Shigella flexneri is a Gram negative facultative anaerobe that infects millions world-wide each year. The route for infection of a host is through the intestinal and rectal epithelium layers, but it also can survive in the environment. Different genes have been found to be up regulated depending upon its presence in the intracellular or extracellular environment, as shown in previous work in the lab. This thesis seeks to examine the role these upregulated genes, bioA and lysA, play in the intracellular activity of S. flexneri. Knock-out mutations in the bioA and lysA genes were created using P1 transduction. To test the effects of these mutations on S. flexneri, plaque, invasion, and attachment assays were performed. It was found that the bioA mutation resulted in fewer plaques being formed, while the lysA mutation resulted in slower forming and incompletely lysed plaques being formed. / text

The gram negative bacterium Shigella flexneri is an etiological agent of bacillary dysentery, and causes destruction of the human intestinal epithelium. S. flexneri is primarily transmitted via the fecal-oral route to its primary infective site in the colon. The bacterium invades and replicates within colonic epithelial cells, ultimately ulcerating the mucosal epithelium. To successfully establish infection, S. flexneri must quickly adapt to different environments in the host, including adjusting metabolism in response to changes in available carbon sources. In this study, the importance of the glycolytic and gluconeogenic pathways in S. flexneri pathogenesis was examined. The metabolic regulators CsrA and Cra reciprocally regulate the glycolytic and gluconeogenic pathways. The post-transcriptional regulator Cra activates expression of genes involved in gluconeogenesis and represses glycolysis. Conversely, CsrA activates glycolysis and represses gluconeogenesis. The absence of Cra increased S. flexneri attachment and invasion of cultured epithelial cells. In contrast, the csrA mutant was significantly impaired in both adherence and invasion. Both the csrA and cra mutants formed small, turbid plaques, suggesting that both regulators are required for plaque formation. The opposing phenotypes of the csrA and cra mutants suggested a correlation between invasion and glycolysis. The role of glycolysis in S. flexneri pathogenesis was confirmed by directly examining the first committed step in the pathway. The glycolytic enzyme phosphofructokinase I (PfkI, encoded by pfkA) is repressed by Cra and activated by CsrA. Glycolysis was critical for S. flexneri pathogenesis, as a mutation in pfkA rendered the bacterium noninvasive. The invasion defect of the csrA and pfkA mutants was due to reduced expression and secretion of the Shigella invasion plasmid antigen (Ipa) effectors. Expression of the master virulence regulators virF and virB was significantly reduced in the pfkA mutant, and is the principle reason for decreased invasion. The data presented show that glycolysis is required for invasion, but that plaque formation requires both glycolysis and gluconeogenesis. Because expression of the master virulence regulators is repressed in the pfkA mutant, S. flexneri may use carbon as an environmental regulator of virulence gene expression. / text

