DNA is subject to attack by DNA damaging agents from both environmental and endogenous sources. In response to DNA damage, living organisms enhance expression of many related genes to facilitate DNA repair and survival. The SOS response is a well-understood prokaryotic regulatory cascade that controls the expression of more than 30 genes in response to DNA damage. However, in eukaryotic organisms from simple budding yeast to human, such a regulatory network has not been reported.<p>Previous research in our laboratory found that among DNA repair mutants of <i>Saccharomyces cerevisiae</i>, only rad6 and rad18 defective in the post-replication repair pathway significantly affected DNA damage induction of several genes examined. Rad6 and Rad18 form a ubiquitin conjugation-ligase complex and are required for the cellular tolerance to damaged DNA. Since the Rad6-Rad18 complex binds to single-stranded DNA, it may act as a DNA damage sensor required for the activation of DNA damage-induced transcription. We performed microarray analysis and found that the induction of up to 379 genes, including those involved in DNA repair, control of replication and transcription, regulation of the cell cycle and cell metabolism, are compromised in the rad6 and rad18 mutants. Although Rad6/Rad18 monoubiquitinates proliferating cell nuclear antigen (PCNA) following DNA damage to initiate a damage tolerance response, PCNA ubiquitination is not required for DNA damage induction. In budding yeast, cell-cycle checkpoints are involved in the control of DNA damage induction of gene expression through phosphorylation of a protein kinase Rad53 by two pathways represented by Rad24 and Sgs1. The Rad6-Rad18 complex appears to function in the Rad24 pathway and parallel to Sgs1. We further demonstrated that the Rad17 subunit of the 9-1-1 complex is subject to Rad6/Rad18- and DNA damage-dependent mono-ubiquitination and that the Rad17-Lys197 residue with flanking sequences homologous to Lys164 of PCNA is absolutely required for the DNA damage induction by Rad6-Rad18. Hence, by ubiquitinating two DNA clamps, PCNA and 9-1-1, the Rad6-Rad18 complex plays a central role in the cellular response to DNA damage by coordinating translesion synthesis, error-free bypass, homologous recombination, as well as transcriptional regulation, reminiscent of roles of RecA in <i>E. coli</i> cells.<p>Several individual genes have also been examined in this study to elucidate the regulatory mechanisms acting on specific DNA damage-inducible genes. In the microarray analysis, DDI2 and DDI3, two identical genes located in duplicated chromosomal regions, were identified due to the highest induction ratio (122-fold) after MMS treatment. Interestingly, DDI2/DDI3 can only be highly induced by SN2-type alkylating agents. Promoter deletion analysis mapped the putative upstream acting sequence (UASDDI2) responsible for 40% of basal expression and 90% of induced expression by MMS.<p>The CRT10 gene was identified through screening of the yeast deletion library for hydroxyurea (HU) resistance. CRT10 encodes a putative 957 amino acid, 110 kDa protein with a leucine repeat and a WD40 repeat near the N-terminus. Deletion of CRT10 resulted in an enhanced resistance to HU reminiscent of the inactivation of two other ribonucleotide reductase (Rnr) suppressors, CRT1 and SML1, which regulate Rnr activity at transcriptional and translational levels, respectively. Epistasis analysis indicates that CRT10 belongs to the CRT1 pathway but not the SML1 pathway. Indeed, deletion of CRT10 enhanced the survival of the mec1 null mutant and increased basal level and DNA damage-induced expression of RNR2 and RNR3, suggesting that Crt10 regulates RNR genes at the transcriptional level. Furthermore, the dun1 mutation is epistatic to crt10 with respect to both HU sensitivity and RNR gene expression. Interestingly, the expression of CRT10 itself is induced by DNA damaging agents and this induction requires DUN1, suggesting that CRT10 plays a role in cellular response to DNA damage and replication blocks. The CRT10 function appears to be achieved by positive regulation of the CRT1 transcript level, indicating that CRT10 is a component of the regulatory circuit.

