Due to the emergence and dissemination of multidrug resistance, bacterial pathogens have been causing a serious public health problem in recent years. To address the existing drug resistant problem, there is an urgent need to find new antimicrobials, especially those against drug-resistant bacteria. SecA is the central component of Sec-dependent secretion pathway, which is responsible for the secretion of many essential proteins as well as many toxins and virulence factors. Two SecA homologues are indentified in some important Gram-positive pathogens. SecA1 is involved in general secretion pathway and essential for viability, whereas SecA2 contribute to secretion of specific virulence factors. The high conservation among a wide range of bacteria and no human counterpart make SecA homologues attractive targets for exploring novel antimicrobials. We hypothesize that inhibition of these SecA homologues could reduce virulence, inhibit bacteria growth, and kill bacteria. SecA1 and SecA2 from four different species were cloned, purified, and characterized. All these SecA homologues show ATPase activities, thus screening ATPase inhibitors might help to develop new antimicrobials. In this study, three structurally different classes of SecA inhibitors were developed and optimized: 1) Rose Bengal (RB) and RB analogs derived from systematical dissection RB and Structure-Activity relationship (SAR) study; 2) pyrimidine analogs derived from virtual screening based on the ATP binding pocket of EcSecA and SAR study; and 3) bistriazole analogs derived from random screening and SAR study. Several potent SecA inhibitors show promising enzymatic inhibition against SecA homologues as well as bacteriostatic and bactericidal effects. Two major efflux pumps of S. aureus, NorA and MepA, have little negative effect on the antimicrobial activities of SecA inhibitors, suggesting that targeting SecA could by-pass efflux pumps. Moreover, these inhibitors impair the secretion of important toxins of S. aureus and B. anthracis, indicating the inhibition of in vivo SecA function could reduce virulence. Target identification assays confirm that these inhibitors could directly bind to SecA homologues, and specifically identify SecA from whole cell lysate of E. coli and S. aureus, suggesting that these inhibitors are really targeting on SecA. These studies validate that SecA is a good target for development antimicrobials.

Due to the emergence and dissemination of multidrug resistance, bacterial pathogens have been causing a serious public health problem in recent years. To address the existing drug resistant problem, there is an urgent need to find new antimicrobials, especially those against drug-resistant bacteria. SecA is the central component of Sec-dependent secretion pathway, which is responsible for the secretion of many essential proteins as well as many toxins and virulence factors. Two SecA homologues are indentified in some important Gram-positive pathogens. SecA1 is involved in general secretion pathway and essential for viability, whereas SecA2 contribute to secretion of specific virulence factors. The high conservation among a wide range of bacteria and no human counterpart make SecA homologues attractive targets for exploring novel antimicrobials. We hypothesize that inhibition of these SecA homologues could reduce virulence, inhibit bacteria growth, and kill bacteria. SecA1 and SecA2 from four different species were cloned, purified, and characterized. All these SecA homologues show ATPase activities, thus screening ATPase inhibitors might help to develop new antimicrobials. In this study, three structurally different classes of SecA inhibitors were developed and optimized: 1) Rose Bengal (RB) and RB analogs derived from systematical dissection RB and Structure-Activity relationship (SAR) study; 2) pyrimidine analogs derived from virtual screening based on the ATP binding pocket of EcSecA and SAR study; and 3) bistriazole analogs derived from random screening and SAR study. Several potent SecA inhibitors show promising enzymatic inhibition against SecA homologues as well as bacteriostatic and bactericidal effects. Two major efflux pumps of S. aureus, NorA and MepA, have little negative effect on the antimicrobial activities of SecA inhibitors, suggesting that targeting SecA could by-pass efflux pumps. Moreover, these inhibitors impair the secretion of important toxins of S. aureus and B. anthracis, indicating the inhibition of in vivo SecA function could reduce virulence. Target identification assays confirm that these inhibitors could directly bind to SecA homologues, and specifically identify SecA from whole cell lysate of E. coli and S. aureus, suggesting that these inhibitors are really targeting on SecA. These studies validate that SecA is a good target for development antimicrobials.

