Examinando por Autor "Gil, Fernando"
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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 Clostridioides difficile Persister Cells and Their Role in Antibiotic Tolerance(Multidisciplinary Digital Publishing Institute (MDPI), 2024-07) Inostroza, Osvaldo; Fuentes, Juan A.; Yáñez, Paulina; Espinoza, Giovanni; Fica, Omar; Queraltó, Camila; Rodríguez, José; Flores, Isidora; González, Ruth; Soto, Jorge A.; Calderón, Iván L.; Gil, FernandoClostridioides difficile is a Gram-positive pathogen known for its toxin production and spore formation. It is primarily responsible for most cases of antibiotic-associated diarrhea. Bacterial persisters are a small subset of the population that exhibits transient tolerance to bactericidal substances, and they are of significant medical concern due to their association with the emergence of antibiotic resistance and difficult-to-treat chronic or recurrent infections. Vancomycin, the predominant antibiotic utilized in the management of C. difficile infection, is extensively applied in the realm of clinical practice. Previous studies have demonstrated a persister-like phenotype with treatments involving this antibiotic. However, the mechanism in C. difficile remains largely unknown, primarily due to the challenge of isolating this small population at any given time. To better characterize C. difficile persister cells, we present a study that enables the enrichment and characterization of persister cells from bacterial cultures in both the exponential and stationary phases. Moreover, we could differentiate between triggered (induced using antibiotics such as vancomycin) and spontaneous (stochastic) persister cells. Additionally, we observed the involvement of toxin-antitoxin systems and Clp proteases in persister cell formation.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 Genome sequence of clostridium paraputrificum 373-A1 isolated in Chile from a patient infected with Clostridium difficile(American Society for Microbiology, 2016) Guerrero-Araya, Enzo; Plaza-Garrido, Angela; Díaz-Yañez, Fernando; Pizaro-Guajardo, Marjorie; Valenzuela, Sandro L.; Meneses, Claudio; Gil, Fernando; Castro-Nallar, Eduardo; Paredes-Sabja, DanielClostridium paraputrificum is a gut microbiota member reported in several cases of bacteremia and coinfections. So far, only one genome sequence of a C. paraputrificum (AGR2156) isolate is available. Here, we present the draft genome of C. paraputrificum strain 373-A1, isolated from stools from a patient with C. difficile infection.Ítem Identification of genes involved in biogenesis of Outer Membrane Vesicles (OMVs) in Salmonella enterica Serovar Typhi(Frontiers Media S.A., 2019-02) Nevermann, Jan; Silva, Andrés; Otero, Carolina; Oyarzún, Diego P.; Barrera, Boris; Gil, Fernando; Calderón, Iván L.; Fuentes, Juan A.Outer membrane vesicles (OMVs) are nano-sized proteoliposomes discharged from the cell envelope of Gram-negative bacteria. OMVs normally contain toxins, enzymes and other factors, and are used as vehicles in a process that has been considered a generalized, evolutionarily conserved delivery system among bacteria. Furthermore, OMVs can be used in biotechnological applications that require delivery of biomolecules, such as vaccines, remarking the importance of their study. Although it is known that Salmonella enterica serovar Typhi (S. Typhi), the etiological agent of typhoid fever in humans, delivers toxins (e.g., HlyE) via OMVs, there are no reports identifying genetic determinants of the OMV biogenesis in this serovar. In the present work, and with the aim to identify genes participating in OMV biogenesis in S. Typhi, we screened 15,000 random insertion mutants for increased HlyE secretion. We found 9 S. Typhi genes (generically called zzz genes) determining an increased HlyE secretion that were also involved in OMV biogenesis. The genes corresponded to ompA, nlpI, and tolR (envelope stability), rfaE and waaC (LPS synthesis), yipP (envC), mrcB (synthesis and remodeling of peptidoglycan), degS (stress sensor serine endopeptidase) and hns (global transcriptional regulator). We found that S. Typhi Δzzz mutants were prone to secrete periplasmic, functional proteins with a relatively good envelope integrity. In addition, we showed that zzz genes participate in OMV biogenesis, modulating different properties such as OMV size distribution, OMV yield and OMV protein cargo. Copyright © 2019 Nevermann, Silva, Otero, Oyarzún, Barrera, Gil, Calderón and Fuentes. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.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 “One for All”: Functional Transfer of OMV-Mediated Polymyxin B Resistance From Salmonella enterica sv. Typhi ΔtolR and ΔdegS to Susceptible Bacteria(Frontiers Media S.A., 2021-05) Marchant, Pedro; Carreño, Alexander; Vivanco, Eduardo; Silva, Andrés; Nevermann, Jan; Otero, Carolina; Araya, Eyleen; Gil, Fernando; Calderón, Iván L.; Fuentes, Juan A.The appearance of multi-resistant strains has contributed to reintroducing polymyxin as the last-line therapy. Although polymyxin resistance is based on bacterial envelope changes, other resistance mechanisms are being reported. Outer membrane vesicles (OMVs) are nanosized proteoliposomes secreted from the outer membrane of Gram-negative bacteria. In some bacteria, OMVs have shown to provide resistance to diverse antimicrobial agents either by sequestering and/or expelling the harmful agent from the bacterial envelope. Nevertheless, the participation of OMVs in polymyxin resistance has not yet been explored in S. Typhi, and neither OMVs derived from hypervesiculating mutants. In this work, we explored whether OMVs produced by the hypervesiculating strains Salmonella Typhi ΔrfaE (LPS synthesis), ΔtolR (bacterial envelope) and ΔdegS (misfolded proteins and σE activation) exhibit protective properties against polymyxin B. We found that the OMVs extracted from S. Typhi ΔtolR and ΔdegS protect S. Typhi WT from polymyxin B in a concentration-depending manner. By contrast, the protective effect exerted by OMVs from S. Typhi WT and S. Typhi ΔrfaE is much lower. This effect is achieved by the sequestration of polymyxin B, as assessed by the more positive Zeta potential of OMVs with polymyxin B and the diminished antibiotic’s availability when coincubated with OMVs. We also found that S. Typhi ΔtolR exhibited an increased MIC of polymyxin B. Finally, we determined that S. Typhi ΔtolR and S. Typhi ΔdegS, at a lesser level, can functionally and transiently transfer the OMV-mediated polymyxin B resistance to susceptible bacteria in cocultures. This work shows that mutants in genes related to OMVs biogenesis can release vesicles with improved abilities to protect bacteria against membrane-active agents. Since mutations affecting OMV biogenesis can involve the bacterial envelope, mutants with increased resistance to membrane-acting agents that, in turn, produce protective OMVs with a high vesiculation rate (e.g., S. Typhi ΔtolR) can arise. Such mutants can functionally transfer the resistance to surrounding bacteria via OMVs, diminishing the effective concentration of the antimicrobial agent and potentially favoring the selection of spontaneous resistant strains in the environment. This phenomenon might be considered the source for the emergence of polymyxin resistance in an entire bacterial community. © Copyright © 2021 Marchant, Carreño, Vivanco, Silva, Nevermann, Otero, Araya, Gil, Calderón and Fuentes.Ítem Participation of S. Typhimurium cysJIH operon in the H2S-mediated ciprofloxacin resistance in presence of sulfate as sulfur source(MDPI AG, 2015-07) Álvarez, Ricardo; Frávega, Jorge; Rodas, Paula; Fuentes, Juan A.; Paredes-Sabja, Daniel; Calderón, Iván L.; Gil, FernandoH2S production has been proposed as a mechanism to explain bacterial resistance to antibiotics. In this work, we present evidence for the role of the cysJIH operon in resistance to ciprofloxacin mediated by H2S production with different sulfate as the only sulfur source. We found that the products of the cysJIH operon are involved in ciprofloxacin resistance by increasing both, the levels of H2S and reduced thiols apparently counteracting antimicrobial-induced reactive oxygen species (ROS). This protective effect was observed only when bacteria were cultured in the presence of sulfate, but not with cysteine, as the sole sulfur source. © 2015, by the authors.