Examinando por Autor "Bonomo, Robert A."
Mostrando 1 - 18 de 18
Resultados por página
Opciones de ordenación
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.Ítem Acceso Abierto A general reaction mechanism for carbapenem hydrolysis by mononuclear and binuclear metallo-β-lactamases(Nature, 2017-09-14) Lisa, María Natalia; Palacios, Antonela R.; Aitha, Mahesh; González, Mariano M.; Moreno, Diego M.; Crowder, Michael W.; Bonomo, Robert A.; Spencer, James; Tierney, David L.; Llarrull, Leticia Irene; Vila, Alejandro J.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 Characterisation of ST25 NDM-1-producing Acinetobacter spp. strains leading the increase in NDM-1 emergence in Argentina(Elsevier, 2020-09-02) Rodgers, Deja; Pasteran, Fernando; Calderon, Manuel; Jaber, Sara; Traglia, German M.; Albornoz, Ezequiel; Corso, Alejandra; Vila, Alejandro J.; Bonomo, Robert A.; Adams, Mark D.; Ramírez, María SoledadÍtem Acceso Abierto Deciphering the evolution of metallo-β-lactamases: A journey from the test tube to the bacterial periplasm(American Society for Biochemistry and Molecular Biology Inc., 2022-02-01) López, Carolina; Delmonti, Juliana; Bonomo, Robert A.; Vila, Alejandro J.Ítem Acceso Abierto Histone-like nucleoid-structuring protein (H-NS) regulatory role in antibiotic resistance in Acinetobacter baumannii(Nature Research, 2021) Rodgers, Deja; Le, Casin; Pimentel, Camila; Tuttobene, Marisel Romina; Subils, Tomás; Escalante, Jenny; Nishimura, Brent; García Véscovi, Eleonora; Sieira, Rodrigo; Bonomo, Robert A.; Tolmasky, Marcelo E.; Ramírez, María SoledadÍtem Acceso Abierto Host-specific enzyme-substrate interactions in SPM-1 metallo-β-lactamase are modulated by second sphere residues(Public Library of Science (PLOS), 2014-01-02) González, Lisandro Javier; Moreno, Diego M.; Bonomo, Robert A.; Vila, Alejandro J.Pseudomonas aeruginosa is one of the most virulent and resistant non-fermenting Gram-negative pathogens in the clinic. Unfortunately, P. aeruginosa has acquired genes encoding metallo-β-lactamases (MβLs), enzymes able to hydrolyze most β-lactam antibiotics. SPM-1 is an MβL produced only by P. aeruginosa, while other MβLs are found in different bacteria. Despite similar active sites, the resistance profile of MβLs towards β-lactams changes from one enzyme to the other. SPM-1 is unique among pathogen-associated MβLs in that it contains “atypical” second sphere residues (S84, G121). Codon randomization on these positions and further selection of resistance-conferring mutants was performed. MICs, periplasmic enzymatic activity, Zn(II) requirements, and protein stability was assessed. Our results indicated that identity of second sphere residues modulates the substrate preferences and the resistance profile of SPM-1 expressed in P. aeruginosa. The second sphere residues found in wild type SPM-1 give rise to a substrate selectivity that is observed only in the periplasmic environment. These residues also allow SPM-1 to confer resistance in P. aeruginosa under Zn(II)-limiting conditions, such as those expected under infection. By optimizing the catalytic efficiency towards β-lactam antibiotics, the enzyme stability and the Zn(II) binding features, molecular evolution meets the specific needs of a pathogenic bacterial host by means of substitutions outside the active site.Ítem Acceso Abierto Human serum albumin (HSA) regulates the expression of histone-like nucleoid structure protein (H-NS) in Acinetobacter baumannii(Nature Research, 2022-08-27) Escalante, Jenny; Nishimura, Brent; Tuttobene, Marisel Romina; Subils, Tomás; Pimentel, Camila; Georgeos, Nardin; Sieira, Rodrigo; Bonomo, Robert A.; Tolmasky, Marcelo E.; Ramírez, María SoledadÍtem Acceso Abierto Human Serum Proteins and Susceptibility of Acinetobacter baumannii to Cefiderocol: Role of Iron Transport(MDPI, 2022-03-03) Le, Casin; Pimentel, Camila; Pasteran, Fernando; Tuttobene, Marisel Romina; Subils, Tomás; Escalante, Jenny; Nishimura, Brent; Arriaga, Susana; Carranza, Aimee; Mezcord, Vyanka; Vila, Alejandro J.; Corso, Alejandra; Actis, Luis A.; Tolmasky, Marcelo E.; Bonomo, Robert A.; Ramírez, María SoledadÍtem Acceso Abierto Induced Heteroresistance in Carbapenem-Resistant Acinetobacter baumannii (CRAB) via Exposure to Human Pleural Fluid (HPF) and Its Impact on Cefiderocol Susceptibility(MDPI, 2023-07-21) Mezcord, Vyanka; Escalante, Jenny; Nishimura, Brent; Traglia, German M.; Sharma, Rajnikant; Vallé, Quentin; Tuttobene, Marisel Romina; Subils, Tomás; Marin, Ingrid; Pasteran, Fernando; Actis, Luis A.; Tolmasky, Marcelo E.; Bonomo, Robert A.; Rao, Gauri; Ramírez, María Soledad; https://orcid.org/0000-0003-1896-8450; https://orcid.org/0000-0002-4780-5311; https://orcid.org/0000-0001-5840-5869; https://orcid.org/0000-0001-9644-9088; https://orcid.org/0000-0002-6298-7811; https://orcid.org/0000-0002-9904-7890Infections caused by Carbapenem-resistant Acinetobacter baumannii (CRAB) isolates, such as hospital-acquired pneumonia (HAP), bacteremia, and skin and soft tissue infections, among others, are particularly challenging to treat. Cefiderocol, a chlorocatechol-substituted siderophore antibiotic, was approved by the U.S. Food and Drug Administration (FDA) in 2019 and prescribed for the treatment of CRAB infections. Despite the initial positive treatment outcomes with this antimicrobial, recent studies reported a higher-than-average all-cause mortality rate in patients treated with cefiderocol compared to the best available therapy. The cause(s) behind these outcomes remains unconfirmed. A plausible hypothesis is heteroresistance, a phenotype characterized by the survival of a small proportion of cells in a population that is seemingly isogenic. Recent results have demonstrated that the addition of human fluids to CRAB cultures leads to cefiderocol heteroresistance. Here, we describe the molecular and phenotypic analyses of CRAB heteroresistant bacterial subpopulations to better understand the nature of the less-than-expected successful outcomes after cefiderocol treatment. Isolation of heteroresistant variants of the CRAB strain AMA40 was carried out in cultures supplemented with cefiderocol and human pleural fluid (HPF). Two AMA40 variants, AMA40 IHC1 and IHC2, were resistant to cefiderocol. To identify mutations and gene expression changes associated with cefiderocol heteroresistance, we subjected these variants to whole genome sequencing and global transcriptional analysis. We then assessed the impact of these mutations on the pharmacodynamic activity of cefiderocol via susceptibility testing, EDTA and boronic acid inhibition analysis, biofilm formation, and static time-kill assays. Heteroresistant variants AMA40 IHC1 and AMA40 IHC2 have 53 chromosomal mutations, of which 40 are common to both strains. None of the mutations occurred in genes associated with high affinity iron-uptake systems or β-lactam resistance. However, transcriptional analyses demonstrated significant modifications in levels of expression of genes associated with iron-uptake systems or β-lactam resistance. The blaNDM-1 and blaADC-2, as well as various iron-uptake system genes, were expressed at higher levels than the parental strain. On the other hand, the carO and ompA genes’ expression was reduced. One of the mutations common to both heteroresistant strains was mapped within ppiA, a gene associated with iron homeostasis in other species. Static time-kill assays demonstrated that supplementing cation-adjusted Mueller–Hinton broth with human serum albumin (HAS), the main protein component of HPF, considerably reduced cefiderocol killing activity for all three strains tested. Notably, collateral resistance to amikacin was observed in both variants. We conclude that exposing CRAB to fluids with high HSA concentrations facilitates the rise of heteroresistance associated with point mutations and transcriptional upregulation of genes coding for β-lactamases and biofilm formation. The findings from this study hold significant implications for understanding the emergence of CRAB resistance mechanisms against cefiderocol treatment. This understanding is vital for the development of treatment guidelines that can effectively address the challenges posed by CRAB infections.Ítem Acceso Abierto Interaction of Acinetobacter baumannii with Human Serum Albumin: Does the host hetermine the outcome?(MDPI, 2021-07-08) Pimentel, Camila; Le, Casin; Tuttobene, Marisel Romina; Subils, Tomás; Bonomo, Robert A.; Tolmasky, Marcelo E.; Ramírez, María SoledadÍtem 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 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 Longitudinal evolution of the Pseudomonas-Derived Cephalosporinase (PDC) structure and activity in a cystic fibrosis patient treated with b-Lactams(American Society for Microbiology, 2022-09-08) Colque, Claudia A.; Albarracín Orio, Andrea G.; Tomatis, Pablo E.; Dotta, Gina; Moreno, Diego M.; Hedemann, Laura G.; Hickman, Rachel A.; Sommer, Lea M.; Feliziani, Sofía; Moyano, Alejandro José; Bonomo, Robert A.; Johansen, Helle K.; Molin, Soren; Vila, Alejandro J.; Smania, Andrea M.Ítem Acceso Abierto Protein determinants of dissemination and host specificity of metallo-β-lactamases(Nature, 2019-08-09) López, Carolina; Ayala, Juan A.; Bonomo, Robert A.; González, Lisandro Javier; Vila, Alejandro J.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 The iron content of human serum albumin nodulates the susceptibility of Acinetobacter baumannii to Cefiderocol(MDPI, 2023-02-20) Escalante, Jenny; Nishimura, Brent; Tuttobene, Marisel Romina; Subils, Tomás; Mezcord, Vyanka; Actis, Luis A.; Tolmasky, Marcelo E.; Bonomo, Robert A.; Ramírez, María SoledadÍtem Acceso Abierto The urgent need for metallo-β-lactamase inhibitors: an unattended global threat(Elsevier Ltd, 2021-07-08) Mojica, Maria F.; Rossi, María Agustina; Vila, Alejandro J.; Bonomo, Robert A.