Secretion of GOB Metallo-β-Lactamase in Escherichia coli Depends Strictly on the Cooperation between the Cytoplasmic DnaK Chaperone System and the Sec Machinery: Completion of Folding and Zn(II) Ion Acquisition Occur in the Bacterial Periplasm ▿

Metallo-β-lactamases (MβLs) are zinc-dependent enzymes produced by many clinically relevant gram-negative pathogens that can hydrolyze most β-lactam antibiotics. MβLs are synthesized in the bacterial cytoplasm as precursors and are secreted into the periplasm. Here, we report that the biogenesis process of the recently characterized MβL GOB-18 demands cooperation between a main chaperone system of the bacterial cytoplasm, DnaK, and the Sec secretion machinery. Using the expression of the complete gob-18 gene from the gram-negative opportunistic pathogen Elizabethkingia meningoseptica in Escherichia coli as a model system, we found that the precursor of this metalloenzyme is secreted by the Sec pathway and reduces cell susceptibility to different β-lactam antibiotics. Moreover, acting with different J proteins such as cytoplasmic DnaJ and membrane-associated DjlA as cochaperones, DnaK plays an essential role in the cytoplasmic transit of the GOB-18 precursor to the Sec translocon. Our studies also revealed a less relevant role, that of assisting in GOB-18 secretion, for trigger factor, while no significant functions were found for other main cytoplasmic chaperones such as SecB or GroEL/ES. The overall findings indicate that the biogenesis of GOB-18 involves cytoplasmic interaction of the precursor protein mainly with DnaK, secretion by the Sec system, and final folding and incorporation of Zn(II) ions into the bacterial periplasm

Site-specific recombination at XerC/D sites mediates the formation and resolution of plasmid co-integrates carrying a blaOXA-58- and TnaphA6-resistance module in Acinetobacter baumannii

Members of the genus Acinetobacter possess distinct plasmid types which provide effective platforms for the acquisition, evolution, and dissemination of antimicrobial resistance structures. Many plasmid-borne resistance structures are bordered by short DNA sequences providing potential recognition sites for the host XerC and XerD site-specific tyrosine recombinases (XerC/D-like sites). However, whether these sites are active in recombination and how they assist the mobilization of associated resistance structures is still poorly understood. Here we characterized the plasmids carried by Acinetobacter baumannii Ab242, a multidrug-resistant clinical strain belonging to the ST104 (Oxford scheme) which produces an OXA-58 carbapenem-hydrolyzing class-D β-lactamase (CHDL). Plasmid sequencing and characterization of replication, stability, and adaptive modules revealed the presence in Ab242 of three novel plasmids lacking self-transferability functions which were designated pAb242_9, pAb242_12, and pAb242_25, respectively. Among them, only pAb242_25 was found to carry an adaptive module encompassing an ISAba825-blaOXA-58 arrangement accompanied by a TnaphA6 transposon, the whole structure conferring simultaneous resistance to carbapenems and aminoglycosides. Ab242 plasmids harbor several XerC/D-like sites, with most sites found in pAb242_25 located in the vicinity or within the adaptive module described above. Electrotransformation of susceptible A. nosocomialis cells with Ab242 plasmids followed by imipenem selection indicated that the transforming plasmid form was a co-integrate resulting from the fusion of pAb242_25 and pAb242_12. Further characterization by cloning and sequencing studies indicated that a XerC/D site in pAb242_25 and another in pAb242_12 provided the active sister pair for the inter-molecular site-specific recombination reaction mediating the fusion of these two plasmids. Moreover, the resulting co-integrate was found also to undergo intra-molecular resolution at the new pair of XerC/D sites generated during fusion thus regenerating the original pAb242_25 and pAb242_12 plasmids. These observations provide the irst evidence indicating that XerC/D-like sites in A. baumannii plasmids can provide active pairs for site-specific recombination mediating inter-molecular fusions and intra molecular resolutions. The overall results shed light on the evolutionary dynamics of A. baumannii plasmids and the underlying mechanisms of dissemination of genetic structures responsible for carbapenem and other antibiotics resistance among the Acinetobacter clinical population.Para citar este articulo: Cameranesi MM, Morán-Barrio J, Limansky AS, Repizo GD and Viale AM (2018) Site-Specific Recombination at XerC/D Sites Mediates the Formation and Resolution of Plasmid Co-integrates Carrying a blaOXA-58- and TnaphA6-Resistance Module in Acinetobacter baumannii. Front. Microbiol. 9:66. doi: 10.3389/fmicb.2018.00066Fil: Cameranesi, Marcela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Cameranesi, Marcela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología; Argentina.Fil: Morán-Barrio, Jorgelina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Morán-Barrio, Jorgelina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología; Argentina.Fil: Limansky, Adriana S. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Limansky, Adriana S. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología; Argentina.Fil: Repizo, Guillermo Daniel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Repizo, Guillermo Daniel. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología; Argentina.Fil: Viale, Alejandro M. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR -CONICET); Argentina.Fil: Viale, Alejandro M. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología; Argentina

Draft Genome Sequence of Acinetobacter bereziniae HPC229, a Carbapenem-Resistant Clinical Strain from Argentina Harboring blaNDM-1

We report here the draft genome sequence of a NDM-1-producing Acinetobacter bereziniae clinical strain, HPC229. This strain harbors both plasmid and chromosomal resistance determinants towards different β-lactams and aminoglycosides as well as several types of multidrug efflux pumps, most likely representing an adaptation strategy for survival under different environments.Fil: Brovedan, Marco. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Marchiaro, Patricia M. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Morán-Barrio, Jorgelina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Revale, Santiago. Instituto de Agrobiotecnología Rosario (INDEAR); Argentina.Fil: Cameranesi, Marcela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Brambilla, Luciano. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Viale, Alejandro M. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina.Fil: Limansky, Adriana S. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Biología Molecular y Celular de Rosario (IBR-CONICET); Argentina

Dynamic state of plasmid genomic architectures resulting from XerC/D-mediated site-specific recombination in Acinetobacter baumannii Rep_3 superfamily resistance plasmids carrying blaOXA-58- and TnaphA6-resistance modules

The acquisition of blaOXA genes encoding different carbapenem-hydrolyzing class-D β-lactamases (CHDL) represents a main determinant of carbapenem resistance in the nosocomial pathogen Acinetobacter baumannii. The blaOXA-58 gene, in particular, is generally embedded in similar resistance modules (RM) carried by plasmids unique to the Acinetobacter genus lacking self-transferability. The ample variations in the immediate genomic contexts in which blaOXA-58-containing RMs are inserted among these plasmids, and the almost invariable presence at their borders of nonidentical 28-bp sequences potentially recognized by the host XerC and XerD tyrosine recombinases (pXerC/D-like sites), suggested an involvement of these sites in the lateral mobilization of the gene structures they encircle. However, whether and how these pXerC/D sites participate in this process is only beginning to be understood. Here, we used a series of experimental approaches to analyze the contribution of pXerC/D-mediated site-specific recombination to the generation of structural diversity between resistance plasmids carrying pXerC/D-bounded blaOXA-58- and TnaphA6-containing RM harbored by two phylogenetically- and epidemiologicallyclosely related A. baumannii strains of our collection, Ab242 and Ab825, during adaptation to the hospital environment. Our analysis disclosed the existence of different bona fide pairs of recombinationally-active pXerC/D sites in these plasmids, some mediating reversible intramolecular inversions and others reversible plasmid fusions/resolutions. All of the identified recombinationally-active pairs shared identical GGTGTA sequences at the cr spacer separating the XerC- and XerD-binding regions. The fusion of two Ab825 plasmids mediated by a pair of recombinationally-active pXerC/D sites displaying sequence differences at the cr spacer could be inferred on the basis of sequence comparison analysis, but no evidence of reversibility could be obtained in this case. The reversible plasmid genome rearrangements mediated by recombinationally-active pairs of pXerC/D sites reported here probably represents an ancient mechanism of generating structural diversity in the Acinetobacter plasmid pool. This recursive process could facilitate a rapid adaptation of an eventual bacterial host to changing environments, and has certainly contributed to the evolution of Acinetobacter plasmids and the capture and dissemination of blaOXA-58 genes among Acinetobacter and non-Acinetobacter populations co-residing in the hospital niche.Fil: Giacone, Lucía. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología. Instituto de Biología Molecular y Celular de Rosario (IBR); Argentina.Fil: Cameranesi, María Marcela. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología. Instituto de Biología Molecular y Celular de Rosario (IBR); Argentina.Fil: Sánchez, Rocio Inés. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología. Instituto de Biología Molecular y Celular de Rosario (IBR); Argentina.Fil: Limansky, Adriana S. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología. Instituto de Biología Molecular y Celular de Rosario (IBR); Argentina.Fil: Morán Barrio, Jorgelina. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología. Instituto de Biología Molecular y Celular de Rosario (IBR); Argentina.Fil: Viale, Alejandro M. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Departamento de Microbiología. Instituto de Biología Molecular y Celular de Rosario (IBR); Argentina

Crystal Structure of the Metallo-β-Lactamase GOB in the Periplasmic Dizinc Form Reveals an Unusual Metal Site.

International audienceMetallo-beta-lactamases (MBLs) are broad-spectrum, Zn(II)-dependent lactamases able to confer resistance to virtually every β-lactam antibiotic currently available. The large diversity of active-site structures and metal content among MBLs from different sources has limited the design of a pan-MBL inhibitor. GOB-18 is a divergent MBL from subclass B3 that is expressed by the opportunistic Gram-negative pathogen Elizabethkingia meningoseptica This MBL is atypical, since several residues conserved in B3 enzymes (such as a metal ligand His) are substituted in GOB enzymes. Here, we report the crystal structure of the periplasmic di-Zn(II) form of GOB-18. This enzyme displays a unique active-site structure, with residue Gln116 coordinating the Zn1 ion through its terminal amide moiety, replacing a ubiquitous His residue. This situation contrasts with that of B2 MBLs, where an equivalent His116Asn substitution leads to a di-Zn(II) inactive species. Instead, both the mono- and di-Zn(II) forms of GOB-18 are active against penicillins, cephalosporins, and carbapenems. In silico docking and molecular dynamics simulations indicate that residue Met221 is not involved in substrate binding, in contrast to Ser221, which otherwise is conserved in most B3 enzymes. These distinctive features are conserved in recently reported GOB orthologues in environmental bacteria. These findings provide valuable information for inhibitor design and also posit that GOB enzymes have alternative functions

Virulence role of the outer membrane protein CarO in carbapenem-resistant Acinetobacter baumannii

© 2020 The Author(s)Novel approaches to treat carbapenem-resistant Acinetobacter baumannii (CRAB) infections are urgently needed and anti-virulence drugs represent promising alternatives, but our knowledge on potential targets is scarce. We searched for potential A. baumannii virulence factors by whole-genome sequencing-based comparisons of CRAB clinical isolates causing bloodstream infections secondary to ventilator-associated pneumonia from demographics and clinically homogeneous patients, who received optimal treatment but with different clinical outcomes. Thus, the carO gene was interrupted in CRAB isolates from surviving patients, while it was intact in isolates from non-surviving patients, and proteomic/immunoblot techniques corroborated it. When the virulence role of A. baumannii CarO was analyzed in model systems, isogenic ΔcarO mutants and a CRAB clinical isolate with truncated CarO, showed lower ability to adhere and invade A549 cells and in vivo virulence. This unnoticed virulence role for CarO postulate this A. baumannii outer membrane protein as a potential target for new therapies against CRAB infections.This work was supported by the Instituto de Salud Carlos III, Proyectos de Investigación en Salud under grant [PI12/0255]; Plan Nacional de I+D+i 2013-2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades under grant [BFU2016-77297-P]; Plan Nacional de I+D+i 2013-2016 and Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades, Spanish Network for Research in Infectious Diseases under grant [REIPI RD16/0016/0009] - co-financed by European Development Regional Fund “A way to achieve Europe,” Operative program Intelligent Growth 2014-2020; and Agencia Nacional de Promoción Científica y Tecnológica, ANPCyT, Argentina, under grants [PICT-2015-1072 and PICT-2017-3536]. G.L.H. was supported by the program i-PFIS (Doctorados IIS-Empresa en Ciencias y Tecnologías de la Salud), Instituto de Salud Carlos III, Subdirección General de Redes y Centros de Investigación Cooperativa, Ministerio de Ciencia, Innovación y Universidades under grant [IFI15/ 00128]. T.C.C. is supported by the V Plan Propio of the University of Seville with a postdoctoral contract. M.E.P. I. is a postdoctoral researcher belonging to the program “Nicolás Monardes” (C1-0038-2019), Servicio Andaluz de Salud, Junta de Andalucía, Spain