Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas. Programa de Pós-Graduação em Biotecnologia / Made available in DSpace on 2012-10-22T13:49:09Z (GMT). No. of bitstreams: 1 225390.pdf: 1106891 bytes, checksum: 2bb5cae7c975eda6d81cab7a182b1ea4 (MD5) / Devido ao hábito alimentar filtrador dos moluscos bivalves, estes organismos podem concentrar, em seus tecidos, microrganismos patogênicos presentes nas águas de cultivo, sendo associados com intoxicações alimentares, como a salmonelose. O Estado de Santa Catarina, por suas características geográficas e da qualidade das águas litorâneas, tornou-se um ambiente ideal para o cultivo de organismos marinhos, especialmente moluscos bivalves, como ostras da espécie Crassostrea gigas. Considerando registros recentes de contaminação por patógenos entéricos em alguns locais de cultivo de moluscos na Ilha de Santa Catarina, a produção e comércio de moluscos necessitam ser monitorados. Com o objetivo de expandir a maricultura na costa do Estado de Santa Catarina e garantir um produto final com alto valor comercial, um sistema de depuração de moluscos foi desenvolvido pela empresa Blue Water Aquaculture e avaliado em relação à eliminação de Salmonella enterica serovar Typhimurium por ostras da espécie C.gigas. Inicialmente, foi padronizado o método para detecção da presença de S. Typhimurium em tecido digestivo de ostras. Esta metodologia consistiu em dissecação do tecido digestivo de ostras, pré-enriquecimento da amostra em meio de cultura não seletivo, extração do DNA bacteriano, e PCR seguida de hibridização molecular utilizando como sonda o produto de PCR de S.Typhimurium marcado com digoxigenina. Como resultado da padronização da metodologia de detecção molecular, foi desenvolvido um método sensível para detectar baixos níveis de S. Typhimurium em tecido digestivo de ostras (0.1UFC/g). Simultaneamente aos trabalhos de depuração, um dos pontos de cultivo de ostras de Florianópolis foi monitorado, no período entre março e outubro de 2005, em relação à qualidade microbiológica da água e da carne dos moluscos ali cultivados. Como resultado foi observado um alto índice de contaminação fecal nas águas e presença de Salmonella spp. nas ostras, enfatizando ainda mais esta necessidade de validação de um sistema de depuração. O sistema de depuração utilizado foi um sistema fechado, no qual 1000 L de água recircularam por 24h. Parâmetros como o tempo de depuração e o tratamento da água utilizada na recirculação foram também avaliados. Durante a recirculação, a água foi esterilizada com luz Ultra Violeta, cloro e associação dos dois tratamentos. Para a validação do sistema de depuração, ostras cultivadas em Santa Catarina (C.gigas) foram inicialmente contaminadas com S. Typhimurium. As ostras foram amostradas após 0, 6, 12, 18 e 24h para avaliar a dinâmica de depuração. Após cada coleta, as amostras foram analisadas por métodos clássicos de microbiologia para quantificar as bactérias viáveis no tecido digestivo das ostras. Os ensaios de depuração com água tratada com UV e Cloro, separadamente, mostraram uma crescente eliminação de bactérias viáveis durante os períodos de depuração, com 90% da contaminação eliminada em 24h. A associação destes tratamentos mostrou a total eliminação das bactérias dentro de 12h. A PCR apresentou uma variável detecção do genoma bacteriano em todos os métodos para esterilização da água, demonstrando que a detecção molecular, nesse caso, não é uma boa metodologia, já que não é indicadora da viabilidade bacteriana, podendo detectar fragmentos de DNA de bactérias inviáveis. Este tipo de trabalho é pioneiro no Brasil e, considerando que o Estado de Santa Catarina apresenta liderança na produção de moluscos no Brasil e que até o momento não há um programa sanitário para regulamentar a exportação da produção, os resultados encontrados apresentam uma grande contribuição para a economia e o desenvolvimento sustentável do país.