DNA is subject to attack by DNA damaging agents from both environmental and endogenous sources. In response to DNA damage, living organisms enhance expression of many related genes to facilitate DNA repair and survival. The SOS response is a well-understood prokaryotic regulatory cascade that controls the expression of more than 30 genes in response to DNA damage. However, in eukaryotic organisms from simple budding yeast to human, such a regulatory network has not been reported.<p>Previous research in our laboratory found that among DNA repair mutants of <i>Saccharomyces cerevisiae</i>, only rad6 and rad18 defective in the post-replication repair pathway significantly affected DNA damage induction of several genes examined. Rad6 and Rad18 form a ubiquitin conjugation-ligase complex and are required for the cellular tolerance to damaged DNA. Since the Rad6-Rad18 complex binds to single-stranded DNA, it may act as a DNA damage sensor required for the activation of DNA damage-induced transcription. We performed microarray analysis and found that the induction of up to 379 genes, including those involved in DNA repair, control of replication and transcription, regulation of the cell cycle and cell metabolism, are compromised in the rad6 and rad18 mutants. Although Rad6/Rad18 monoubiquitinates proliferating cell nuclear antigen (PCNA) following DNA damage to initiate a damage tolerance response, PCNA ubiquitination is not required for DNA damage induction. In budding yeast, cell-cycle checkpoints are involved in the control of DNA damage induction of gene expression through phosphorylation of a protein kinase Rad53 by two pathways represented by Rad24 and Sgs1. The Rad6-Rad18 complex appears to function in the Rad24 pathway and parallel to Sgs1. We further demonstrated that the Rad17 subunit of the 9-1-1 complex is subject to Rad6/Rad18- and DNA damage-dependent mono-ubiquitination and that the Rad17-Lys197 residue with flanking sequences homologous to Lys164 of PCNA is absolutely required for the DNA damage induction by Rad6-Rad18. Hence, by ubiquitinating two DNA clamps, PCNA and 9-1-1, the Rad6-Rad18 complex plays a central role in the cellular response to DNA damage by coordinating translesion synthesis, error-free bypass, homologous recombination, as well as transcriptional regulation, reminiscent of roles of RecA in <i>E. coli</i> cells.<p>Several individual genes have also been examined in this study to elucidate the regulatory mechanisms acting on specific DNA damage-inducible genes. In the microarray analysis, DDI2 and DDI3, two identical genes located in duplicated chromosomal regions, were identified due to the highest induction ratio (122-fold) after MMS treatment. Interestingly, DDI2/DDI3 can only be highly induced by SN2-type alkylating agents. Promoter deletion analysis mapped the putative upstream acting sequence (UASDDI2) responsible for 40% of basal expression and 90% of induced expression by MMS.<p>The CRT10 gene was identified through screening of the yeast deletion library for hydroxyurea (HU) resistance. CRT10 encodes a putative 957 amino acid, 110 kDa protein with a leucine repeat and a WD40 repeat near the N-terminus. Deletion of CRT10 resulted in an enhanced resistance to HU reminiscent of the inactivation of two other ribonucleotide reductase (Rnr) suppressors, CRT1 and SML1, which regulate Rnr activity at transcriptional and translational levels, respectively. Epistasis analysis indicates that CRT10 belongs to the CRT1 pathway but not the SML1 pathway. Indeed, deletion of CRT10 enhanced the survival of the mec1 null mutant and increased basal level and DNA damage-induced expression of RNR2 and RNR3, suggesting that Crt10 regulates RNR genes at the transcriptional level. Furthermore, the dun1 mutation is epistatic to crt10 with respect to both HU sensitivity and RNR gene expression. Interestingly, the expression of CRT10 itself is induced by DNA damaging agents and this induction requires DUN1, suggesting that CRT10 plays a role in cellular response to DNA damage and replication blocks. The CRT10 function appears to be achieved by positive regulation of the CRT1 transcript level, indicating that CRT10 is a component of the regulatory circuit.