Alternative lipid synthesis in response to phosphate limitation promotes antibiotic tolerance in Gram-negative ESKAPE pathogens.

The Gram-negative outer membrane protects bacterial cells from environmental toxins such as antibiotics. The outer membrane lipid bilayer is asymmetric; while glycerophospholipids compose the periplasmic facing leaflet, the surface layer is enriched with phosphate-containing lipopolysaccharides. The anionic phosphates that decorate the cell surface promote electrostatic interactions with cationic antimicrobial peptides such as colistin, allowing them to penetrate the bilayer, form pores, and lyse the cell.

PBP1A Directly Interacts with the Divisome Complex to Promote Septal Peptidoglycan Synthesis in Acinetobacter baumannii.

The class A penicillin-binding proteins (aPBPs), PBP1A and PBP1B, are major peptidoglycan synthases that synthesize more than half of the peptidoglycan per generation in Escherichia coli. Whereas aPBPs have distinct roles in peptidoglycan biosynthesis during growth (i.e.

Peptidoglycan Recycling Promotes Outer Membrane Integrity and Carbapenem Tolerance in Acinetobacter baumannii.

β-Lactam antibiotics exploit the essentiality of the bacterial cell envelope by perturbing the peptidoglycan layer, typically resulting in rapid lysis and death. Many Gram-negative bacteria do not lyse but instead exhibit "tolerance," the ability to sustain viability in the presence of bactericidal antibiotics for extended periods. Antibiotic tolerance has been implicated in treatment failure and is a stepping-stone in the acquisition of true resistance, and the molecular factors that promote intrinsic tolerance are not well understood.

High-level carbapenem tolerance requires antibiotic-induced outer membrane modifications.

Antibiotic tolerance is an understudied potential contributor to antibiotic treatment failure and the emergence of multidrug-resistant bacteria. The molecular mechanisms governing tolerance remain poorly understood. A prominent type of β-lactam tolerance relies on the formation of cell wall-deficient spheroplasts, which maintain structural integrity via their outer membrane (OM), an asymmetric lipid bilayer consisting of phospholipids on the inner leaflet and a lipid-linked polysaccharide (lipopolysaccharide, LPS) enriched in the outer monolayer on the cell surface.

In , the Stringent Factor Rel Regulates Metabolism but Is Not the Only (p)ppGpp Synthase.

The stringent response is a broadly conserved stress response system that exhibits functional variability across bacterial clades. Here, we characterize the role of the stringent factor Rel in the nontuberculous mycobacterial pathogen, Mycobacterium abscessus (). We found that deletion of does not ablate (p)ppGpp synthesis and that does not provide a survival advantage in several stress conditions or in antibiotic treatment.

A nematode-derived, mitochondrial stress signaling-regulated peptide exhibits broad antibacterial activity.

A dramatic rise of infections with antibiotic-resistant bacterial pathogens continues to challenge the healthcare field due to the lack of effective treatment regimes. As such, there is an urgent need to develop new antimicrobial agents that can combat these multidrug-resistant superbugs. Mitochondria are central regulators of metabolism and other cellular functions, including the regulation of innate immunity pathways involved in the defense against infection.

Septal Class A Penicillin-Binding Protein Activity and ld-Transpeptidases Mediate Selection of Colistin-Resistant Lipooligosaccharide-Deficient Acinetobacter baumannii.

Despite dogma suggesting that lipopolysaccharide/lipooligosaccharide (LOS) was essential for viability of Gram-negative bacteria, several clinical isolates produced LOS colonies after colistin selection. Inactivation of the conserved class A penicillin-binding protein, PBP1A, was a compensatory mutation that supported isolation of LOS, but the impact of PBP1A mutation was not characterized. Here, we show that the absence of PBP1A causes septation defects and that these, together with ld-transpeptidase activity, support isolation of LOS PBP1A contributes to proper cell division in , and its absence induced cell chaining.

Generating Transposon Insertion Libraries in Gram-Negative Bacteria for High-Throughput Sequencing.

Transposon sequencing (Tn-seq) is a powerful method that combines transposon mutagenesis and massive parallel sequencing to identify genes and pathways that contribute to bacterial fitness under a wide range of environmental conditions. Tn-seq applications are extensive and have not only enabled examination of genotype-phenotype relationships at an organism level but also at the population, community and systems levels. Gram-negative bacteria are highly associated with antimicrobial resistance phenotypes, which has increased incidents of antibiotic treatment failure.

Discovery and characterization of New Delhi metallo-β-lactamase-1 inhibitor peptides that potentiate meropenem-dependent killing of carbapenemase-producing Enterobacteriaceae.

Objectives: Metallo-β-lactamases (MBLs) are an emerging class of antimicrobial resistance enzymes that degrade β-lactam antibiotics, including last-resort carbapenems. Infections caused by carbapenemase-producing Enterobacteriaceae (CPE) are increasingly prevalent, but treatment options are limited. While several serine-dependent β-lactamase inhibitors are formulated with commonly prescribed β-lactams, no MBL inhibitors are currently approved for combinatorial therapies.

