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Ítem Acceso Abierto Cerebrospinal fuid (CSF) augments metabolism and virulence expression factors in Acinetobacter baumannii(Nature Research, 2021-02-26) Martínez, Jasmine; Razo Gutiérrez, Chelsea; Razo Gutiérrez, Chelsea; Courville, Robert; Pimentel, Camila; Liu, Christine; Fung, Sammie E.; Tuttobene, Marisel Romina; Phan, Kimberly; Vila, Alejandro J.; Shahrestani, Parvin; Jimenez, Veronica; Tolmasky, Marcelo E.; Becka, Scott A.; Papp Wallace, Krisztina M.; Bonomo, Robert A.; Soler Bistue, Alfonso; Sieira, Rodrigo; Ramírez, María SoledadIn a recent report by the Centers for Disease Control and Prevention (CDC), multidrug resistant (MDR) Acinetobacter baumannii is a pathogen described as an “urgent threat.” Infection with this bacterium manifests as diferent diseases such as community and nosocomial pneumonia, bloodstream infections, endocarditis, infections of the urinary tract, wound infections, burn infections, skin and soft tissue infections, and meningitis. In particular, nosocomial meningitis, an unwelcome complication of neurosurgery caused by extensivelydrug resistant (XDR) A. baumannii, is extremely challenging to manage. Therefore, understanding how A. baumannii adapts to diferent host environments, such as cerebrospinal fuid (CSF) that may trigger changes in expression of virulence factors that are associated with the successful establishment and progress of this infection is necessary. The present invitro work describes, the genetic changes that occur during A. baumannii infltration into CSF and displays A. baumannii’s expansive versatility to persist in a nutrient limited environment while enhancing several virulence factors to survive and persist. While a hypervirulent A. baumannii strain did not show changes in its transcriptome when incubated in the presence of CSF, a lowvirulence isolate showed signifcant diferences in gene expression and phenotypic traits. Exposure to 4% CSF caused increased expression of virulence factors such as fmbriae, pilins, and iron chelators, and other virulence determinants that was confrmed in various model systems. Furthermore, although CSF’s presence did not enhance bacterial growth, an increase of expression of genes encoding transcription, translation, and the ATP synthesis machinery was observed. This work also explores A. baumannii’s response to an essential component, human serum albumin (HSA), within CSF to trigger the diferential expression of genes associated with its pathoadaptibility in this environment.Ítem Acceso Abierto Effects of lactoferrin, a protein present in the female reproductive tract, on parameters of human sperm capacitation and gamete interaction(Wiley, 2015-10-07) Zumoffen, Carlos María; Massa, Estefanía; Caille, Adriana María; Munuce, María José; Ghersevich, Sergio AlbinoIn a recent study, lactoferrin (LF) was detected in human oviductal secretion. The protein was able to bind to oocytes and sperm, and modulated gamete interaction. The aim of the present study was to investigate the effect of LF on parameters related to human sperm capacitation and sperm–zona pellucida interaction. Semen samples were obtained from healthy normozoospermic donors (n = 7). Human follicular fluids and oocytes were collected from patients undergoing in vitro fertilization. Motile sperm obtained by swim-up were incubated for 6 or 22 h under capacitating conditions with LF (0–100 μg/mL). After incubations, viability, motility, presence of α-d-mannose receptors (using a fluorescent probe on mannose coupled to bovine serum albumin), spontaneous and induced acrosome reaction (assessed with Pisum sativum agglutinin conjugated to fluorescein isothiocyanate), and tyrosine phosphorylation of sperm proteins were evaluated. Sperm–zona pellucida interaction in the presence of LF was investigated using the hemizone assay. The presence of LF did not affect sperm viability or motility, but caused a dose-dependent significant decrease in sperm α-d-mannose-binding sites, and the effect was already significant with the lowest concentration of the protein used after 22 h incubation. Dose-dependent significant increases in both induced acrosome reaction and tyrosine phosphorylation of sperm proteins were observed in the presence of LF. The present data indicate that LF modulates parameters of sperm function. The inhibition of gamete interaction by LF could be partially explained by the decrease in sperm d-mannose-binding sites. The presence of the LF promoted sperm capacitation in vitro.