Ítem Participation of the Salmonella OmpD porin in the infection of RAW264.7 macrophages and BALB/c mice(Public Library of Science, 2014) Ipinza, Francisco; Collao, Bernardo; Monsalva, Debbie; Bustamante, Victor H.; Luraschi, Roberto; Alegría-Arcos, Melissa; Almonacid, Daniel E.; Aguayo, Daniel; Calderón, Iván L.; Gil, Fernando; Santiviago, Carlos A.; Morales, Eduardo H.; Calva, Edmundo; Saavedra, Claudia P.Salmonella Typhimurium is the etiological agent of gastroenteritis in humans and enteric fever in mice. Inside these hosts, Salmonella must overcome hostile conditions to develop a successful infection, a process in which the levels of porins may be critical. Herein, the role of the Salmonella Typhimurium porin OmpD in the infection process was assessed for adherence, invasion and proliferation in RAW264.7 mouse macrophages and in BALB/c mice. In cultured macrophages, a ΔompD strain exhibited increased invasion and proliferation phenotypes as compared to its parental strain. In contrast, overexpression of ompD caused a reduction in bacterial proliferation but did not affect adherence or invasion. In the murine model, the ΔompD strain showed increased ability to survive and replicate in target organs of infection. The ompD transcript levels showed a down-regulation when Salmonella resided within cultured macrophages and when it colonized target organs in infected mice. Additionally, cultured macrophages infected with the ΔompD strain produced lower levels of reactive oxygen species, suggesting that down-regulation of ompD could favor replication of Salmonella inside macrophages and the subsequent systemic dissemination, by limiting the reactive oxygen species response of the host. © 2014 Ipinza et al.Ítem The chaperone ClpC participates in sporulation, motility, biofilm, and toxin production of Clostridioides difficile(Elsevier Ltd, 2023-06) Queraltó, Camila; Ortega, Constanza; Díaz-Yáñez, Fernando; Inostroza, Osvaldo; Espinoza, Giovanni; Álvarez, Ricardo; González, Ruth; Parra, Francisco; Paredes-Sabja, Daniel; Acuña, Lillian G.; Calderón, Iván L.; Fuentes, Juan A.; Gil, FernandoObjectives: Clostridioides difficile is a nosocomial pathogen that is associated with the use of antibiotics. One of the most worrying aspects of C. difficile infection is its ability to resist antimicrobial therapies, owing to spore formation. In several bacterial pathogens, proteases of the Clp family participate in phe notypes associated with persistence and virulence. This suggests that these proteins could be involved in virulence-related traits. In this study, we analysed the role of ClpC chaperone-protease of C. difficile in virulence-related traits by comparing the phenotypes of wild-type and mutant strains lacking the clpC gene ( clpC). Methods: We performed biofilm, motility, spore formation, and cytotoxicity assays. Results: Our results show significant differences between the wild-type and clpC strains in all analysed parameters. Conclusions: Based on these findings, we conclude that clpC plays a role in the virulence properties of C. difficile. © 2023 The Author(s). Published by Elsevier Ltd on behalf of International Society for Antimicrobial Chemotherapy. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)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.Ítem The RNA Chaperone Hfq Participates in Persistence to Multiple Antibiotics in the Fish Pathogen Yersinia ruckeri(MDPI AG, 2021-06) Calderón, Iván; Barros, María José; Montt, Fernanda; Gil, Fernando; Fuentes, Juan; Acuña, LillianYersinia ruckeri causes outbreaks of enteric redmouth disease in salmon aquaculture all over the world. The transient antibiotic tolerance exhibited by bacterial persisters is commonly thought to be responsible for outbreaks; however, the molecular factors underlying this behavior have not been explored in Y. ruckeri. In this study, we investigated the participation of the RNA chaperone Hfq from Y. ruckeri in antibiotic persistence. Cultures of the hfq-knockout mutant (∆hfq) exhibited faster replication, increased ATP levels and a more reductive environment than the wild type. The growth curves of bacteria exposed to sublethal concentrations of ampicillin, oxolinic acid, ciprofloxacin and polymyxin B revealed a greater susceptibility for the ∆hfq strain. The time-kill curves of bacteria treated with the antibiotics mentioned above and florfenicol, using inoculums from exponential, stationary and biofilm cultures, demonstrated that the ∆hfq strain has significant defects in persister cells production. To shed more light on the role of Hfq in antibiotic persistence, we analyzed its dependence on the (p)ppGpp synthetase RelA by determining the persister cells production in the absence of the relA gene. The ∆relA and ∆relA∆hfq strains displayed similar defects in persister cells formation, but higher than ∆hfq strain. Similarly, stationary cultures of the ∆relA and ∆relA∆hfq strains exhibited comparable levels of ATP but higher than that of the ∆hfq strain, indicating that relA is epistatic over hfq. Taken together, our findings provide valuable information on antibiotic persistence in Y. ruckeri, shedding light on the participation of Hfq in the persistence phenomenon. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.Ítem The small RNA RyhB homologs from Salmonella typhimurium participate in the response to S-nitrosoglutathione-induced stress(Academic Press Inc., 2014-07) Calderón, Paulina F.; Morales, Eduardo H.; Acuña, Lillian G.; Fuentes, Danitza N.; Gil, Fernando; Porwollik, S.; McClelland, Michael; Saavedra, Claudia P.; Calderón, Iván L.Typically, the expression of sRNAs is activated in response to environmental stimuli in order to regulate gene expression through post-transcriptional mechanisms. In the present work we show that the Salmo nella typhimurium paralogous sRNAs RyhB-1 and RyhB-2 are induced in response to the nitrosating agent S-nitrosoglutathione (GSNO). Inactivation of these sRNAs decreased S. typhimurium resistance to GSNO and increased the levels of nitrosylated proteins. These results prompted us to evaluate a possible role of these sRNAs in nitrosative stress resistance. RNA profiling was used as a screen to identify novel RyhB-1 and RyhB-2 regulated targets. A subset of genes was filtered based on their potential role in the response to nitrosative stress and their expression was analyzed by quantitative RT-PCR in wild type, single and double mutant strains (DryhB1, DryhB2 and DryhB1 DryhB2) treated with GSNO. In response to GSNO RyhB-1 and RyhB-2 negatively regulate the expression of the genes cyoABC (cytochrome bo oxi dase), cydB (cytochrome bd oxidase), cybC (cytochrome b-562), and positively regulate the nirBCD operon (nitrite reductase system). Together, these results suggest that RyhB-1 and RyhB-2 finely tune the expres sion of genes coding for cytochrome oxidases and the nitrate reductase system, allowing the cell to cope with GSNO-induced stress. 2014 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).Ítem β-lactam-induced OMV release promotes polymyxin tolerance in Salmonella enterica sv. Typhi(Frontiers Media SA, 2024-04) Marchant, Pedro; Vivanco, Erika; Silva, Andrés; Nevermann, Jan; Fuentes, Ignacio; Barrera, Boris; Otero, Carolina; Calderón, Iván L.; Gil, Fernando; Fuentes, Juan A.The rise of multidrug-resistant bacteria is a global concern, leading to a renewed reliance on older antibiotics like polymyxins as a last resort. Polymyxins, cationic cyclic peptides synthesized nonribosomally, feature a hydrophobic acyl tail and positively charged residues. Their antimicrobial mechanism involves initial interaction with Gram-negative bacterial outer-membrane components through polar and hydrophobic interactions. Outer membrane vesicles (OMVs), nano-sized proteoliposomes secreted from the outer membrane of Gram-negative bacteria, play a crucial role in tolerating harmful molecules, including cationic peptides such as polymyxins. Existing literature has documented environmental changes’ impact on modulating OMV properties in Salmonella Typhimurium. However, less information exists regarding OMV production and characteristics in Salmonella Typhi. A previous study in our laboratory showed that S. Typhi ΔmrcB, a mutant associated with penicillin-binding protein (PBP, a β-lactam antibiotic target), exhibited hypervesiculation. Consequently, this study investigated the potential impact of β-lactam antibiotics on promoting polymyxin tolerance via OMVs in S. Typhi. Our results demonstrated that sub-lethal doses of β-lactams increased bacterial survival against polymyxin B in S. Typhi. This phenomenon stems from β-lactam antibiotics inducing hypervesiculation of OMVs with higher affinity for polymyxin B, capturing and diminishing its biologically effective concentration. These findings suggest that β-lactam antibiotic use may inadvertently contribute to decreased polymyxin effectivity against S. Typhi or other Gram-negative bacteria, complicating the effective treatment of infections caused by these pathogens. This study emphasizes the importance of evaluating the influence of β-lactam antibiotics on the interaction between OMVs and other antimicrobial agents.