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Escherichia coli enteroagregativa (EAEC) é um patógeno relacionado ao desenvolvimento de quadros de diarréia aguda ou persistente. A resposta inflamatória induzida por EAEC está relacionada à liberação de interleucina 8, que atua estimulando a transmigração de neutrófilos através do epitélio. Os macrófagos, de forma similar aos neutrófilos, são células fagocíticas que produzem espécies reativas de oxigênio (ERO), como o peróxido de hidrogênio (H2O2). Neste trabalho, avaliamos as consequências da interação de diferentes cepas clínicas com macrófagos humanos ativados da linhagem U-937. Todas as cepas testadas apresentaram filamentos nos testes de aderência aos macrófagos, diferentemente do que ocorre na interação com outras linhagens celulares como HEp-2, T84 e Caco-2. O ferro é um microelemento essencial para bactérias, sendo utilizado como cofator de enzimas e que também pode participar da geração de ERO através da reação de Fenton. Considerando-se a possibilidade de que o H2O2 produzido pelos macrófagos possa gerar radical hidroxil através da reação de Fenton, testes de aderência foram realizados com as amostras cultivadas na presença do captador de ferro 2,2-dipiridil. Tal fato não suprimiu a formação de filamentos, entretanto diminuiu a aderência das cepas EAEC 042 e 17-2. Com o objetivo de produzir uma resposta adaptativa ao H2O2, as culturas bacterianas foram pré-tratadas com uma dose sub-letal de H2O2 por 60 minutos antes de aderirem aos macrófagos. Nossos resultados mostraram que o pré-tratamento também não inibiu o aparecimento de filamentos em relação às culturas não tratadas. Além disto, foi observado que o pré-tratamento com o H2O2 reduziu a aderência das amostras de EAEC ao tapete celular. A filamentação é uma das respostas SOS, induzida pela presença de danos e/ou bloqueio na síntese da molécula de DNA. Com o objetivo de verificar se o H2O2 produzido pelos macrófagos estaria causando danos induzindo o sistema SOS e a filamentação bacteriana, foram realizados testes de viabilidade com mutantes derivados de E. coli K12 deficientes em enzimas do reparo por excisão de bases (BW535) e na resposta SOS (DM49). Nossos resultados mostram que os mutantes apresentaram os níveis de sobrevivência semelhantes ao observado para cepa selvagem isogênica (AB1157). Todos estes resultados em conjunto indicam que o H2O2 não é o indutor da filamentação nos testes de aderência. Macrófagos ativados apresentam ação microbicida através da ação da enzima indolamina dioxigenase (IDO), associada à redução do aminoácido L-triptofano. Desta forma, realizamos testes qualitativos de aderência de EAEC aos macrófagos suplementando o meio de interação com este aminoácido. Nossos resultados mostram que a adição de triptofano ao meio de interação reduz o número de filamentos por campo. Desta forma, aventamos a hipótese de que a depleção do triptofano seja responsável pela indução de resposta SOS, tendo como conseqüência a filamentação das bactérias. / Enteroaggregative Escherichia coli is a pathogen related to cases development of acute or persistent diarrhea. The inflammatory response induced by EAEC is linked to induction of IL-8 release, which acts stimulating neutrophils diapedesis through the epithelium. Macrophages, similarly to neutrophils, are phagocytic cells that produce reactive oxygen species (ROS), such as H2O2. Iron is an essential microelement to bacteria, been used as cofactor of enzymes in fundamental cellular processes but it is involved in ROS generation through Fenton reaction. On this report we evaluated the consequences of different EAEC strains interaction with activated human macrophages (U937 lineage). All tested strains showed filaments on the adherence tests with macrophages, unlike to what occurs in the interaction with other cellular lineages as HEp-2, T84 e Caco-2. Considering the possibility of H2O2 macrophage produced generates hydroxyl radical through Fenton reaction, the strains were grown in medium containing iron chelator 2,2-dipiridil. Iron chelation did not suppress filamentation, however decreased the adherence of 042 e 17-2 strains. Strains pretreated with a H2O2 subletal dose (60 M) by 60 minutes, which resulted in a bacterial adaptative response, also did not decrease the filamentation associated to adherence beside H2O2 pretreatment decreased the adherence of EAEC strains tested. In order to verify if H2O2 induces filamentation through DNA lesions and SOS induction, we evaluated the survival of a triple mutant deficient in three enzymes involved in BER and a mutant deficient in SOS response induction, both E. coli K12 derived. Both mutants presented similar survival levels like wild strain. This result suggest that H2O2 is not involved in SOS induction and filamentation response. Activated macrophages show microbicidal action, which is related to enzymes such as IDO (indoleamine dioxygenase), associated to reduction of L-tryptophan available to microorganism. In this way, we performed adhrence macrophages assays supplementing the interaction medium with L-tryptophan. Our results showed that tryptophan addition reduced the filamentation of adhered EAEC strains. Thus, we suggested that L-tryptophan reduction could be responsible for SOS response induction and bacterial filamentation.

