Bacteria have the ability to sense different environmental signals. When an environmental stress is detected, bacteria rapidly adjust their gene expression profile to be able to survive and thrive. The transduction of such environmental signals often requires the coordinated involvement of several factors that constitute complex regulatory networks. Hence, depending on the combination of signals, a unique gene expression profile required to adapt to the specific stress conditions is generated. Proteins are the best-known regulatory factors. However, non-proteinaceous molecules are also important in signal-responsive control of bacterial gene expression. Alarmones are low molecular weight non-proteinaceous regulatory factors which can characteristically be rapidly turned-over to mediate instant changes in gene expression. One such alarmone is the modified nucleotide ppGpp, which directly binds to RNA polymerase to alter its activity. The levels of this alarmone are expected to rapidly increase in response to any environmental stress that result in slow proliferation. DksA, a putative ppGpp co-regulator that likewise directly targets RNA polymerase, has been suggested to be required for both the positive and negative regulation mediated by ppGpp in Escherichia coli. This thesis describes dissection of the role of ppGpp and DksA on transcriptional regulation, primarily using the fim genetic determinant that encodes for the type 1 fimbriae. Type 1 fimbriae are involved in adhesion to abiotic surface and initial adhesion/invasion of bladder cells, as well as in biofilm formation. We found that ppGpp regulates phase variation by increasing the sub-population of cells that express the fimbriae. The effect of ppGpp was ultimately traced to its role in transcription of the fimB gene that encodes a recombinase involved in the phase variation process (paper 1). In contrast, we unexpectedly found that lack of DksA causes an increase, rather than a decrease, in transcription from the fimB P2 promoter in vivo. However, in vitro transcription studies demonstrated that ppGpp and DksA, both independently and co-dependently, stimulate transcription from the fimB P2 promoter. These seemingly contradictory results from the in vivo and in vitro transcriptional studies were shown to be, at least in part, a consequence of the increased association of Gre-factors with RNA polymerase that can occur in the absence of DksA in vivo (paper 2). The results outlined above have implications for the role of ppGpp and/or DksA in global gene expression. Using gene expression profile (microarray analysis) during the transition from logarithmic to stationary phase of E. coli, we found that while most of the genes regulated by ppGpp and DksA are regulated in the same direction by the two factors, many were not. In addition to the fim genes, genes involved in flagella functioning, taxis responses, and a few genes encoding different transport systems are also differentially regulated in ppGpp- and DksA-deficient strains in vivo. Our results clearly indicate that the effect of deficiencies in ppGpp and DksA is far more complex than phenotypic similarity of the corresponding mutants anticipated by the proposed concerted action of ppGpp and DksA on gene expression (paper 2 & 3).

