A pouca solubilidade do ferro impõe desafios para sua captação por bactérias e outros organismos. Uma solução eficaz para este problema é a utilização de sideróforos próprios ou exógenos para solubilizar o ferro do ambiente ou de proteínas do hospedeiro e transportá-lo para o interior da célula bacteriana. Neste trabalho, identificamos vias de produção e captação de ferro por sideróforos e definimos o papel destas moléculas na virulência da bactéria Chromobacterium violaceum, um abundante componente da microbiota de solo e água que ocasionalmente causa graves infecções em humanos. Por meio de análises in silico, vários genes relacionados com a síntese e captação de sideróforos foram encontrados no genoma de C. violaceum ATCC 12472 em dois clusters de síntese de metabólitos secundários. Obtenção de linhagens mutantes de vários destes genes e caracterização destas linhagens por ensaios de CAS, curvas de crescimento em carência de ferro e ensaios de estimulação de crescimento revelaram que C. violaceum produz sideróforos endógenos. Essa produção mostrou-se dependente do percursor comum 2,3-DHBA produzido pelas enzimas codificadas pelos genes entCEBA (CV_1485-84-83-82) e de duas enzimas sintetases de peptídeo não ribossomais (NRPSs CV_1486 e CV_2233), as quais provavelmente montam dois sideróforos distintos do tipo catecolato. Cada sideróforo foi captado por um receptor dependente de TonB (RDTB) específico, com o sideróforo produzido via NRPS CV_1486 sendo captado pelo RDTB CV_1491, e o sideróforo produzido via NRPS CV_2233 sendo captado pelo RDTB CV_2230, uma vez que mutantes sem esses RDTBs acumularam sideróforos no meio externo no ensaio de CAS. Além de seus sideróforos endógenos, C. violaceum foi capaz de utilizar xenosideróforos do tipo catecolato de outras bactérias via o RDTB CV_1491. Ensaios de infecção em camundongos revelaram que tanto a síntese quanto a captação de seus sideróforos endógenos são importantes para a virulência de C. violaceum, pois as linhagens mutantes que não produzem sideróforos (?CV_1485-84- 83-82, ?CV_1485-84-83-82/1486::pNPT e ?CV_1486/2233::pNPT) ou são incapazes8 de captá-los (?CV_2230/1491) tiveram sua virulência diminuída em relação a linhagem selvagem. Os dados mostrando que o mutante que não capta ambos sideróforos de C. violaceum teve atenuação mais acentuada da virulência e induziu menor produção de NET em ensaios com neutrófilos in vitro sugerem que o acúmulo de sideróforos na infecção pode ser benéfico para o hospedeiro. Por fim, demonstramos a possibilidade de gerar mutantes de transposon em C. violaceum e ao realizarmos varredura de uma coleção destes mutantes identificamos ao menos um potencial novo fator de transcrição envolvido na regulação da síntese de sideróforo nesta bactéria. Portanto, os dados obtidos neste trabalho revelaram que C. violaceum utiliza-se de diferentes sideróforos endógenos para captação de ferro e que estas moléculas são importantes para seu estabelecimento no hospedeiro. / The low solubility of iron imposes challenges for its uptake by bacteria and other organisms. An effective solution to this problem is the use of own or exogenous siderophores to solubilize the iron from environmental or host sources and transport it into the bacterial cell. In this work, we identified pathways for production and uptake of siderophores, and we defined the role of these molecules in virulence of the bacterium Chromobacterium violaceum, an abundant component of the microbiota of soil and water, which occasionally causes serious infections in humans. By performing an in silico analysis, we found several genes related with synthesis and uptake of siderophores in the genome of C. violaceum ATCC 12472 within two secondary metabolite biosynthesis gene clusters. Obtaining mutant strains from several of these genes and characterizing these strains by CAS assays, growth curves under iron deficiency and growth stimulation assays revealed that C. violaceum produces endogenous siderophores. This production was shown to be dependent on the common precursor 2,3-DHBA produced by the enzymes encoded by the genes entCEBA (CV_1485-84-83-82) and on two non-ribosomal peptide synthetase enzymes (NRPSs CV_1486 and CV_2233), which probably build two distinct catecholate siderophores. Each siderophore was picked up by a specific TonB-dependent receptor (RDTB), with the siderophore produced via NRPS CV_1486 being picked up by RDTB CV_1491, and the siderophore produced via NRPS CV_2233 being picked up by RDTB CV_2230, since mutants without those RDTBs accumulated siderophores in the external environment in the CAS assays. In addition to its endogenous siderophores, C. violaceum was able to use catecholate-type xenosiderophores from other bacteria via the RDTB CV_1491. Infection assays in mice revealed that both the synthesis and the uptake of its endogenous siderophores are important for the virulence of C. violaceum, since mutant strains that do not produce siderophores (?CV_1485-84-83- 82, ?CV_1485-84-83- 82/1486 :: pNPT and ?CV_1486/2233 :: pNPT) or are unable to uptake them (?CV_2230/1491) had their virulence decreased relative to the wild type strain. The data showing that the mutant strain unable to uptake both siderophores of10 C. violaceum had more pronounced attenuation of virulence and induced lower NET production in in vitro neutrophil assays suggest that the accumulation of siderophores in the infection may be beneficial to the host. Finally, we demonstrated the possibility of generating transposon mutants in C. violaceum, and by screening a collection of these mutants we identified at least one potential novel transcription factor involved in the regulation of siderophore synthesis in this bacterium. Therefore, the data obtained in this work revealed that C. violaceum uses different endogenous siderophores for iron uptake and that these molecules are important for its establishment in the host.

