separation-cell-envelope-for-gram-negative-bacteria-into-inner-outer

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for <em>Acinetobacter baumannii</em>

This method works by partitioning the envelope of Gram-negative bacteria into total, inner, and outer membrane (OM) fractions and concludes with assays to ...

University of Oklahoma Health Sciences Center

 serovar Typhimurium ( Typhimurium) relies upon the inner membrane protein PbgA to enhance outer membrane (OM) integrity and promote virulence in mice. The PbgA transmembrane domain (residues 1 to 190) is essential for viability, while the periplasmic domain (residues 191 to 586) is dispensable. Residues within the basic region (residues 191 to 245) bind acidic phosphates on polar phospholipids, like for cardiolipins, and are necessary for salmonella OM integrity.  Typhimurium bacteria increase their OM cardiolipin concentrations during activation of the PhoPQ regulators. The mechanism involves PbgA's periplasmic globular region (residues 245 to 586), but the biological role of increasing cardiolipins on the surface is not understood. Nonsynonymous polymorphisms in three essential lipopolysaccharide (LPS) synthesis regulators,  (also known as ), , and , variably suppressed the defects in OM integrity, rifampin resistance, survival in macrophages, and systemic colonization of mice in the Δ mutant (in which the PbgA periplasmic domain from residues 191 to 586 is deleted). Compared to the OMs of the wild-type salmonellae, the OMs of the  mutants had increased levels of lipid A-core molecules, cardiolipins, and phosphatidylethanolamines and decreased levels of specific phospholipids with cyclopropanated fatty acids. Complementation and substitution mutations in LapB and LpxC generally restored the phospholipid and LPS assembly defects for the  mutants. During bacteremia, mice infected with the  mutants survived and cleared the bacteria, while animals infected with wild-type salmonellae succumbed within 1 week. Remarkably, wild-type mice survived asymptomatically with  salmonellae in their livers and spleens for months, but Toll-like receptor 4-deficient animals succumbed to these infections within roughly 1 week. In summary,  Typhimurium uses PbgA to influence LPS assembly during stress in order to survive, adapt, and proliferate within the host environment.

Salmonella enterica Serovar Typhimurium Uses PbgA/YejM To Regulate Lipopolysaccharide Assembly during Bacteremia.

The outer membrane (OM) of Gram-negative bacteria is an asymmetric lipid bilayer that consists of inner-leaflet phospholipids and outer leaflet lipopolysaccharides (LPS). The asymmetric character and the unique biochemistry of LPS molecules contribute to the OM's ability to function as a molecular permeability barrier that protects the bacterium against hazards in the environment. Assembly and regulation of the OM has been extensively studied for understanding mechanisms of antibiotic resistance and bacterial defense against host immunity; however, sparse knowledge exists for how Gram-negative bacteria release their OMs into their environment to manipulate their hosts. Discoveries in bacterial-lipid trafficking, OM-lipid homeostasis, and host recognition of microbial patterns have shed new light on how microbes secrete OM vesicles (OMVs) to influence inflammation, cell death, and disease pathogenesis. Pathogens release OMVs that contain phospholipids, like cardiolipins, and components of LPS molecules, like lipid-A endotoxins. These multi-acylated lipid amphiphiles are molecular patterns that are differentially detected by host receptors like the Toll-like receptor 4/myeloid differentiation factor 2 complex (Tlr4/MD-2) and the mouse caspase-11, human caspases-4 and -5, receptor. We discuss how lipid ligands on OMVs engage these pattern recognition receptors on the membranes and in the cytosol of mammalian cells. We then detail how bacteria regulate OM-lipid asymmetry, negative-membrane curvature, and the phospholipid-to-LPS ratio to control OMV formation. The goal is to highlight intersections between OM-lipid regulation and host immunity and to provide working models for how bacterial lipids influence vesicle formation.

Nicole P. Giordano

Department of Microbiology and Immunology

University of Oklahoma Health Sciences Center

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

Nicole P. Giordano

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Microbiology and Immunology

University of Oklahoma Health Sciences Center

Separation of the Cell Envelope for Gram-negative Bacteria into Inner and Outer Membrane Fractions with Technical Adjustments for Acinetobacter baumannii

Department of Microbiology and Immunology