Made available in DSpace on 2014-06-11T19:32:53Z (GMT). No. of bitstreams: 0 Previous issue date: 2006-11-30Bitstream added on 2014-06-13T20:44:12Z : No. of bitstreams: 1 santo_e_dr_jabo.pdf: 2769623 bytes, checksum: 67ca8d21675f50ba7c4d00ceff7f7038 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Esta pesquisa foi realizada em 23 açougues da cidade de T aquaritinga, estado de São Paulo, durante um período de 10 meses. Foram isoladas duzentas e oitenta e sete cepas de Escheríchía calí de carne moída, moedor de carne e mãos de manipuladores de carne. Cinco destas cepas foram caracterizadas como E.coli patogênica extra-intestinal (ExPEC). Investigou-se a presença de fímbrias, produção de hemolisina, aerobactina e colicina. Também foi analisada a presença dos genes (pap, afa, sfa) relacionados com a expressão de fímbrias, através da reação em cadeia da polimerase (PCR). Das amostras analisadas 100% apresentavam aerobactina e fímbria do tipo 1, 80% produziam hemolisina, e 60% expressaram colicina e fímbria P. Também foi verificado que 60% das cepas de ExPEC apresentavam o genótipo pap e 40% o genótipo pap-sfa concomitantemente. Quanto ao nível de resistência aos 12 antimicrobianos testados, observou-se que 80% das cepas eram resistentes a múltiplos antimicrobianos (3). Os antimicrobianos mais eficientes foram: ceftriaxona e amoxicilina-ácido clavulânico (0%) de resistência, seguidos de amicacina, amoxicilina, ciprofloxacina e gentamicina com resultado, considerado satisfatório, de 20% de resistência. Em contraste, houve elevada resistência (80%) para tetraciclina e estreptomicina . Conclui-se que retalhos de carne podem ser um importante veículo para disseminação na comunidade de cepas ExPEC. Este trabalho chama a atenção para os retalhos de carne como fonte potencial de cepas de ExPEC, que não são reconhecidas como patógeno de origem alimentar, o que pode representar um motivo de preocupação para as autoridades da vigilância epidemiológica. / For ten months, at Taquaritinga city, São Paulo State, we have conducted an malysis over meat conditions, at 23 butcheries. In this survey we collected. two hundred eighty seven generic Escherichia coli from ground beef, mincers and of the hands of neat manipulators. Five of these isolates were recognized as Extraintestinal Pathogenic :.coli (ExPEC) strains. The presence of fimbriae, hemolysin production, aerobactin and colicin. were lVestigated, as well the existence of genes (pap, ata, sfa) related to fimbriae !xpression, using a Polimerase Chain Reaction (PCR). In this ExPEC strains, we have verified the presence of aerobactin and fimbriae Ipe 1 (100%), hemolysin (80%) and related results for colicin and P fimbriae (60%). We Iso confirmed that 60% of ExPEC strains exhibited pap, and 40% were simultaneously, ap-sfa.. From twelve antimicrobial agents tested, we found a resistance levei (80%) to lultiple antimicrobial agents (~3). The most efficient antimicrobial agents were: 3ftriaxone and amoxacilin-clavulanic acid (0%) resistance, followed by a satisfactory resistance for amicacin, amoxacilin, ciprofloxacin and gentamicin (20%). In contrast, we lund a high levei of resistance (80%) for tetracycline and streptomycin. From this study, we have toncluded that meat can be a very important vehicle for community dissemination of ExPEC, which may represent a reason of concern.

A paracoccidioidomicose (PCM) é uma micose granulomatosa sistêmica, que tem como agentes etiológicos fungos dimórficos do gênero Paracoccidioides. A produção de melanina por vários fungos interfere no mecanismo da patogênese, como ocorre na paracoccidioidomicose. O presente projeto visou avaliar a produção de melanina pelos isolados de P. lutzii, e seus efeitos in vitro, avaliando o processo de fagocitose. Os resultados obtidos durante este trabalho mostraram que os isolados de P. lutzii (Pb01, Pb66, ED01, Pb1578 e Pb8334) melanizam de formas diferenciadas entre eles e quando comparados com isolados de P. brasiliensis (Pb60855, Pb18 e Pbcão). Ensaios de fagocitose, com os isolados Pb18, Pb60855 e Pb01, mostraram que a melanina reduziu a porcentagem de fagocitose das leveduras não tratadas de Pb18 e Pb60855, e o contrário aconteceu com Pb01. As análises dos perfis proteicos e enzimáticos dos isolados Pb18, Pb60855 e Pb01, permitiram determinar que o isolado Pb01 produz uma menor quantidade de proteínas que Pb18 e Pb60855 nas condições ensaiadas. / Paracoccidioidomycosis (PCM) is a granulomatous systemic mycosis, whose etiological agents are dimorphic fungi of the genus Paracoccidioides. Melanin production by various fungi interferes in the mechanism of pathogenesis, as in paracoccidioidomycosis. Thus, this project aimed to evaluate the production of melanin by isolates of P. lutzii and its in vitro effects by assessing the process of phagocytosis. The results obtained during this work showed that the isolates of P. lutzii (Pb01, Pb66, ED01, Pb1578 and Pb8334) produce melanin in different ways between them and also when compared with isolates of P. brasiliensis (Pb60855, Pb18 and Pbcão). Phagocytosis assays, with Pb18, Pb60855 and Pb01, showed that melanin reduced the percentage of phagocytosis of untreated yeast of Pb60855 and Pb18 and the opposite happened with Pb01. The analysis of the protein and enzymatic profiles, of the isolates Pb18, Pb60855 and Pb01, enabled to determine that the isolated Pb01 produces a smaller amount of proteins compared with Pb18 and Pb60855 in the tested conditions.