Made available in DSpace on 2014-12-17T14:05:20Z (GMT). No. of bitstreams: 1 VivianeKSM_TESE.pdf: 2717769 bytes, checksum: dc3d156a1c1b3c1791c93a0e2266556f (MD5) Previous issue date: 2011-12-13 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Chromobacterium violaceum is a free-living bacillus, Gram-negative commonly found in water and sand of tropical and subtropical regions. One of its main characteristic it's the ability to produce the purple pigment named violacein, that shows countless biological activities. In 2003, the genome of this organism was totally sequenced and revealed important informations about the physiology of this bacteria. However, few post-genomics studies had been accomplished. This work evaluated the protein profile of C. violaceum cultivated in LB medium at 28?C that allowed the identification and characterization of proteins related to a possible secretion system that wasn't identified and characterized yet in C. violaceum, to the quorum sensing system, to regulatory process of transcription and translation, stress adaptation and biotechnological potential. Moreover, the response of the bacteria to UVC radiation was evaluated. The comparison of the protein profile, analyzed through 2-D electrophoresis, of the control group versus the treatment group allowed the identification of 52 proteins that arose after stress induction. The obtained results enable the elaboration of a stress response pathway in C. violaceum generated by the UVC light. This pathway, that seems to be a general stress response, involves the expression of proteins related to cellular division, purine and pirimidine metabolism, heat chock or chaperones, energy supply, regulation of biofilm formation, transport, regulation of lytic cycle of bacteriophages, besides proteins that show undefined function. Despite the response present similarities with the classic SOS response of E. coli, we still cannot assert that C. violaceum shows a SOS-like response, mainly due to the absence of characterization of a LexA-like protein in this organism / Chromobacterium violaceum ? um bacilo de vida-livre, Gram-negativo comumente encontrado no solo e nas ?guas de regi?es tropicais e subtropicais. Uma das principais caracter?sticas deste organismo ? sua capacidade de produzir o pigmento violace?na, o qual apresenta in?meras atividades biol?gicas. Em 2003, o genoma deste organismo foi completamente sequenciado e revelou informa??es importantes sobre a fisiologia desta bact?ria. Por?m, poucos estudos p?s-gen?micos tem sido realizados. Este trabalho avaliou o perfil proteico de C. violaceum cultivada em meio LB a 28?C, o que permitiu a identifica??o de prote?nas relacionadas a um poss?vel sistema de secre??o ainda n?o identificado e caracterizado em C. violaceum, ao sistema quorum sensing, a processos regulat?rios da transcri??o e tradu??o, adapta??o ao estresse e ao potencial biotecnol?gico. Al?m disso, a resposta desta bact?ria ? radia??o UVC foi avaliada. A compara??o do perfil prot?ico, analisado por eletroforese 2-D, do controle versus tratado possibilitou a identifica??o de 52 prote?nas que surgiram ap?s a indu??o do estresse. Os resultados obtidos permitiram a elabora??o de uma via de resposta de C. violaceum ao estresse gerado pela luz UVC. Esta via, que parece ser de resposta geral ao estresse, envolve a express?o de prote?nas relacionada ? divis?o celular, metabolismo de purinas e pirimidinas, choque t?rmico ou chaperonas, fornecimento de energia, regula??o da forma??o de biofilme, transporte, regula??o do ciclo l?tico de bacteri?fagos, al?m de prote?nas que ainda n?o apresentam fun??o caracterizada. Apesar da reposta apresentar similaridades com a SOS cl?ssica de E. coli, ainda n?o podemos afirmar que C. violaceum apresenta uma resposta SOS-like, principalmente devido a aus?ncia da caracteriza??o de um prote?na LexA-like neste organismo

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Escherichia coli enteroagregativa (EAEC) é um patógeno relacionado ao desenvolvimento de quadros de diarréia aguda ou persistente. A resposta inflamatória induzida por EAEC está relacionada à liberação de interleucina 8, que atua estimulando a transmigração de neutrófilos através do epitélio. Os macrófagos, de forma similar aos neutrófilos, são células fagocíticas que produzem espécies reativas de oxigênio (ERO), como o peróxido de hidrogênio (H2O2). Neste trabalho, avaliamos as consequências da interação de diferentes cepas clínicas com macrófagos humanos ativados da linhagem U-937. Todas as cepas testadas apresentaram filamentos nos testes de aderência aos