainda não elucidado. A compreensão destes processos é essencial para o entendimento da relação patógeno-hospedeir... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Salmonella Typhimurium is a pathogen that promotes gastroenteritis in humans and mammals, and its ability to invade epithelial cells and replicate inside macrophages makes Salmonella an important model for the study of virulence and host-pathogen interactions. This interaction is based on chemical signaling via host and pathogen hormones, and during this investigation a novel protein named VisP was described. This protein is related to diverse cell processes as membrane maintenance, metabolism, virulence and stress response. Flagellar motility, which is important for colonization and exploration of new niches, is one of the bacterial pathogenicity mechanisms that occurs during infection. The aim of the study was to investigate flagellar motility and its operation in S. Typhimurium and verify the participation of VisP protein during this process. Motility assays, gene expression analysis of target genes important for pathogenesis, expression of FliC protein and microscopy were performed with the isogenic mutants comparative to the wild type. The results demonstrated that the visP gene impacts motility in S. Typhimurium, promoting a misbalance in cellular membrane and increasing levels of flagellin via an unknown mechanism. The elucidation of these processes is essential to understanding host-pathogen associations, and contributes to the development of novel technologies and therapies. / Mestre

Following starvation, Dictyostelium discoideum cells aggregate, a response that requires chemotaxis to cyclic AMP (cAMP) and the relay of the cAMP signal by the activation of adenylyl cyclase (ACA). Insertional inactivation of the rasG gene resulted in delayed aggregation and a partial inhibition of early gene expression, suggesting that RasG does have a role in early development. When the responses of rasG⁻ cells to cAMP were compared with the responses of rasC⁻ strain, these studies revealed that signal transduction through RasG is more important in chemotaxis and early gene expression, but that signal transduction through RasC is more important in ACA activation. Characterization of a rasC⁻/rasG⁻ mutant revealed that both cAMP chemotaxis and adenylyl cyclase (ACA) activation were negligible in this strain. The ectopic expression of carA from the actin 15 promoter restored early developmental gene expression to the rasC⁻/rasG⁻ strain, rendering it suitable for an analysis of cAMP signal transduction. Since there was negligible signaling through either the cAMP chemotactic pathway or the adenylyl cyclase activation pathway in this strain, it is clear that RasG and RasC are the only two Ras subfamily proteins that directly control these pathways. The mutational analysis of Switch I and Switch II regions also defined the key residues that generate functional differences between RasC and RasG. Rap1 is also activated in response to cAMP but its position in the signal transduction cascade was clarified by the finding that its activation was totally abolished in rasC⁻/rasG⁻/[act15]:carA and in rasG⁻ cells, but only slightly reduced in rasC⁻ cells. The finding that in vitro guanylyl cyclase activation is also abolished in the rasC/rasG⁻4act15]:carA strain identifies RasG⁻/RasC⁻ as the presumptive monomeric GTPases required for this activation. The phenotypes of the vegetative ras null mutants were also examined. The results indicate that RasG plays an important role in cytokinesis. The partial absence of chemotaxis to folate in rase cells compared to the total absence of chemotaxis to folate in rasC⁻/rasG⁻, and rasC⁻/rasG⁻/[act15]:carA cells suggests a compensatory role of RasC for RasG during this process, a similar phenomenon to that observed for cAMP chemotaxis by aggregating cells. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Migrating cells have two basic ways of extending their leading edge: by dendritic actin polymerization beneath the membrane, or by fluid pressure, which produces blebs. Most cells are believed to move using actin-driven projections, but in more physiological conditions, blebbing motility is also apparent. It has been shown that certain cells even can switch between these two modes of motility, although it is not known how this switch is triggered. Besides, it is unclear whether blebbing can be regulated by chemotactic stimuli, and generally, how blebbing is controlled in the cell. In this study I employed a popular model organism – Dictyostelium discoideum – to investigate the role of blebbing in chemotaxis. Here I confirm that in standard conditions Dictyostelium cells move by a combination of F-actin-driven protrusions and blebs. Blebbing is characterized by the rapid projection of hemispherical patches of plasma membrane at 2-4 times the speed of an actin-driven projection, and leaves transient scars of F-actin marking the original cortex in the base of blebs. I demonstrate that Dictyostelium cells can adjust their mode of movement according to the conditions: in a resistive environment they switch almost entirely to “bleb mode”. I show that in chemotaxing cells, blebs are mainly restricted to the leading edge, and they often lead the way when a cell is forced to re-orientate. Bleb location appears to be controlled directly by chemotactic gradients. To investigate how chemoattractant induces blebbing, I have screened signal transduction mutants for altered blebbing. I have found that blebbing is unaffected in many chemotactic mutants, but unexpectedly depends on PI3-kinases and two downstream PIP3-binding proteins of unknown function – PhdA and CRAC. I conclude that Dictyostelium cells move using a hybrid motor in which hydrostatic pressure-driven bleb formation is as important as F-actin-driven membrane extension, and that cells can change the balance between modes as required. I propose that blebbing motility of Dictyostelium cells is a direct response to mechanical resistance of environment. More generally, bleb-driven motility may be a ‘”high-force” mode of movement that is suited to penetrating tissues. Blebs are chemotactic and their induction may involve branches of the chemotactic signal transduction pathway distinct from F-actin regulation.