The overall aim of this project was to determine the molecular mechanisms by which the flagellin gene from Salmonella enterica serovar Typhimurium (S. Typhimurium) activates the NAIP/NLRC4 inflammasome and its contribution to the host protective immune response against salmonellosis. Inflammasomes are multi-protein complexes formed in response to the activation of pattern recognition receptors (PRRs). The NOD-like receptor (NLR)-family of inflammasome complexes are formed from the cytosolic NLR receptors, ASC adaptor and caspase-1 in response to pathogen- associated molecules or danger-associated signals. The NAIP/NLRC4 inflammasome is activated by the S. enterica flagellar filament protein (FliC), the SPI-1 type III secretion system needle (PrgI) and inner rod proteins (PrgJ). Recognition of these bacterial ligands by the NAIP receptors allows oligomerisation with NLRC4 and subsequent recruitment of caspase-1. Caspase-1 mediates pyroptosis, while recruitment of ASC is also required for cleavage of pro-IL-1β and pro-IL-18 to their active forms by caspase-1. Differential recognition of the flagellar filament proteins (flagellin) by the NAIP/NLRC4 inflammasome forms an important part of my thesis. Here, I have looked at the molecular mechanisms and immunological consequences of the differential recognition of flagellin by the NAIP/NLRC4 inflammasome using S. Typhimurium SL1344 and the non-pathogenic E. coli strain K12-MG1655. An important part of my work was to try and determine which regions of fliC are required for NAIP/NLRC4 inflammasome activation and whether they can be mutated while preserving motility. To do this a panel of ten strains expressing chimeric fliC genes were created and characterised in macrophage infection experiments and bacterial motility assays. My results confirm the C-terminus of FliC is critical for both inflammasome activation and motility in agreement with published reports. To further investigate this differential recognition by the NAIP/NLRC4 inflammasome I modified S. Typhimurium strain of moderate virulence (M525P) to express flagellin from E. coli K12-MG1655. This strain (M525PΔfliC::fliCK12-MG1655CmR) retained motility and both in vitro and in vivo characterisation was carried out in macrophages and using a murine model of sublethal salmonellosis respectively. Activation of the NAIP/NLRC4 inflammasome was impaired in murine macrophages infected with M525PΔfliC::fliCK12-MG1655CmR when compared to those infected with M525P. Mice infected with M525PΔfliC::fliCK12-MG1655CmR had increased liver and spleen bacterial burdens compared to those infected with M525P, indicating that optimal NAIP/NLRC4 inflammasome activation is key for efficient control of microbial spread in vivo. The role of NAIP receptors in inflammasome formation was further investigated with the use of CRISPR/Cas9 to generate mutant murine macrophage cell lines. To investigate the consequence of gene deletions cell lines were designed to lack NAIP 1, 2, 5 and 6, while others were designed to express tagged NAIP proteins to elucidate the cellular localisation of the NAIP proteins during inflammasome formation by microscopy. Characterisation of these cell lines is ongoing, with extensive optimisation of the CRISPR/Cas9 technique undertaken during this study.

Orientador: Wanderley Dias da Silveira / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-07-24T22:45:50Z (GMT). No. of bitstreams: 1 Mendonca_Sergiode_D.pdf: 5307853 bytes, checksum: de9b6f00c4099ec1ab0d2f50163feb4e (MD5) Previous issue date: 1999 / Doutorado / Genetica / Doutor em Ciências Biológicas

Salmonella enterica species are intracellular bacteria causative agents of gastroenteritis and typhoid fever in humans. Pregnancy poses an increased risk of severe Salmonellosis in many mammalian species contributing to miscarriage and/or maternal illness. Previous studies indicated that Salmonella infection in pregnant mice caused rapid fetal and maternal death due to massive bacterial proliferation in the placenta. However, the susceptibility of human primary trophoblast cells (cTBCs) to Salmonella infection was not known. We hypothesized that human placental trophoblast cells are productively infected and provide a unique intracellular niche that permits uncontrolled Salmonella replication due to an ineffective maternal innate immune response to the virulent bacteria resulting in placental death. Firstly, we observed that S.Tm strains defective in the