O grupo de estafilococos não-aureus (SNA), frequentemente isolados de quartos mamários com mastite subclínica, ápice do teto e ambiente, possue variabilidade ecológica que desafia a compreensão da patogenia a estes atribuída. Os fatores espécie-específicos associados à essa infecção ainda não foram identificados e a susceptibilidade difere entre vacas e quartos e promove diferentes perfis de infecção. Com o objetivo de avaliar a resistência à fagocitose e atividade microbicida, comparou-se a viabilidade, a produção intracelular de espécies reativas de oxigênio (ERO) e a fagocitose de neutrófilos sanguíneos de vacas primíparas e multíparas frente a distintos isolados viáveis de estafilococos. Utilizou-se doze vacas sadias (seis primíparas e seis multíparas) em terço médio de lactação e SO isolados viáveis de estafilococos (38 SNA e 12 Staphylococcus aureus) de diferentes nichos ecológicos. A viabilidade de neutrófilos (P = 0,55), produção de ERO (P = 0,12) e atividade funcional dos fagócitos (P = 0,33) foram semelhantes entre as primíparas e multíparas testadas . Contudo, foram observadas diferenças (P &le;0,05) entre os distintos grupos de espécies e estirpes de estafilococos quanto ao estímulo da produção intracelular de ERO pelos neutrófilos e à fagocitose. S. chromogenes de origens distintas, ápice do teto (P = 0,01), infecção intramamária transiente (P < 0,01) e infecção intramamárias persistente (P < 0,01) estimularam mais a produção de ERO pelos neutrófilos do que as outras espécies. Todos isolados foram fagocitados pelos neutrófilos, mas S. chromogenes resistiram mais eficientemente que as outras espécies de SNA, principalmente, S. chromogenes isolados do ápice do teto (P < 0,01). S. haemolyticus isolados do ápice do teta (P = 0,02) e infecção intramamária transiente (P < 0,01), assim como, S. fleurettii (P < 0,01), foram substancialmente fagocitados do mesmo modo que S. aureus isolado de suabe nasa\\ (P = 0,03). Mais evidente do que possíveis variações entre as respostas mamárias de primíparas e multíparas é a variação entre os SNA. Quanto mais adaptado à mama, maior resistência à fagocitose. / The group of non-aureus staphylococci (NAS), often isolated from mammary quarters with subclinical mastitis, teat apex and environment, has ecological variability that challenges the understanding of the pathogenesis attributed to them. The species-specific factors associated with this infection have not yet been identified and the susceptibility differs between cows and quarters and promotes different infection profiles. In order to evaluate the resistance to phagocytosis and I or microbicidal activity of these pathogens, the viability , intracellular production of reactive oxygen species (ROS) and blood neutrophil phagocytosis of primiparous and multiparous cows were compared to different viable isolates of staphylococci. Twelve healthy cows (six primiparous and six multiparous) were used in the middle third of lactation and 50 viable isolates of staphylococci (38 SNA and 12 Staphylococcus aureus) from different ecological niches. Neutrophil viability (P = 0.55), ROS production (P = 0.12) and phagocyte functional activity (P = 0.33) were similar among the primiparous and multiparous groups tested. However, differences (P &lt;0.05) between the different groups of species and strains of staphylococci were observed for the stimulation of intracellular ROS production by neutrophils and phagocytosis. S. chromogenes of different origins, ceiling apex (P =0.01), transient intramammary infection (P &lt;0.01) and persistent intramammary infection (P &lt;0 .01) further stimulated the production of ROS by neutrophils than species. All isolates were phagocytosed by neutrophils, but S. chromogenes resisted more efficiently than the other SNA species, especially S. chromogenes isolated from the apex of the ceiling (P &lt;0.01). S. haemolyticus isolated from apex to ceiling (P =0.02) and transient (P &lt;0.01) intramammary infection, as well as S. fleurettii (P &lt;0.01), were substantially phagocytosed in the same manner as S. aureus isolated from nasal swab (P = 0.03). More evident than possible variations between mammary responses of primiparous and multiparous is the variation between ANS. The more adapted to the breast, the greater resistance to phagocytosis.