Ureases (EC 3.5.1.5) são metaloenzimas dependentes de Ni2+que hidrolisam ureia para produzir amônia e CO2. Estas enzimas são encontradas em fungos, bactérias e plantas, compartilhando estruturas similares. Nosso grupo vem demonstrando que ureases possuem propriedades biológicas independentes da atividade ureolítica que potencialmente contribuem para a patogenicidade de micro-organismoss produtores de urease. A presença de urease em bactérias patogênicas (p.e. Helicobacter pylori, Proteus mirabilis) está correlacionada com a patogênese em algumas doenças humanas. Alguns fungos de importância médica também são produtores de urease entre eles Cryptococcus neoformans, Coccidioides immitis, Histoplasma capsulatum, Sporothrix schenckii. Cryptococcus gattii é um dos agentes etiológicos da criptococose em humanos e animais e acomete mais frequentemente indivíduos imunocompetentes. A maioria dos isolados produzem urease e vários autores sugerem que a liberação de amônia pela atividade da urease de Cryptococcus tem papel importante na patogenia da doença favorecendo uma maior permeabilidade que proporciona a transmigração das leveduras para o sistema nervoso central (SNC). No presente trabalho, para analisar o potencial de virulência da urease de C. gattii foram construídos mutantes com inativação do gene estrutural URE (ure1) e dos genes que codificam as proteínas acessórias (URED, UREF – ure4 e ure6 respectivamente). Assim como já descrito para H. pylori, a urease de C. gattii desempenha papel importante na virulência independente da atividade enzimática. Esta função ocorre anterior a invasão do SNC diminuindo a multiplicação da levedura em macrófagos, aumentando a carga infecciosa no sangue e atenuando a mortalidade tanto no mutante ure1Δ como no mutante ure6Δ em camundongos infectados por via intranasal. / Ureases (EC 3.5.1.5) are metalloenzymes Ni2+ dependents that hydrolyze urea to produce ammonia and CO2. These enzymes are found in fungi, bacteria and plants, show very similar structures. Our group has shown that plant and bacterial ureases display biological properties independent of their ureolytic activity that may contribute to the pathogenesis of urease-producing microrganisms. The presence of urease in pathogenic bacteria (e.g. Helicobacter pylori, Proteus mirabilis) strongly correlates with pathogenesis in some human diseases. Many medically important fungi also produce urease, among which are Cryptococcus neoformans, Coccidioides immitis, Histoplasma capsulatum, Sporothrix schenckii. Cryptococcus gattii is one of the etiologic agent that causes criptococcosis in human and animals, which often affects immunocompromised patients. The majority of clinical isolates produce large amounts of urease, and several authors suggest that the ammonia realease from urease activity might introduce a local damage of the endothelium, thus increasing permeability which provides yeast transmigration to central nervous system (CNS). To analyse virulence potential of C. gattii urease, mutants inactivating structural URE (ure1) gene and coding genes for accessory proteins required to assemble the Ni2+-containing active site (URED, UREF – ure4 and ure6 respectively ). As already described to H. pylori urease, the C. gattii urease play important roles in virulence independent of ureolytic activity before CNS invasion, reducing yeast multiplication in macrophage, decreasing blood burden and also attenuating mortality either ure1Δ and accessory ure6Δ mutant in mice intranasal infection.