Salmonella pathogenicity island (SPI)-1 type III secretion system (TTSS) (S.Tm-ΔinvA) were unable to enter epithelial cells, but efficiently infected placental choriocarcinoma cell lines through scavenger receptor-mediated endocytosis. Next, we observed that S.Tm failed to grow vigorously in macrophages, but replicated rapidly within epithelial and placental trophoblast cells. Further examination of intracellular localization of S.Tm indicated that bacteria were arrested in early Rab5 expressing phagosomal vesicles within trophoblast cells, whereas phagosomal maturation progressed steadily in macrophages (with expression of lysosomal-associated membrane protein-1 (LAMP-1) and cathepsin D). Moreover, human primary cTBCs harboring S.Tm underwent rapid death of the cells. Infected cTBCs expressed phosphorylated-receptor-interacting serine/threonine-protein kinase (RIPK)-1 protein and phosphorylated-mixed lineage kinase domain-like (MLKL), suggesting induction of the necroptosis pathway of cell death. Furthermore, specific inhibition of necroptosis rescued S.Tm-induced death of cTBCs. Finally, S.Tm infected trophoblast cells produced interleukin (IL)-10, and signal transducer and activator of transcription (STAT)-3 signalling. This correlated to delayed phagosomal maturation which consequently facilitated intracellular pathogen proliferation. Overall, human trophoblast cells may act as reservoirs for S.Tm survival and may aid dissemination in the pregnant host.

Magíster en Bioquímica en el área de especialización de Bioquímica Toxicológica y Diagnóstico Molecular / Memoria para optar al Título de Bioquímica / El género Salmonella comprende dos especies, S. enterica y S. bongori, que en conjunto agrupan a más de 2.500 serovares. De éstos, los pertenecientes a S. enterica subespecie enterica son responsables de aproximadamente el 99% de los casos de salmonelosis en animales de sangre caliente. A nivel mundial se producen anualmente millones de casos de salmonelosis en el ser humano y miles de muertes, principalmente en países subdesarrollados. En esta tesis se propuso identificar un conjunto de genes requeridos para la colonización sistémica de un hospedero murino por tres serovares de Salmonella: S. Typhi, S. Typhimurium y S. Enteritidis. Este estudio se realizó mediante un análisis masivo de mutantes bajo selección negativa in vivo. La detección de aquellas mutantes con defectos en la colonización sistémica aguda de ratones BALB/c se realizó mediante hibridaciones comparativas utilizando un microarray genómico de Salmonella. El posterior análisis comparativo de las mutantes bajo selección negativa in vivo en los tres serovares, nos permitió identificar que mutantes en 131 genes serían atenuadas in vivo. Dentro de este grupo identificamos genes codificados en islas de patogenicidad conservadas del género Salmonella, genes necesarios para la biosíntesis de purinas y compuestos aromáticos (aro, pur y gua), genes relacionados con la biosíntesis y modificación del LPS (rfa, rfb) y genes que codifican reguladores globales asociados a patogenicidad (phoP, envZ, rpoN, dam y rsd). Otros genes identificados corresponden a los que codifican el sistema transportador de proteínas Twin-Arginine (tatABC), genes que codifican las diferentes subunidades de una NADH deshidrogenasa (genes nuo); un locus que corresponde a un transportador de péptidos del tipo ABC (sapBF). También pudimos detectar que mutantes en genes involucrados en el transporte de solutos se encuentran bajo selección, como trkH que codifica un transportador de potasio. El sistema de transporte Twin-Arginine corresponde a una de las dos vías de translocación de proteínas hacia el espacio periplasmático en bacterias Gram negativo. La participación de este sistema en la patogenicidad de Salmonella se confirmó mediante ensayos de competencia in vivo entre mutantes definidas del operón y la respectiva cepa silvestre en los tres serovares estudiados. El análisis global de mutantes en tres serovares nos permitió determinar un conjunto de genes comunes necesarios para establecer la colonización sistémica aguda en un hospedero murino. Posteriormente, se confirmó la participación del sistema de transporte de proteínas Tat en la patogenicidad de Salmonella. Los resultados de los ensayos de competencia nos permitieron confirmar la predicción obtenida en el análisis de masivo de mutantes bajo selección negativa in vivo. / The Salmonella genus comprises two species: S. bongori and