L’ostréiculture française repose essentiellement sur l’élevage de l’huître creuse, Crassostrea gigas confronté cependant à des épisodes de mortalités anormales, touchant les différents stades de vie de l'huître. Plusieurs études ont démontré l’implication d’agents infectieux comme des bactéries du genre Vibrio dans ces mortalités. En France, V. aestuarianus est une bactérie connue depuis les années 2000 pour impacter la survie des huîtres. Sa fréquence de détection dans les cas de mortalités d’huîtres adultes analysés par le réseau REPAMO (REseau de PAthologie des Mollusques) est cependant en augmentation depuis 2011. Dans ce contexte, afin d’étudier le développement de la maladie induite par V. aestuarianus chez C. gigas, un modèle d’expérimentation par balnéation dans de l’eau de mer contenant des bactéries fraichement excrétées, au plus proche des modes de contaminations naturelles, a été développé. Le suivi de la présence de la souche 12/016 (souche virulente) et son mutant 12/016ΔvarS (souche non-virulente) dans l’eau de mer, dans les différents tissus et dans l’hémolymphe des animaux vivants et moribonds a montré que le cycle infectieux est constitué de I) une phase de pénétration rapide de la bactérie dans l’hôte (moins de 24h) et de colonisation initiale de l’hémolymphe et des branchies, II) une phase d’incubation de 3-4 jours au cours de laquelle la souche virulente se multiplie dans l'ensemble des tissus d'huître et III) une phase de mortalités aiguës (mort de l'animal par septicémie). A ce stade, le recrutement et la lyse hémocytaire ainsi que différentes lésions tissulaires comme la lyse du tissu conjonctif sous-épithélial au niveau du manteau et l’atrophie de diverticules digestives ont été observés. D'autre part, l'étude d’expression relative de 18 gènes de virulence connus chez d’autres Vibrion a montré que l’expression des facteurs de virulence de V. aestuarianus est régulée différemment au cours de différentes étapes de l'infection et nous avons observé que la métalloprotéase vam est significativement sur-exprimée dans l’hémolymphe des animaux contaminés à j4 post infection (étape intermédiaire de l’infection) par rapport à son niveau d’expression au premier jour de l’infection (étape précoce). / Oyster-farming in France is mainly based on pacific cupped oysters, Crassostrea gigas culture. Currently, oyster culture is confronted by several abnormal episodes of mass mortality affecting all life stages. These outbeaks involve, among other factors, infectious agents including bacteria of the genus Vibrio. In France, since 2000, V. aestuarianus is known as a bacterium that impacts the survival of C. gigas. Since 2011, its detection frequency in adult oyster mortalities cases reported by REPAMO network (REseau de PAthologie des Mollusques), is constantly increasing. In this context, to study V. aestuarianus disease development in C. gigas, an experimental infection model based on immersion in sea water containing freshly shed bacteria was developed. By monitoring the presence of strain 12/016 (virulent strain) and its mutant 12/016 ΔvarS (non-virulent strain) in the seawater, in the different tissues and in the haemolymph of live and moribund animals, we showed that the infectious cycle consists of several successive phases: I) rapid penetration of the bacterium into the host (less than 24 hours) and initial colonization of the haemolymph and gills, II) 3-4 days of incubation during which the virulent strain multiplies in whole oyster tissues and III) acute mortalities (animal death due to septicemia). At this stage, recruitment and haemocyte lysis as well as different tissue lesions such as lysis of the sub-epithelial connective tissue in the mantle and atrophy of digestive diverticula were observed. On the other hand, relative expression of 18 virulence genes (known in other Vibrion) were analyzed by RT-QPCR. Virulence factors are regulated differently during different stages of infection and vam metalloprotease is significantly over-expressed in the haemolymph of infected animals at day 4 post infection (intermediate stage of infection) compared to its level of expression at day 1 post infection (early stage).

Type IV pili (T4P) are bacterial biomolecular machines that mediate interactions with the environment. Bacterial pathogens such as Pseudomonas aeruginosa require T4P for virulence. Significant progress has been made in recent years towards our understanding of how the proteins in the T4P system interact and function. While over 50 different proteins are involved in T4P biogenesis, the two outer membrane components, PilF and PilQ, are the focus of the work presented in this thesis. PilF was found to be required for assembly of PilQ into secretins, the outer membrane channels through which T4P fibers exit the cell. The functions of PilF are consistent with a family of lipoproteins called pilotins, to which the roles of secretin assembly and/or localization are attributed. Structure determination by X-ray crystallography revealed that PilF is composed of six tetratricopeptide (TPR) protein-protein interaction motifs. Functional mapping of PilF indicated that a hydrophobic groove on the first TPR is involved in secretin assembly. Secretin localization correlated directly with that of PilF. The effects of pilF mutations and the structural data led to the hypothesis that PilF and PilQ interact directly. We propose that PilF and PilQ interact at the inner membrane and are co-transported to the outer membrane by the Lol lipoprotein sorting system. PilQ multimerizes into secretins upon outer membrane insertion and aligns with inner membrane T4P proteins to form a complete molecular machine. PilQ mutagenesis mapping showed that: the N-terminal “system specific” domain is important but not essential for secretin function; the central “multimerization” domain is critical for secretin assembly and function; and the C-terminal tail implicated in secretin-pilotin interactions is dispensable for PilQ function. Purified PilQ enabled copurification of PilF from cell lysates, providing the first evidence for their interaction. These data provide a framework for future exploration of T4P assembly in P. aeruginosa.

