Browsing by Subject "Lipid A"
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Item Elucidating the LPS modification repertoire of Pseudomonas aeruginosa(2015-05) Nowicki, Emily Marie; Trent, Michael Stephen; Whiteley, Marvin; Upton, Jason; Davies, Bryan W.; Kirisits, Mary JoGram-negative bacteria enhance their survival in harmful environments by outer membrane remodeling, particularly at the lipid A moiety of LPS. We recently identified a functional ortholog of the lipid A kinase, lpxT, in Pseudomonas aeruginosa. LpxT[subscript Pa] is unique from previously characterized LpxT enzymes in that it is able to phosphorylate both lipid A phosphate groups as well as generate a novel 1-triphosphate species. Low Mg²⁺ results in modulation of LpxT[subscript Pa] activity and is influenced by transcription of lipid A aminoarabinose (L-Ara4N) transferase ArnT, which is induced when Mg²⁺ is limiting (Nowicki et al., Mol Micro, 2014). We have also revealed the identity of a functional phosphoethanolamine (pEtN) transferase, EptA[subscript Pa], in P. aeruginosa, and the first report of pEtN-modified lipid A in this organism. EptA[subscript Pa] adds pEtN strictly to the non-canonical position of lipid A. Transcription of EptA[subscript Pa] is regulated by Zn²⁺ via the ColRS twocomponent system, contrasting from EptA regulation in enteric bacteria such as Salmonella enterica and Escherichia coli. Further, although L-Ara4N modification readily occurs at the same site of pEtN addition under several environmental conditions, Zn²⁺exclusively induces pEtN addition to lipid A and downregulates transcription of the L-Ara4N transferase gene (Nowicki et al., Mol Micro, 2015). The existence and specificity of these modification enzymes suggests that coordinated regulation of P. aeruginosa outer membrane remodeling occurs to permit adaptation to a changing environment.Item Membrane remodeling in epsilon proteobacteria and its impact on pathogenesis(2012-05) Cullen, Thomas Wilson; Trent, Michael Stephen; Whiteley, Marvin; Harshey, Rasika M.; Stevens, Scott W.; O'Halloran, Terry J.Bacterial pathogens assemble complex surface structures in an attempt to circumvent host immune detection. A great example is the glycolipid known as lipopolysaccharide or lipooligosaccharide (LPS), the major surface molecule in nearly all gram-negative organisms. LPS is anchored to the bacterial cell surface by a anionic hydrophobic lipid known as lipid A, the major agonist of the mammalian TLR4-MD2 receptor and likely target for cationic antimicrobial peptides (CAMPs) secreted by host cells (i.e. defensins). In this work we investigate LPS modification machinery in related ε-proteobacteria, Helicobacter pylori and Campylobacter jejuni, two important human pathogens, and demonstrate that enzymes involved in LPS modification not only play a role in evasion of host defenses but also an unexpected role in bacterial locomotion. More specifically, we identify the enzyme responsible for 4'-dephosphorylation of H. pylori lipid A, LpxF. Demonstrating that lipid A depohsphorylation at the 1 and 4'-positions by LpxE and LpxF, respectively, are the primary mechanisms used by H. pylori for CAMP resistance, contribute to attenuated TRL4-MD2 activation and are required for colonization of a the gastric mucosa in murine host. Similarly in C. jejuni, we identify an enzyme, EptC, responsible for modification of lipid A at both the 1 and 4'-positions with phosphoethanolamine (pEtN), also required for CAMP resistance in this organism. Suprisingly, EptC was found to serve a dual role in modifying not only lipid A with pEtN but also the flagellar rod protein FlgG at residue Thr75, required for motility and efficient flagella production. This work links membrane biogenesis with flagella assembly, both shown to be required for colonization of a host and adds to a growing list of post-translational modifications found in prokaryotes. Understanding how pathogens evade immune detection, interphase with the surrounding environment and assemble major surface features is key in the development of novel treatments and vaccines.Item Modulating the innate immune response and bacterial fitness by combinatorial engineering of endotoxin(2014-08) Needham, Brittany Dawn; Trent, Michael Stephen; Whiteley, Marvin; Georgiou, George; Croyle, Maria; Ehrlich, LaurenGram-negative