Sara C. McGrath

Virulence factors of Yersinia pestis are overcome by a strong lipopolysaccharide response

At mammalian body temperature, the plague bacillus Yersinia pestis synthesizes lipopolysaccharide (LPS)–lipid A with poor Toll-like receptor 4 (TLR4)–stimulating activity. To address the effect of weak TLR4 stimulation on virulence, we modified Y. pestis to produce a potent TLR4-stimulating LPS. Modified Y. pestis was completely avirulent after subcutaneous infection even at high challenge doses. Resistance to disease required TLR4, the adaptor protein MyD88 and coreceptor MD-2 and was considerably enhanced by CD14 and the adaptor Mal. Both innate and adaptive responses were required for sterilizing immunity against the modified strain, and convalescent mice were protected from both subcutaneous and respiratory challenge with wild-type Y. pestis. Despite the presence of other established immune evasion mechanisms, the modified Y. pestis was unable to cause systemic disease, demonstrating that the ability to evade the LPS-induced inflammatory response is critical for Y. pestis virulence. Evading TLR4 activation by lipid A alteration may contribute to the virulence of various Gram-negative bacteria.

The Lipid A 1-Phosphatase of Helicobacter pylori Is Required for Resistance to the Antimicrobial Peptide Polymyxin

Modification of the phosphate groups of lipid A with amine-containing substituents, such as phosphoethanolamine, reduces the overall net negative charge of gram-negative bacterial lipopolysaccharide, thereby lowering its affinity to cationic antimicrobial peptides. Modification of the 1 position of Helicobacter pylori lipid A is a two-step process involving the removal of the 1-phosphate group by a lipid A phosphatase, LpxEHP (Hp0021), followed by the addition of a phosphoethanolamine residue catalyzed by EptAHP (Hp0022). To demonstrate the importance of modifying the 1 position of H. pylori lipid A, we generated LpxEHP-deficient mutants in various H. pylori strains by insertion of a chloramphenicol resistance cassette into lpxEHP and examined the significance of LpxE with respect to cationic antimicrobial peptide resistance. Using both mass spectrometry analysis and an in vitro assay system, we showed that the loss of LpxEHP activity in various H. pylori strains resulted in the loss of modification of the 1 position of H. pylori lipid A, thus confirming the function of LpxEHP. Due to its unique lipid A structure, H. pylori is highly resistant to the antimicrobial peptide polymyxin (MIC > 250 microg/ml). However, disruption of lpxEHP in H. pylori results in a dramatic decrease in polymyxin resistance (MIC, 10 microg/ml). In conclusion, we have characterized the first gram-negative LpxE-deficient mutant and have shown the importance of modifying the 1 position of H. pylori lipid A for resistance to polymyxin.

An Outer Membrane Enzyme Encoded by Salmonella typhimurium lpxR That Removes the 3′-Acyloxyacyl Moiety of Lipid A

The Salmonella and related bacteria modify the structure of the lipid A portion of their lipopolysaccharide in response to environmental stimuli. Some lipid A modifications are required for virulence and resistance to cationic antimicrobial peptides. We now demonstrate that membranes of Salmonella typhimurium contain a novel hydrolase that removes the 3′-acyloxyacyl residue of lipid A in the presence of 5 mm Ca2+. We have identified the gene encoding the S. typhimurium lipid A 3′-O-deacylase, designated lpxR, by screening an ordered S. typhimurium genomic DNA library, harbored in Escherichia coli K-12, for expression of Ca2+-dependent 3′-O-deacylase activity in membranes. LpxR is synthesized with an N-terminal type I signal peptide and is localized to the outer membrane. Mass spectrometry was used to confirm the position of lipid A deacylation in vitro and the release of the intact 3′-acyloxyacyl group. Heterologous expression of lpxR in the E. coli K-12 W3110, which lacks lpxR, resulted in production of significant amounts of 3′-O-deacylated lipid A in growing cultures. Orthologues of LpxR are present in the genomes of E. coli 0157:H7, Yersinia enterocolitica, Helicobacter pylori, and Vibrio cholerae. The function of LpxR is unknown, but it could play a role in pathogenesis because it might modulate the cytokine response of an infected animal.

Expression cloning and periplasmic orientation of the Francisella novicida lipid A 4'-phosphatase LpxF.

Francisella tularensis and related intracellular pathogens synthesize lipid A molecules that differ from their Escherichia coli counterparts. Although a functional orthologue of lpxK, the gene encoding the lipid A 4′-kinase, is present in Francisella, no 4′-phosphate moiety is attached to Francisella lipid A. We now demonstrate that a membrane-bound phosphatase present in Francisella novicida U112 selectively removes the 4′-phosphate residue from tetra- and pentaacylated lipid A molecules. A clone that expresses the F. novicida 4′-phosphatase was identified by assaying lysates of E. coli colonies, harboring members of an F. novicida genomic DNA library, for 4′-phosphatase activity. Sequencing of a 2.5-kb F. novicida DNA insert from an active clone located the structural gene for the 4′-phosphatase, designated lpxF. It encodes a protein of 222 amino acid residues with six predicted membrane-spanning segments. Rhizobium leguminosarum and Rhizobium etli contain functional lpxF orthologues, consistent with their lipid A structures. When F. novicida LpxF is expressed in an E. coli LpxM mutant, a strain that synthesizes pentaacylated lipid A, over 90% of the lipid A molecules are dephosphorylated at the 4′-position. Expression of LpxF in wild-type E. coli has no effect, because wild-type hexaacylated lipid A is not a substrate. However, newly synthesized lipid A is not dephosphorylated in LpxM mutants by LpxF when the MsbA flippase is inactivated, indicating that LpxF faces the outer surface of the inner membrane. The availability of the lpxF gene will facilitate re-engineering lipid A structures in diverse bacteria.

A Novel 3-Deoxy-d-manno-Octulosonic Acid (Kdo) Hydrolase That Removes the Outer Kdo Sugar of Helicobacter pylori Lipopolysaccharide

The lipid A domain anchors lipopolysaccharide (LPS) to the outer membrane and is typically a disaccharide of glucosamine that is both acylated and phosphorylated. The core and O-antigen carbohydrate domains are linked to the lipid A moiety through the eight-carbon sugar 3-deoxy-D-manno-octulosonic acid known as Kdo. Helicobacter pylori LPS has been characterized as having a single Kdo residue attached to lipid A, predicting in vivo a monofunctional Kdo transferase (WaaA). However, using an in vitro assay system we demonstrate that H. pylori WaaA is a bifunctional enzyme transferring two Kdo sugars to the tetra-acylated lipid A precursor lipid IV(A). In the present work we report the discovery of a Kdo hydrolase in membranes of H. pylori capable of removing the outer Kdo sugar from Kdo2-lipid A. Enzymatic removal of the Kdo group was dependent upon prior removal of the 1-phosphate group from the lipid A domain, and mass spectrometric analysis of the reaction product confirmed the enzymatic removal of a single Kdo residue by the Kdo-trimming enzyme. This is the first characterization of a Kdo hydrolase involved in the modification of gram-negative bacterial LPS.

A Leptospira interrogans enzyme with similarity to yeast Ste14p that methylates the 1-phosphate group of lipid A.

Distinct from other spirochetes, cells of Leptospira interrogans contain orthologues of all the Escherichia coli lpx genes required for lipid A biosynthesis, but they synthesize a modified form of lipopolysaccharide that supposedly activates toll-like receptor 2 (TLR2) instead of TLR4. The recent determination of the L. interrogans lipid A structure revealed an unprecedented O-methylation of its 1-phosphate group (Que-Gewirth, N. L. S., Ribeiro, A. A., Kalb, S. R., Cotter, R. J., Bulach, D. M., Adler, B., Saint Girons, I., Werts, C., and Raetz, C. R. H. (2004) J. Biol. Chem. 279, 25420-25429). The enzymatic activity responsible for selective 1-phosphate methylation has not been previously explored. A membrane enzyme that catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to the 1-phosphate moiety of E. coli Kdo2-[4′-32P]lipid A has now been discovered. The gene encoding this enzyme was identified based on the hypothesis that methylation of a phosphate group is chemically analogous to methylation of a carboxylate moiety at a membrane-water interface. Database searching revealed a candidate gene (renamed lmtA) in L. interrogans showing distant homology to the yeast isoprenylcysteine carboxyl methyltransferase, encoded by sterile-14, which methylates the a-type mating factor. Orthologues of lmtA were not present in E. coli, the lipid A of which normally lacks the 1-phosphomethyl group, or in other spirochetes, which do not synthesize lipid A. Expression of the lmtA gene behind the lac promoter on a low copy plasmid resulted in the appearance of SAM-dependent methyltransferase activity in E. coli inner membranes and methylation of about 30% of the endogenous E. coli lipid A. Inactivation of the ABC transporter MsbA did not inhibit methylation of newly synthesized lipid A. Methylated E. coli lipid A was analyzed by mass spectrometry and NMR spectroscopy to confirm the location of the phosphomethyl group at the 1-position. In human cells, engineered to express the individual TLR subtypes, 1-phosphomethyl-lipid A purified from lmtA-expressing E. coli potently activated TLR4 but not TLR2.

Association between α4 integrin cytoplasmic tail and non-muscle myosin IIA regulates cell migration

α4β1 integrin regulates cell migration via cytoplasmic interactions. Here, we report an association between the cytoplasmic tail of α4 integrin (α4 tail) and non-muscle myosin IIA (MIIA), demonstrated by co-immunoprecipitation of the MIIA heavy chain (HC) with anti-α4-integrin antibodies and pull-down of MIIA-HC with recombinant α4 tail from cell lysates. The association between the α4 tail and MIIA does not require paxillin binding or phosphorylation at Ser988 in the α4 tail. We found that substituting Glu982 in the α4 tail with alanine (E982A) disrupts the α4–MIIA association without interfering with the paxillin binding or Ser988 phosphorylation. By comparing stably transfected CHO cells, we show that the E982A mutation reduces the ability of α4β1 integrin to mediate cell spreading and to promote front–back polarization. In addition, we show that E982A impairs shear-flow-induced migration of the α4-integrin-expressing CHO cells by reducing their migration speed and directional persistence. The E982A mutation also leads to defects in the organization of MIIA filament bundles. Furthermore, when cells are plated on fibronectin and simulated with shear flow, α4β1 integrin forms filament-like patterns that co-align with MIIA filament bundles. These results provide a new mechanism for linking integrins to the actomyosin cytoskeleton and for regulating cell migration by integrins and non-muscle myosin II.