Browsing by Author "Vanetti, Maria Cristina Dantas"

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    Acyl homoserine lactone changes the abundance of proteins and the levels of organic acids associated with stationary phase in Salmonella Enteritidis.
    (2017) Almeida, Felipe Alves de; Pimentel Filho, Natan de Jesus; Carrijo, Lanna Clícia; Bento, Cláudia Braga Pereira; Pereira, Maria Cristina Baracat; Pinto, Uelinton Manoel; Oliveira, Leandro Licursi de; Vanetti, Maria Cristina Dantas
    Quorum sensing (QS) is cell-cell communication mechanism mediated by signaling molecules known as autoinducers (AIs) that lead to differential gene expression. Salmonella is unable to synthesize the AI-1 acyl homoserine lactone (AHL), but is able to recognize AHLs produced by other microorganisms through SdiA protein. Our study aimed to evaluate the influence of AI-1 on the abundance of proteins and the levels of organic acids of Salmonella Enteritidis. The presence of N-dodecyl-homoserine lactone (C12- HSL) did not interfere on the growth or the total amount of extracted proteins of Salmonella. However, the abundance of the proteins PheT, HtpG, PtsI, Adi, TalB, PmgI (or GpmI), Eno, and PykF enhanced while the abundance of the proteins RplB, RplE, RpsB, Tsf, OmpA, OmpC, OmpD, and GapA decreased when Salmonella Enteritidis was anaerobically cultivated in the presence of C12-HSL. Additionally, the bacterium produced less succinic, lactic, and acetic acids in the presence of C12-HSL. However, the concentration of extracellular formic acid reached 20.46 mM after 24 h and was not detected when the growth was in the absence of AI-1. Considering the cultivation period for protein extraction, their abundance, process and function, as well as the levels of organic acids, we observed in cells cultivated in presence of C12-HSL a correlation with what is described in the literature as entry into the stationary phase of growth, mainly related to nitrogen and amino acid starvation and acid stress. Further studies are needed in order to determine the specific role of the differentially abundant proteins and extracellular organic acids secreted by Salmonella in the presence of quorum sensing signaling molecules.
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    Acyl homoserine lactone‑based quorum sensing stimulates biofilm formation by Salmonella Enteritidis in anaerobic conditions.
    (2017) Almeida, Felipe Alves de; Pimentel Filho, Natan de Jesus; Pinto, Uelinton Manoel; Mantovani, Hilário Cuquetto; Oliveira, Leandro Licursi de; Vanetti, Maria Cristina Dantas
    Quorum sensing regulates a variety of phenotypes in bacteria including the production of virulence factors. Salmonella spp. have quorum sensing systems mediated by three autoinducers (AI-1, AI-2, and AI-3). The AI-1-mediated system is incomplete in that the bacterium relies on the synthesis of signaling molecules by other microorganisms. This study aimed to evaluate the influence of the AI-1 N-dodecanoyl-DL-homoserine lactone (C12- HSL) on the growth, motility, adhesion, and biofilm formation of Salmonella enterica serovar Enteritidis PT4 578 on a polystyrene surface. Experiments were conducted at 37 °C in anaerobic tryptone soy broth supplemented with C12-HSL and/or a mixture of four synthetic furanones, at the concentration of 50 nM each. The planktonic growth, adhesion, swarming, and twitching motility were not altered in the presence of C12-HSL and/or furanones under anaerobic conditions. However, C12-HSL induced biofilm formation after 36 h of cultivation as determined by quantification of biofilm formation, by enumeration of adhered cells to polystyrene coupons, and finally by imaging the presence of multilayered cells on an epifluorescence microscope. When furanones were present in the medium, an antagonistic effect against C12-HSL on the biofilm development was observed. The results demonstrate an induction of biofilm formation in Salmonella Enteritidis by AI-1 under anaerobic conditions. Considering that Salmonella does not produce AI-1 but respond to it, C12-HSL synthesized by other bacterial species could trigger biofilm formation by this pathogen in conditions that are relevant for its pathogenesis.
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    Induction of the viable but nonculturable state of Salmonella enterica serovar Enteritidis deficient in (p)ppGpp synthesis.
