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<p>Environmental transitions cause bacteria to simultaneously alter lipopolysaccharide
(LPS) structure, release LPS-binding proteins, and increase outer membrane vesicle
(OMV) production. However, any relationship between these events is unknown. The
research described here aims to fill this gap in knowledge by examining the effect
of environmental membrane remodeling factors and LPS-binding proteins on the regulation
of OMV production and composition. </p><p>The ability of Gram-negative bacteria to
carefully modulate outer membrane (OM) composition is essential to their survival.
However, the asymmetric and heterogeneous structure of the Gram-negative OM poses
unique challenges to the cell’s successful adaption to rapid environmental transitions.
Although mechanisms to recycle and degrade OM phospholipid material exist, there is
no known mechanism to remove unfavorable lipopolysaccharide (LPS) glycoforms except
by slow dilution through cell growth. As all Gram-negative bacteria constitutively
shed outer membrane vesicles (OMVs), we propose that cells may utilize OMV formation
as a way to selectively remove environmentally-disadvantageous LPS species. We examined
the native kinetics of OM composition during physiologically relevant environmental
changes in Salmonella enterica¸a well-characterized model system for activation of
PhoP/Q and PmrA/B two component systems (TCS). In response to acidic pH, toxic metals,
antimicrobial peptides, and lack of divalent cations, these TCS modify the LPS lipid
A and core, lengthen the O-antigen, and up-regulate specific OM proteins. An environmental
change to PhoP/Q- and PmrA/B-activating conditions simultaneously induced the addition
of modified species of LPS to the OM, down-regulation of previously dominant species
of LPS, greater OMV production, and increased OMV diameter. Comparison of the relative
abundance of lipid A species present in the OM and the newly-budded OMVs following
two sets of rapid environmental shifts revealed the retention of lipid A species with
modified phosphate moieties in the OM concomitant with the selective loss of palmitoylated
species via vesiculation following exposure to moderately acidic environmental conditions.
A polymorphic model for regulation of OM LPS composition may explain the propensity
of different LPS structures, with varied geometries and biophysical properties, to
be retained in the OM or to be shed via OMVs.</p><p>Many pathogenic bacteria secrete
toxins which are also lectins, and an increasing number of these proteins have been
found to bind to LPS in addition to the host receptor. To investigate the effect of
LPS-binding proteins on OM dynamics, we first defined the binding affinity and specificity
of heat-labile enterotoxin-LPS binding. We then characterized the ability of the toxin
to modulate membrane properties and stimulate OMV production in enterotoxigenic E.
coli. Finally, we present a model wherein external toxin binds to LPS and crowds upon
the OM to stimulate OMV formation.</p>
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