In-cell kinetic stability is an essential trait in metallo-β-lactamase evolution.

Protein stability is an essential property for biological function. In contrast to the vast knowledge on protein stability in vitro, little is known about the factors governing in-cell stability. Here we show that the metallo-β-lactamase (MBL) New Delhi MBL-1 (NDM-1) is a kinetically unstable protein on metal restriction that has evolved by acquiring different biochemical traits that optimize its in-cell stability.

Metallo-β-lactamases and a tug-of-war for the available zinc at the host-pathogen interface.

Metallo-β-lactamases (MBLs) are zinc-dependent hydrolases that inactivate virtually all β-lactam antibiotics. The expression of MBLs by Gram-negative bacteria severely limits the therapeutic options to treat infections. MBLs bind the essential metal ions in the bacterial periplasm, and their activity is challenged upon the zinc starvation conditions elicited by the native immune response.

On the Offensive: the Role of Outer Membrane Vesicles in the Successful Dissemination of New Delhi Metallo-β-lactamase (NDM-1).

The 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.

Specific Protein-Membrane Interactions Promote Packaging of Metallo-β-Lactamases into Outer Membrane Vesicles.

Outer membrane vesicles (OMVs) act as carriers of bacterial products such as plasmids and resistance determinants, including metallo-β-lactamases. The lipidated, membrane-anchored metallo-β-lactamase NDM-1 can be detected in Gram-negative OMVs. The soluble domain of NDM-1 also forms electrostatic interactions with the membrane.

Metallo-β-lactamases in the Age of Multidrug Resistance: From Structure and Mechanism to Evolution, Dissemination, and Inhibitor Design.

Antimicrobial resistance is one of the major problems in current practical medicine. The spread of genes coding for resistance determinants among bacteria challenges the use of approved antibiotics, narrowing the options for treatment. Resistance to carbapenems, last resort antibiotics, is a major concern.

Molecular Bases of the Membrane Association Mechanism Potentiating Antibiotic Resistance by New Delhi Metallo-β-lactamase 1.

Resistance to last-resort carbapenem antibiotics is an increasing threat to human health, as it critically limits therapeutic options. Metallo-β-lactamases (MBLs) are the largest family of carbapenemases, enzymes that inactivate these drugs. Among MBLs, New Delhi metallo-β-lactamase 1 (NDM-1) has experienced the fastest and largest worldwide dissemination.

The H-NS Regulator Plays a Role in the Stress Induced by Carbapenemase Expression in Acinetobacter baumannii.

Disruption 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-β-lactamases (MBLs), known to elicit envelope stress by the accumulation of toxic precursors in the periplasm, to interrogate the role of H-NS in , together with other stressors. Using a multidrug-resistant 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.

Distinct Mechanisms of Dissemination of NDM-1 Metallo-β-Lactamase in Species in Argentina.

A 4-year surveillance of carbapenem-resistant spp. isolates in Argentina identified 40 strains carrying Genome sequencing revealed that most were , whereas seven represented other spp. The genomes were closely related, suggesting recent spread.

Human pleural fluid triggers global changes in the transcriptional landscape of Acinetobacter baumannii as an adaptive response to stress.

Acinetobacter baumannii is a feared, drug-resistant pathogen, characterized by its ability to resist extreme environmental and nutrient-deprived conditions. Previously, we showed that human serum albumin (HSA) can increase foreign DNA acquisition specifically and alter the expression of genes associated with pathogenicity. Moreover, in a recent genome-wide transcriptomic study, we observed that pleural fluid (PF), an HSA-containing fluid, increases DNA acquisition, can modulate cytotoxicity, and control immune responses by eliciting changes in the A.

A simple protocol to characterize bacterial cell-envelope lipoproteins in a native-like environment.

Physiological conditions in living cells are strictly regulated to allow, optimize, and coordinate biological processes. The bacterial cell envelope is the compartment where the communication with the external environment takes place. This involves membrane proteins, key players in many biological processes that ensure bacterial survival.

Protein determinants of dissemination and host specificity of metallo-β-lactamases.

The worldwide dissemination of metallo-β-lactamases (MBLs), mediating resistance to carbapenem antibiotics, is a major public health problem. The extent of dissemination of MBLs such as VIM-2, SPM-1 and NDM among Gram-negative pathogens cannot be explained solely based on the associated mobile genetic elements or the resistance phenotype. Here, we report that MBL host range is determined by the impact of MBL expression on bacterial fitness.

Shaping Substrate Selectivity in a Broad-Spectrum Metallo-β-Lactamase.

Metallo-β-lactamases (MBLs) are the major group of carbapenemases produced by bacterial pathogens. The design of MBL inhibitors has been limited by, among other issues, incomplete knowledge about how these enzymes modulate substrate recognition. While most MBLs are broad-spectrum enzymes, B2 MBLs are exclusive carbapenemases.

Clinical Evolution of New Delhi Metallo-β-Lactamase (NDM) Optimizes Resistance under Zn(II) Deprivation.

Carbapenem-resistant (CRE) are rapidly spreading and taking a staggering toll on all health care systems, largely due to the dissemination of genes coding for potent carbapenemases. An important family of carbapenemases are the Zn(II)-dependent β-lactamases, known as metallo-β-lactamases (MBLs). Among them, the New Delhi metallo-β-lactamase (NDM) has experienced the fastest and widest geographical spread.

Membrane anchoring stabilizes and favors secretion of New Delhi metallo-β-lactamase.

Carbapenems, 'last-resort' β-lactam antibiotics, are inactivated by zinc-dependent metallo-β-lactamases (MBLs). The host innate immune response withholds nutrient metal ions from microbial pathogens by releasing metal-chelating proteins such as calprotectin. We show that metal sequestration is detrimental for the accumulation of MBLs in the bacterial periplasm, because those enzymes are readily degraded in their nonmetallated form.

Triton Hodge Test: Improved Protocol for Modified Hodge Test for Enhanced Detection of NDM and Other Carbapenemase Producers.

Accurate detection of carbapenemase-producing Gram-negative bacilli is of utmost importance for the control of nosocomial spread and the initiation of appropriate antimicrobial therapy. The modified Hodge test (MHT), a carbapenem inactivation assay, has shown poor sensitivity in detecting the worldwide spread of New Delhi metallo-β-lactamase (NDM). Recent studies demonstrated that NDM is a lipoprotein anchored to the outer membrane in Gram-negative bacteria, unlike all other known carbapenemases.

Host-specific enzyme-substrate interactions in SPM-1 metallo-β-lactamase are modulated by second sphere residues.

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.

Carbapenem resistance in Elizabethkingia meningoseptica is mediated by metallo-β-lactamase BlaB.

Elizabethkingia meningoseptica, a Gram-negative rod widely distributed in the environment, is resistant to most β-lactam antibiotics. Three bla genes have been identified in E. meningoseptica, coding for the extended-spectrum serine-β-lactamase CME (class D) and two unrelated wide-spectrum metallo-β-lactamases, BlaB (subclass B1) and GOB (subclass B3).

Engineered mononuclear variants in Bacillus cereus metallo-beta-lactamase BcII are inactive.

Metallo-beta-lactamases (MbetaLs) are zinc enzymes able to hydrolyze almost all beta-lactam antibiotics, rendering them inactive, at the same time endowing bacteria high levels of resistance. The design of inhibitors active against all classes of MbetaLs has been hampered by their structural diversity and by the heterogeneity in metal content in enzymes from different sources. BcII is the metallo-beta-lactamase from Bacillus cereus, which is found in both the mononuclear and dinuclear forms.