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

Salmonellae regulate membrane lipids during infection, but the exact proteins and mechanisms that promote their survival during bacteremia remain largely unknown. Mutations in genes encoding the conserved  serovar Typhimurium ( Typhimurium) Tol-Pal apparatus caused the outer membrane (OM) sensor lipoprotein, RcsF, to become activated. The capsule activation phenotype for the mutants suggested that Tol-Pal might influence envelope lipid homeostasis. The mechanism involves reducing OM glycerophospholipid (GPL) levels, since the mutant salmonellae similarly accumulated phosphatidylglycerols (PGl) and phosphatidylethanolamines (PE) within the OM in comparison to the wild type. The data support the  model, whereby Tol-Pal directs retrograde GPL translocation across the periplasm. The  Typhimurium mechanism involves contributions from YbgC, a cytoplasmic acyl coenzyme A (acyl-CoA) thioesterase, and CpoB, a periplasmic TolA-binding protein. The functional relationship between Tol-Pal and YbgC and CpoB was previously unresolved. The  Typhimurium Tol-Pal proteins contribute similarly toward promoting OM-GPL homeostasis and Rcs signaling inactivity but differently toward promoting bacterial morphology, rifampin resistance, survival in macrophages, and survival in mice. For example, , , , and  mutants were significantly more attenuated than , , and  mutants in a systemic mouse model of disease. Therefore, key roles exist for TolQ, TolR, TolA, and CpoB during murine bacteremia, which are independent of maintaining GPL homeostasis. The ability of TolQR to channel protons across the inner membrane (IM) is necessary for  Typhimurium TolQRA function, since mutating conserved channel-facing residues rendered TolQ ineffective at rescuing deletion mutant phenotypes. Therefore, Tol-Pal promotes  Typhimurium survival during bacteremia, in part, by reducing OM GPL concentrations, while TolQRA and CpoB enhance systemic virulence by additional mechanisms.

Salmonella Tol-Pal Reduces Outer Membrane Glycerophospholipid Levels for Envelope Homeostasis and Survival during Bacteremia.

 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.

Melina B. Cian

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

Melina B. Cian

.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