Ítem Acceso Abierto On the offensive: the role of outer membrane vesicles in the successful dissemination of New Delhi Metallo-β-lactamase (NDM-1)(American Society for Microbiology, 2021-09-28) Martínez, Melina María Belén; Bonomo, Robert A.; Vila, Alejandro J.; Maffía, Paulo César; González, Lisandro Javier; https://orcid.org/0000-0002-4007-8721; https://orcid.org/0000-0002-3299-894X; https://orcid.org/0000-0002-7978-3233; https://orcid.org/0000-0001-7423-2646; https://orcid.org/0000-0002-0575-1810The emergence and worldwide dissemination of carbapenemase-producing Gram-negative bacteria are a major public health threat. Metallo-β-lactamases (MBLs) represent the largest family of carbapenemases. Regrettably, these resistance determinants are spreading worldwide. Among them, the New Delhi metallo-β-lactamase (NDM-1) is experiencing the fastest and largest geographical spread. NDM-1 β-lactamase is anchored to the bacterial outer membrane, while most MBLs are soluble, periplasmic enzymes. This unique cellular localization favors the selective secretion of active NDM-1 into outer membrane vesicles (OMVs). Here, we advance the idea that NDM-containing vesicles serve as vehicles for the local dissemination of NDM-1. We show that OMVs with NDM-1 can protect a carbapenem-susceptible strain of Escherichia coli upon treatment with meropenem in a Galleria mellonella infection model. Survival curves of G. mellonella revealed that vesicle encapsulation enhances the action of NDM-1, prolonging and favoring bacterial protection against meropenem inside the larva hemolymph. We also demonstrate that E. coli cells expressing NDM-1 protect a susceptible Pseudomonas aeruginosa strain within the larvae in the presence of meropenem. By using E. coli variants engineered to secrete variable amounts of NDM-1, we demonstrate that the protective effect correlates with the amount of NDM-1 secreted into vesicles. We conclude that secretion of NDM-1 into OMVs contributes to the survival of otherwise susceptible nearby bacteria at infection sites. These results disclose that OMVs play a role in the establishment of bacterial communities, in addition to traditional horizontal gene transfer mechanisms. IMPORTANCE: Resistance to carbapenems, last-resort antibiotics, is spreading worldwide, raising great concern. NDM-1 is one of the most potent and widely disseminated carbapenem-hydrolyzing enzymes spread among many bacteria and is secreted to the extracellular medium within outer membrane vesicles. We show that vesicles carrying NDM-1 can protect carbapenem-susceptible strains of E. coli and P. aeruginosa upon treatment with meropenem in a live infection model. These vesicles act as nanoparticles that encapsulate and transport NDM-1, prolonging and favoring its action against meropenem inside a living organism. Secretion of NDM-1 into vesicles contributes to the survival of otherwise susceptible nearby bacteria at infection sites. We propose that vesicles play a role in the establishment of bacterial communities and the dissemination of antibiotic resistance, in addition to traditional horizontal gene transfer mechanisms.Ítem Acceso Abierto Slow protein dynamics elicits new enzymatic functions by means of epistatic interactions(Oxford University Press, 2022-09-22) Rossi, María Agustina; Palzkill, Timothy; Almeida, Fabio C. L.; Vila, Alejandro J.; https://orcid.org/0000-0002-7978-3233Protein evolution depends on the adaptation of these molecules to different functional challenges. This occurs by tuning their biochemical, biophysical, and structural traits through the accumulation of mutations. While the role of protein dynamics in biochemistry is well recognized, there are limited examples providing experimental evidence of the optimization of protein dynamics during evolution. Here we report an NMR study of four variants of the CTX-M β-lactamases, in which the interplay of two mutations outside the active site enhances the activity against a cephalosporin substrate, ceftazidime. The crystal structures of these enzymes do not account for this activity enhancement. By using NMR, here we show that the combination of these two mutations increases the backbone dynamics in a slow timescale and the exposure to the solvent of an otherwise buried β-sheet. The two mutations located in this β-sheet trigger conformational changes in loops located at the opposite side of the active site. We postulate that the most active variant explores alternative conformations that enable binding of the more challenging substrate ceftazidime. The impact of the mutations in the dynamics is context-dependent, in line with the epistatic effect observed in the catalytic activity of the different variants. These results reveal the existence of a dynamic network in CTX-M β-lactamases that has been exploited in evolution to provide a net gain-of-function, highlighting the role of alternative conformations in protein evolution.Ítem Acceso Abierto Fibroblast growth factor receptors (FGFRs) in human sperm: expression, functionality and involvement in motility regulation(Public Library of Science (PLOS), 2015-05-13) Saucedo, Lucía; Buffa, Gabriela Natalia; Rosso, Marina; Guillardoy, Tomás; Góngora, Adrián; Munuce, María José; Vázquez Levin, Mónica Hebe; Marín Briggiler, Clara; Dr. Baldi, Alberto provide the FGF2Fibroblast growth factors receptors (FGFRs) have been widely characterized in somatic cells, but there is scarce evidence of their expression and function in mammalian gametes. The objective of the present study was to evaluate the expression of FGFRs in human male germ cells, to determine sperm FGFR activation by the FGF2 ligand and their participation in the regulation of sperm motility. The