Type IV pili (T4P) are bacterial biomolecular machines that mediate interactions with the environment. Bacterial pathogens such as Pseudomonas aeruginosa require T4P for virulence. Significant progress has been made in recent years towards our understanding of how the proteins in the T4P system interact and function. While over 50 different proteins are involved in T4P biogenesis, the two outer membrane components, PilF and PilQ, are the focus of the work presented in this thesis. PilF was found to be required for assembly of PilQ into secretins, the outer membrane channels through which T4P fibers exit the cell. The functions of PilF are consistent with a family of lipoproteins called pilotins, to which the roles of secretin assembly and/or localization are attributed. Structure determination by X-ray crystallography revealed that PilF is composed of six tetratricopeptide (TPR) protein-protein interaction motifs. Functional mapping of PilF indicated that a hydrophobic groove on the first TPR is involved in secretin assembly. Secretin localization correlated directly with that of PilF. The effects of pilF mutations and the structural data led to the hypothesis that PilF and PilQ interact directly. We propose that PilF and PilQ interact at the inner membrane and are co-transported to the outer membrane by the Lol lipoprotein sorting system. PilQ multimerizes into secretins upon outer membrane insertion and aligns with inner membrane T4P proteins to form a complete molecular machine. PilQ mutagenesis mapping showed that: the N-terminal “system specific” domain is important but not essential for secretin function; the central “multimerization” domain is critical for secretin assembly and function; and the C-terminal tail implicated in secretin-pilotin interactions is dispensable for PilQ function. Purified PilQ enabled copurification of PilF from cell lysates, providing the first evidence for their interaction. These data provide a framework for future exploration of T4P assembly in P. aeruginosa.

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 5 uppsatser.

Submitted by DIEGO FELIPE ALVES BATISTA null (diegofelipe_vet@hotmail.com) on 2017-07-23T15:53:00Z No. of bitstreams: 1 Tese final.pdf: 4005234 bytes, checksum: 457b822652d4193c9c8e25953f4d3dc1 (MD5) / Rejected by Luiz Galeffi (luizgaleffi@gmail.com), reason: Solicitamos que realize uma nova submissão seguindo a orientação abaixo: Incluir o número do processo de financiamento FAPESP nos agradecimentos da dissertação/tese. Corrija esta informação e realize uma nova submissão com o arquivo correto. Agradecemos a compreensão. on 2017-07-26T13:34:20Z (GMT) / Submitted by DIEGO FELIPE ALVES BATISTA null (diegofelipe_vet@hotmail.com) on 2017-07-26T14:07:28Z No. of bitstreams: 1 Tese_Diego_Felipe_Alves_Batista.pdf: 4004591 bytes, checksum: 1de74c2da3ba5ba3e56c6bcf6f9ba6f2 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-07-26T19:26:28Z (GMT) No. of bitstreams: 1 batista_dfa_dr_jabo.pdf: 4004591 bytes, checksum: 1de74c2da3ba5ba3e56c6bcf6f9ba6f2 (MD5) / Made available in DSpace on 2017-07-26T19:26:28Z (GMT). No. of bitstreams: 1 batista_dfa_dr_jabo.pdf: 4004591 bytes, checksum: 1de74c2da3ba5ba3e56c6bcf6f9ba6f2 (MD5) Previous issue date: 2017-07-04 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / O tifo aviário, causado por Salmonella Gallinarum biotipo Gallinarum, é uma infecção caracterizada pela alta mortalidade nos lotes de aves suscetíveis acometidos, enquanto S. Gallinarum biotipo Pullorum, o agente da pulorose, infecta as aves de produção industrial com as quais desenvolve relação mais branda. Ainda é escasso o conhecimento sobre os mecanismos moleculares que sustentam essas diferentes interações patógeno-hospedeiro. Nesse estudo, objetivou-se investigar o efeito de deleção parcial das sequências codificantes dos genes idnT (transportador de L-idonato ou D-gluconato), idnO (5-cetogluconato redutase) e ccmH (heme liase necessária na montagem de citocromos do tipo C) sobre a patogenicidade de S. Gallinarum 287/91 (SG287/91), uma vez que seus ortólogos são pseudogenes conservados em S. Pullorum. Os clones mutantes SG∆idnTO, SG∆ccmH e SG∆ccmHidnTO foram obtidos por meio da técnica de mutação sítio-dirigida, denominada de recombinação Lambda-Red e testados em dois experimentos independentes com aves comerciais semipesadas de postura suscetíveis ao tifo aviário. No 1º experimento não se observou alteração da patogenicidade dos clones mutantes após inoculação oral, pois todos os animais infectados desenvolveram sinais clínicos típicos do tifo aviário e vieram a óbito ao longo de 12 dias pós-infecção (dpi). Apesar dos 100% de mortalidade, as infecções desenvolvidas pelos clones SG∆idnTO e SG∆ccmHidnTO levaram os animais a óbito dentro de 48 horas desde o aparecimento dos sinais clínicos, enquanto SG287/91 o fez em 6 dias, sugerindo aumento da virulência dos clones mutantes. No 2º experimento observou-se que as