macrófagos, diferentemente do que ocorre na interação com outras linhagens celulares como HEp-2, T84 e Caco-2. O ferro é um microelemento essencial para bactérias, sendo utilizado como cofator de enzimas e que também pode participar da geração de ERO através da reação de Fenton. Considerando-se a possibilidade de que o H2O2 produzido pelos macrófagos possa gerar radical hidroxil através da reação de Fenton, testes de aderência foram realizados com as amostras cultivadas na presença do captador de ferro 2,2-dipiridil. Tal fato não suprimiu a formação de filamentos, entretanto diminuiu a aderência das cepas EAEC 042 e 17-2. Com o objetivo de produzir uma resposta adaptativa ao H2O2, as culturas bacterianas foram pré-tratadas com uma dose sub-letal de H2O2 por 60 minutos antes de aderirem aos macrófagos. Nossos resultados mostraram que o pré-tratamento também não inibiu o aparecimento de filamentos em relação às culturas não tratadas. Além disto, foi observado que o pré-tratamento com o H2O2 reduziu a aderência das amostras de EAEC ao tapete celular. A filamentação é uma das respostas SOS, induzida pela presença de danos e/ou bloqueio na síntese da molécula de DNA. Com o objetivo de verificar se o H2O2 produzido pelos macrófagos estaria causando danos induzindo o sistema SOS e a filamentação bacteriana, foram realizados testes de viabilidade com mutantes derivados de E. coli K12 deficientes em enzimas do reparo por excisão de bases (BW535) e na resposta SOS (DM49). Nossos resultados mostram que os mutantes apresentaram os níveis de sobrevivência semelhantes ao observado para cepa selvagem isogênica (AB1157). Todos estes resultados em conjunto indicam que o H2O2 não é o indutor da filamentação nos testes de aderência. Macrófagos ativados apresentam ação microbicida através da ação da enzima indolamina dioxigenase (IDO), associada à redução do aminoácido L-triptofano. Desta forma, realizamos testes qualitativos de aderência de EAEC aos macrófagos suplementando o meio de interação com este aminoácido. Nossos resultados mostram que a adição de triptofano ao meio de interação reduz o número de filamentos por campo. Desta forma, aventamos a hipótese de que a depleção do triptofano seja responsável pela indução de resposta SOS, tendo como conseqüência a filamentação das bactérias. / Enteroaggregative Escherichia coli is a pathogen related to cases development of acute or persistent diarrhea. The inflammatory response induced by EAEC is linked to induction of IL-8 release, which acts stimulating neutrophils diapedesis through the epithelium. Macrophages, similarly to neutrophils, are phagocytic cells that produce reactive oxygen species (ROS), such as H2O2. Iron is an essential microelement to bacteria, been used as cofactor of enzymes in fundamental cellular processes but it is involved in ROS generation through Fenton reaction. On this report we evaluated the consequences of different EAEC strains interaction with activated human macrophages (U937 lineage). All tested strains showed filaments on the adherence tests with macrophages, unlike to what occurs in the interaction with other cellular lineages as HEp-2, T84 e Caco-2. Considering the possibility of H2O2 macrophage produced generates hydroxyl radical through Fenton reaction, the strains were grown in medium containing iron chelator 2,2-dipiridil. Iron chelation did not suppress filamentation, however decreased the adherence of 042 e 17-2 strains. Strains pretreated with a H2O2 subletal dose (60 M) by 60 minutes, which resulted in a bacterial adaptative response, also did not decrease the filamentation associated to adherence beside H2O2 pretreatment decreased the adherence of EAEC strains tested. In order to verify if H2O2 induces filamentation through DNA lesions and SOS induction, we evaluated the survival of a triple mutant deficient in three enzymes involved in BER and a mutant deficient in SOS response induction, both E. coli K12 derived. Both mutants presented similar survival levels like wild strain. This result suggest that H2O2 is not involved in SOS induction and filamentation response. Activated macrophages show microbicidal action, which is related to enzymes such as IDO (indoleamine dioxygenase), associated to reduction of L-tryptophan available to microorganism. In this way, we performed adhrence macrophages assays supplementing the interaction medium with L-tryptophan. Our results showed that tryptophan addition reduced the filamentation of adhered EAEC strains. Thus, we suggested that L-tryptophan reduction could be responsible for SOS response induction and bacterial filamentation.