expression of FGFR1, 2, 3 and 4 mRNAs and proteins in human testis and localization of these receptors in germ cells of the seminiferous epithelium was demonstrated. In ejaculated sperm, FGFRs were localized to the acrosomal region and flagellum. Sperm exposure to FGF2 caused an increase in flagellar FGFR phosphorylation and activation of extracellular signal-regulated kinase (ERK) and protein kinase B (PKB or Akt) signaling pathways. Incubation with FGF2 led to a significant increase in the percentage of total and progressive sperm motility, as well as in sperm kinematics. All responses were prevented by sperm preincubation with BGJ398, a specific inhibitor of FGFR tyrosine kinase activity. In addition to confirming the expression of FGFRs in germ cells of the human testis, our study describes for the first time the presence, localization and functionality of human sperm FGFRs, and provides evidence of the beneficial effect of FGF2 upon sperm motility.Ítem Acceso Abierto Gating interactions steer loop conformational changes in the active site of the L1 metallo-β-lactamase(eLife Sciences Publications, 2023-02-24) Zhao, Zhuoran; Shen, Xiayu; Chen, Shuang; Gu, Jing; Wang, Haun; Mojica, María F.; Samanta, Moumita; Bhowmik, Debsindhu; Vila, Alejandro J.; Bonomo, Robert A.; Haider, Shozeb; http://orcid.org/0000-0002-1380-9824; http://orcid.org/0000-0001-7770-9091; http://orcid.org/0000-0002-7978-3233; http://orcid.org/0000-0003-2650-2925β-Lactam antibiotics are the most important and widely used antibacterial agents across the world. However, the widespread dissemination of β-lactamases among pathogenic bacteria limits the efficacy of β-lactam antibiotics. This has created a major public health crisis. The use of β-lactamase inhibitors has proven useful in restoring the activity of β-lactam antibiotics, yet, effective clinically approved inhibitors against class B metallo-β-lactamases are not available. L1, a class B3 enzyme expressed by Stenotrophomonas maltophilia, is a significant contributor to the β-lactam resistance displayed by this opportunistic pathogen. Structurally, L1 is a tetramer with two elongated loops, α3-β7 and β12-α5, present around the active site of each monomer. Residues in these two loops influence substrate/inhibitor binding. To study how the conformational changes of the elongated loops affect the active site in each monomer, enhanced sampling molecular dynamics simulations were performed, Markov State Models were built, and convolutional variational autoencoder-based deep learning was applied. The key identified residues (D150a, H151, P225, Y227, and R236) were mutated and the activity of the generated L1 variants was evaluated in cellbased experiments. The results demonstrate that there are extremely significant gating interactions between α3-β7 and β12-α5 loops. Taken together, the gating interactions with the conformational changes of the key residues play an important role in the structural remodeling of the active site. These observations offer insights into the potential for novel drug development exploiting these gating interactions.Ítem Acceso Abierto Arabidopsis thaliana Hcc1 is a Sco-like metallochaperonefor Cu A assembly in Cytochrome c Oxidase(Wiley, 2020-12) Llases, María Eugenia; Lisa, María Natalia; Morgada, Marcos Nicolás; Giannini, Estefanía; Alzari, Pedro M.; Vila, Alejandro J.The assembly of the CuA site in Cytochrome c Oxidase (COX) is a criticalstep for aerobic respiration in COX-dependent organisms. Several geneproducts have been associated with the assembly of this copper site, themost conserved of them belonging to the Sco family of proteins, whichhave been shown to perform different roles in different organisms. Plantsexpress two orthologs of Sco proteins: Hcc1 and Hcc2. Hcc1 is known tobe essential for plant development and for COX maturation, but its precisefunction has not been addressed until now. Here, we report the biochemi-cal, structural and functional characterization of Arabidopsis thaliana Hcc1protein (here renamed Sco1). We solved the crystal structure of the Cu+1-bound soluble domain of this protein, revealing a tri coordinated environ-ment involving a CxxxCxnH motif. We show that AtSco1 is able to workas a copper metallochaperone, inserting two Cu+1 ions into the CuA site ina model of CoxII. We also show that AtSco1 does not act as a thiol-disul-fide oxido-reductase. Overall, this information sheds new light on the bio-chemistry of Sco proteins, highlighting the diversity of functions amongthem despite their high structural similarities.