mutantes invadiram o hospedeiro a partir do intestino, embora as quantidades recuperadas de SG∆idnTO e SG∆ccmHidnTO nos fígados e de SG∆idnTO nos baços, no 5º dpi, foram superiores a de SG287/91, reforçando a hipótese de aumento da virulência dos clones contendo a alteração idnTO. Apesar disso, os níveis de transcrição das citocinas CXCLi2 e IL6 produzidos à infecção por SG∆idnTO e SG∆ccmHidnTO não diferiram nas tonsilas cecais nos 1º e 3º dpi e nos baços no 3º dpi em relação à infecção por SG287/91. Somente SG∆ccmH inclinou-se a estimular a transcrição de CXCLi2 e IL6 nas tonsilas cecais no 1° dpi em relação ao grupo controle, enquanto SG287/91 tendeu a suprimi-la. Porém, não houve suporte estatístico para essa observação. Os níveis de mRNA do IFNγ estavam aumentados para todas as estirpes de S. Gallinarum, mutantes ou não, porém sem diferença estatística entre eles. Os resultados do presente estudo indicam que a ruptura nos genes idnTO, e em menor grau do gene ccmH, poderiam levar a perda de “fitness” em S. Gallinarum, lhes justificando a permanência no genoma desse micro-organismo, ao contrário do que ocorre com S. Pullorum. O estudo da patogenicidade de estirpe de S. Pullorum tendo reconstituídos os genes idnTO e ccmH no seu genoma poderia esclarecer os motivos pelos quais esses foram negativamente selecionados por esse micro-organismo. / Fowl typhoid, caused by Salmonella Gallinarum biovar Gallinarum, is an infectious disease which elicits high mortality into a flock of susceptible birds whereas S. Gallinarum biovar Pullorum, the aetiological agent of pullorum disease, infects poultry of commercial importance with which such a bacterium sets off a more permissive host-pathogen interaction. Little is known about the molecular mechanisms driving these distinct interplays with the host. Herein, we aimed at investigating the effect of partial deletions in the idnT (L-idonate / D-gluconate transporter), idnO (5-ketogluconase reductase) and ccmH (heme liase involved in the c-type cytochrome maturation) coding sequences on S. Gallinarum 287/91 (SG287/91) pathogenicity since they are conserved pseudogenes in S. Pullorum genomes. SG∆idnTO, SG∆ccmH and SG∆ccmHidnTO mutant strains were constructed through a one-step inactivation technique, known as Lambda-Red-mediated recombination, and tested on two independent experiments by using a commercial brown egg-producing layer line susceptible to fowl typhoid. On the experiment 1, no changing was observed in the pathogenicity of the mutant strains upon oral inoculation as the infected animals developed typical fowl typhoid clinical signs and died along 12 days post-infection (dpi). In spite of causing 100% mortality, SG∆idnTO and SG∆ccmHidnTO killed all the animals within 48 hours since the clinical signs appearance while SG287/91 did so in 6 days, indicating an increased virulence by these mutant strains. On the experiment 2 every mutant strain were able to invade the host system from the intestine albeit SG∆idnTO and SG∆ccmHidnTO were recovered from livers and SG∆idnTO alone from spleens at higher numbers than was SG287/91, supporting the hypothesis of increased virulence for those clones harbouring the idnTO mutation. Despite the results above, CXCLi2 and IL6 transcription levels during infection by SG∆idnTO and SG∆ccmHidnTO were similar to that induced by SG287/91 in caecal tonsils at 1 and 3 dpi and in spleens at 3 dpi. In contrast, SG∆ccmH trended to stimulate CXCLi2 and IL6 transcription in caecal tonsils at 1 dpi when compared to the negative, control group whereas SG287/91 tended to suppress it, but no statistical significance was found for such an observation. IFNγ mRNA were augmented for all S. Gallinarum strains, mutant or not, but without statistical difference amongst them. These findings indicate that gene decay into idnTO, and at a lesser extent, into ccmH sequences might lead to the loss of fitness by S. Gallinarum, raising an explanation for their maintenance on this bacterium chromosome when the opposite happens to S. Pullorum. Studying the pathogenicity of a S. Pullorum strain possessing both the idnTO and ccmH genes in its genome could bring to light the reasons whereby such genes were negatively selected by this microorganism. / FAPESP: 2013/22920-4 / FAPESP: 2013/26127-7

