Outer membrane engineering through lipid A modification allows modulation of Burkholderia pseudomallei membrane permeability and innate immune responsivity

Burkholderia pseudomallei causes the tropical disease melioidosis. Host mortality primarily results from sepsis-related complications and associated cytokine release. Lipopolysaccharide (LPS), a major virulence factor and mediator of sepsis, is modified by B. pseudomallei in response to external stimuli. LpxO and PagL are lipid A-modifying proteins encoded by the B. pseudomallei genome to decrease recognition by host defenses. The contribution of lpxO and pagL- dependent lipid A modifications to outer membrane permeability and host response has not been clearly demonstrated. Mono-phosphorylated lipid A (MPLA) is known to maintain LPS-specific immunogenicity while reducing endotoxicity and has been used extensively as a vaccine adjuvant. Generation of MPLA can be mediated through LpxE. In this work, a panel of defined lipid A modification strains in B. pseudomallei was generated to study effects on bacterial physiology and cytokine expression in macrophages. Knockout of lpxO and pagL resulted in dehydroxylation and penta-acylation of lipid A, respectively, and reduced membrane permeability of B. pseudomallei . Expression of lpxE removed one phosphate group as measured by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and caused increased membrane permeability. Outer membrane vesicles (OMVs) from the generated mutants were used to study the immunological effect of lipid A modifications on human monocyte-derived macrophages. Macrophages treated with OMVs isolated from lpxE -expressing strains showed significant increases in gene expression of proinflammatory cytokines tumor necrosis factor and interferon gamma compared to wild-type B. pseudomallei OMVs. These results show the importance of lipid A modifiers LpxO and PagL for B. pseudomallei physiology and highlight the immunostimulatory effect of lpxE -modified OMVs that could enhance OMV vaccine technology targeting melioidosis.