Tumorstroma und Tumor-assoziierte Makrophagen (TAMs) spielen in neoplastischen Erkrankungen eine wichtige Rolle im Bezug auf Tumorwachstum und Progression. In einigen Krebsarten besteht zwischen den Tumor-assoziierten Makrophagen und den Krebszellen eine intensive Interaktion, welche zu vermehrter Angiogenese und zur Unterdrückung lokaler Immunantworten führt. Aus diesem Grund stellen TAMs einen vielversprechenden Angriffspunkt für eine Krebstherapie da. In dieser Arbeit wird gezeigt, dass intrazelluläre Bakterien wie Salmonella und Shigella hauptsächlich TAMs im Tumorgewebe infizierten. Um dieses Verhalten näher zu untersuchen, konstruierten wir einen im Wachstum abgeschwächten Shigella Stamm, welcher jedoch noch die Fähigkeit hat, Apoptose in Makrophagen zu induzieren. Shigellen sind invasive Bakterien, die in das Darmgewebe einwandern und dort eine massive Inflammation induzieren. Intrazelluläre Shigellen aktivieren Caspase-1 und induzieren dadurch Apoptose in Makrophagen durch den sekretierten Virulenzfaktor IpaB. Durch eine Deletion des genomischen aroA-Gens, wurde ein Shigella Stamm konstruiert, der Defekte im intrazellulären Wachstum aufweist. Dennoch war dieser Stamm noch fähig eukaryotische Zellen zu infizieren, sich interzellulär fortzubewegen, Caspase-1 zu aktivieren und Apoptose in Makrophagen zu induzieren. Es wurde gezeigt, dass dieser Shigellen Stamm nach i.v. Injektion hauptsächlich die TAMs im 4T1-induzierten und transgenen MMTV-HER2/neu Brustkrebsmodel infizieren. Diese attenuierten Shigellen wurden im Tumorgewebe hauptsächlich intrazellulär detektiert, im Gegensatz dazu wurden attenuierte Salmonellen zu späten Zeitpunkten (7 d p.i.) auch extrazellulär im Tumorgewebe aufgefunden. Der metabolisch aber nicht in der Virulenz attenuierte Shigella Stamm konnte in beiden Brustkrebsmodellen zu allen Zeitpunkten (4 h, 6h and 7 d p.i.) Caspase-1 in TAMs aktivieren und Apoptose induzieren. Diese Apoptose führte in beiden Brustkrebsmodellen zu einer langandauernden und hoch signifikanten Reduktion der TAMs-Anzahl (bis zu 70 %). Im Gegensatz dazu konnten Salmonellen nur zu frühen Zeitpunkten (6 h p.i.) Apoptose in TAMs induzieren und dies führte in beiden Modellen zu keiner Reduzierung der TAMs-Anzahl. In dem 4T1-induzierten Tumormodel wurden die Mäuse mit dem attenuierten Shigella Stamm behandelt, was zu einer kompletten Blockierung des Tumorwachstums führte, dies traf aber nicht für den avirulenten Stamm zu. Darüberhinaus infizierte Shigella hauptsächlich die Makrophagen Fraktion eines Ovarkarzinoms ex vivo und induzierte in diesen Zellen Caspase- 1 Aktivierung und Apoptose. Zusammenfassend zeigen diese Daten, dass im Wachstum attenuierte intrazelluläre Bakterien dazu fähig sind Apoptose in TAMs zu induzieren. Dadurch werden sie zu einem vielversprechenden Therapeutikum zur Behandlung von betimmten Krebserkrankungen, bei denen TAMs eine erwiesene Rolle im Tumorwachstum und der Tumorprogression spielen. / In neoplastic diseases the tumor stroma and especially tumor-associated macrophages (TAMs) play an important role in tumor growth and progression. TAMs exhibit an intensive cross-talk with tumor cells resulting in the promotion of angiogenesis and the inhibition of local protective immune responses in certain tumor entities. Therefore, TAMs are a potential target for tumor therapy. Here it was shown that intravenously applied intracellular bacteria like Salmonella and Shigella primarily target TAMs. To exploit this feature a growth attenuated Shigella strain with the capacity to induce apoptosis in macrophages was designed. Shigella are invasive bacteria that penetrate the colonic tissue and initiate an acute inflammation. In macrophages, Shigella rapidly induces caspase-1 processing and apoptosis via the virulence factor IpaB. By genomic deletion of the aroA-locus a metabolically attenuated strain defective in intracellular growth but with retained capacity of infection, cell-to-cell spread, caspase-1 processing and apoptosis induction in macrophages was designed. It was shown that this strain primarily targets TAMs in 4T1 cell induced and transgenic MMTV-HER2/new breast cancer models. Shigella were almost exclusively found intracellularly, whereas growth attenuated Salmonella were also found extracellularly at late time points. The metabollically attenuated Shigella strain with retained virulence, but not avirulent Shigella strains, was able to activate caspase-1 and induce apoptosis in TAMs at all time points (4 h, 6 h and 7 d p.i.) in both breast cancer models. This unrestricted apoptosis induction translated into a substantial, long-lasting and highly significant reduction of TAMs number (up to 70 %) in both models. In contrast, Salmonella could only induce apoptosis in TAMs at early time points (6 h p.i.) and failed to reduce TAMs in both models. In the 4T1 model, the effect on tumor size was monitored and treatment of the mice with the attenuated Shigella strain resulted in a complete block of tumor growth. Finally, Shigella primarily infected the macrophage fraction, activated caspase-1 and induced apoptosis in cells derived from a human ovarian carcinoma ex vivo. Taken together, this data suggests that growth attenuated intracellular bacteria capable of inducing apoptosis in TAMs are a promising therapeutic option for certain cancer diseases where TAMs have a proven role for tumor growth or progression.

