Examinando por Autor "Rodas, Paula I."
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Ítem A feed-forward loop between SroC and MgrR small RNAs modulates the expression of eptB and the susceptibility to polymyxin B in Salmonella Typhimurium(Microbiology Society, 2016-11) Acuña, Lillian G.; Barros, M. José; Peñaloza, Diego; Rodas, Paula I.; Paredes-Sabja, Daniel; Fuentes, Juan A.; Gil, Fernando; Calderón, Iván L.Base-pairing small RNAs (sRNAs) regulate gene expression commonly by direct interaction with cognate mRNAs. Nevertheless, recent studies have expanded this knowledge with the discovery of the RNA ‘sponges’ which are able to interact and repress the functions of classical base-pairing sRNAs. In this work, we present evidence indicating that the sponge RNA SroC from Salmonella enterica serovar Typhimurium base pairs with the MgrR sRNA, thereby antagonizing its regulatory effects on both gene expression and resistance to the antimicrobial peptide polymyxin B (PMB). By a predictive algorithm, we determined putative SroC–MgrR base-pairing regions flanking the interaction area between MgrR and its target mRNA, eptB, encoding a LPS-modifying enzyme. With a two-plasmid system and compensatory mutations, we confirmed that SroC directly interacts and down-regulates the levels of MgrR, thus relieving the MgrR-mediated repression of eptB mRNA. Since it was previously shown that an Escherichia coli strain carrying an mgrR deletion is more resistant to PMB, we assessed the significance of SroC in the susceptibility of S. Typhimurium to PMB. Whereas the sroC deletion increased the sensitivity to PMB, as compared to the wild-type, the resistance phenotypes between the DmgrR and DsroCDmgrR strains were comparable, evidencing that mgrR mutation is epistatic to the sroC mutation. Together, these results indicate that both SroC and MgrR sRNAs compose a coherent feed-forward loop controlling the eptB expression and hence the LPS modification in S. Typhimurium.Ítem Characterization of the Adherence of Clostridium difficile Spores: The Integrity of the Outermost Layer Affects Adherence Properties of Spores of the Epidemic Strain R20291 to Components of the Intestinal Mucosa(FRONTIERS MEDIA SA, 2016-09) Mora-Uribe, Paola; Miranda-Cárdenas, Camila; Castro-Córdova, Pablo; Gil, Fernando; Calderón, Iván; Fuentes, Juan A.; Rodas, Paula I.; Banawas, Saeed; Sarker, Mahfuzur R.; Paredes-Sabja, DanielClostridium difficile is the causative agent of the most frequently reported nosocomial diarrhea worldwide. The high incidence of recurrent infection is the main clinical challenge of C. difficile infections (CBI). Formation of C. difficile spores of the epidemic strain R20291 has been shown to be essential for recurrent infection and transmission of the disease in a mouse model. However, the underlying mechanisms of how these spores persist in the colonic environment remains unclear. In this work, we characterized the adherence properties of epidemic R20291 spores to components of the intestinal mucosa, and we assessed the role of the exosporium integrity in the adherence properties by using cdeC mutant spores with a defective exosporium layer. Our results showed that spores and vegetative cells of the epidemic R20291 strain adhered at high levels to monolayers of Caco-2 cells and mucin. Transmission electron micrographs of Caco-2 cells demonstrated that the hair-like projections on the surface of R20291 spores are in close proximity with the plasma membrane and microvilli of undifferentiated and differentiated monolayers of Caco-2 cells. Competitive-binding assay in differentiated Caco-2 cells suggests that spore-adherence is mediated by specific binding sites. By using spores of a cdeC mutant we demonstrated that the integrity of the exosporium layer determines the affinity of adherence of C. difficile spores to Caco-2 cells and mucin. Binding of fibronectin and vitronectin to the spore surface was concentration-dependent, and depending on the concentration, spore-adherence to Caco-2 cells was enhanced. In the presence of an aberrantly-assembled exosporium (cdeC spores), binding of fibronectin, but not vitronectin, was increased. Notably, independent of the exosporium integrity, only a fraction of the spores had fibronectin and vitronectin molecules binding to their surface. Collectively, these results demonstrate that the integrity of the exosporium layer of strain R20291 contributes to selective spore adherence to components of the intestinal mucosa.