O sistema SOS controla a expressão de diversos genes, muitos envolvidos com o reparo de DNA. Caulobacter crescentus vem emergindo como um modelo alternativo interessante para o estudo de mecanismos de reparo de DNA. Temos como objetivos realizar uma análise funcional de genes de função desconhecida regulados por SOS, e investigar a indução de SOS por antibióticos beta-lactâmicos em C. crescentus. Análises funcionais dos genes CC_3424 e CC_3467 mostraram que deleções nestes genes resultam em fenótipo de sensibilidade à mitomicina C (MMC). CC_3424 possui similaridade com glioxalases e CC_3467 com endonucleases. Acreditamos que CC_3467 atue no reparo de ligações intercadeia no DNA, e que CC_3424 atue detoxificando a MMC das células. Estudos dos efeitos biológicos da indução do sistema SOS mostram que a cefalexina (CFE) induz este regulon em concentrações subinibitórias. Células tratadas com CFE apresentam mais danos oxidativos do tipo 8-oxoguanina. Estes resultados mostram que concentrações subinibitórias de CFE resultam em estresse oxidativo em C. crescentus. / The SOS response controls the expression of several genes, many of which are involved in DNA repair mechanisms. Caulobacter crescentus has emerged as an alternative bacterial model for DNA repair. As aims, we will undertake a functional analysis of some of the genes regulated by the SOS response, and will investigate the SOS induction by beta-lactam antibiotics in C. crescentus. Functional analysis of the genes CC_3424 and CC_3467 showed that deletions in these genes result in a phenotype of sensitivity to mitomycin C (MMC). CC_3424 has similarity to glyoxalase and CC_3467 to endonucleases. We believe that the CC_3467 gene plays a role in the repair of interstrand crosslinks in the DNA, while CC_3424 acts in MMC cellular detoxification. Studies of biological effects of SOS induction showed that subinibitory concentrations of cephalexin (CFE) induce the SOS regulon. Cells treated with CFE have higher concentrations of 8-oxoG oxidative damage. These results show that subinibitory concentrations of cephalexin leads to cellular oxidative stress in C. crescentus.

L’émergence des déterminants de résistances plasmidiques aux quinolones (PMQR), auxquels appartient le gène qnrD, participent de manière significative à la sélection des résistances de haut-niveau aux permet les réparations de l’ADN lors des stress soumis aux bactéries, et d’autre part, que les aminosides, une autre classe d’antibiotiques que les fluoroquinolones, induisaient la réponse SOS chez Escherichia coli. En effet, nous avons montré que les petits plasmides-qnrD chez E. coli, induisent la formation de monoxyde de nitrogène et l’inhibition de la voie de détoxification Hmp-dépendante. Ces processus génèrent des lésions à l’ADN qui s’ajoutent à celles occasionnées par les aminosides concourant à activer la réponse SOS chez E. coli. L’ensemble de nos résultats montrent que l’émergence de la résistance aux fluoroquinolones peut être occasionnée par l’exposition d’E. coli à une autre classe d’antibiotiques, ici les aminosides. / The emerging plasmid-mediated quinolones resistance (PMQR) determinants significantly participate in the selection of high-level of resistance to the major antibiotics fluoroquinolones, leading to numerous clinical failures. In this study, we reported for the first time that PMQR expression could be triggered by the fluoroquinolones but also by another major class of antibiotics, the aminoglycosides. We were able to show that this unique cross selection of antibiotic resistance was the consequence of the PMQR determinant qnrD being SOS-regulated in a RecA-LexA dependent manner. We demonstrated that sub inhibitory concentration of aminoglycoside induced nitric oxide formation associated with the repression of the Hmp-mediated detoxification pathway, resulting in the induction of the SOS response and thus up-regulation of the PMQR. Overall, our findings revealed an unexpected antibiotic resistance cross-selection with low aminoglycosides concentrations promoting emergence of fluoroquinolones resistance.