bacteria decorate their outermost surface structure, lipopolysaccharide, with elaborate chemical moieties, which effectively disguises them from immune surveillance and protects them from the onslaught of host defenses. Many of these changes occur on the lipid A component of lipopolysaccharide, which is crucial for host recognition of Gram-negative infection. Despite its highly inflammatory nature, LPS is a molecule with remarkable therapeutic potential. Lipid A is a glycolipid that serves as the hydrophobic anchor of LPS and constitutes a potent ligand of the TLR4/MD2 receptor of the innate immune system. A less toxic mixture of mono-phosphorylated lipid A species (MPL) recently became the first new FDA-approved adjuvant in over 70 years. Whereas wild-type E. coli LPS provokes strong inflammatory MyD88-mediated TLR4 signaling, MPL preferentially induces less inflammatory TRIF-mediated responses. Here, we developed a system for combinatorial structural diversification of E. coli lipid A yielding a spectrum of bioactive variants that display distinct TLR4 agonist activities and cytokine induction. Mice immunized with engineered lipid A/antigen emulsions exhibited robust IgG titers indicating the efficacy of these molecules as adjuvants. Other types of modification to the lipid A domain, such as altering the length of the fatty acyl chains that anchor LPS to the cell membrane, were found to affect bacterial fitness but not drastically influence detection by the TLR4/MD2 receptor. Overall, this combinatorial approach demonstrates how engineering lipid A can be exploited to generate a spectrum of immunostimulatory molecules for vaccine and therapeutics development as well as for a deeper understanding of bacterial membrane biogenesis.Item Unexpected biochemistry determines endotoxin structure in two enteric gram-negatives(2015-05) Di Pierro, Erica Jacqueline; Trent, Michael Stephen; Davies, Bryan W; Ehrlich, Lauren; Maynard, Jennifer; Payne, ShelleyMost gram-negative organisms require lipopolysaccharide and its membrane anchor, lipid A, for growth and survival. Also known as endotoxin, lipid A is synthesized via a nine-step enzymatic process, culminating in a conserved hexa-acylated, bis-phosphorylated disaccharide of glucosamine. This framework is often altered by condition- or species-specific lipid A modifications, which change the biochemical properties of the molecule in response to and to defend against environmental stress signals. Here, we expound on two stories in different gram-negative organisms, both involving novel or unanticipated biochemistry that impacts lipid A structure. First, the missing acyltransferase in the Epsilonproteobacterium Helicobacter pylori lipid A biosynthesis pathway is identified. This enzyme transfers a secondary acyl chain to the 3'-linked primary acyl chain of lipid A like E. coli LpxM, but shares almost no sequence similarity with the E. coli acyltransferase. It is reannotated as LpxJ and demonstrated to possess an unprecedented ability to act before the 2'-secondary acyltransferase, LpxL, as well as the 3-deoxy-D-manno-octulosonic acid transferase, KdtA. LpxJ is one member of a large class of acyltransferases found in a diverse range of organisms that lack an E. coli LpxM homolog, suggesting that LpxJ participates in lipid A biosynthesis in place of an LpxM homolog. The second story focuses on regulation of modifications to endotoxin structure that occur after the conserved biosynthesis pathway. E. coli pmrD is shown to be required for PmrAB-dependent lipid A modifications in conditions that exclusively activate PhoPQ; this result proves that PmrD connects PhoPQ and PmrAB despite previous reports that it is an inactive connector in this organism. Further, RNA sequencing and polymyxin B survival assays solidify the role of E. coli pmrD in influencing expression of pmrA and its target genes and promoting survival during exposure to cationic antimicrobial peptides. Notably, the presence of an unknown factor or system capable of activating pmrD to promote lipid A modification in the absence of the PhoPQ system is also revealed. In all, the findings presented here expand our understanding of alternative approaches to lipid A biosynthesis and the complex systems that regulate modifications of this dynamic molecule.