Os fatores de transcrição da família MarR atuam como sensores diretos de sinais intracelulares e regulam vários processos em bactérias, incluindo virulência e degradação de compostos aromáticos. Neste trabalho, identificamos de modo global os fatores de transcrição da família MarR envolvidos na virulência do patógeno oportunista de humanos Chromobacterium violaceum. Usando mutagênese por troca alélica, geramos mutantes nulos não polares para doze dos quinze reguladores da família MarR encontrados no genoma de C. violaceum. Em ensaios de virulência, quando injetados por via intraperitoneal em camundongos BALB/c, os mutantes ?CV_0210 (?ohrR), ?CV_0577 e ?CV_2726 foram menos virulentos, enquanto o mutante ?CV_1776 foi mais virulento, quando comparados à linhagem selvagem. Para os demais nove mutantes MarR não houve diferença na virulência. Para definir o regulon de alguns destes reguladores da família MarR, os perfis de expressão gênica foram determinados por ensaios de microarranjo de DNA e Northern blot para as linhagens mutantes ?CV_0210 (?ohrR), ?CV_1776, ?CV_1810 e ?CV_2726, para a linhagem selvagem superexpressando CV_2726 e para a linhagem selvagem em estresse oxidativo com hidroperóxido de cumeno (CHP). O regulon do repressor CV_1810 compreendeu dois operons divergentes, que codificam enzimas que possivelmente metabolizam compostos aromáticos, mas produtos do catabolismo destes compostos não funcionaram como ligantes capazes de antagonizar a repressão de CV_1810 no gene CV_1801. O regulon do ativador CV_2726, definido como quatorze genes comuns diferencialmente expressos em ensaios na ausência e na condição de superexpressão do gene CV_2726, revelou poucos genes (cstA) com potencial de estar envolvidos no fenótipo de menor virulência do mutante ?CV_2726. Os reguladores CV_0577 e CV_1776 foram alocados na subfamília UrtR de resposta a urato e provavelmente influenciam a virulência de C. violaceum com regulons sobrepostos. O regulon de CV_1776 abrangeu dezenas de genes, muitos deles relacionados ao catabolismo de aminoácidos, mas há poucos candidatos a fatores de 10 virulência clássicos (pecM, escU). Alguns genes do catabolismo/utilização de purinas (CV_0578 e CV_3771) foram regulados tanto por CV_1776 quanto por CV_0577 e responderam a presença de urato. O perfil transcricional da resposta adaptativa de C. violaceum a CHP, um ligante que oxida o regulador OhrR, revelou aumento na expressão de genes relacionados à detoxificação de peróxidos (enzimas antioxidantes e sistemas redutores de tiol), degradação da porção aromática do CHP (oxigenases) e proteção contra estresses secundários (reparo de DNA, choque térmico, limitação de ferro e nitrogênio). O regulon de OhrR revelou-se pequeno, incluindo dois genes com expressão aumentada, CV_0209 (ohrA) e CV_0208 (possível diguanilato ciclase), e três genes com expressão diminuída (hemolisina, quitinase e colagenase) no mutante ?ohrR. Assim, a virulência atenuada do mutante ?ohrR deve estar relacionada ao aumento da produção do segundo mensageiro cíclico di-GMP (c-diGMP) e à diminuição da expressão de enzimas degradativas extracelulares. Em conclusão, definimos a resposta transcricional à CHP, identificamos potenciais fatores de virulência, como a diguanilato ciclase, no regulon OhrR, e mostramos que C. violaceum utiliza os fatores de transcrição da família MarR CV_0577, CV_1776, CV_2726 e OhrR para modular sua virulência. / Transcription factors belonging to the MarR family act as direct intracellular sensors of signals and control many processes in bacteria, including virulence and degradation of aromatic compounds. In this work, we identify and characterize MarR family transcription factors controlling virulence in Chromobacterium violaceum, an opportunistic pathogen of humans. Using allelic exchange mutagenesis, we generate non-polar null mutants for twelve of the fifteen MarR family regulators found in the C. violaceum genome. In virulence tests, when introduced by intraperitoneal injection in BALB/c mice, the ?CV_0210 (?ohrR), ?CV_0577 and ?CV_2726 mutant strains were less virulent, while the ?CV_1776 was more virulent, when compared to the wild-type strain. The other nine MarR mutants showed no difference in virulence tests. To define the regulon of some MarR family transcription factors, the gene expression profiles were determined by DNA microarray analysis and Northern blot assays for the ?CV_0210 (?ohrR), ?CV_1776, ?CV_1810 and ?CV_2726 mutant strains, for the wild-type strain overexpressing CV_2726 and for the wild-type strain exposed to oxidative stress generated by cumene hydroperoxide (CHP). The CV_1810 is a repressor of a regulon that comprised two divergent operons encoding enzymes that possibly metabolize aromatic compounds, but catabolic products of these compounds did not function as ligands capable of antagonizing the repression of CV_1810 on the CV_1801 gene. The regulon of the activator CV_2726, defined as fourteen differentially expressed genes commonly found in assays in the absence and overexpression of the CV_2726 gene, revealed few genes (cstA) with potential to be involved in the phenotype of lower virulence of the ?CV_2726 mutant strain. Regulators CV_0577 and CV_1776 were allocated in the urate-responsive UrtR subfamily and probably afect the virulence of C. violaceum with overlapping regulons. The CV_1776 regulon contains dozens of genes, many of them related to amino acid catabolism, but there are few candidates for classical virulence factors (pecM, escU). Some genes related to catabolism/utilization of purine (CV_0578 and