Inducers and autoinducers on Salmonella enterica serovar Typhimurium motility, growth and gene expression / Indutores e autoindutores na motilidade, no crescimento e na expressão gênica de Salmonella enterica serovar Typhimurium

Genus Salmonella bacteria are among the major pathogenic microorganisms in food. This bacterium pathogenicity is related to a number of virulence factors, among which its flagella. Flagellum expression is one of the virulence factors modulated by Quorum Sensing. Epinephrine produced by mammals uses the same signaling pathway of the 3 bacteria autoinducer. This study evaluated the effect of molecules inducer (epinephrine) and autoinducers (autoinducer 2 and autoinducer 3) and their association with the motility, growth and expression genes flhC, fliA, fliY, motA, motB e fliC of Salmonella Typhimurium (ST). Initially, ST was inoculated in BHI. Then, motility assays, growth curves and gene expression were performed by testing different concentrations of epinephrine (50, 125, 250, 500µM), conditioned medium (10 and 50%) and a combination of these. ST was exposed to different concentrations of epinephrine, conditioned medium and an association of both. Following, motility assays, bacterial growth and gene expression were performed. The results obtained showed that the combination of 500uM epinephrine with 50% conditioned medium increased ST bacterial motility by increasing the expression of genes involved in flagellum assembly.

Salmonella está entre os principais micro-organismos patogênicos veiculados por alimentos. A patogenicidade dessa bactéria está relacionada a uma série de fatores de virulência e, dentre estes, podemos citar os flagelos. A expressão do flagelo está entre os fatores de virulência modulados por Quorum Sensing. A adrenalina produzida pelos mamíferos utiliza a mesma via de sinalização do autoindutor 3 das bactérias. Nesse sentido, este trabalho teve como objetivo avaliar o efeito de moléculas indutora (adrenalina) e autoindutoras (auto-indutor 2 e auto-indutor 3) e a associação destas na motilidade, no crescimento celular e na expressão dos genes flhC, fliA, fliY, motA, motB e fliC de SalmonellaTyphimurium (ST). Inicialmente, ST foi semeada em caldo BHI. Após, ensaios de motilidade, curvas de crescimento e expressão gênica foram feitos, testando diferentes concentrações de adrenalina (50, 125, 250, 500µM), meio condicionado (10 e 50%) e a associação destes. A partir dos resultados obtidos, observou-se que o tratamento que utilizou 50% de meio condicionado + 500µM de adrenalina aumentou a motilidade de ST, em decorrência do aumento de genes envolvidos com montagem do flagelo.

In vitro biosynthesis of autoinducer 2 of Streptococcus mutans UA1 59 and observation of the influence factors / 实用口腔医学杂志

Objective:To synthesize autoinducer-2 by the clone and prokaryotic expression of Streptococcus mutans(S.mutans)UAl59 luxS gene and to observe the influence factors.Methods:The expression vector pET21 a(+)-luxS of S.mutans UAl59 was transformed into Escheriehia coli BL2l(DE3).The S-ribosylhomocysteinase(Luxs)expression was induced by IPTG.The His tag fusion protein was isolated by Ni-chelating column and identified by Western blotting.Finally the protein was renatured by dialysis method.S-ribosylhomo-cysteine (SAH)was catalyzed by s-adenosylhomocysteine nucleosidas (Pfs)and LuxS,and then AI-2 was syntheszed.The AI-2 activi-ty was examined by luminescence of Vibrio harveyi BB1 70 when the concentration of LuxS protein or pH(4 -1 2)or the concentration of sodium fluoride was changed in reaction mixes of AI-2 synthesis.Results:Compared with the control group,with the increase of LuxS protein concentration,the relative activity of in vitro synthesized AI-2 increased gradually(P <0.001 ).When pH was between 6 -1 0, the relative activity of AI-2 were the highest,beyond the range of pH,the relative activity of AI-2 decreased(P <0.001 ).When a final concentration of sodium fluoride was more than 0.3%,the luminescence values decreased(P <0.05).Conclusion:LuxS fusion protein can promote the production of AI-2.Optimum pH for AI-2 biosynthesis in vitro must be between 6-1 0.Biosynthesis of AI-2 is inhibited by sodium fluoride with final concentration of more than 0.3%.

SmcR, the Quorum-sensing Master Regulator, Is Partially Involved in Temperature/Salinity-mediated Changes in Metalloprotease vvpE Expression in Vibrio vulnificus

Vibrio vulnificus, a gram-negative halophilic marine bacterium and opportunistic human pathogen, must withstand various environmental changes, especially simultaneous changes in temperature and salinity, from 25degrees C/2.5% to 37degrees C/0.9% (SCTS) upon entering the human body. In our previous study, SCTS stimulated vvpE expression even in the background of a mutation in luxS encoding LuxS enzyme for the biosynthesis of quorum-sensing (QS) signal molecule autoinducer-2 (AI-2), suggesting that the A1-2-mediated QS system is partially involved in the SCTS-mediated change of vvpE expression. In this study, we examined the effects of the QS master regulator SmcR on SCTS-mediated changes in vvpE expression and extracellular VvpE production. SCTS stimulated V. vulnificus growth, but with no increase in maximal growth levels. The SCTS-mediated prolongation of the stationary growth phase resulted in a significant increase in growth phase-dependent smcR and vvpE expressions. A mutation in smcR seriously repressed vvpE expression, but had no significant effect on V. vulnificus growth. However, the smcR mutation only partially attenuates SCTS-mediated changes in vvpE expression. These results indicate that SCTS stimulates the expressions of smcR and vvpE by stimulating V. vulnificus growth, and that SmcR is only partially involved in SCTS-mediated changes in vvpE expression.