High molecular weight mouse nerve growth factor(H M W-NGF), in addition to its effects on certain neural elements, is also chemotactic for human polymorphonuclear leukocytes. One of the subunits of H M W-NGF is a protease of the serine family and its active site contains a serine residue and a closely-neighboring histidine residue that are both essential for proteolysis. Elimination of enzyme activity by irreversibly blocking the single serine has no effect on leukotaxis, but blocking the histidine abolishes leukotaxis. These results suggest the possibility that part of the proteolytic active site of this enzyme may have evolved to perform more than one, completely different, biologic function - proteolysis as well as nonproteolytically mediated chemotaxis.

<p>    </p> <p>Migration is a ubiquitous phenomenon in biology and is relevant to all scales ranging from bacteria to human beings. It is relevant to fundamental biological processes like bacterial chemotaxis, development, disease progression, etc. So, understanding migration is pivotal to addressing fundamental questions in biology. We address three broad questions relevant to cell migration using models from physics: (i) What are the critical features of cancer cell migration? (ii) Is it possible to explain complex cell migration data using minimal bio- chemical networks? And (iii) how does cell-to-cell communication affect its migration at the population level? To address these questions we performed (i) mathematical analysis using the Cellular Potts model, simulations using the Biased Persistent random walk model, and steady-state analysis of cell response to graded signals to explain cancer cell migration in response to single and multiple chemical and mechanical signals, (ii) rigorous network anal- ysis of ∼ 500,000 minimal networks having features of fundamental biochemical processes like regulation, conversion or molecular binding to understand the origin of antagonism in multiple cue cancer cell migration experiments and (iii) the steady-state analysis of Keller- Segel equations mimicking collective cell migration to understand the role of cell to cell communication on chemotaxis of a bacterial population. From our analysis, we found that (i) persistence and bias in cancer cell migration are decoupled from each other owing to a lack of memory about past movements and for any general cell migration they are inherently constrained to take only a fixed set of values. (ii) Bias in cancer cell migration in response to a combination of chemoattractant gradients can be less than the response to individual gradients (antagonism in bias) while the speed remains unaltered. This antagonism in bias and lack thereof in speed can be explained by several minimal networks having molecular regulation, conversion, or binding as its central feature and all these distinct mechanisms show convergence and saturation of an internal molecule common to both the chemoattrac- tants. (iii) By analyzing the role of cell-cell communication in bacterial chemotaxis using the Keller-Segel model we find that communication enhances chemotaxis only when it is adaptive to its external surroundings and cell-to-cell variability helps in increasing the chemotactic drift in the bacterial population. </p>

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Bacteria perform biological nitrogen fixation (BNF) which leads to conversion of N2 to ammonia. One of the best studied models of BNF is the symbiotic association of Sinorhizobium meliloti - Medicago sativa (alfalfa). Since alfalfa is a major source of animal feed and the fourth largest crop grown in the USA, enhanced understanding of this symbiosis can have implications for increasing crop yields, reducing environmental contamination and food costs. Studies discussed here focus on two symbiotically important bacterial traits, type IVb pili and chemotaxis. Chapter 2 characterizes S. meliloti type IVb pili encoded by flp-1 and establishes their role in nodulation. Bundle-forming pili were visualized in wild-type cells, while cells lacking pilA1, the pilin-encoding gene, showed an absence of pili. Competitive nodulation assays with alfalfa concluded that cells lacking pili had a significant nodulation defect. Regulation of pilA1 expression via a quorum sensing regulator, ExpR, was confirmed. Chapter 3 describes the role of the flp-2 cluster in establishing symbiosis. PilA2 is a pilin subunit encoded from flp-2. The pilA2 deletion strain was defective in nodulation by 31% as compared to the wild type. A non-significant change in nodulation was seen in pilA1pilA2 strain. Thus, both flp-1 and flp-2 have a significant role in establishing symbiosis. Chapter 4 focuses on the deviations of S. meliloti chemotaxis from the enterobacterial paradigm. Transcriptional fusions showed that S. meliloti chemoreceptors (MCPs) are class III genes and regulated by FlbT. Quantitative immunoblots determined the cellular amounts of chemoreceptors. Chemoreceptors were grouped in three classes; high, low, and extremely-low abundance, similar to the high and low abundance chemoreceptors of Escherichia coli. Importantly, the MCP:CheA ratio in an S. meliloti cell was observed to be 37:1, similar to that in Bacillus subtilis of 24:1, but quite different from that in E. coli of 3.4:1. In conclusion, our data indicates that soil bacteria may have optimized their chemotaxis system based on their milieu, which is different from enteric bacteria. These studies have enhanced our understanding of two symbiotically important processes in S. meliloti, and pave the way for future manipulations of the system to increase symbiosis and reduce our dependence on synthetic fertilizers. / Ph. D.