    (2015) Rodrigues, Ramila Cristiane; Martins, Evandro; Vanetti, Maria Cristina Dantas; Pinto, Uelinton Manoel; Santos, Míriam Teresinha dos
    Salmonella enterica enters a viable but nonculturable (VBNC) state in adverse environmental conditions. Under nutritional stress, RelA and SpoT proteins synthesize (p)ppGpp, a molecule that controls the expression of genes important for the survival of a cell under those conditions. This study aimed to verify the involvement of (p)ppGpp in the entry of S. enterica serovar Enteritidis PT4 578 cells into the VBNC state by evaluating ΔrelA and ΔrelAΔspoT mutants. The wild-type and mutant strains deficient in (p)ppGpp synthesis were subjected to osmotic, nutritional and cold stress, after which the cells entered a VBNC state at different time periods, concurrent with reductions in cell diameter, volume and length, and conversion from a bacillary to a coccoid form. No difference in the culturability or cell viability of the wild-type and single and double mutants was observed. Expression of the rpoS gene was increased in the double mutant, while both mutant strains presented a reduction in mreB gene expression after 25 days under conditions of nutritional and cold stress. Surprisingly, (p)ppGpp was not necessary for induction of the VBNC state in Salmonella PT4 578 cells, but may be associated with regulation of genes that control septum formation during cell division, maintaining the bacillary cell morphology (mreB) and stress response (rpoS). Our findings contribute to the understanding of the mechanisms of resistance and survival of Salmonella under adverse conditions.
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    Lack of AHL-based quorum sensing in Pseudomonas fluorescens isolated from milk.
    (2014) Martins, Maurílio Lopes; Pinto, Uelinton Manoel; Riedel, Kathrin; Vanetti, Maria Cristina Dantas; Mantovani, Hilário Cuquetto; Araújo, Elza Fernandes de
    Numerous bacteria coordinate gene expression in response to small signalling molecules in many cases known as acylhomoserine lactones (AHLs), which accumulate as a function of cell density in a process known as quorum sensing. This work aimed to determine if phenotypes that are important to define microbial activity in foods such as biofilm formation, swarming motility and proteolytic activity of two Pseudomonas fluorescens strains, isolated from refrigerated raw milk, are influenced by AHL molecules. The tested P. fluorescens strains did not produce AHL molecules in none of the evaluated media. We found that biofilm formation was dependent on the culture media, but it was not influenced by AHLs. Our results indicate that biofilm formation, swarming motility and proteolytic activity of the tested P. fluorescens strains are not regulated by acyl-homoserine lactones. It is likely that AHL-dependent quorum sensing system is absent from these strains.
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    Milk-deteriorating exoenzymes from Pseudomonas fluorescens 041 isolated from refrigerated raw milk.
    (2015) Martins, Maurílio Lopes; Pinto, Uelinton Manoel; Riedel, Katharina; Vanetti, Maria Cristina Dantas
    The practice of refrigerating raw milk at the farm has provided a selective advantage for psychrotrophic bacteria that produce heat-stable proteases and lipases causing severe quality problems to the dairy industry. In this work, a protease (AprX) and a lipase (LipM) produced by Pseudomonas fluorescens 041, a highly proteolytic and lipolytic strain isolated from raw milk obtained from a Brazilian farm, have been purified and characterized. Both enzymes were purified as recombinant proteins from Escherichia coli. The AprX metalloprotease exhibited activity in a broad temperature range, including refrigeration, with a maximum activity at 37 °C. It was active in a pH range of 4.0 to 9.0. This protease had maximum activity with the substrates casein and gelatin in the presence of Ca+2. The LipM lipase had a maximum activity at 25 °C and a broad pH optimum ranging from 7.0 to 10. It exhibited the highest activity, in the presence of Ca+2, on substrates with long-chain fatty acid residues. These results confirm the spoilage potential of strain 041 in milk due to, at least in part, these two enzymes. The work highlights the importance of studies of this kind with strains isolated in Brazil, which has a recent history on the implementation of the cold chain at the dairy farm.
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    Quorum sensing regulated phenotypes in Aeromonas hydrophila ATCC 7966 deficient in AHL production.
    (2016) Rossi, Adriana dos Reis Ponce; Pinto, Uelinton Manoel; Ribon, Andrea de Oliveira Barros; Bazzolli, Denise Mara Soares; Vanetti, Maria Cristina Dantas
    The virulence of Aeromonas hydrophila is positively regulated by a quorum sensing (QS) system based on the ahyRI genes. The aim of this study was to evaluate the phenotypic characteristics related to virulence in the A. hydrophila strain ATCC 7966 in order to check if virulence traits are still functioning, despite the absence of a well characterized QS system. As expected, we were not able to detect quorum signaling AHL molecules in A. hydrophila ATCC 7966 by cross-streaking or thin layer chromatography with AHL biosensor strains. Although A. hydrophila ATCC 7966 did not produce AHLs, transcription of the ahyI and ahyR genes was observed by RT-PCR. Phenotypes generally recognized as QS-regulated in A. hydrophila were maintained in ATCC 7966; these phenotypes included proteolytic activity on casein and gelatin and amylolytic, lipolytic, and β- hemolytic activities, as well as the ability to adhere to surfaces and to form biofilms. Moreover, the addition of exogenous AHLs did not induce changes in the evaluated phenotypes. The results suggest that although this strain does not produce AHL as evaluated in this study, it maintains the phenotypes commonly associated with QS in this species indicating that other regulatory mechanisms independent of ahyRI may be in place.