El impacto biológico de los autoinductores bacterianos / The biological impact of bacterial autoinducers

Las bacterias, a pesar de ser organismos unicelulares, presentan una gran complejidad. Durante mucho tiempo fueron consideradas como organismos asociales cuyas funciones principales eran el nutrirse y el reproducirse. Sin embargo, se ha observado que las bacterias son los microorganismos con la mayor capacidad de adaptación a ambientes diversos, además responden a múltiples estímulos, tanto nutricionales como ambientales (pH, disponibilidad de oxígeno, osmolaridad, etc.). En las últimas décadas se ha reportado que tanto las bacterias grampositivas como las gramnegativas son capaces de comunicarse entre si mediante sistemas especializados de comunicación celular. A tales sistemas se les ha denominado “sistemas de señalización” y “autoinductores” a las moléculas señal que desencadenan diferentes respuestas celulares, como la formación de biopelículas, la transformación bacteriana, la producción de bioluminiscencia, la producción de antibióticos o de factores de virulencia, entre otras. En este trabajo se presentan los aspectos más relevantes relacionados a los autoinductores de bacterias grampositivas y gramnegativas, así como su participación en diferentes procesos biológicos.

Bacteria, in spite of being unicellular organisms, present great complexity. During a long time they were considered as asocial organisms whose main functions were feeding and reproducing. Nevertheless, it has been observed that bacteria are the microorganisms with the greatest capacity for adapting to diverse environments, also responding to multiple stimuli, both nutritional and environmental (pH, oxygen availability, osmolarity, etc.). During the last decades it has been reported that bacteria, both gram negative and gram positive, are capable of communicating among them through specialized cell-communication systems. These systems have been called “signaling systems” and the signaling molecules which unchain the various cell responses such as biofilm formation, bacterial transformation, luminescence production, antibiotic production, or virulence factor production, among others, have been called “autoinducers”. This paper presents the most relevant aspects related with gram positive and gram negative bacteria autoinducers, as well as their participation in different biological processes.

Change of Vibrio vulnificus Metalloprotease VvpE Production by Temperature and Salinity

Vibrio vulnificus, a gram-negative halophilic marine bacterium and opportunistic pathogen, must withstand various environmental changes, especially the simultaneous change of temperature and salinity (SCTS) from 25degrees C/2.5% to 37degrees C/0.9% upon entering the human body. Previous studies have suggested that temperature and salinity may affect the production of metalloprotease VvpE via the LuxS-mediated autoinducer-2 quorum sensing system (AI-2-QSS). However, this hypothesis remains to be verified through coherent experiments. In this study, SCTS stimulated V. vulnificus growth with no increase in total growth levels. The SCTS-mediated prolongation of the stationary growth phase resulted in a significant increase in growth phase-dependent luxS and vvpE transcriptions; however, SCTS did not affect luxS or vvpE transcription levels during the exponential growth phase. SCTS also advanced extracellular VvpE production, which was consistent with vvpE transcription and V. vulnificus growth. SCTS-mediated modulation of vvpE expression was slightly attenuated but still observed in the background of a luxS mutation which seriously repressed vvpE expression. These results indicate that SCTS stimulates luxS and vvpE expression by stimulating V. vulnificus growth; however, the LuxS-mediated AI-2-QSS plays only a minor role, if any, in the SCTS-mediated modulation of vvpE expression.

Effects of Pseudomonas aeruginosa quorum sensing molecule OdDHL on murine mast cells / 中华微生物学和免疫学杂志

Objective To investigate the biological effects of Pseudomonas aeruginosa quorum sensing molecule OdDHL on murine mast cells. Methods The molecule structure and purity of synthesized OdDHL were confirmed by mass spectrum or proton nuclear magnetic resonance (NMR) and high-performance liquid chromatography, respectively. Its biological activity was checked using a quorum sensing sensor bacterial strain. The viability, apoptosis and intracellular calcium changes of P815 cell line in response to different concentration of OdDHL were determined. Results The biological active OdDHL was synthesized successfully. OdDHL inhibited proliferation of P815 cells in a dose, and time dependent manner. It also induced apoptasis and intracellular calcium release in P815 cells. Conclusion Psendomonas aeruginosa quorum sensing molecule OdDHL induces apoptosis and intracellular calcium release in murine mast cell line P815.