Ítem Acceso Abierto Assessing endocrine and immune parameters in human immunodeficiency virus-infected patients before and after the immune reconstitution inflammatory syndrome(Brazilian Society of Endocrinology and Metabolism, 2018-02) Rateni, Liliana Beatriz; Lupo, Sergio; Racca, Liliana; Palazzi, Jorge; Ghersevich, Sergio AlbinoObjective: The present study compares immune and endocrine parameters between HIV-infected patients who underwent the Immune Reconstitution Inflammatory Syndrome (IRIS-P) during antiretroviral therapy (ART) and HIV-patients who did not undergo the syndrome (non-IRIS-P). Materials and methods: Blood samples were obtained from 31 HIV-infected patients (15 IRIS-P and 16 non-IRIS-P) before ART (BT) and 48 ± 2 weeks after treatment initiation (AT). Plasma Interleukin-6 (IL-6) and Interleukin-18 (IL-18) were determined by ELISA. Cortisol, dehydroepiandrosterone sulfate (DHEA-S) and thyroxin concentrations were measured using chemiluminescence immune methods. Results: Concentrations of IL-6 (7.9 ± 1.9 pg/mL) and IL-18 (951.5 ± 233.0 pg/mL) were significantly higher (p < 0.05) in IRIS-P than in non-IRIS-P (3.9 ± 1.0 pg/mL and 461.0 ± 84.4 pg/mL, respectively) BT. Mean T4 plasma level significantly decreased in both groups of patients after treatment (p < 0.05). In both groups cortisol levels were similar before and after ART (p > 0.05). Levels of DHEA-S in IRIS-P decreased AT (1080.5 ± 124.2 vs. 782.5 ± 123.8 ng/mL, p < 0.05) and they were significantly lower than in non-IRIS-P (782.5 ± 123.8 vs. 1203.7 ± 144.0 ng/mL, p < 0.05). IRIS-P showed higher values of IL-6 and IL-18 BT and lower levels of DHEA-S AT than in non-IRIS-P. Conclusion: These parameters could contribute to differentiate IRIS-P from non-IRIS-P. The significant decrease in DHEA-S levels in IRIS-P after ART might suggest a different adrenal responseÍtem Acceso Abierto Identification of key sequence features required for microRNA biogenesis in plants(Nature Research, 2020-10-21) Rojas, Arantxa María Larisa; Drusin, Salvador Iván; Chorostecki, Uciel Pablo; Mateos, Julieta L.; Moro, Belén; Bologna, Nicolás G.; Bresso, Edgardo Gabriel; Schapire, Arnaldo L.; Rasia, Rodolfo M.; Moreno, Diego M.; Palatnik, Javier F.; http://orcid.org/0000-0003-4316-4518; http://orcid.org/0000-0001-5350-514X; http://orcid.org/0000-0003-2229-6853; http://orcid.org/0000-0002-1156-6662; http://orcid.org/0000-0002-2810-3533; http://orcid.org/0000-0002-2161-7910; http://orcid.org/0000-0002-9798-459X; http://orcid.org/0000-0003-3940-067X; http://orcid.org/0000-0001-5493-8537; http://orcid.org/0000-0001-7996-5224MicroRNAs (miRNAs) are endogenous small RNAs of ∼21 nt that regulate multiple biological pathways in multicellular organisms. They derive from longer transcripts that harbor an imperfect stem-loop structure. In plants, the ribonuclease type III DICER-LIKE1 assisted by accessory proteins cleaves the precursor to release the mature miRNA. Numerous studies highlight the role of the precursor secondary structure during plant miRNA biogenesis; however, little is known about the relevance of the precursor sequence. Here, we analyzed the sequence composition of plant miRNA primary transcripts and found specifically located sequence biases. We show that changes in the identity of specific nucleotides can increase or abolish miRNA biogenesis. Most conspicuously, our analysis revealed that the identity of the nucleotides at unpaired positions of the precursor plays a crucial role during miRNA biogenesis in Arabidopsis.Ítem Acceso Abierto Interactions of hydrolyzed β-lactams with the L1 metallo-β-lactamase: crystallography supports stereoselective binding of cephem/carbapenem products(Elsevier, 2023-03-15) Hinchliffe, Philip; Calvopiña, Karina; Rabe, Patrick; Mojica, María F.; Schofield, Christopher J.; Dmitrienko, Gary I.; Bonomo, Robert A.; Vila, Alejandro J.; Spencer, James; https://orcid.org/0000-0002-1380-9824; https://orcid.org/0000-0002-7978-3233L1 is a dizinc subclass B3 metallo-β-lactamase (MBL) that hydrolyzes most β-lactam antibiotics and is a key resistance determinant in the Gram-negative pathogen Stenotrophomonas maltophilia, an important cause of nosocomial infections in immunocompromised patients. L1 is not usefully inhibited by MBL inhibitors in clinical trials, underlying the need for further studies on L1 structure and mechanism. We describe kinetic studies and crystal structures of L1 in complex with hydrolyzed β-lactams from the penam (mecillinam), cephem (cefoxitin/cefmetazole), and carbapenem (tebipenem, doripenem, and panipenem) classes. Despite differences in their structures, all the β-lactam-derived products hydrogen bond to Tyr33, Ser221, and Ser225 and are stabilized by interactions with a conserved hydrophobic pocket. The carbapenem products were modeled as Δ1-imines, with (2S)-stereochemistry. Their binding mode is determined by the presence of a 1β-methyl substituent: the Zn-bridging hydroxide either interacts with the C-6 hydroxyethyl group (1β-hydrogen-containing carbapenems) or is displaced by the C-6 carboxylate (1β-methyl-containing carbapenems). Unexpectedly, the mecillinam product is a rearranged N-formyl amide rather than penicilloic acid, with the N-formyl oxygen interacting with the Zn-bridging hydroxide. NMR studies imply mecillinam rearrangement can occur nonenzymatically in solution. Cephem-derived imine products are bound with (3R)-stereochemistry and retain their 3′ leaving groups, likely representing stable endpoints, rather than intermediates, in MBL-catalyzed hydrolysis. Our structures show preferential complex formation by carbapenem- and cephem-derived species protonated on the equivalent (β) faces and so identify interactions that stabilize diverse hydrolyzed antibiotics. These results may be exploited in developing antibiotics, and β-lactamase inhibitors, that form long-lasting complexes with dizinc MBLs.