Ureases (EC 3.5.1.5) são metaloenzimas dependentes de Ni2+que hidrolisam ureia para produzir amônia e CO2. Estas enzimas são encontradas em fungos, bactérias e plantas, compartilhando estruturas similares. Nosso grupo vem demonstrando que ureases possuem propriedades biológicas independentes da atividade ureolítica que potencialmente contribuem para a patogenicidade de micro-organismoss produtores de urease. A presença de urease em bactérias patogênicas (p.e. Helicobacter pylori, Proteus mirabilis) está correlacionada com a patogênese em algumas doenças humanas. Alguns fungos de importância médica também são produtores de urease entre eles Cryptococcus neoformans, Coccidioides immitis, Histoplasma capsulatum, Sporothrix schenckii. Cryptococcus gattii é um dos agentes etiológicos da criptococose em humanos e animais e acomete mais frequentemente indivíduos imunocompetentes. A maioria dos isolados produzem urease e vários autores sugerem que a liberação de amônia pela atividade da urease de Cryptococcus tem papel importante na patogenia da doença favorecendo uma maior permeabilidade que proporciona a transmigração das leveduras para o sistema nervoso central (SNC). No presente trabalho, para analisar o potencial de virulência da urease de C. gattii foram construídos mutantes com inativação do gene estrutural URE (ure1) e dos genes que codificam as proteínas acessórias (URED, UREF – ure4 e ure6 respectivamente). Assim como já descrito para H. pylori, a urease de C. gattii desempenha papel importante na virulência independente da atividade enzimática. Esta função ocorre anterior a invasão do SNC diminuindo a multiplicação da levedura em macrófagos, aumentando a carga infecciosa no sangue e atenuando a mortalidade tanto no mutante ure1Δ como no mutante ure6Δ em camundongos infectados por via intranasal. / Ureases (EC 3.5.1.5) are metalloenzymes Ni2+ dependents that hydrolyze urea to produce ammonia and CO2. These enzymes are found in fungi, bacteria and plants, show very similar structures. Our group has shown that plant and bacterial ureases display biological properties independent of their ureolytic activity that may contribute to the pathogenesis of urease-producing microrganisms. The presence of urease in pathogenic bacteria (e.g. Helicobacter pylori, Proteus mirabilis) strongly correlates with pathogenesis in some human diseases. Many medically important fungi also produce urease, among which are Cryptococcus neoformans, Coccidioides immitis, Histoplasma capsulatum, Sporothrix schenckii. Cryptococcus gattii is one of the etiologic agent that causes criptococcosis in human and animals, which often affects immunocompromised patients. The majority of clinical isolates produce large amounts of urease, and several authors suggest that the ammonia realease from urease activity might introduce a local damage of the endothelium, thus increasing permeability which provides yeast transmigration to central nervous system (CNS). To analyse virulence potential of C. gattii urease, mutants inactivating structural URE (ure1) gene and coding genes for accessory proteins required to assemble the Ni2+-containing active site (URED, UREF – ure4 and ure6 respectively ). As already described to H. pylori urease, the C. gattii urease play important roles in virulence independent of ureolytic activity before CNS invasion, reducing yeast multiplication in macrophage, decreasing blood burden and also attenuating mortality either ure1Δ and accessory ure6Δ mutant in mice intranasal infection.