S. enterica, which can be grouped into more than 2,500 serotypes. Serovars within S. enterica subspecies enterica account for ~99% of all salmonellosis in warm-blooded animals. Worldwide, these organisms are responsible for hundreds of millions of salmonellosis cases and hundreds of thousands of deaths, mainly in underdeveloped countries. In this thesis, we aimed to identify a group of genes required for systemic colonization of a murine host by three Salmonella serotypes: S. Typhi, S. Typhimurium and S. Enteritidis. We used a high-throughput microarray-based screening for mutants with defects in systemic colonization of BALB/c mice. Subsequent comparative analysis of mutants under negative selection in vivo allowed us to identify that mutants in 131 genes are attenuated in the three serotypes under study. Within this group we found genes encoded in some of the pathogenicity islands conserved in the Salmonella genus, genes required for biosynthesis of purines and aromatic compounds (aro, pur and gua), genes related to LPS biosynthesis (rfa and rfb) and genes encoding regulators previously associated with virulence (phoP, envZ, rpoN, dam and rsd). Other genes identified are those encoding the Twin-Arginine transport system (tatABC), genes coding the different subunits of a NADH dehydrogenase (nuo genes) and a locus encoding an ABC peptide transporter (sapBF). We also identified that mutants in genes involved in solute transport (i.e: trkH, that encodes a potassium transporter) are under negative selection in vivo. The Twin-Arginine transport system corresponds to one of the two pathways used by Gram-negative bacteria to translocate proteins to the periplasmatic space. Participation of this system in Salmonella pathogenicity was confirmed in the three serotypes under study by means of in vivo competition assays between targeted mutants of the operon and the corresponding wild-type strains. Overall, the global analysis of mutants under negative selection in vivo in three serotypes of Salmonella allowed us to identify a common group of genes required to establish acute systemic colonization of a murine host. We confirmed the participation of the Tat transport system in the pathogenicity of Salmonella using in vivo competition assays. These results further support the predictions obtained in our global analysis. / Fondecyt

Analiza el efecto de la lactoferrina en la cinética de crecimiento de Salmonella typhimurium cepa SL 1344 ΔhilA, evalúa el efecto citotóxico del tratamiento con gentamicina a células HEp-2, determina el efecto in vitro de la lactoferrina sobre la adhesión de Salmonella typhimurium SL 1344 ΔhilA y especifica el tratamiento y concentración de lactoferrina que permita mayor disminución de la adherencia e invasión a las células HEp-2. / Tesis

Salmonella typhimurium (ST) infection not only causes salmonellosis in humans, but also can result in great economic losses in the typically narrow-margin, high-volume broiler business due to reduced growth rates and mortalities. Over the last decade, the use of antibiotics and attenuated vaccines to restrain or prevent bacterial infections in domestic animals has been criticised because of the possible development of antibiotic resistance and the potential dangers of residual antibiotics and vaccines in animal-derived food products for human consumption. For these reasons, many countries have begun phasing out growth promoting antibiotics in broiler diets. It is therefore essential for the poultry production industry to develop feed additives and processing techniques as alternatives for sub-therapeutic dietary supplementation of antibiotics. However, innovative research is needed to evaluate the efficacy of new and existing alternative products. The general aim of this trial was to determine the effects of Salmonella typhimurium colonisation of the gastrointestinal tract of broiler chicks on gastrointestinal health and production performance. The effect of Zinc-Bacitracin (Zn-BC), a commonly used antibiotic growth promoter in the poultry industry, on Salmonella colonisation was also measured. A pilot trial was first conducted to determine the level of Salmonella typhimurium required to infect broiler chicks, and the necessity of administering an immunosuppressive agent in order to obtain infection. The main trial followed to determine the effects of Salmonella typhimurium on gastrointestinal health and function. The ultimate aim of the study was to obtain