Ítem Acceso Abierto The H-NS regulator plays a role in the stress induced by carbapenemase expression in acinetobacter baumannii(American Society for Microbiology, 2020-08-26) Huang, Fanny; Fitchett, Noelle; Razo Gutiérrez, Chelsea; Le, Casin; Martínez, Jasmine; Ra, Grace; López, Carolina; González, Lisandro Javier; Sieira, Rodrigo; Vila, Alejandro J.; Bonomo, Robert A.; Ramírez, María Soledad; https://orcid.org/0000-0002-9904-7890Disruption of the histone-like nucleoid structuring protein (H-NS) was shown to affect the ability of Gram-negative bacteria to regulate genes associated with virulence, persistence, stress response, quorum sensing, biosynthesis pathways, and cell adhesion. Here, we used the expression of metallo--lactamases (MBLs), known to elicit envelope stress by the accumulation of toxic precursors in the periplasm, to interrogate the role of H-NS in Acinetobacter baumannii, together with other stressors. Using a multidrug-resistant A. baumannii strain, we observed that H-NS plays a role in alleviating the stress triggered by MBL toxic precursors and counteracts the effect of DNA-damaging agents, supporting its role in stress response.Ítem Acceso Abierto Structural determinants of Arabidopsis thaliana Hyponastic Leaves 1 function in vivo(Public Library of Science, 2014-11-19) Burdisso, Paula; Milia, Fernando; Schapire, Arnaldo L.; Bologna, Nicolás G.; Palatnik, Javier F.; Rasia, Rodolfo M.MicroRNAs have turned out to be important regulators of gene expression. These molecules originate from longer transcripts that are processed by ribonuclease III (RNAse III) enzymes. Dicer proteins are essential RNAse III enzymes that are involved in the generation of microRNAs (miRNAs) and other small RNAs. The correct function of Dicer relies on the participation of accessory dsRNA binding proteins, the exact function of which is not well-understood so far. In plants, the double stranded RNA binding protein Hyponastic Leaves 1 (HYL1) helps Dicer Like protein (DCL1) to achieve an efficient and precise excision of the miRNAs from their primary precursors. Here we dissected the regions of HYL1 that are essential for its function in Arabidopsis thaliana plant model. We generated mutant forms of the protein that retain their structure but affect its RNA-binding properties. The mutant versions of HYL1 were studied both in vitro and in vivo, and we were able to identify essential aminoacids/residues for its activity. Remarkably, mutation and even ablation of one of the purportedly main RNA binding determinants does not give rise to any major disturbances in the function of the protein. We studied the function of the mutant forms in vivo, establishing a direct correlation between affinity for the pri-miRNA precursors and protein activity.Ítem Acceso Abierto Acute diarrhoea in children: determination of duration using a combined bismuth hydroxide gel and oral rehydration solution therapy vs. oral rehydration solution(MDPI, 2016-12-21) Oviedo, Adriana; Díaz, Mirna; Valenzuela, María Laura; Vidal, Victoria; Racca, Liliana; Bottai, Hebe; Priore, Graciela; Peluffo, Graciela; Di Bartolomeo, Susana; Cabral, Graciela; Toca, María del CarmenOral rehydration salt (ORS) treatment in young children with acute diarrhoea (AD) has contributed to decrease mortality associated with dehydration although effective strategies to reduce morbidity associated with this disease are required. The aim of this study was to evaluate the diarrhoea duration when using combined colloidal bismuth hydroxide gel (CBHG) and oral rehydration salt treatment compared with ORS therapy in children with AD. We designed a double-blind, randomised prospective study with treatment and control groups. Patients aged one to 12 years, with no prior pathology and with AD of less than 48 h were included. The Chi-squared and Mann-Whitney tests were used, as well as the Cox proportional hazards model and the Kaplan-Meier estimator. Patients were randomised into an ORS and CBHG treatment group and a control group for ORS plus placebo. (Average age: 3.2 years). The result of the post-treatment evaluation with respect to the average duration of AD was 25.5 h for the treated group vs. 41.5 h for the control group (p = 0.015). The average number of stools was 4.8 in the treated group and 8.2 in the control group (p = 0.032). We conclude that the use of CBHG plus ORS significantly reduced the duration of AD, the number of stools and the percentage of children with persistent AD after 24 h of treatment compared to the control group. AD remitted almost twice as fast in patients treated with CBHG and ORS compared to those who received ORS plus placebo.