CV_3771) were 12 regulated by both CV_1776 and CV_0577 and responded to the presence of urate. The transcriptional profile of the adaptive response of C. violaceum to CHP, a ligand that oxidizes the OhrR regulator, revealed the upregulation of genes related to the detoxification of peroxides (antioxidant enzymes and thiol-reducing systems), degradation of the aromatic moiety of CHP (oxygenases), and protection against other secondary stresses (DNA repair, heat shock, iron limitation, and nitrogen starvation responses). The OhrR regulon was shown to be small, including two upregulated genes, CV_0209 (ohrA) and CV_0208 (putative diguanylate cyclase), and three downregulated genes (hemolysin, chitinase, and collagenase) in the ?ohrR mutant. Thus, the attenuated virulence of the ?ohrR mutant might be related to the increased production of the second messenger cyclic di-GMP (c-di-GMP) and the decreased expression of extracellular enzymes required for tissue dissemination, in this mutant strain. In conclusion, we have defined the transcriptional response to CHP, identified potential virulence factors such as diguanylate cyclase as members of the OhrR regulon, and shown that C. violaceum uses the transcription factors of the MarR family CV_0577, CV_1776, CV_2726 and OhrR to modulate its virulence.

Chapter -I Introduction Salmonella is a pathogen well-known for its ability to infect a wide variety of hosts and causes disease ranging from mild gastroenteritis to typhoid fever. During infection, it is exposed to a myriad of conditions; from the aquatic environment, the gut lumen to the phagolysosome. The success of Salmonella as a pathogen lies in its ability to sense each of these environments and adapt itself for survival and proliferation accordingly. This is done mainly via the action of specific two-component systems (TCSs) which sense cues specific to each of these niches and trigger the appropriate transcriptional reprogramming. This reprogramming is best studied for the genes directly known to be involved in virulence. In the case of Salmonella, most of these genes are a part of specific clusters, acquired through horizontal gene transfer, known as Salmonella Pathogenicity Islands (SPIs). Of the various SPIs, the two most important are SPI-1 and SPI-2. SPI-1 is classically involved in orchestrating bacterial invasion of non-phagocytic cells in the gut, allowing the pathogen to invade the host. Furthermore, its role is well characterized in the classic inflammation associated with gastroenteritis. On the other hand, SPI-2 is specialized for survival within the harsh intracellular environment of host cells such as macrophages and epithelial cells. Other important virulence determinants include motility, chemotaxis as well as adhesins. The transcription of these virulence genes is under tight regulation and responsive to environmental conditions. Many small molecules such as short chain fatty acids, pp(p)Gpp, bile and acyl homoserine lactones among others are known to be potent regulators of virulence in Salmonella. Furthermore, the metabolic products of the normal flora in the gut also affect its virulence. Thus the metabolic status, of both the host as well as the pathogen, plays an important role in determining the outcome of the infection. Many metabolic enzymes and their products are now known to directly or indirectly affect virulence gene expression. In this study, we explore one such class of metabolic enzymes viz amino acid racemases. They catalyze the chiral conversion of L-amino acids to D-amino acids and vice versa. We have studied the biochemical properties of two such non-canonical racemases as well as their role in bacterial survival and pathogenesis. Chapter-II Identification and characterization of putative aspartate racemases in Salmonella Amino acid racemases, such as alanine and glutamate racemases, are ubiquitously found in all bacteria and they play an essential role in cell wall biosynthesis. Recently it has been found, that bacteria possess other amino acid racemases which produce non-canonical D-amino acids. These D-amino acids, upon secretion, further orchestrate various phenotypes such as cell wall remodeling and biofilm dispersal. In this study, we have explored the ability of Salmonella to produce such non-canonical D-amino acids. The genome of S. Typhimurium possesses genes encoding two putative aspartate racemases; ygeA and aspR. These genes were maximally expressed in mid-log phase of bacterial growth and their corresponding proteins ar localized in the outer membrane of the bacterium. The biochemical characterization of the proteins YgeA and AspR revealed that only the latter is catalytically active under in vitro conditions. AspR could catalyze the conversion of L-Aspartate to D-Aspartate and vice versa, however was unable to use any other amino acid as its substrate. With atleast one of the racemases showing catalytic activity, the profiling