baseline values of various parameters that could be used in future trials for the evaluation of antibiotic alternative products. The results obtained from the pilot trial showed that it was not necessary to administer cyclophosphamide as the Salmonella typhimurium proved to be highly virulent. The cloacal swabs taken in the second trial showed that the use of Zn-BC as an antibiotic did not inhibit Salmonella colonisation in the challenged birds. The inclusion of Zn-BC in this trial inhibited the growth of the gut microflora allowing the Salmonella to proliferate in the body of the chicken, which lead to the conclusion that the routine inclusion of Zn-BC at sub-clinical levels as a growth promoter may be detrimental when the bird gets exposed to Gram(-) bacteria, such as Salmonella. In both of the trials, Salmonella challenge resulted in enlargement of the organs with a consequent increase in the organ weights. In the pilot trial there was a significant difference (P<0.0033) of the control weights for the duodenum, ileum, caeca and liver and those of the Salmonella infected birds. Control birds that did not receive CY had duodenum weights of 1.00 (±0.236) while the birds infected with 1 x 108 CFU/mL had weights of 1.99 (±0.310), while the control birds that did receive CY had duodenum weights of 0.98 (±0.244) with the Salmonella infected birds having weights of between 1.79 (±0.299) and 2.13 (±0.006). Significant results (P<0.016) in the main trial were found to occur predominantly at 7 days of age for the duodenum, jejenum, ileum and caeca weights. Control birds in the group that did receive antibiotics had 7 day duodenum weights of 1.80 (±0.301) compared to the Salmonella infected bird which had weights of between 2.33 (±0.376) and 2.51 (±0.424). In general Salmonella did not affect the growth and performance of the challenged birds. Birds challenged with Salmonella showed a tendency to have enlarged livers, possibly due to hepatic damage. In the main trial there was a significant difference (P<0.016) in liver weights at 28 days of age between the control and Salmonella infected groups regardless of whether the birds received antibiotics or not. The control birds that received antibiotics had liver weights of 3.24 (±0.234) while the birds infected with the higher level of Salmonella had weights of 3.86 (±0.542). This finding, together with the noticeable, although mainly insignificant, trend of decreased serum albumin levels and increased serum globulin and total serum protein levels noted in infected birds can be used in conjunction to measure the effect of ST on liver damage. Salmonella colonisation resulted in an increase in the severity of lesions seen in the gastrointestinal tract (P<0.0016). Histopathology results proved to be inconsistent and did not provide any conclusive evidence on the effect of Salmonella on the organs. Villi measurements taken in the second trial showed that Salmonella significantly (P<0.016) shortened the length of the villi in the duodenum and jejenum of challenged birds when measured at 28 days of age. Control birds had duodenum villi length of 662.5 (±56.79) while those birds infected with Salmonella had lengths of between 558.9 (±77.74) and 537.0 (±51.66). There was a significant difference in the duodenum villi length regardless of antibiotic inclusion into the diet. In the birds that did receive antibiotics, there was a significant difference (P<0.016) in the jejenum villi length with the control birds having the longest villi 725.7 (±90.92) while the birds infected with the higher level of Salmonella having the shortest villi 557.2 (±124.5). It would appear that using all of the information and results obtained for liver weights, broiler performance, serum biochemical level, lesion scoring, histopathology and villous morphological measurements should be used in conjunction with one another to measure the effect of Salmonella on the broiler chicken. The results obtained in this trial clearly show just how significant a problem Salmonella infection can be in the poultry industry due to seemingly healthy adult birds displaying little or no systemic disease being non-symptomatic carriers. Many of the Para-typhoid salmonellae do not always produce clinical signs in chicks, and their presence in the poultry industry may go unrecognised for this reason. / Dissertation (MSc(Agric))--University of Pretoria, 2011. / Animal and Wildlife Sciences / unrestricted

No description available.