Ítem Evaluation of ceftiofur-PHBV microparticles in rats(Dove Medical Press Ltd., 2014-05) Vilos, Cristian; Constandil, Luis; Rodas, Paula I.; Cantin, Mario; Zepeda, Katherine; Herrera, Natalia; Velasquez, Luis A.Despite the high number of antibiotics used for the treatment of infectious disease in animals, the development of slow release formulations presents a significant challenge, particu larly in using novel biomaterials with low cost. In this report, we studied the pharmacokinetics, toxicity, and therapeutic activity of ceftiofur–PHBV (ceftiofur–poly(3-hydroxybutyrate-co 3-hydroxyvalerate)) in rats. The pharmacokinetic study demonstrated a sustained release of ceftiofur into the bloodstream, with detectable levels over the minimum inhibitory concentration for at least 17 days after a single intramuscular injection of ceftiofur–PHBV (10 mg/kg weight). In addition, the toxicological evaluation of biochemical, hematological, and coagulation blood parameters at the therapeutic dose demonstrated the safety of ceftiofur–PHBV, with no adverse effects. In addition, ceftiofur–PHBV exhibited a therapeutic effect for a longer time period than the nonencapsulated ceftiofur in rats challenged with Salmonella Typhimurium. The slow release of ceftiofur from the ceftiofur–PHBV, its low toxicity in the blood parameters evaluated, and the efficacy in the rats infected with Salmonella Typhimurium make ceftiofur–PHBV a strong candidate for biotechnological applications in the veterinary industry.Ítem Modified profile of matrix metalloproteinase 2 and 9 production by human fallopian tube epithelial cells after infection in vitro with neisseria gonorrhoeae(Oxford University Press, 2017) Rodas, Paula I.; Jauffret, Claudia; Pérez, Doris; González, Yaquelin; Carreño, Carolina; Tapia, Cecilia V.; Osorio, Eduardo; Velasquez, Luis A.; Christodoulides, MyronEpithelial shedding and scarring of fallopian tube mucosa are the main consequences of sexually transmitted Neisseria gonorrhoeae infection and probably involve an imbalance of host extracellular matrix components and their regulators such as matrix metalloproteinases (MMPs). In the current study, primary human fallopian tube epithelial cells were infected with N. gonorrhoeae, and MMP patterns were examined. Gonococcal infection induced a significant increase in secreted MMP-9 and an accumulation of cytoplasmic MMP-2 over time, but no significant MMP-3 or MMP-8 production was observed. Thus, MMP-9 in particular could play a role in tubal scarring in response to gonococcal infection. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society.Ítem Motility modulation by the small non-coding RNA SroC in Salmonella Typhimurium(Oxford University Press, 2015-08) Fuentes, Danitza N.; Calderón, Paulina F.; Acuña, Lillian G.; Rodas, Paula I.; Paredes-Sabja, Daniel; Fuentes, Juan A.; Gil, Fernando; Calderón, Iván L.Bacterial regulatory networks of gene expression include the interaction of diverse types of molecules such as the small non-coding RNAs (sRNAs) and their cognate messenger RNAs (mRNAs). In this study, we demonstrated that the Salmonella Typhimurium sRNA SroC is significantly expressed between the late-exponential and stationary phase of growth in an rpoS-dependent manner. The expression of flagellar genes predicted as targets of this sRNA was quantitatively analyzed in both a ΔsroC mutant and a SroC-overexpressing (pSroC) strain. Deletion of sroC increased flagellar gene expression (i.e. flhBAE and fliE). Conversely, overexpression of SroC reduced flhBAE and fliE expression. These observations correlated with phenotypic evaluation of motility, where sroC deletion slightly increased motility, which in turn, was drastically reduced upon overexpression of SroC. The effects of deletion and overexpression of sroC in biofilm formation were also examined, where the ΔsroC and pSroC strains exhibited a reduced and increased ability to form biofilm, respectively. Furthermore, electron microscopy revealed that the wild-type strain overexpressing SroC had a non-flagellated phenotype. Taken together, our results showed that S. Typhimurium sRNA SroC modulates the flagellar synthesis by down-regulating the expression of flhBAE and fliE genes. © FEMS 2015. All rights reserved.Ítem Preclinical development and in vivo efficacy of ceftiofur-PLGA microparticles(Public Library of Science, 2015-04) Vilos, Cristian; Velasquez, Luis A.; Rodas, Paula I.; Zepeda, Katherine; Bong, Soung-Jae; Herrera, Natalia; Cantin, Mario; Simon, Felipe; Constandil, LuisDrug delivery systems based on polymeric microparticles represent an interesting field of development for the treatment of several infectious diseases for humans and animals. In this work, we developed PLGA microparticles loaded with ceftiofur (PLGA-cef), a third-generation cephalosporin that is used exclusively used in animals. PLGA-cef was prepared by the double emulsion w/o/w method, and exhibited a diameter in the range of 1.5-2.2 μm, and a negative ζ potential in the range of -35 to -55 mV. The loading yield of PLGA-cef was ∼7% and encapsulation efficiency was approximately 40%. The pharmacokinetic study demonstrated a sustained release profile of ceftiofur for 20 days. PLGA-cef administrated in a single dose was more effective than ceftiofur non-encapsulated in rats challenged with S. Typhimurium. The in vivo toxicological evaluation showed that PLGA-cef did not affect the blood biochemical, hematological and hemostasis parameters. Overall, the PLGA-cef showed slow in vivo release profile, high antibacterial efficacy, and low toxicity. The results obtained supports the safe application of PLGA-cef as sustained release platform in the veterinary industry. © 2015 Vilos et al.Ítem The NarE protein of neisseria gonorrhoeae catalyzes ADP-ribosylation of several ADP-ribose acceptors despite an N-terminal deletion(Oxford University Press, 2016-09) Rodas, Paula I.; Álamos-Musre, A. Said; Álvarez, Francisca P.; Escobar, Alejandro; Tapia, Cecilia V.; Osorio, Eduardo; Otero, Carolina; Calderón, Iván L.; Fuentes, Juan A.; Gil, Fernando; Paredes-Sabja, Daniel; Christodoulides, MyronThe ADP-ribosylating enzymes are encoded in many pathogenic bacteria in order to affect essential functions of the host. In this study, we show that Neisseria gonorrhoeae possess a locus that corresponds to the ADP-ribosyltransferase NarE, a previously characterized enzyme in N. meningitidis. The 291 bp coding sequence of gonococcal narE shares 100% identity with part of the coding sequence of the meningococcal narE gene due to a frameshift previously described, thus leading to a 49-amino-acid deletion at the N-terminus of gonococcal NarE protein. However, we found a promoter region and a GTG start codon, which allowed expression of the protein as demonstrated by RT-PCR and western blot analyses. Using a gonococcal NarE–6xHis fusion protein, we demonstrated that the gonococcal enzyme underwent auto-ADP-ribosylation but to a lower extent than meningococcal NarE. We also observed that gonoccocal NarE exhibited ADP-ribosyltransferase activity using agmatine and cell-free host proteins as ADP-ribose acceptors, but its activity was inhibited by human β-defensins. Taken together, our results showed that NarE of Neisseria gonorrhoeae is a functional enzyme that possesses key features of bacterial ADP-ribosylating enzymes.