of the secreted D-amino acids in Salmonella conditioned medium was undertaken using LC-MS. It was observed that the bacterium actively secreted specific D-amino acids such as D-Ala and D-Met into the culture medium in a growth-phase dependent manner. Furthermore, analysis of the secreted D-amino acid profile of the strains lacking either one or both the racemases revealed that atleast a subset of the secreted D-amino acids were dependent on the activity of YgeA and AspR. Thus, D-amino acids secreted by S. Typhimurium might represent a novel class of signaling molecules. Chapter – III Role of aspartate racemases in growth and survival of S. Typhimurium In order to understand the role of ygeA and aspR in vivo, we created knockouts of these genes (both single as well as double knockout) in S. Typhimurium using λ Red recombinase strategy. These knockouts were then assessed for their growth and morphology. The aspartate racemase knockouts behave similar to the wild type during growth in LB as well as M9 minimal medium. While their gross morphology remained the same as the wild type, the size distribution of the racemase knockouts was slightly different in the stationary phase. Unlike the wild type bacteria, the mutants did not exhibit the characteristic reduction in cell size upon entry into stationary phase. In addition, the survival of the mutants in the presence of cell wall damaging agents such as bile and Triton-X 100 was compromised as compared to the wild type. This can be ascribed to changes in the cell wall of the bacterium, wherein the mutants accumulated peptidoglycan in the stationary phase of growth. This suggests that aspartate racemases might have an effect on cell wall biosynthesis in Salmonella in the stationary phase. Another important strategy employed by bacteria to survive in stress conditions is biofilm formation. It was seen that the mutants were compromised in their ability to form a biofilm at the liquid-air interface in vitro. This defect is due to a transcriptional downregulation of the genes required for biofilm formation. These results demonstrate that, contrary to the established inhibitory effects of D-amino acids on biofilms of various bacteria, the aspartate racemases appear to act as positive regulators of biofilm formation in Salmonella. Chapter – IV Involvement of aspartate racemases in the regulation of Salmonella pathogenesis Salmonella’s success as a pathogen can be broadly assessed by its ability to invade and replicate within two major cell types: epithelial cells and macrophage-like cells. We have studied the fate of the aspartate racemase knockout strains in both these cell types. While the mutants replicate as well as the wild type in macrophage cell lines, their ability to invade epithelial cell lines is highly compromised. This defect can be ascribed to the downregulation of the Salmonella Pathogenicity Island-1 (SPI-1) in the racemase knockouts at the transcriptional level. One of the major pathways that regulate SPI-1 activation is the flagellar pathway. It was observed that in addition to SPI-1, the motility of the racemase mutants was also highly compromised. The mutants did not possess any flagella and showed a high transcriptional downregulation of all the three classes of flagellar genes. Transcriptome analysis revealed a global reprogramming in the aspartate racemase mutants, resulting in the differential regulation of motility, adhesion, amino acid transport, cell wall biosynthesis and other pathways. Of the genes upregulated in the knockouts, FimZ is known for its negative effect on motility and might be responsible for the observed downregulation of the flagellar regulon. This suggests that ygeA and aspR might be repressors of fimbrial gene expression. In totality, the racemases affected the pathogenesis of Salmonella, where the double knockout was severely compromised in the colitis model of infection. Overall the study is the first to identify secretion of non-canonical D-amino acids by Salmonella and suggests that YgeA and AspR might be the source of the same. This is supported in part by in vitro studies with the purified proteins. Studies in vivo further highlight the possible substrates that might be utilized by these enzymes. Physiologically, the aspartate racemases appear to regulate cell wall remodeling and biofilm formation. In contrast to the established literature, aspartate racemases (and their possible D-amino acid products) seem to be essential for formation of biofilms and regulate this phenotype at the transcriptional level. Furthermore, our studies put forth aspartate racemases as novel positive regulators of Flagella and SPI-1, affecting the success of Salmonella in the colitis model of infection in mice. Transcriptome analysis hints at the pleiotropic effects of aspartate racemases in Salmonella, bringing forth hitherto unexplored roles for this class of enzymes in the biology of this pathogen.