The interaction of bacterial pathogens and the human host is a complex process that has shaped both organisms on a molecular, cellular and population level. When pathogenic bacteria infect the human body, a battle ensues between the host immune system and the pathogen. In order to escape an immune response and to colonize the host, pathogenic bacteria have developed diverse virulence strategies and some pathogens even replicate within host cells. For survival and propagation within the dynamic environment of a host cell, these bacteria interfere with the regulation of host pathways, such as the cell cycle, for their own benefit. The intracellular pathogen Salmonella Typhimurium invades eukaryotic cells and resides and replicates in a modified vacuolar compartment in which it is protected from the innate immune response. To this end, it employs a set of virulence factors that help to invade cells (SPI-1 effectors) and to hijack and modify the host endolysosomal system, in order to stabilize and mature its vacuolar niche (SPI-2 effectors). Previous studies have shown that Salmonella arrests host cells in G2/M phase and that Salmonella infected cells progress faster from G1 into S phase, suggesting that the G1 phase is disadvantageous for Salmonella infection. In fact, it has already been observed that Salmonella replication is impaired in G1 arrested cells. However, the reason for this impairment remained unclear. The current study addressed this question for the first time and revealed that the highly adapted, intracellular lifestyle of Salmonella is drastically altered upon G1 arrest of the host cell. It is shown that proteasomal degradation in G1 arrested cells is delayed and endolysosomal and autophagosomal trafficking is compromised. Accordingly, processing of lysosomal proteins is insufficient and lysosomal activity is decreased; resulting in uneven distribution and accumulation of endolysosomes and autophagosomes, containing undegraded cargo. The deregulation of these cellular signaling pathways affects maturation of the Salmonella containing vacuole (SCV). For the first time it is shown that acidification of SCVs is impaired upon G1 arrest. Thus, an important environmental factor for the switch from SPI-1 to SPI-2 gene expression is missing and the SPI-2 system is not activated. Consequently, targeting and modification of host cell structures by SPI-2 effectors e.g. recruitment of endolysosomal membrane proteins, like LAMP1, or exchange of endosomal cargo, is compromised. In addition, degradation of Salmonella SPI-1 effectors by the host proteasome is delayed. Their prolonged presence sustained the recruitment of early endosomes and contributed to the SCV remaining in an early, vulnerable maturation stage. Finally, it was shown that SCV membrane integrity is compromised; the early SCV ruptures and bacteria are released into the cytoplasm. Depending on the host cell type, SPI-2 independent, cytoplasmic replication is promoted. This might favor bacterial spreading, dissemination into the tissue and provide an advantage in host colonization. Overall, the present study establishes a link between host cell cycle regulation and the outcome of Salmonella infection. It fills the gap of knowledge as to why the host cell cycle stage is of critical importance for Salmonella infection and sheds light on a key aspect of host-pathogen interaction. / Die Interaktion zwischen bakteriellen Krankheitserregern und dem menschlichen Wirt ist ein komplexer Prozess, der beide Organismen auf molekularer, zellulärer und Populationsebene geprägt hat. Wenn pathogene Bakterien den menschlichen Körper infizieren, kommt es zu einem Kampf zwischen dem Immunsystem des Wirtes und dem Krankheitserregers. Um einer Immunantwort zu entgehen und den Wirt zu besiedeln, haben pathogene Bakterien diverse Strategien entwickelt und einige Erreger vermehren sich sogar innerhalb von Wirtszellen. Zum Überleben und zur Vermehrung innerhalb der dynamischen Umgebung einer Wirtszelle, manipulieren diese Bakterien die Regulation zellulärer Netzwerke, wie zum Beispiel den Zellzyklus, zu ihrem eigenen Vorteil. Salmonella Typhimurium, ein intrazelluläres Bakterium, dringt in eukaryotische Wirtszellen ein und vermehrt sich in einem modifizierten, vakuolären Kompartiment, welches gleichzeitig vor der angeboren