title: Toxic Accumulation of LPS Pathway Intermediates Underlies the Requirement of LpxH for Growth of Acinetobacter baumannii ATCC 19606.
abstract: The lipid A moiety of lipopolysaccharide (LPS) is the main constituent of the outer leaflet of the Gram-negative bacterial outer membrane (OM) and is essential in many Gram-negative pathogens. An exception is Acinetobacter baumannii ATCC 19606, where mutants lacking enzymes occurring early in lipid A biosynthesis (LpxA, LpxC or LpxD), and correspondingly lacking LPS, can grow. In contrast, we show here that LpxH, an enzyme that occurs downstream of LpxD in the lipid A biosynthetic pathway, is essential for growth in this strain. Multiple attempts to disrupt lpxH on the genome were unsuccessful, and when LpxH expression was controlled by an isopropyl β-d-1-thiogalactopyranoside (IPTG) inducible promoter, cell growth under typical laboratory conditions required IPTG induction. Mass spectrometry analysis of cells shifted from LpxH-induced to uninduced (and whose growth was correspondingly slowing as LpxH was depleted) showed a large cellular accumulation of UDP-2,3-diacyl-GlcN (substrate of LpxH), a C14:0(3-OH) acyl variant of the LpxD substrate (UDP-3-O-[(R)-3-OH-C14]-GlcN), and disaccharide 1-monophosphate (DSMP). Furthermore, the viable cell counts of the LpxH depleted cultures dropped modestly, and electron microscopy revealed clear defects at the cell (inner) membrane, suggesting lipid A intermediate accumulation was toxic. Consistent with this, blocking the synthesis of these intermediates by inhibition of the upstream LpxC enzyme using CHIR-090 abrogated the requirement for IPTG induction of LpxH. Taken together, these data indicate that LpxH is essential for growth in A. baumannii ATCC19606, because, unlike earlier pathway steps like LpxA or LpxC, blockage of LpxH causes accumulation of detergent-like pathway intermediates that prevents cell growth.
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, CA, United States of America.
authors.forename: Daryl L
authors.initials: DL
authors.lastname: Richie
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, CA, United States of America.
authors.forename: Kenneth T
authors.initials: KT
authors.lastname: Takeoka
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, CA, United States of America.
authors.forename: Jade
authors.initials: J
authors.lastname: Bojkovic
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, CA, United States of America.
authors.forename: Louis E
authors.initials: LE
authors.lastname: Metzger
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, CA, United States of America.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Rath
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, CA, United States of America.
authors.forename: William S
authors.initials: WS
authors.lastname: Sawyer
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, CA, United States of America.
authors.forename: Jun-Rong
authors.initials: JR
authors.lastname: Wei
authors.affiliation: Novartis Institutes for BioMedical Research, Emeryville, CA, United States of America.
authors.forename: Charles R
authors.initials: CR
authors.lastname: Dean
mesh_terms: D040981:Acinetobacter baumannii / Q000235:genetics / Q000254:growth & development* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008050:Lipid A / Q000378:metabolism* / Q000633:toxicity
chemical_list: D001426:Bacterial Proteins; D008050:Lipid A