Ítem Acceso Abierto An experiment-informed signal transduction model for the role of the Staphylococcus aureus MecR1 protein in β-lactam resistance(Springer Nature, 2019-12-20) Belluzo, Bruno Salvador; Abriata, Luciano Andrés; Giannini, Estefanía; Mihovilcevic, Damila; Dal Peraro, Matteo; Llarrull, Leticia IreneThe treatment of hospital- and community-associated infections by methicillin-resistant Staphylococcus aureus (MRSA) is a perpetual challenge. This Gram-positive bacterium is resistant specifically to β-lactam antibiotics, and generally to many other antibacterial agents. Its resistance mechanisms to β-lactam antibiotics are activated only when the bacterium encounters a β-lactam. This activation is regulated by the transmembrane sensor/signal transducer proteins BlaR1 and MecR1. Neither the transmembrane/metalloprotease domain, nor the complete MecR1 and BlaR1 proteins, are isolatable for mechanistic study. Here we propose a model for full-length MecR1 based on homology modeling, residue coevolution data, a new extensive experimental mapping of transmembrane topology, partial structures, molecular simulations, and available NMR data. Our model defines the metalloprotease domain as a hydrophilic transmembrane chamber effectively sealed by the apo-sensor domain. It proposes that the amphipathic helices inserted into the gluzincin domain constitute the route for transmission of the β-lactam-binding event in the extracellular sensor domain, to the intracellular and membrane-embedded zinc-containing active site. From here, we discuss possible routes for subsequent activation of proteolytic action. This study provides the first coherent model of the structure of MecR1, opening routes for future functional investigations on how β-lactam binding culminates in the proteolytic degradation of MecI.Ítem Acceso Abierto Neurite outgrowth induced by stimulation of angiotensin II AT2 receptors in SH-SY5Y neuroblastoma cells involves c-Src activation(Elsevier, 2023) Blanco, Helga M.; Perez, Celia N.; Banchio, Claudia; Alvarez, Sergio E.; Ciuffo, Gladys M.Neuroblastoma, the most common extracranial solid tumor occurring in childhood, originates from the aberrant proliferation of neural crest cells. Accordingly, the mechanism underling neuronal differentiation could provide new strategies for neuroblastoma treatment. It is well known that neurite outgrowth could be induced by Angiotensin II (Ang II) AT2 receptors; however, the signaling mechanism and its possible interaction with NGF (neural growth factor) receptors remain unclear. Here, we show that Ang II and CGP42112A (AT2 receptor agonist) promote neuronal differentiation by inducing neurite outgrowth and βIII-tubulin expression in SH-SY5Y neuroblastoma cells. In addition, we demonstrate that treatment with PD123319 (AT2 receptor antagonist) reverts Ang II or CGP42112A-induced differentiation. By using specific pharmacological inhibitors we established that neurite outgrowth induced by CGP42112A requires the activation of MEK (mitogen-activated protein kinase kinase), SphK (sphingosine ki- nase) and c-Src but not PI3K (phosphatidylinositol 3-kinase). Certainly, CGP42112A stimulated a rapid and transient (30 s, 1 min) phosphorylation of c-Src at residue Y416 (indicative of activation), following by a Src deactivation as indicated by phosphorylation of Y527. Moreover, inhibition of the NGF receptor tyrosine kinase A (TrkA) reduced neurite outgrowth induced by Ang IIand CGP42112A. In summary, we demonstrated that AT2 receptor-stimulated neurite outgrowth in SH-SY5Y cells involves the induction of MEK, SphK and c-Src and suggests a possible transactivation of TrkA. In that regard, AT2 signaling pathway is a key player in neuronal differentiation and might be a potential target for therapeutic treatments.Ítem Acceso Abierto Involvement of the histone-like nucleoid structuring protein (H-NS) in Acinetobacter baumannii’s natural transformation(MDPI, 2021-08-26) Le, Casin; Pimentel, Camila; Tuttobene, Marisel Romina; Subils, Tomás; Escalante, Jenny; Nishimura, Brent; Arriaga, Susana; Rodgers, Deja; Bonomo, Robert A.; Sieira, Rodrigo; Tolmasky, Marcelo E.; Ramírez, María Soledad; https://orcid.org/0000-0003-0234-4643; https://orcid.org/0000-0002-4495-563X; https://orcid.org/0000-0002-6298-7811; https://orcid.org/0000-0002-9904-7890Most Acinetobacter baumannii strains are naturally competent. Although some information is available about factors that enhance or reduce the frequency of the transformation of this bacterium, the regulatory elements and mechanisms are barely understood. In this article, we describe studies on the role of the histone-like nucleoid structuring protein, H-NS, in the regulation of the expression of genes related to natural competency and the ability to uptake foreign DNA. The expression levels of the natural transformation-related genes pilA, pilT, pilQ, comEA, comEC, comF, and drpA significantly increased in a ∆hns derivative of A. baumannii A118. The complementation of the mutant with a recombinant plasmid harboring hns restored the expression levels of six of these genes (pilT remained expressed at high levels) to those of the wild-type strain. The transformation frequency of the A. baumannii A118 ∆hns strain was significantly higher than that of the wild-type. Similar, albeit not identical, there were consequences when hns was deleted from the hypervirulent A. baumannii AB5075 strain. In the AB5075 complemented strain, the reduction in gene expression in a few cases was not so pronounced that it reached wild-type levels, and the expression of comEA was enhanced further. In conclusion, the expression of all seven transformation-related genes was enhanced after deleting hns in A. baumannii A118 and AB5075, and these modifications were accompanied by an increase in the cells’ transformability. The results highlight a role of H-NS in A. baumannii’s natural competence.