Ureases (EC 3.5.1.5) são metaloenzimas dependentes de Ni2+que hidrolisam ureia para produzir amônia e CO2. Estas enzimas são encontradas em fungos, bactérias e plantas, compartilhando estruturas similares. Nosso grupo vem demonstrando que ureases possuem propriedades biológicas independentes da atividade ureolítica que potencialmente contribuem para a patogenicidade de micro-organismoss produtores de urease. A presença de urease em bactérias patogênicas (p.e. Helicobacter pylori, Proteus mirabilis) está correlacionada com a patogênese em algumas doenças humanas. Alguns fungos de importância médica também são produtores de urease entre eles Cryptococcus neoformans, Coccidioides immitis, Histoplasma capsulatum, Sporothrix schenckii. Cryptococcus gattii é um dos agentes etiológicos da criptococose em humanos e animais e acomete mais frequentemente indivíduos imunocompetentes. A maioria dos isolados produzem urease e vários autores sugerem que a liberação de amônia pela atividade da urease de Cryptococcus tem papel importante na patogenia da doença favorecendo uma maior permeabilidade que proporciona a transmigração das leveduras para o sistema nervoso central (SNC). No presente trabalho, para analisar o potencial de virulência da urease de C. gattii foram construídos mutantes com inativação do gene estrutural URE (ure1) e dos genes que codificam as proteínas acessórias (URED, UREF – ure4 e ure6 respectivamente). Assim como já descrito para H. pylori, a urease de C. gattii desempenha papel importante na virulência independente da atividade enzimática. Esta função ocorre anterior a invasão do SNC diminuindo a multiplicação da levedura em macrófagos, aumentando a carga infecciosa no sangue e atenuando a mortalidade tanto no mutante ure1Δ como no mutante ure6Δ em camundongos infectados por via intranasal. / Ureases (EC 3.5.1.5) are metalloenzymes Ni2+ dependents that hydrolyze urea to produce ammonia and CO2. These enzymes are found in fungi, bacteria and plants, show very similar structures. Our group has shown that plant and bacterial ureases display biological properties independent of their ureolytic activity that may contribute to the pathogenesis of urease-producing microrganisms. The presence of urease in pathogenic bacteria (e.g. Helicobacter pylori, Proteus mirabilis) strongly correlates with pathogenesis in some human diseases. Many medically important fungi also produce urease, among which are Cryptococcus neoformans, Coccidioides immitis, Histoplasma capsulatum, Sporothrix schenckii. Cryptococcus gattii is one of the etiologic agent that causes criptococcosis in human and animals, which often affects immunocompromised patients. The majority of clinical isolates produce large amounts of urease, and several authors suggest that the ammonia realease from urease activity might introduce a local damage of the endothelium, thus increasing permeability which provides yeast transmigration to central nervous system (CNS). To analyse virulence potential of C. gattii urease, mutants inactivating structural URE (ure1) gene and coding genes for accessory proteins required to assemble the Ni2+-containing active site (URED, UREF – ure4 and ure6 respectively ). As already described to H. pylori urease, the C. gattii urease play important roles in virulence independent of ureolytic activity before CNS invasion, reducing yeast multiplication in macrophage, decreasing blood burden and also attenuating mortality either ure1Δ and accessory ure6Δ mutant in mice intranasal infection.

Previous research in our laboratory showed that when knockout mutations were created in the pyrB and pyrC genes of the pyrimidine pathway in Pseudomonas aeruginosa, not only were the resultant mutants auxotrophic for pyrimidines but they were also impaired in virulence factor production. Such a correlation had not been previously reported for P. aeruginosa, a ubiquitous opportunistic pathogen in humans. In an earlier study it was reported that mutants blocked in one of the first three enzymes of the pyrimidine pathway in the non-pathogenic strain P. putida M produced no pyoverdin pigment while mutants blocked in the later steps produced copious amounts of pigment, just like the wild type. This study probed for the same connection between pyrimidine auxotrophy and pigment production applied in P. aeruginosa. To that end a knockout mutation was created in pyrD, the fourth step in the pyrimidine pathway which encodes dihydroorotate dehydrogenase. The resulting mutant required pyrimidines for growth but produced wild type pigment levels. Since the pigment pyoverdin is a siderophore it may also be considered a virulence factor, other virulence factors were quantified in the mutant. These included casein protease, hemolysin, elastase, swimming, swarming and twitching motility, and iron binding capacity. In all cases these virulence factors were significantly decreased in the mutant. Even supplementing with uracil did not attain wild type levels. Starvation of the pyrimidine mutant for uracil caused increased specific activity of the pyrimidine enzymes, suggesting that regulation of the pyrimidine pathway occurred at the level of transcription. This effect has also been reported for P. oleovorans. The present research consolidates the idea that pyrimidine auxotrophs cause decreased pathogenicity in P. aeruginosa. Such a finding may open the search for chemotherapy targets in cystic fibrosis and burn victims where P. aeruginosa is an infecting agent.