Shigellosis, or bacillary dysentery, is a rectocolitis caused by the gram-negative, enteroinvasive bacteria of the genus Shigella. Shigellosis still remains a major public health burden with an estimated 80 million cases of bloody diarrhoea and 700.000 deaths per year, primarily in children under the age of 5. Shigella disrupts, invades, and causes inflammatory destruction of the colonic epithelium in humans through virulence effectors secreted by the type III secretion apparatus (TTSA). In contrast to the Shigella-induced manipulation of the host innate immune response, the impact of Shigella on the adaptive immunity has been poorly studied thus far. In order to understand why the naturally induced protective humoral response requires several infections to be primed and is of short duration, the work presented here investigates if Shigella is able to directly interact with T cells. Indeed, it has been shown that Shigella was able to invade and proliferate inside T cells. Furthermore, Shigella was able to inhibit T cell migration through a TTSA effector. Moreover, the Shigella effector IpgD, a phosphoinositide 4-phosphatase that specifically dephosphorylates phosphatidylinositol-(4,5)-bisphosphate (PIP2) into phosphatidylinositol-(5)-monophosphate (PI(5)P), was identified as the effector responsible for the observed inhibition. It could be demonstrated that IpgD was responsible for a reduction of intracellular PIP2 levels in T cells. Further experiments showed a reduced level of phosphorylated ezrin, radixin and moesin (ERM) proteins in infected, as well as with IpgD transfected, T cells. The ERM protein family plays an imported role in signal transduction and motility and their activity is closely related to the binding of PIP2. Therefore, the low level of PIP2 leads to a dephosphorylation of the ERM proteins which inhibits T cells response to chemokine stimulation. Indeed, IpgD transfected T cells show a reduced ability to re-localise the ERM proteins upon chemokine stimulation. Targeting T cell motility, via TTSA effectors, could explain the low level of specific T cell priming during Shigella infection. This is the first report of Shigella induced manipulation of T cell function and on the inhibition of T cell migration by a bacterial effector. / Shigellose oder Bakterieruhr ist eine von Bakterien der Gattung Shigella ausgelöste Dysenterie Erkrankung des Dickdarms. Mit jährlich über 80 Millionen Fällen von blutigen Durchfällen und 700000 Todesfällen, hauptsächlich bei Kindern unter 5 Jahren, stellt Shigella immer noch ein ernsthaftes Gesundheitsproblem dar. Shigella destabilisiert das menschliche Dickdarmgewebe und dringt in dieses ein, wo es eine akute Entzündung auslöst, die das Gewebe weiterhin zerstört. Verursacht wird dies durch bakterielle Effektoren, die durch ein Type III Sekretionssytem (TTSA) sekretiert werden. Verglichen mit der Anzahl an Studien über die Manipulation der angeborenen Immunabwehr gibt es nur wenige Studien über die Interaktionen von Shigella mit dem adaptiven Immunsystem. Um zu verstehen, warum für die Entwicklung einer humoralen Immunantwort mehrere Infektionen erforderlich sind, wurde im Rahmen dieser Arbeit untersucht, ob Shigella in der Lage ist, direkt mit TZellen zu interagieren. Es konnte gezeigt werden, dass Shigella in T-Zellen eindringen und sich vermehren kann. Darüber hinaus zeigt sich, dass Shigella in der Lage ist, durch TTSA-Effektoren die T-Zell-Migration zu hemmen. Der Shigella Effektor IpgD konnte als der für die Hemmung verantwortliche Effektor identifiziert werden. Bei IpgD handelt es sich um eine 4-Phosphoinositid-Phosphatase, die Phosphatidylinositol-(4,5)-bisphosphat (PIP2) zu Phosphatidyl-inositol-(5)- monophosphat (PI(5)P) dephosphoryliert. Es wurde deutlich, dass der Effektor IpgD, neben der Menge an PIP2, auch die Menge an phosphorylierten Ezrin, Radixin und Moesin (ERM) Proteinen in T-Zellen reduziert. Die ERM-Protein-Familie spielt in der Signaltransduktion und bei der Motilität von T-Zellen eine wichtige Rolle und ihre Phosphorylierung ist eng an die Bindung von PIP2 gekoppelt. Daher führt eine geringe Menge an PIP2 zu einer Dephosphorylierung der ERM-Proteine, was eine Stimulierung der T-Zellen durch Chemokine hemmt. In der Tat zeigten IpgDtransfizierte T-Zellen eine verminderte Fähigkeit zur Relokalisierung der ERM-Proteine nach einer Chemokine-Stimulation. In dieser Arbeit konnte erstmals die Manipulation von T-Zell-Funktionen durch Shigella und die Hemmung der T-Zell-Migration, ausgelöst durch einen bakteriellen TTSA-Effektor, gezeigt werden.