Ítem Acceso Abierto Bacterially produced metabolites protect C. elegans neurons from degeneration(Public Library of Science, 2020-03-24) Urrutia, Arles; Garcia Angulo, Victor Antonio; Fuentes, Andrés; Caneo, Mauricio; Legüe, Marcela; Urquiza Zurich, Sebastian; Delgado, Scarlett E; Ugalde, Juan; Burdisso, Paula; Calixto, AndreaCaenorhabditis elegans and its cognate bacterial diet comprise a reliable, widespread model to study diet and microbiota effects on host physiology. Nonetheless, how diet influences the rate at which neurons die remains largely unknown. A number of models have been used in C. elegans as surrogates for neurodegeneration. One of these is a C. elegans strain expressing a neurotoxic allele of the mechanosensory abnormality protein 4 (MEC-4d) degenerin/epithelial Na+ (DEG/ENaC) channel, which causes the progressive degeneration of the touch receptor neurons (TRNs). Using this model, our study evaluated the effect of various dietary bacteria on neurodegeneration dynamics. Although degeneration of TRNs was steady and completed at adulthood in the strain routinely used for C. elegans maintenance (Escherichia coli OP50), it was significantly reduced in environmental and other laboratory bacterial strains. Strikingly, neuroprotection reached more than 40% in the E. coli HT115 strain. HT115 protection was long lasting well into old age of animals and was not restricted to the TRNs. Small amounts of HT115 on OP50 bacteria as well as UV-killed HT115 were still sufficient to produce neuroprotection. Early growth of worms in HT115 protected neurons from degeneration during later growth in OP50. HT115 diet promoted the nuclear translocation of DAF-16 (ortholog of the FOXO family of transcription factors), a phenomenon previously reported to underlie neuroprotection caused by down-regulation of the insulin receptor in this system. Moreover, a daf-16 loss-of-function mutation abolishes HT115-driven neuroprotection. Comparative genomics, transcriptomics, and metabolomics approaches pinpointed the neurotransmitter γ-aminobutyric acid (GABA) and lactate as metabolites differentially produced between E. coli HT115 and OP50. HT115 mutant lacking glutamate decarboxylase enzyme genes (gad), which catalyze the conversion of GABA from glutamate, lost the ability to produce GABA and also to stop neurodegeneration. Moreover, in situ GABA supplementation or heterologous expression of glutamate decarboxylase in E. coli OP50 conferred neuroprotective activity to this strain. Specific C. elegans GABA transporters and receptors were required for full HT115-mediated neuroprotection. Additionally, lactate supplementation also increased anterior ventral microtubule (AVM) neuron survival in OP50. Together, these results demonstrate that bacterially produced GABA and other metabolites exert an effect of neuroprotection in the host, highlighting the role of neuroactive compounds of the diet in nervous system homeostasis.Ítem Acceso Abierto The assembly of bacteria living in natural environments shapes neuronal integrity and behavioral outputs in Caenorhabditis elegans(American Society for Microbiology, 2023-04-25) Urquiza Zurich, Sebastian; Garcia Angulo, Victor Antonio; Burdisso, Paula; Palominos, M. Fernanda; Fernandez Hubeid, Lucia; Harcha, Paloma A.; Castillo, Juan P.; Calixto, Andrea; https://orcid.org/0000-0002-6168-7286Bacterivore nematodes are the most abundant animals in the biosphere, largely contributing to global biogeochemistry. Thus, the effects of environmental microbes on the nematodes’ life-history traits are likely to contribute to the general health of the biosphere. Caenorhabditis elegans is an excellent model to study the behavioral and physiological outputs of microbial diets. However, the effects of complex natural bacterial assemblies have only recently been reported, as most studies have been carried out with monoxenic cultures of laboratory-reared bacteria. Here, we quantified the physiological, phenotypic, and behavioral traits of C. elegans feeding on two bacteria that were coisolated with wild nematodes from a soil sample. These bacteria were identified as a putative novel species of Stenotrophomonas named Stenotrophomonas sp. strain Iso1 and a strain of Bacillus pumilus designated Iso2. The distinctive behaviors and developmental patterns observed in animals fed with individual isolates changed when bacteria were mixed. We studied in more depth the degeneration rate of the touch circuit of C. elegans and show that B. pumilus alone is protective, while the mix with Stenotrophomonas sp. is degenerative. The analysis of the metabolite contents of each isolate and their combination identified NAD1 as being potentially neuroprotective. In vivo supplementation shows that NAD1 restores neuroprotection to the mixes and also to individual nonprotective bacteria. Our results highlight the distinctive physiological effects of bacteria resembling native diets in a multicomponent scenario rather than using single isolates on nematodes.