Orientadores: Marcelo Brocchi, Gustavo Bueno Gregoracci / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-24T09:27:26Z (GMT). No. of bitstreams: 1 SartiSprogis_AdrianeCristina_M.pdf: 1852459 bytes, checksum: 397af9ee41d085346189ca76eee83f96 (MD5) Previous issue date: 2014 / Resumo: A salmonelose é uma zoonose que representa um sério problema de saúde pública mundial, devido: à sua alta prevalência, à dificuldade de seu controle, ao seu caráter endêmico, à morbidade e à mortalidade. O conhecimento da ocorrência das diferentes sorovariedades de S. enterica em diferentes regiões e países pode ajudar no rastreamento e reconhecimento de patógenos emergentes, e assim implementar políticas de tratamento e prevenção. A grande maioria das sorovariedades expressa dois tipos diferentes de antígenos flagelares codificados pelos genes fliC (fase 1) e fljB (fase 2), sendo assim denominadas bifásicas. Contudo, algumas sorovariedades expressam apenas uma das fases, e são denominadas monofásicas. É possível que a variação de fase flagelar em S. enterica esteja associada a uma função de escape do sistema imunológico, por aumentar o repertório de antígenos expressos pela célula bacteriana, evitando temporariamente a resposta imune celular. Assim sendo, S. enterica bifásicas possuiriam uma vantagem seletiva sobre as monofásicas, porém isso não é totalmente verificado nos estudos epidemiológicos, pois no Estado de São Paulo, S. enterica I,4,[5],12:i:-, (monofásica) é uma das mais comumente associadas aos casos de diarreia e/ou infecções sistêmicas em pacientes humanos. De fato, a partir da década de 1990 houve um aumento significativo da S. enterica I,4,[5],12:i:- em muitos países. Sequências de profagos são muito comuns em S. enterica, sendo de conhecimento que esses fagos codificam vários fatores que contribuem para patogenicidade, diversidade genética e/ou características que aumentam o fitness. Coculturas experimentais de linhagens de S. enterica podem induzir espontaneamente profagos, que matam bactérias sensíveis, e assim a indução espontânea de fagos em uma população lisogênica acentua a competitividade entre populações. Neste estudo foram analisadas culturas puras de S. enterica Enteritidis (bifásica) adicionadas de fagos líticos induzidos de S. enterica I,4,[5],12:i:-, bem como coculturas entre as duas sorovariedades citadas, nas quais foram observadas induções espontâneas de fagos associados à alta densidade populacional e alterações das taxas de crescimento em ambos os estudos, corroborando a hipótese de que S. enterica monofásica pode alterar a dinâmica populacional a seu favor, pela liberação de fagos líticos à outra sorovariedade, interferindo no crescimento populacional de S. enterica Enteritidis, e que o sucesso evolutivo de S. enterica I,4,[5],12.:i:- pode estar associado a fagos líticos atuando como um regulador na ecologia bacteriana. Esses dados podem mudar nosso conhecimento sobre a interação bactéria-fago de uma simples relação parasita-hospedeiro para uma coevolução de duas vias entre seus genomas / Abstract: Salmonellosis is a zoonosis that is a serious public health problem worldwide, due to its high prevalence, difficulty controlling, their endemicity, morbidity and mortality. The knowledge of the occurrence of different serovars of S. enterica in different regions and countries can help in tracking and recognition of emerging pathogens and thus implement policies for treatment and prevention. The majority of serovars express two different types of flagellar antigens encoded by genes: fliC (phase 1) and fljB (phase 2), so called biphasic. However, some serovars express only one of the phases and are termed monophasic. It is possible that flagellar phase variation of S. enterica is associated with an escape function of the immune system to increase the repertoire of antigens expressed by the bacterial cell temporarily preventing cellular immune response. Thus, S. enterica biphasic would have a selective advantage over monophasic, but this is not fully verified in epidemiologic studies, because in the State of São Paulo, S. enterica I,4,[5],12:i:-, (monophase) is the one most commonly associated with cases of diarrhea and/or systemic infections in human patients, in fact, from the 1990s there was a significant increase of S. enterica I,4,[5],12:i:- in many countries. Prophages sequences are very common in S. enterica, with the knowledge that these phages encode several factors that contribute to pathogenicity, genetic diversity and/or characteristics that increase fitness. Cocultures experimental strains of S. enterica prophages can induce spontaneous, killing susceptible bacteria, and thus the spontaneous induction in a population of lysogenic phage enhances the competitiveness between populations. This study analyzed pure cultures of S. enterica Enteritidis ( biphasic ) added lytic phage induced from S. enterica I,4,[5],12:i:-, as well as cocultures between the two serovars cited where inductions were observed spontaneous phage associated with high population density and changes in growth rates in both studies, supporting the hypothesis that S. enterica monophase can alter the population dynamics to their advantage by releasing lytic phage to another serovar, interfering with the population growth of S. enterica Enteritidis and the evolutionary success of S. enterica I,4,[5],12:i:- may be associated with lytic phages acting as a regulator in bacterial ecology. These data may change our understanding of bacteria- phage from a simple parasite-host coevolution for a two-way between their genomes / Mestrado / Clinica Medica / Mestra em Clínica Médica

No description available.

Haemophilus parainfluenzae (Hp) and H. influenzae (Hi) are closely related members of the Pasteurellaceae family and are common commensal bacteria of the human nasopharynx. Whilst Hi is frequently implicated in meningitis, otitis media and respiratory tract infections, reports of pathogenic behaviour by Hp are very rare. Lipopolysaccharide (LPS) is a key component of the Gram negative cell wall, and its structure influences the ability of Haemophilus to interact with the host and evade immune clearance. A better understanding of the differences in LPS structure between Hi and Hp could help to ascertain which parts of the molecule are important for commensal and pathogenic behaviour. Hi LPS comprises lipid A, a conserved oligosaccharide inner core, and an oligosaccharide outer core that differs between strains. The latter is partly phase variable by the slipped strand mispairing during replication of DNA repeat tracts within several LPS biosynthesis genes. Very little was known about LPS in Hp so we investigated its biosynthesis and structure in a panel of 20 Hp carriage isolates. Using PCR, DNA sequencing and Southern analysis we demonstrated that Hp possesses homologues of the Hi lipid A and inner core LPS synthesis genes and a few of the genes for outer core synthesis; however, homologues of the Hi phase variable outer core genes were largely absent and did not contain repeat tracts. The results of immunoblotting and collaborative structural analysis were consistent with this data. Phosphocholine, a phase variable Hi LPS epitope that has been implicated in otitis media, was found to be absent in Hp LPS due to the lack of four genes required for its biosynthesis and incorporation. The introduction of these genes into Hp led to the phase variable addition of phosphocholine to the LPS, indicating that there is no fundamental reason why Hp could not use a similar mechanism of variation to Hi if it was advantageous to do so. SDS-PAGE data suggested the presence of O-antigens (repeated chains of sugars) in many of the Hp strains, an unusual feature for Haemophilus, and all of the strains were found to contain a potential O-antigen synthesis locus. Each locus encodes homologues of several glycosyltransferases in addition to either the Wzy polymerase- or ABC transporter-dependent mechanisms of O-antigen synthesis and transport. Comparisons of wild type and isogenic mutant strains showed that the O-antigen enhances resistance to complement-mediated killing and appears to affect adhesion to epithelial cells in vitro. Hp is a successful commensal organism but lacks the flexibility of adapting its LPS using repeat-mediated phase variation, potentially limiting its range of host niches.