Thesis advisor: Christina Faherty / Shigella, a Gram-negative bacterial pathogen, induces inflammation and diarrhea by invading the colonic epithelium. Annually, millions of Shigella infections occur globally, mainly in malnourished children. Despite extensive research, no effective vaccine exists. This work explores the mechanisms of Shigella proliferation before colonic infection, where an adverse environment is encountered, including bile salts exposure. One means of bile salts evasion is possibly lipopolysaccharide (LPS) modification. LPS—O-antigen, the polysaccharide core, and the lipid A—is a crucial outer membrane component for virulence. Transposon mutant analysis suggested a role of LPS in bile salts resistance; thus, the goal of this study was to define Shigella LPS modifications following bile salts exposure. LPS mutants were investigated to distinguish crucial components of the LPS structure for bile salts resistance. Mutants were analyzed relative to wild type for growth in bile salts and biofilm formation. The LPS from all strains was purified and analyzed by polyacrylamide gel electrophoresis. Stained gels show modifications in the Oag, lipid A, and core components. Key bands were sent for mass spectrophotometry sequencing. Results indicate that the O-antigen regulates Shigella bile salts resistance, as the complete O-antigen deletion mutant and partial deletion mutants exhibited slow growth in bile salts and failed to form a biofilm in the presence of bile salts. This work highlights the importance of bile salts exposure for Shigella in future targeted antibodies against the pathogen. / Thesis (BS) — Boston College, 2019. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Biology.

Thesis (Ph.D.) -- University of Adelaide, School of Molecular and Biomedical Science, Discipline of Microbiology and Immunology, 2008. / Includes "Thesis Amendments" attached to the inside back pages. "October 2007" Bibliography: leaves 108-121. Also available in print form.

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Shigella is a causative agent of bacillary dysentery in humans. The ability of Shigella to disseminate in the intestinal epithelium is crucial for disease establishment. This process of cell-to-cell spread involves actin-based motility, which allows movement of Shigella through the cytoplasm, and the ability of Shigella to form filopodia-like membrane protrusions that are engulfed by adjacent cells. Compared to the process of Shigella actin tail assembly, which requires recruitment and activation of host actin modulators such as N-WASP and Arp2/3, the mechanism of how Shigella moves from an infected cell into neighboring cells and what host factors are involved remain poorly characterized. In this dissertation, I investigate whether members of the Ena/VASP family, as key actin regulators, or Inverse-BAR (I-BAR) family proteins, as coordinators of membrane curvature and actin dynamics, are required in dissemination of S. flexneri in a cell monolayer. Ena/VASP family proteins regulate cell migration, adhesion, shape, and cell-cell interaction. The members of the family include Vasodilator-Stimulated Phosphoprotein (VASP), Ena-VASP-like (Evl), and Mammalian enabled (Mena). We have previously shown that Mena, despite its localization to the actin tail, has no role in S. flexneri actin-based motility. Here, I investigate the role of Mena, Evl, and VASP in S. flexneri dissemination. I determine that the presence of VASP or Evl restricts cell-to-cell spread of S. flexneri. I further show evidence that the conserved EVH1 domain and phosphorylation of VASP regulate the ability of Shigella to spread. I-BAR proteins, including IRSp53 and IRTKS, contain a conserved domain that directly binds to membrane lipids and induces convex membrane deformation. This unique property and the ability of these proteins to bind F-actin and actin modulators are necessary for the formation of actin pedestals by pathogenic E. coli and filopodia. Using cells with reduced levels of IRTKS or IRSp53, I examine the role of these proteins in cell-to-cell spread and show that neither IRTKS nor IRSp53 is required for S. flexneri spread. Collectively, these results advance our understanding of host proteins that participate in S. flexneri dissemination.

Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1998? / Includes bibliography (27 leaves).