Ítem Acceso Abierto Acinetobacter baumannii response to cefiderocol challenge in human urine(Springer Nature, 2022-05-24) Nishimura, Brent; Escalante, Jenny; Tuttobene, Marisel Romina; Subils, Tomás; Mezcord, Vyanka; Pimentel, Camila; Georgeos, Nardin; Pasteran, Fernando; Rodríguez, Cecilia; Sieira, Rodrigo; Actis, Luis A.; Tolmasky,, Marcelo E.; Bonomo, Robert A.; Ramírez, María SoledadCefiderocol (CFDC) is a novel chlorocatechol-substituted siderophore antibiotic approved to treat complicated urinary tract infections (cUTI) and hospital-acquired and ventilator-acquired pneumonia (HAP/VAP). Previous work determined that albumin-rich human fluids increase the minimum inhibitory concentration (MICs) of Acinetobacter baumannii against CFDC and reduce the expression of genes related to iron uptake systems. This latter effect may contribute to the need for higher concentrations of CFDC to inhibit growth. The presence of human urine (HU), which contains low albumin concentrations, did not modify MIC values of two carbapenem-resistant A. baumannii. Levels of resistance to CFDC were not modified by HU in strain AMA40 but were reduced in strain AB5075. Expanding the studies to other carbapenem-resistant A. baumannii isolates showed that the presence of HU resulted in unmodified or reduced MIC of CDFC values. The expression of piuA, pirA, bauA, and bfnH determined by qRT-PCR was enhanced in A. baumannii AMA40 and AB5075 by the presence of HU in the culture medium. All four tested genes code for functions related to recognition and transport of ferric-siderophore complexes. The effect of HU on expression of pbp1, pbp3, blaOXA-51-like, blaADC, and blaNDM-1, genes associated with resistance to β-lactams, as well as genes coding for efflux pumps and porins was variable, showing dependence with the strain analyzed. We conclude that the lack of significant concentrations of albumin and free iron in HU makes this fluid behave differently from others we tested. Unlike other albumin rich fluids, the presence of HU does not impact the antibacterial activity of CFDC when tested against A. baumannii.Ítem Acceso Abierto 2-Mercaptomethyl-thiazolidines use conserved aromatic–S interactions to achieve broad-range inhibition of metallo-β-lactamases(Royal Society of Chemistry, 2021-01-05) Rossi, María Agustina; Martínez, Verónica; Hinchliffe, Philip; Mojica, María F.; Castillo, Valerie; Moreno, Diego M.; Smith, Ryan; Spellberg, Brad; Drusano, George L.; Banchio, Claudia; Bonomo, Robert A.; Spencer, James; Vila, Alejandro J.; Mahler, Graciela; https://orcid.org/0000-0003-4720-4070; https://orcid.org/0000-0002-3697-5219; https://orcid.org/0000-0001-8611-4743; https://orcid.org/0000-0002-1380-9824; https://orcid.org/0000-0001-5493-8537; https://orcid.org/0000-0002-4602-0571; https://orcid.org/0000-0002-7978-3233; https://orcid.org/0000-0003-0612-0516Infections caused by multidrug resistant (MDR) bacteria are a major public health threat. Carbapenems are among the most potent antimicrobial agents that are commercially available to treat MDR bacteria. Bacterial production of carbapenem-hydrolysing metallo-β-lactamases (MBLs) challenges their safety and efficacy, with subclass B1 MBLs hydrolysing almost all β-lactam antibiotics. MBL inhibitors would fulfil an urgent clinical need by prolonging the lifetime of these life-saving drugs. Here we report the synthesis and activity of a series of 2-mercaptomethyl-thiazolidines (MMTZs), designed to replicate MBL interactions with reaction intermediates or hydrolysis products. MMTZs are potent competitive inhibitors of B1 MBLs in vitro (e.g., Ki = 0.44 μM vs. NDM-1). Crystal structures of MMTZ complexes reveal similar binding patterns to the most clinically important B1 MBLs (NDM-1, VIM-2 and IMP-1), contrasting with previously studied thiol-based MBL inhibitors, such as bisthiazolidines (BTZs) or captopril stereoisomers, which exhibit lower, more variable potencies and multiple binding modes. MMTZ binding involves thiol coordination to the Zn(II) site and extensive hydrophobic interactions, burying the inhibitor more deeply within the active site than D/L-captopril. Unexpectedly, MMTZ binding features a thioether–π interaction with a conserved active-site aromatic residue, consistent with their equipotent inhibition and similar binding to multiple MBLs. MMTZs penetrate multiple Enterobacterales, inhibit NDM-1 in situ, and restore carbapenem potency against clinical isolates expressing B1 MBLs. Based on their inhibitory profile and lack of eukaryotic cell toxicity, MMTZs represent a promising scaffold for MBL inhibitor development. These results also suggest sulphur–π interactions can be exploited for general ligand design in medicinal chemistry.