The spirochete Borrelia burgdorferi is the etiologic agent of Lyme disease. This pathogen has a complex enzootic life cycle that involves passage between the tick vector (Ixodes scapularis) and various vertebrate hosts with humans being inadvertent hosts. There is a pressing need to study the genetic aspects of the B. burgdorferi infectious cycle and particularly spirochete genes involved in mammalian infectivity so as to develop novel therapeutic and diagnostic strategies to combat Lyme disease. The B. burgdorferi genome is fragmented and comprised of a single 900 kb linear chromosome and multiple linear and circular plasmids. It has been observed that plasmids are lost during serial passage and manipulation in vitro and the loss of some of the plasmids has been shown to be related to the loss of infectivity and persistence in the host. One such plasmid is linear plasmid 36 (lp36). lp36 is approximately 36kb in size and carries 56 putative open reading frames a majority of which have no predicted function. B. burgdorferi lacking lp36 show no deficiency in survival in ticks; however, these mutant spirochetes are highly attenuated for mammalian infectivity. The genetic components of this plasmid that contribute to its function in mammalian infectivity have yet to be clearly defined. Using an in vivo expression technology (IVET) based genetic screen the lp36- encoded gene bbk46 was identified as a candidate B. burgdorferi gene that is expressed during mammalian infection. Herein we present evidence that bbk46 is required for B. burgdorferi persistent infection of immunocompetent mice. Our data iii support a molecular model of immune evasion by which bbk46 functions as an RNA to regulate expression of the antigenic variation protein VlsE. These data represent the first demonstration of a regulatory mechanism critical for controlling vlsE gene expression. Moreover these findings further define the critical role of linear plasmid 36 in Borrelia burgdorferi pathogenesis.

Pseudomonas aeruginosa est un pathogène opportuniste responsable du développement de maladies nosocomiales. Il provoque des infections en employant différents facteurs de virulence dont le principal, associé aux infections sévères, est le système de sécrétion de type 3 (SST3). Les souches de type PA7, taxonomiquement marginales, sont dépourvues de SST3 et leur pouvoir pathogène repose sur le nouveau système de virulence ExlBA. Ce SST5b, ou TPS, est composé du transporteur ExlB qui permet la translocation d’ExlA, une toxine induisant la perméabilisation de la membrane plasmique des cellules eucaryotes.Ce travail représente la première investigation des mécanismes de régulation du système ExlBA. Ainsi, il a été mis en évidence que la déplétion en fer est un signal d’activation de l’expression des gènes exlBA. De plus, les deux principaux messagers secondaires, AMPc et di-GMPc, sont impliqués dans la régulation du TPS. En effet, la voie CyaB-AMPc/Vfr, connue pour réguler le SST3, contrôle la toxicité des souches de type PA7 grâce à une activation transcriptionnelle directe des gènes exlBA, qui peut être stimulée par la chélation du calcium extracellulaire. Parallèlement, alors qu’ExlA était supposée être sécrétée pour agir sur les cellules eucaryotes, cette étude a montré que la toxine doit être exposée à la surface de la membrane bactérienne pour provoquer la lyse de ces cellules, ceci par un mécanisme dépendant du di-GMPc. Effectivement, une forte concentration en di-GMPc empêche la sécrétion d’ExlA en induisant de façon post-traductionnelle son maintien au niveau du transporteur ExlB, ce qui favoriserait l’action de la toxine sur les membranes eucaryotes. / Pseudomonas aeruginosa is an opportunistic pathogen responsible for nosocomial diseases. It provokes infections due to several virulence factors. Among them the most aggressive is the type 3 secretion system (T3SS), associated with severe infection. PA7-like strains, that are taxonomic outliers, lack the T3SS but are still pathogenic thanks to the novel virulence system ExlBA. This T5bSS, or TPS, is composed by the transporter ExlB that allows translocation of ExlA toxin to induce permeabilisation of eukaryotic cell membrane.This study is the first investigation of regulatory mechanisms that modulate ExlBA. It provided evidence that iron depletion is an activator signal of exlBA gene expression. Furthermore, the two main second messengers, cAMP and c-di-GMP, are involved in ExlBA regulation. CyaB-cAMP/Vfr pathway, known to regulate T3SS, controls toxicity of PA7-like strains through direct transcriptional activation of exlBA. This pathway may be stimulated by an extracellular calcium chelation. At the same time, while ExlA was supposed to be secreted to kill eukaryotic cells, this work showed that the toxin must be exposed at the surface of the bacterial membrane to cause lysis of these cells, by a mechanism dependent on c-di-GMP. Indeed, a c-di-GMP high concentration prevents ExlA secretion by inducing its maintenance at the ExlB transporter, that would promote the action of the toxin on eukaryotic membranes.