David K. Giles

Modulating the innate immune response by combinatorial engineering of endotoxin

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 Toll-like receptor (TLR)4/myeloid differentiation factor 2 receptor of the innate immune system. A less toxic mixture of monophosphorylated lipid A species (MPL) recently became the first new Food and Drug Administration-approved adjuvant in over 70 y. Whereas wild-type Escherichia coli LPS provokes strong inflammatory MyD88 (myeloid differentiation primary response gene 88)-mediated TLR4 signaling, MPL preferentially induces less inflammatory TRIF (TIR-domain–containing adaptor-inducing IFN-β)-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. This approach demonstrates how combinatorial engineering of lipid A can be exploited to generate a spectrum of immunostimulatory molecules for vaccine and therapeutics development.

Helicobacter pylori versus the Host: Remodeling of the Bacterial Outer Membrane Is Required for Survival in the Gastric Mucosa

Modification of bacterial surface structures, such as the lipid A portion of lipopolysaccharide (LPS), is used by many pathogenic bacteria to help evade the host innate immune response. Helicobacter pylori, a gram-negative bacterium capable of chronic colonization of the human stomach, modifies its lipid A by removal of phosphate groups from the 1- and 4′-positions of the lipid A backbone. In this study, we identify the enzyme responsible for dephosphorylation of the lipid A 4′-phosphate group in H. pylori, Jhp1487 (LpxF). To ascertain the role these modifications play in the pathogenesis of H. pylori, we created mutants in lpxE (1-phosphatase), lpxF (4′-phosphatase) and a double lpxE/F mutant. Analysis of lipid A isolated from lpxE and lpxF mutants revealed lipid A species with a 1 or 4′-phosphate group, respectively while the double lpxE/F mutant revealed a bis-phosphorylated lipid A. Mutants lacking lpxE, lpxF, or lpxE/F show a 16, 360 and 1020 fold increase in sensitivity to the cationic antimicrobial peptide polymyxin B, respectively. Moreover, a similar loss of resistance is seen against a variety of CAMPs found in the human body including LL37, β-defensin 2, and P-113. Using a fluorescent derivative of polymyxin we demonstrate that, unlike wild type bacteria, polymyxin readily associates with the lpxE/F mutant. Presumably, the increase in the negative charge of H. pylori LPS allows for binding of the peptide to the bacterial surface. Interestingly, the action of LpxE and LpxF was shown to decrease recognition of Helicobacter LPS by the innate immune receptor, Toll-like Receptor 4. Furthermore, lpxE/F mutants were unable to colonize the gastric mucosa of C57BL/6J and C57BL/6J tlr4 -/- mice when compared to wild type H. pylori. Our results demonstrate that dephosphorylation of the lipid A domain of H. pylori LPS by LpxE and LpxF is key to its ability to colonize a mammalian host.

Amino acid addition to Vibrio cholerae LPS establishes a link between surface remodeling in Gram-positive and Gram-negative bacteria

Historically, the O1 El Tor and classical biotypes of Vibrio cholerae have been differentiated by their resistance to the antimicrobial peptide polymyxin B. However, the molecular mechanisms associated with this phenotypic distinction have remained a mystery for 50 y. Both Gram-negative and Gram-positive bacteria modify their cell wall components with amine-containing substituents to reduce the net negative charge of the bacterial surface, thereby promoting cationic antimicrobial peptide resistance. In the present study, we demonstrate that V. cholerae modify the lipid A anchor of LPS with glycine and diglycine residues. This previously uncharacterized lipid A modification confers polymyxin resistance in V. cholerae El Tor, requiring three V. cholerae proteins: Vc1577 (AlmG), Vc1578 (AlmF), and Vc1579 (AlmE). Interestingly, the protein machinery required for glycine addition is reminiscent of the Gram-positive system responsible for d-alanylation of teichoic acids. Such machinery was not thought to be used by Gram-negative organisms. V. cholerae O1 El Tor mutants lacking genes involved in transferring glycine to LPS showed a 100-fold increase in sensitivity to polymyxin B. This work reveals a unique lipid A modification and demonstrates a charge-based remodeling strategy shared between Gram-positive and Gram-negative organisms.

Elucidation of a novel Vibrio cholerae lipid A secondary hydroxy-acyltransferase and its role in innate immune recognition

Similar to most Gram‐negative bacteria, the outer leaflet of the outer membrane of Vibrio cholerae is comprised of lipopolysaccharide. Previous reports have proposed that V. cholerae serogroups O1 and O139 synthesize structurally different lipid A domains, which anchor lipopolysaccharide within the outer membrane. In the current study, intact lipid A species of V. cholerae O1 and O139 were analysed by mass spectrometry. We demonstrate that V. cholerae serogroups associated with human disease synthesize a similar asymmetrical hexa‐acylated lipid A species, bearing a myristate (C14:0) and 3‐hydroxylaurate (3‐OH C12:0) at the 2′‐ and 3′‐positions respectively. A previous report from our laboratory characterized the V. cholerae LpxL homologue Vc0213, which transfers a C14:0 to the 2′‐position of the glucosamine disaccharide. Our current findings identify V. cholerae Vc0212 as a novel lipid A secondary hydroxy‐acyltransferase, termed LpxN, responsible for transferring the 3‐hydroxylaurate (3‐OH C12:0) to the V. cholerae lipid A domain. Importantly, the presence of a 3‐hydroxyl group on the 3′‐linked secondary acyl chain was found to promote antimicrobial peptide resistance in V. cholerae; however, this functional group was not required for activation of the innate immune response.

The Polysaccharide Capsule of Campylobacter jejuni Modulates the Host Immune Response

ABSTRACT Campylobacter jejuni is a major cause of bacterial diarrheal disease worldwide. The organism is characterized by a diversity of polysaccharide structures, including a polysaccharide capsule. Most C. jejuni capsules are known to be decorated nonstoichiometrically with methyl phosphoramidate (MeOPN). The capsule of C. jejuni 81-176 has been shown to be required for serum resistance, but here we show that an encapsulated mutant lacking the MeOPN modification, an mpnC mutant, was equally as sensitive to serum killing as the nonencapsulated mutant. A nonencapsulated mutant, a kpsM mutant, exhibited significantly reduced colonization compared to that of wild-type 81-176 in a mouse intestinal colonization model, and the mpnC mutant showed an intermediate level of colonization. Both mutants were associated with higher levels of interleukin 17 (IL-17) expression from lamina propria CD4+ cells than from cells from animals infected with 81-176. In addition, reduced levels of Toll-like receptor 4 (TLR4) and TLR2 activation were observed following in vitro stimulation of human reporter cell lines with the kpsM and mpnC mutants compared to those with wild-type 81-176. The data suggest that the capsule polysaccharide of C. jejuni and the MeOPN modification modulate the host immune response.

Remodelling of the Vibrio cholerae membrane by incorporation of exogenous fatty acids from host and aquatic environments

The Gram‐negative bacteria Vibrio cholerae poses significant public health concerns by causing an acute intestinal infection afflicting millions of people each year. V. cholerae motility, as well as virulence factor expression and outer membrane protein production, has been shown to be affected by bile. The current study examines the effects of bile on V. cholerae phospholipids. Bile exposure caused significant alterations to the phospholipid profile of V. cholerae but not of other enteric pathogens. These changes consisted of a quantitative increase and migratory difference in cardiolipin, decreases in phosphatidylglycerol and phosphatidylethanolamine, and the dramatic appearance of an unknown phospholipid determined to be lyso‐phosphatidylethanolamine. Major components of bile were not responsible for the observed changes, but long‐chain polyunsaturated fatty acids, which are minor components of bile, were shown to be incorporated into phospholipids of V. cholerae. Although the bile‐induced phospholipid profile was independent of the V. cholerae virulence cascade, we identified another relevant environment in which V. cholerae assimilates unique fatty acids into its membrane phospholipids – marine sediment. Our results suggest that Vibrio species possess unique machinery conferring the ability to take up a wider range of exogenous fatty acids than other enteric bacteria.

EptC of Campylobacter jejuni mediates phenotypes involved in host interactions and virulence.

ABSTRACT Campylobacter jejuni is a natural commensal of the avian intestinal tract. However, the bacterium is also the leading cause of acute bacterial diarrhea worldwide and is implicated in development of Guillain-Barré syndrome. Like many bacterial pathogens, C. jejuni assembles complex surface structures that interface with the surrounding environment and are involved in pathogenesis. Recent work in C. jejuni identified a gene encoding a novel phosphoethanolamine (pEtN) transferase, EptC (Cj0256), that plays a promiscuous role in modifying the flagellar rod protein, FlgG; the lipid A domain of lipooligosaccharide (LOS); and several N-linked glycans. In this work, we report that EptC catalyzes the addition of pEtN to the first heptose sugar of the inner core oligosaccharide of LOS, a fourth enzymatic target. We also examine the role pEtN modification plays in circumventing detection and/or killing by host defenses. Specifically, we show that modification of C. jejuni lipid A with pEtN results in increased recognition by the human Toll-like receptor 4–myeloid differentiation factor 2 (hTLR4-MD2) complex, along with providing resistance to relevant mammalian and avian antimicrobial peptides (i.e., defensins). We also confirm the inability of aberrant forms of LOS to activate Toll-like receptor 2 (TLR2). Most exciting, we demonstrate that strains lacking eptC show decreased commensal colonization of chick ceca and reduced colonization of BALB/cByJ mice compared to wild-type strains. Our results indicate that modification of surface structures with pEtN by EptC is key to its ability to promote commensalism in an avian host and to survive in the mammalian gastrointestinal environment.

The Outer Surface Lipoprotein VolA Mediates Utilization of Exogenous Lipids by Vibrio cholerae

ABSTRACT Previous work from our laboratory showed that the Gram-negative aquatic pathogen Vibrio cholerae can take up a much wider repertoire of fatty acids than other Gram-negative organisms. The current work elaborated on the ability of V. cholerae to exploit an even more diverse pool of lipid nutrients from its environment. We have demonstrated that the bacterium can use lysophosphatidylcholine as a metabolite for growth. Using a combination of thin-layer chromatography and mass spectrometry, we also showed that lysophosphatidylcholine-derived fatty acid moieties can be used for remodeling the V. cholerae membrane architecture. Furthermore, we have identified a lysophospholipase, VolA (Vibrio outer membrane lysophospholipase A), required for these activities. The enzyme is well conserved in Vibrio species, is coexpressed with the outer membrane fatty acid transporter FadL, is one of very few surface-exposed lipoprotein enzymes to be identified in Gram-negative bacteria and the first instance of a surface lipoprotein phospholipase. We propose a model whereby the bacterium efficiently couples the liberation of fatty acid from lysophosphatidylcholine to its subsequent metabolic uptake. An expanded ability to scavenge diverse environmental lipids at the bacterial surface increases overall bacterial fitness and promotes homeoviscous adaptation through membrane remodeling. IMPORTANCE Our understanding of how bacteria utilize environmental lipid sources has been limited to lipids such as fatty acids and cholesterol. This narrow scope may be attributed to both the intricate nature of lipid uptake mechanisms and the diversity of lipid substrates encountered within an ecological niche. By examining the ability of the pathogen Vibrio cholerae to utilize exogenous lipids, we uncovered a surface-exposed lipoprotein (VolA) that is required for processing the prevalent host lipid lysophosphatidylcholine. VolA functions as a lipase liberating a fatty acid from exogenous lysophospholipids. The freed fatty acid is then transported into the cell, serving as a carbon source, or shunted into phospholipid synthesis for membrane assembly. A limited number of surface-exposed lipoproteins have been found in Gram-negative organisms, and few have enzymatic function. This work highlights the ability of bacteria to exploit exogenous lipids for both maintenance of the membrane and carbon source acquisition. Our understanding of how bacteria utilize environmental lipid sources has been limited to lipids such as fatty acids and cholesterol. This narrow scope may be attributed to both the intricate nature of lipid uptake mechanisms and the diversity of lipid substrates encountered within an ecological niche. By examining the ability of the pathogen Vibrio cholerae to utilize exogenous lipids, we uncovered a surface-exposed lipoprotein (VolA) that is required for processing the prevalent host lipid lysophosphatidylcholine. VolA functions as a lipase liberating a fatty acid from exogenous lysophospholipids. The freed fatty acid is then transported into the cell, serving as a carbon source, or shunted into phospholipid synthesis for membrane assembly. A limited number of surface-exposed lipoproteins have been found in Gram-negative organisms, and few have enzymatic function. This work highlights the ability of bacteria to exploit exogenous lipids for both maintenance of the membrane and carbon source acquisition.

High-throughput Database Search and Large-scale Negative Polarity Liquid Chromatography–Tandem Mass Spectrometry with Ultraviolet Photodissociation for Complex Proteomic Samples

The use of ultraviolet photodissociation (UVPD) for the activation and dissociation of peptide anions is evaluated for broader coverage of the proteome. To facilitate interpretation and assignment of the resulting UVPD mass spectra of peptide anions, the MassMatrix database search algorithm was modified to allow automated analysis of negative polarity MS/MS spectra. The new UVPD algorithms were developed based on the MassMatrix database search engine by adding specific fragmentation pathways for UVPD. The new UVPD fragmentation pathways in MassMatrix were rigorously and statistically optimized using two large data sets with high mass accuracy and high mass resolution for both MS1 and MS2 data acquired on an Orbitrap mass spectrometer for complex Halobacterium and HeLa proteome samples. Negative mode UVPD led to the identification of 3663 and 2350 peptides for the Halo and HeLa tryptic digests, respectively, corresponding to 655 and 645 peptides that were unique when compared with electron transfer dissociation (ETD), higher energy collision-induced dissociation, and collision-induced dissociation results for the same digests analyzed in the positive mode. In sum, 805 and 619 proteins were identified via UVPD for the Halobacterium and HeLa samples, respectively, with 49 and 50 unique proteins identified in contrast to the more conventional MS/MS methods. The algorithm also features automated charge determination for low mass accuracy data, precursor filtering (including intact charge-reduced peaks), and the ability to combine both positive and negative MS/MS spectra into a single search, and it is freely open to the public. The accuracy and specificity of the MassMatrix UVPD search algorithm was also assessed for low resolution, low mass accuracy data on a linear ion trap. Analysis of a known mixture of three mitogen-activated kinases yielded similar sequence coverage percentages for UVPD of peptide anions versus conventional collision-induced dissociation of peptide cations, and when these methods were combined into a single search, an increase of up to 13% sequence coverage was observed for the kinases. The ability to sequence peptide anions and cations in alternating scans in the same chromatographic run was also demonstrated. Because ETD has a significant bias toward identifying highly basic peptides, negative UVPD was used to improve the identification of the more acidic peptides in conjunction with positive ETD for the more basic species. In this case, tryptic peptides from the cytosolic section of HeLa cells were analyzed by polarity switching nanoLC-MS/MS utilizing ETD for cation sequencing and UVPD for anion sequencing. Relative to searching using ETD alone, positive/negative polarity switching significantly improved sequence coverages across identified proteins, resulting in a 33% increase in unique peptide identifications and more than twice the number of peptide spectral matches.

Chlamydial Hsp60-2 Is Iron Responsive in Chlamydia trachomatis Serovar E-Infected Human Endometrial Epithelial Cells In Vitro

ABSTRACT Chlamydial 60-kDa heat shock proteins (cHsp60s) are known to play a prominent role in the immunopathogenesis of disease. It is also known that several stress-inducing growth conditions, such as heat, iron deprivation, or exposure to gamma interferon, result in the development of persistent chlamydial forms that often exhibit enhanced expression of cHsp60. We have shown previously that the expression of cHsp60 is greatly enhanced in Chlamydia trachomatis serovar E propagated in an iron-deficient medium. The objective of this work was to determine which single cHsp60 or combination of the three cHsp60 homologs encoded by this organism responds to iron limitation. Using monospecific polyclonal peptide antisera that recognize only cHsp60-1, cHsp60-2, or cHsp60-3, we found that expression of cHsp60-2 is responsive to iron deprivation. Overall, our studies suggest that the expression of cHsp60 homologs differs among the mechanisms currently known to induce persistence.