Crystal structure and biochemical characterization of the transmembrane PAP2 type phosphatidylglycerol phosphate phosphatase from Bacillus subtilis.

Type 2 phosphatidic acid phosphatases (PAP2s) can be either soluble or integral membrane enzymes. In bacteria, integral membrane PAP2s play major roles in the metabolisms of glycerophospholipids, undecaprenyl-phosphate (C55-P) lipid carrier and lipopolysaccharides. By in vivo functional experiments and biochemical characterization we show that the membrane PAP2 coded by the Bacillus subtilis yodM gene is the principal phosphatidylglycerol phosphate (PGP) phosphatase of B. subtilis. We also confirm that this enzyme, renamed bsPgpB, has a weaker activity on C55-PP. Moreover, we solved the crystal structure of bsPgpB at 2.25 Å resolution, with tungstate (a phosphate analog) in the active site. The structure reveals two lipid chains in the active site vicinity, allowing for PGP substrate modeling and molecular dynamic simulation. Site-directed mutagenesis confirmed the residues important for substrate specificity, providing a basis for predicting the lipids preferentially dephosphorylated by membrane PAP2s.

Role of the Outer Membrane and Porins in Susceptibility of ß-Lactamase-Producing Enterobacteriaceae to Ceftazidime-Avibactam.

This study examined the activity of the novel antimicrobial combination ceftazidime-avibactam against Enterobacteriaceae exhibiting different outer membrane permeability profiles, specifically with or without porins and with or without expression of the main efflux pump (AcrAB-TolC). The addition of the outer membrane permeabilizer polymyxin B nonapeptide increased the antibacterial activities of avibactam alone, ceftazidime alone, and ceftazidime-avibactam against the characterized clinical isolates of Escherichia coli, Enterobacter aerogenes, and Klebsiella pneumoniae. This enhancement of activities was mainly due to increased passive penetration of compounds since inhibition of efflux by the addition of phenylalanine-arginine ß-naphthylamide affected the MICs minimally. OmpF (OmpK35) or OmpC (OmpK36) pores were not the major route by which avibactam crossed the outer membranes of E. coli and K. pneumoniae. In contrast, Omp35 and Omp36 allowed diffusion of avibactam across the outer membrane of E. aerogenes, although other diffusion channels for avibactam were also present in that species. It was clear that outer membrane permeability and outer membrane pore-forming proteins play a key role in the activity of ceftazidime-avibactam. Nevertheless, the MICs of ceftazidime-avibactam (with 4 mg/liter avibactam) against the ceftazidime-resistant clinical isolates of the three species of Enterobacteriaceae studied were ≤ 8 mg/liter, regardless of outer membrane permeability changes resulting from an absence of defined porin proteins or upregulation of efflux.

Validation of MICA Legionella for Enumeration of Legionella pneumophila in Sanitary Waters and Cooling Tower Waters: AOAC Performance Tested MethodSM 032201.

BACKGROUND: Frequent testing for Legionella concentration in water is required by most health risk monitoring organizations worldwide. Domestic hot water and cooling tower water networks must be regularly controlled to prevent Legionnaires' disease, a potentially deadly lung infection. MICA Legionella is the fastest culture-based detection method for all serogroups of Legionella pneumophila, with automatic enumeration in 48 h and no need for confirmation. OBJECTIVE: This study compares the performance and robustness of MICA Legionella with the reference method ISO 11731:2017 for the enumeration of culturable L. pneumophila. METHODS: MICA Legionella and ISO 11731:2017 results were compared for domestic hot water and cooling tower water. Inclusivity and exclusivity were tested on reference and environmental strains. Ruggedness, lot-to-lot consistency, and stability of the reagents kit were also studied. RESULTS: Enumeration of L. pneumophila by MICA Legionella was statistically equivalent to ISO 11731:2017 in both matrixes. In cooling tower waters, MICA Legionella showed better sensitivity than ISO 11731:2017. It presented a 94% sensitivity and a 97% specificity. CONCLUSION: MICA Legionella is a highly sensitive and specific method for culturable L. pneumophila enumeration. It presents, in 48 hours, equivalent or better results than ISO 11731:2017. Its protocol is robust to variations. Its reagents kit is stable for up to 18 months. HIGHLIGHTS: MICA Legionella is a robust and reliable method for the enumeration of culturable L. pneumophila in domestic and cooling tower water. It reduces significantly the number of sample pretreatments required in ISO 11731:2017. Automatic identification and enumeration of L. pneumophila microcolonies eliminates the requirement to have skilled analysts and limits the results variability. It also greatly reduces the time to results to 48 h instead of 7-10 days with ISO 11731:2017 while providing statistically equivalent results.

Cytosolic and Secreted Peptidoglycan-Degrading Enzymes in Drosophila Respectively Control Local and Systemic Immune Responses to Microbiota.

Gut-associated bacteria produce metabolites that both have a local influence on the intestinal tract and act at a distance on remote organs. In Drosophila, bacteria-derived peptidoglycan (PGN) displays such a dual role. PGN triggers local antimicrobial peptide production by enterocytes; it also activates systemic immune responses in fat-body cells and modulates fly behavior by acting on neurons. How these responses to a single microbiota-derived compound are simultaneously coordinated is not understood. We show here that the PGRP-LB locus generates both cytosolic and secreted PGN-cleaving enzymes. Through genetic analysis, we demonstrate that the cytosolic PGRP-LB isoforms cell-autonomously control the intensity of NF-κB activation in enterocytes, whereas the secreted isoform prevents massive and detrimental gut-derived PGN dissemination throughout the organism. This study explains how Drosophila are able to uncouple the modulation of local versus systemic responses to a single gut-bacteria-derived product by using isoform-specific enzymes.