Colistin heteroresistance in Enterobacter cloacae is regulated by PhoPQ-dependent 4-amino-4-deoxy-l-arabinose addition to lipid A.

The Enterobacter cloacae complex (ECC) consists of closely related bacteria commonly associated with the human microbiota. ECC are increasingly isolated from healthcare-associated infections, demonstrating that these Enterobacteriaceae are emerging nosocomial pathogens. ECC can rapidly acquire multidrug resistance to conventional antibiotics.

A penicillin-binding protein inhibits selection of colistin-resistant, lipooligosaccharide-deficient Acinetobacter baumannii.

The Gram-negative bacterial outer membrane fortifies the cell against environmental toxins including antibiotics. Unique glycolipids called lipopolysaccharide/lipooligosaccharide (LPS/LOS) are enriched in the cell-surface monolayer of the outer membrane and promote antimicrobial resistance. Colistin, which targets the lipid A domain of LPS/LOS to lyse the cell, is the last-line treatment for multidrug-resistant Gram-negative infections.

The Power of Asymmetry: Architecture and Assembly of the Gram-Negative Outer Membrane Lipid Bilayer.

Determining the chemical composition of biological materials is paramount to the study of natural phenomena. Here, we describe the composition of model gram-negative outer membranes, focusing on the predominant assembly, an asymmetrical bilayer of lipid molecules. We also give an overview of lipid biosynthetic pathways and molecular mechanisms that organize this material into the outer membrane bilayer.

Outer membrane vesicles displaying engineered glycotopes elicit protective antibodies.

The O-antigen polysaccharide (O-PS) component of lipopolysaccharides on the surface of gram-negative bacteria is both a virulence factor and a B-cell antigen. Antibodies elicited by O-PS often confer protection against infection; therefore, O-PS glycoconjugate vaccines have proven useful against a number of different pathogenic bacteria. However, conventional methods for natural extraction or chemical synthesis of O-PS are technically demanding, inefficient, and expensive.

UVliPiD: A UVPD-Based Hierarchical Approach for De Novo Characterization of Lipid A Structures.

The lipid A domain of the endotoxic lipopolysaccharide layer of Gram-negative bacteria is comprised of a diglucosamine backbone to which a variable number of variable length fatty acyl chains are anchored. Traditional characterization of these tails and their linkages by nuclear magnetic resonance (NMR) or mass spectrometry is time-consuming and necessitates databases of pre-existing structures for structural assignment. Here, we introduce an automated de novo approach for characterization of lipid A structures that is completely database-independent.

Reinforcing Lipid A Acylation on the Cell Surface of Acinetobacter baumannii Promotes Cationic Antimicrobial Peptide Resistance and Desiccation Survival.

Unlabelled: Acinetobacter baumannii is an emerging Gram-negative pathogen found in hospitals and intensive care units. In order to persist in hospital environments, A. baumannii withstands desiccative conditions and can rapidly develop multidrug resistance to conventional antibiotics.

Crystallographic study of the phosphoethanolamine transferase EptC required for polymyxin resistance and motility in Campylobacter jejuni.

The foodborne enteric pathogen Campylobacter jejuni decorates a variety of its cell-surface structures with phosphoethanolamine (pEtN). Modifying lipid A with pEtN promotes cationic antimicrobial peptide resistance, whereas post-translationally modifying the flagellar rod protein FlgG with pEtN promotes flagellar assembly and motility, which are processes that are important for intestinal colonization. EptC, the pEtN transferase required for all known pEtN cell-surface modifications in C.

Defining gene-phenotype relationships in Acinetobacter baumannii through one-step chromosomal gene inactivation.

Rates of infection with hospital-acquired Acinetobacter baumannii have exploded over the past decade due to our inability to limit persistence and effectively treat disease. A. baumannii quickly acquires antibiotic resistance, and its genome encodes mechanisms to tolerate biocides and desiccation, which enhance its persistence in hospital settings.

A regulatory checkpoint during flagellar biogenesis in Campylobacter jejuni initiates signal transduction to activate transcription of flagellar genes.

Unlabelled: Many polarly flagellated bacteria require similar two-component regulatory systems (TCSs) and σ(54) to activate transcription of genes essential for flagellar motility. Herein, we discovered that in addition to the flagellar type III secretion system (T3SS), the Campylobacter jejuni flagellar MS ring and rotor are required to activate the FlgSR TCS. Mutants lacking the FliF MS ring and FliG C ring rotor proteins were as defective as T3SS mutants in FlgSR- and σ(54)-dependent flagellar gene expression.

A specificity determinant for phosphorylation in a response regulator prevents in vivo cross-talk and modification by acetyl phosphate.

Bacterial two-component systems (TCSs) sense stimuli and transduce signals intracellularly through phosphotransfer between cognate histidine kinases (HKs) and response regulators (RRs) to alter gene expression or behavioral responses. Without high phosphotransfer specificity between cognate HKs and RRs, cross-phosphorylation or cross-talk between different TCSs may occur and diminish responses to appropriate stimuli. Some mechanisms to reduce cross-talk involve HKs controlling levels of cognate RR phosphorylation.