Immunantwort des Wirtes schützt. Zu diesem Zweck entwickelten Salmonellen eine Reihe von Virulenzfaktoren. Diese sind zum einen für die Invasion von Zellen verantwortlich (SPI-1 Faktoren), zum anderen greifen sie das endolysosomale System der Wirtszelle an und modifizieren es, mit dem Ziel die intrazelluläre Salmonellen-enthaltende Vakuole (SCV) zu stabilisieren und reifen zu lassen (SPI-2 Faktoren). Frühere Studien haben gezeigt, dass Salmonellen ihre Wirtszellen in der G2/M Phase blockieren. Zudem gehen Salmonellen-infizierte Zellen schneller von der G1 in die S-Phase über, was auf einen Nachteil der G1-Phase für die Salmonelleninfektion hindeutet. In der Tat wurde bereits beobachtet, dass die Vermehrung von Salmonellen in G1-arretierten Zellen beeinträchtigt war. Der Grund für diese Beeinträchtigung blieb jedoch unklar. Die vorliegende Studie befasst sich zum ersten Mal mit dieser Frage und zeigt auf, dass der hoch angepasste, intrazelluläre Lebensstil von Salmonellen während des G1-Arrest der Wirtszelle dramatisch verändert wird. Im Rahmen der hier vorgelegten Arbeit wurde gezeigt, dass der proteasomale Abbau in G1-arretierten Zellen verzögert und die endolysosomalen und autophagosomalen Transportnetzwerke beeinträchtigt sind. Dementsprechend ist die Prozessierung lysosomaler Proteine unzulänglich und die lysosomale Aktivität herabgesetzt; was zu einer ungleichmäßigen Verteilung und Anreicherung von Endolysosomen und Autophagosomen führt, die nicht abgebaute Stoffwechselprodukte akkumulieren. Die Deregulierung der genannten zellulären Signalwege beeinflusst die Reifung der SCV. Es konnte hier zum ersten Mal gezeigt werden, dass die Ansäuerung der SCV in G1-arretierten Zellen inhibiert ist. Somit fehlt ein essentieller Faktor für den Wechsel von SPI-1 zu SPI-2-Genexpression und das SPI-2 System wird nicht aktiviert. Folglich findet keine Modifikation der Wirtszelle durch SPI-2-Effektoren, z.B. die Rekrutierung endolysosomaler Membranproteine, wie LAMP1 oder der Austausch endosomaler Fracht statt. Zudem ist der Abbau von bakteriellen SPI-1-Effektoren durch das Wirtsproteasom verzögert. Die verlängerte Präsenz der SPI-1 Effektoren fördert eine anhaltende Rekrutierung von frühen Endosomen und trägt zum Verbleib der SCV in einem frühen, sehr instabilen Reifestadium bei. Schließlich wurde gezeigt, dass die Integrität der SCV Membran kompromittiert ist, die Vakuole aufbricht und die Bakterien ins Zytoplasma entlassen werden. In Abhängigkeit des Wirtszelltyps wird eine SPI-2 unabhängige, zytoplasmatische Vermehrung begünstigt, was möglicherweise die Ausbreitung der Bakterien ins Gewebe erleichtert und somit einen Vorteil bei der Besiedelung des Wirtes darstellt. Insgesamt etabliert die vorliegende Studie einen Zusammenhang zwischen der Regulation des Wirtszellzyklus und dem Ergebnis einer Salmonelleninfektion. Es wird aufgezeigt, warum der Zellzyklus der Wirtszelle von entscheidender Bedeutung für den Verlauf der Salmonelleninfektion ist und beleuchtet somit einen essentiellen Aspekt der Wirt-Pathogen-Interaktion.

Small proteins, often defined as shorter than 50 amino acids, have been implicated in fundamental cellular processes. Despite this, they have been largely understudied throughout all domains of life, since their size often makes their identification and characterization challenging. This work addressed the knowledge gap surrounding small proteins with a focus on the model bacterial pathogen Salmonella Typhimurium. In a first step, new small proteins were identified with a combination of computational and experimental approaches. Infection-relevant datasets were then investigated with the updated Salmonella annotation to prioritize promising candidates involved in virulence. To implement the annotation of new small proteins, predictions from the algorithm sPepFinder were merged with those derived from Ribo-seq. These were added to the Salmonella annotation and used to (re)analyse different datasets. Information regarding expression during infection (dual RNA-seq) and requirement for virulence (TraDIS) was collected for each given coding sequence. In parallel, Grad-seq data were mined to identify small proteins engaged in intermolecular interactions. The combination of dual RNA-seq and TraDIS lead to the identification of small proteins with features of virulence