Skin related injuries are some of the most dangerous forms of wounds. In addition to treating the wound itself, health care providers must be cautious of microbial infections. In this study, we evaluate a novel all-natural antimicrobial gel compound (AMG) designed to kill planktonic bacteria, penetrate bacterial biofilms, and accelerate wound healing. In -vitro experiments demonstrate that AMG is effective in inhibiting planktonic growth and biofilm development of eight common pathogens. LIVE/DEAD staining and confocal microscopy reveal that planktonic growth and three-dimensional structure of biofilms were significantly reduced. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) was used to investigate a small panel of genes (PrsA, Sprx) and showed potential targets for future study. A physiologically relevant wound model was created for treating S. aureus infections by using AMG alone or in combination with a common topical antibiotic, Mupirocin. AMG is a safe and effective treatment option for skin related infection.

Together with the current antibiotic resistance crisis, bacterial persistence appears to play an increasingly important role in the frequent failure of antibiotic treatments. Persister cells are rare bacteria that transiently become drug tolerant, allowing them to survive lethal concentrations of bactericidal antibiotics. Upon antibiotic removal, persister cells are able to resume growth and give rise to a new bacterial population as sensitive to the antibiotic as the original population. Interest in persister cells seriously increased in the past few years as these phenotypic variants were shown to be involved in the recalcitrance of chronic infections, such as tuberculosis and pneumonia and in the well-known biofilm tolerance to antibiotics. Persistence has therefore been extensively studied throughout the last decade, which led to the discovery of large variety of different molecular mechanisms involved in persisters formation. However, the specific physiology of bacterial persisters remains elusive up to now, mainly because of the transient nature and the low frequencies of persister cells in growing bacterial cultures. This work aims to gain a better understanding of the physiology of Escherichia coli persisters by combining population analyses with single-cell observations.In the first part of this thesis, we developed an experimental method allowing for measuring persistence with increased reproducibility. The method was further refined, which allowed us to observe four distinct phases in the ofloxacin time-kill curve, suggesting the existence of a tolerance continuum at the population level at treatment time. Characterization of these four phases notably revealed that the growth rate and the intrinsic antibiotic susceptibility of the strain define the number of surviving cells at the onset of the persistence phase, while persister cells survival mainly relies on active stress responses (SOS and stringent responses in particular).We next investigated the molecular mechanisms underlying the well-known correlation between persistence and the growth rate. Interestingly, we showed that the growth rate determines the number of survival cells at the onset of the persistent phase, whereas it does not affect the death rate of persister cells during antibiotic treatment. Furthermore, slow growth was shown to influence survival to ofloxacin independently of the replication rate, thereby suggesting that target inactivation solely cannot explain this correlation. However, our preliminary data indicate that ppGpp induction upon ofloxacin exposure substantially increases in slow growing bacterial populations, supporting a model in which slow growth would allow bacteria to respond faster to the antibiotic treatment, thereby generating more persisters than fast growing bacterial populations.Finally, both population and single-cell analyses were performed to assess the influence of the SOS response on persistence to ofloxacin. Firstly, population analyses revealed that the SOS response is required for survival of both sensitive and persister cells, but only during recovery, after ofloxacin removal, presumably allowing cells to induce SOS-dependent DNA repair pathways, required to deal with the accumulated ofloxacin-induced DNA lesions. The SOS response therefore appears as a good target for anti-persisters strategies, as shown by the 100-fold decrease in persistence upon co-treatment of a bacterial population with an SOS-inhibitor and ofloxacin. Secondly, single-cell analyses revealed that persister cells sustain similar DNA damages than sensitive cells upon ofloxacin treatment and induce SulA- and SOS-independent filamentation upon antibiotic removal, probably reflecting the presence of remaining cleaved complexes, formed during ofloxacin exposure. Importantly, we showed filamentation to occur in persister cells upon ampicillin treatment as well, thereby suggesting these filaments to be part of a more general survival pathway, which molecular basis remains unknown. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished