Clinical isolate Acinetobacter baumannii CLA-1 was resistant to a series of antibiotic molecules, including carbapenems. Cloning and expression of the -lactamase gene content of this isolate in Escherichia coli DH10B identified a chromosome-encoded oxacillinase, OXA-40, that differed by one or two amino acid changes from OXA-24, -25, and -26 and an AmpC-type cephalosporinase. The OXA-40 -lactamase had a mainly narrowspectrum hydrolytic profile, but it included ceftazidime and imipenem. Its activity was resistant to inhibition by clavulanic acid, tazobactam, sulbactam, and, like most of the other carbapenem-hydrolyzing oxacillinases, NaCl. OXA-40 had an FGN triad replacing a YGN motif at class D -lactamase (DBL) positions 144 to 146. Site-directed DNA mutagenesis leading to a Phe-to-Tyr change at DBL position 144 in OXA-40 gave a mutant enzyme with increased hydrolytic activity against most -lactams, including imipenem. Conversely, with a gene encoding the narrow-spectrum oxacillinase OXA-1 as the template, a nucleotide substitution leading to a Tyr-to-Phe change in the YGN motif of OXA-1 gave a mutant enzyme with decreased hydrolytic activity without an increase in carbapenem-hydrolyzing activity. Thus, the Phe residue in the FGN motif was not associated with carbapenem-hydrolyzing activity by itself but instead was associated with weak overall hydrolytic activity. Finally, this Phe residue in OXA-40 explained resistance to inhibition by NaCl whereas a Tyr residue in motif YGN was related to susceptibility to NaCl.Acinetobacter baumannii is an important cause of nosocomial infections such as pneumonia, septicemia, urinary tract infections, and wound infections (6). In many cases, carbapenems have become the drugs of choice for treatment of infections due to multiple-drug-resistant A. baumannii strains (6). Nevertheless, there are growing reports of carbapenem resistance in this species. The early reports described A. baumannii isolates with -lactamase-independent carbapenem resistance (13,16,30), but the most recent reports have described -lactamase-mediated resistance (1-3, 8, 10, 15, 22, 25, 26, 28, 31). In rare cases, IMP-like and VIM-like metallo--lactamases (Ambler class B) have been described in Southeast Asian and Italian isolates of the species (1,12,25,26,31).Five oxacillinases (Ambler class D) with carbapenem-hydrolyzing activity have been characterized recently in that species, several strains of which were responsible for nosocomial outbreaks (3,8,10,15,22). OXA-23 (also named ARI-1 [15,22,28]) and OXA-27 (3), have 99% amino acid identity, whereas they have only 60% identity with a second group of oxacillinases consisting of OXA-24, -25, and -26, which differ by a few amino acid substitutions (3, 10).We have characterized the genetics and biochemistry of the carbapenem-hydrolyzing oxacillinase OXA-40, which belongs to the subgroup of oxacillinases containing OXA-24, -25, and -26. Like the other carbapenem-hydrolyzing oxacillinases, OXA-40 has an FGN motif in place of the classical YGN motif of...
Burkholderia cenocepacia is an important opportunistic pathogen causing serious chronic infections in patients with cystic fibrosis (CF). Adaptation of B. cenocepacia to the CF airways may play an important role in the persistence of the infection. We have identified a sensor kinase-response regulator (BCAM0379) named AtsR in B. cenocepacia K56-2 that shares 19% amino acid identity with RetS from Pseudomonas aeruginosa. atsR inactivation led to increased biofilm production and a hyperadherent phenotype in both abiotic surfaces and lung epithelial cells. Also, the atsR mutant overexpressed and hypersecreted an Hcp-like protein known to be specifically secreted by the type VI secretion system (T6SS) in other gram-negative bacteria. Amoeba plaque assays demonstrated that the atsR mutant was more resistant to Dictyostelium predation than the wild-type strain and that this phenomenon was T6SS dependent. Macrophage infection assays also demonstrated that the atsR mutant induces the formation of actin-mediated protrusions from macrophages that require a functional Hcp-like protein, suggesting that the T6SS is involved in actin rearrangements. Three B. cenocepacia transposon mutants that were found in a previous study to be impaired for survival in chronic lung infection model were mapped to the T6SS gene cluster, indicating that the T6SS is required for infection in vivo. Together, our data show that AtsR is involved in the regulation of genes required for virulence in B. cenocepacia K56-2, including genes encoding a T6SS.
Burkholderia cenocepacia is an opportunistic pathogen of the cystic fibrosis lung that elicits a strong inflammatory response. B. cenocepacia employs a type VI secretion system (T6SS) to survive in macrophages by disarming Rho-type GTPases, causing actin cytoskeletal defects. Here, we identified TecA, a non-VgrG T6SS effector responsible for actin disruption. TecA and other bacterial homologs bear a cysteine protease-like catalytic triad, which inactivates Rho GTPases by deamidating a conserved asparagine in the GTPase switch-I region. RhoA deamidation induces caspase-1 inflammasome activation, which is mediated by the familial Mediterranean fever disease protein Pyrin. In mouse infection, the deamidase activity of TecA is necessary and sufficient for B. cenocepacia-triggered lung inflammation and also protects mice from lethal B. cenocepacia infection. Therefore, Burkholderia TecA is a T6SS effector that modifies a eukaryotic target through an asparagine deamidase activity, which in turn elicits host cell death and inflammation through activation of the Pyrin inflammasome.
Although the carbapenem-hydrolyzing -lactamase (CHL) BlaB-1 is known to be in Chryseobacterium meningosepticum NCTC 10585, a second CHL gene, bla GOB-1 , was cloned from another C. meningosepticum clinical isolate (PINT). The G؉C content of bla GOB-1 (36%) indicated the likely chromosomal origin of this gene. Its expression in Escherichia coli DH10B yields a mature CHL with a pI of 8.7 and a relative molecular mass of 28.2 kDa. In E. coli, GOB-1 conferred resistance to narrow-spectrum cephalosporins and reduced susceptibility to ureidopenicillins, broad-spectrum cephalosporins, and carbapenems. GOB-1 had a broadspectrum hydrolysis profile including penicillins and cephalosporins (but not aztreonam). The catalytic efficiency for meropenem was higher than for imipenem. GOB-1 had low amino acid identity with the class B CHLs, sharing 18% with the closest, L-1 from Stenotrophomonas maltophilia, and only 11% with BlaB-1. Most of the conserved amino acids that may be involved in the active site of CHLs (His-101, Asp-103, His-162, and His-225) were identified in GOB-1. Sequence heterogeneity was found for GOB-1-like and BlaB-1-like -lactamases, having 90 to 100% and 86 to 100% amino acid identity, respectively, among 10 unrelated C. meningosepticum isolates. Each isolate had a GOB-1-like and a BlaB-1-like gene. The same combination of GOB-1-like and BlaB-1-like -lactamases was not found in two different isolates. C. meningosepticum is a bacterial species with two types of unrelated chromosome-borne class B CHLs that can be expressed in E. coli and, thus, may represent a clinical threat if spread in gram-negative aerobes.
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