factors, namely high intracellular induction and a virulence phenotype upon transposon insertion. As a proof of principle of the power of this approach in highlighting high confidence candidates, two small proteins were characterized in the context of Salmonella infection. MgrB, a known regulator of the PhoPQ two-component system, was shown to be essential for the infection of epithelial cells and macrophages, possibly via its stabilizing effect on flagella or by interacting with other sensor kinases of twocomponent systems. YjiS, so far uncharacterized in Salmonella, had an opposite role in infection, with its deletion rendering Salmonella hypervirulent. The mechanism underlying this, though still obscure, likely relies on the interaction with inner-membrane proteins. Overall, this work provides a global description of Salmonella small proteins in the context of infection with a combinatorial approach that expedites the identification of interesting candidates. Different high-throughput datasets available for a broad range of organisms can be analysed in a similar manner with a focus on small proteins. This will lead to the identification of key factors in the regulation of various processes, thus for example providing targets for the treatment of bacterial infections or, in the case of commensal bacteria, for the modulation of the microbiota composition. / Kleine Proteine, oft definiert als kürzer als 50 Aminosäuren, sind in fundamentale zelluläre Prozesse involviert. Trotzdem sind sie in allen Domänen des Lebens noch weitgehend unerforscht, da ihre Größe ihre Identifizierung und Charakterisierung oft schwierig macht. Diese Arbeit adressiert die Wissenslücke um kleine Proteine mit einem Fokus auf das bakterielle Modellpathogen Salmonella Typhimurium. In einem ersten Schritt wurden neue kleine Proteine mit einer Kombination aus bioinformatischen und experimentellen Ansätzen identifiziert. Anschließend wurden infektionsrelevante Datensätze mit der aktualisierten Salmonella-Annotation untersucht, um vielversprechende Kandidaten zu priorisieren, die an der Virulenz beteiligt sind. Um die Annotation neuer kleiner Proteine zu implementieren, wurden die Vorhersagen aus dem Algorithmus sPepFinder mit denen aus Ribo-seq kombiniert. Diese wurden der Salmonella-Annotation hinzugefügt und zur (Re-)Analyse verschiedener Datensätze verwendet. Für jede gegebene kodierende Sequenz wurden Informationen zur Expression während der Infektion (duale RNA-seq) und zum Beitrag zur Virulenz (TraDIS) gesammelt. Parallel dazu wurden Grad-seq-Daten ausgewertet, um kleine Proteine zu identifizieren, die an intermolekularen Interaktionen beteiligt sind. Die Kombination von dualer RNA-seq und TraDIS führte zur Identifizierung von kleinen Proteinen mit Merkmalen von Virulenzfaktoren, nämlich einer hohen intrazellulären Induktion und einem Virulenz-Phänotyp nach Transposon- Insertion. Als Beweis für die Leistungsfähigkeit dieses Ansatzes Identifikation von vielversprechenden Kandidaten wurden zwei kleine Proteine im Kontext einer Salmonella-Infektion charakterisiert. MgrB, ein bekannter Regulator des PhoPQ-Zweikomponentensystems, erwies sich als ein für die Infektion von Epithelzellen und Makrophagen essentielles Protein, möglicherweise über seine stabilisierende Wirkung von Flagellen oder durch Interaktion mit Sensorkinasen von Zweikomponentensystemen. YjiS, das in Salmonella bisher nicht charakterisiert wurde, hatte eine entgegengesetzte Rolle bei der Infektion, wobei seine Deletion Salmonella hypervirulent macht. Der Mechanismus, der dem zugrunde liegt, ist zwar noch unklar, beruht aber wahrscheinlich auf der Interaktion mit inneneren Membranproteinen. Insgesamt liefert diese Arbeit eine globale Beschreibung der kleinen Salmonella- Proteine im Kontext der Infektion mit einem kombinatorischen Ansatz, der die Identifizierung interessanter Kandidaten beschleunigt. Verschiedene Hochdurchsatz- Datensätze, die für ein breites Spektrum von Organismen verfügbar sind, können auf ähnliche Weise mit einem Fokus auf kleine Proteine analysiert werden. Dies wird zur Identifizierung von Schlüsselfaktoren in der Regulation verschiedener Prozesse führen und damit z. B. Targets für die Behandlung bakterieller Infektionen oder, im Falle kommensaler Bakterien, für die Modulation der Mikrobiota- Zusammensetzung liefern.