lipid-a and Cholera

Following an episode of cholera, a rapidly dehydrating, watery diarrhea caused by the Gram-negative bacterium, Vibrio cholerae O1, humans mount a robust anti-lipopolysaccharide (LPS) antibody response that is associated with immunity to subsequent re-infection. In neonatal mouse and rabbit models of cholera, passively administered anti-LPS polyclonal and monoclonal (MAb) antibodies reduce V. cholerae colonization of the intestinal epithelia by inhibiting bacterial motility and promoting vibrio agglutination. Here we demonstrate that human anti-LPS IgG MAbs also arrest V. cholerae motility and induce bacterial paralysis. A subset of those MAbs also triggered V. cholerae to secrete an extracellular matrix (ECM). To identify changes in gene expression that accompany antibody exposure and that may account for motility arrest and ECM production, we subjected V. cholerae O1 El Tor to RNA-seq analysis after treatment with ZAC-3 IgG, a high affinity MAb directed against the core/lipid A region of LPS. We identified > 160 genes whose expression was altered following ZAC-3 IgG treatment, although canonical outer membrane stress regulons were not among them. ompS (VCA1028), a porin associated with virulence and indirectly regulated by ToxT, and norR (VCA0182), a σ54-dependent transcription factor involved in late stages of infection, were two upregulated genes worth noting.

Topics: Agglutination; Animals; Antibodies, Bacterial; Antibodies, Monoclonal; Bacterial Outer Membrane Proteins; Bacterial Proteins; Cholera; Cholera Toxin; Disease Models, Animal; Gene Deletion; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Genes, Bacterial; Host-Pathogen Interactions; Humans; Immunoglobulin G; Lipid A; Lipopolysaccharides; Mice; Mice, Inbred BALB C; Rabbits; Transcription Factors; Transcriptome; Vibrio cholerae O1; Virulence

In the environment and during infection, the human intestinal pathogen Vibrio cholerae must overcome noxious compounds that damage the bacterial outer membrane. The El Tor and classical biotypes of O1 V. cholerae show striking differences in their resistance to membrane disrupting cationic antimicrobial peptides (CAMPs), such as polymyxins. The classical biotype is susceptible to CAMPs, but current pandemic El Tor biotype isolates gain CAMP resistance by altering the net charge of their cell surface through glycine modification of lipid A. Here we report a second lipid A modification mechanism that only functions in the V. cholerae El Tor biotype. We identify a functional EptA ortholog responsible for the transfer of the amino-residue phosphoethanolamine (pEtN) to the lipid A of V. cholerae El Tor that is not functional in the classical biotype. We previously reported that mildly acidic growth conditions (pH 5.8) downregulate expression of genes encoding the glycine modification machinery. In this report, growth at pH 5.8 increases expression of eptA with concomitant pEtN modification suggesting coordinated regulation of these LPS modification systems. Similarly, efficient pEtN lipid A substitution is seen in the absence of lipid A glycinylation. We further demonstrate EptA orthologs from non-cholerae Vibrio species are functional.

Topics: Antimicrobial Cationic Peptides; Bacterial Proteins; Cholera; Ethanolamines; Glycine; Humans; Lipid A; Lipopolysaccharides; Vibrio cholerae

Cationic antimicrobial peptides (CAMPs), such as polymyxins, are used as a last-line defense in treatment of many bacterial infections. However, some bacteria have developed resistance mechanisms to survive these compounds. Current pandemic O1

Topics: Acyltransferases; Amino Acid Substitution; Aminoacyltransferases; Anti-Bacterial Agents; Bacterial Proteins; Cholera; Drug Resistance, Bacterial; Gene Deletion; Glycine; Humans; Lipid A; Lipopolysaccharides; Models, Molecular; Molecular Structure; Mutation; Pandemics; Phylogeny; Polymyxins; Protein Interaction Domains and Motifs; Recombinant Proteins; Substrate Specificity; Vibrio cholerae

Vibrio cholerae is the causative agent of cholera, an acute diarrheal disease that remains endemic in many parts of the world. The mechanisms underlying immunity to cholera remain poorly defined, though it is increasingly clear that protection is associated with antibodies against lipopolysaccharide (LPS). Here we report that ZAC-3, a monoclonal antibody against the core/lipid A region of V. cholerae LPS is a potent inhibitor of V. cholerae flagellum-based motility in viscous and liquid environments. ZAC-3 arrested motility of the classical Ogawa strain O395, as well as the El Tor Inaba strain C6706. In addition, we demonstrate, in the neonatal mouse model, that ZAC-3 IgG and Fab fragments significantly reduced the ability of both V. cholerae strains O395 and C6706 to colonize the intestinal epithelium, revealing the potential of antibodies against the core/lipid A to contribute to immunity across biotypes, possibly through a mechanism involving motility arrest.

Topics: Animals; Animals, Newborn; Antibodies, Bacterial; Antibodies, Monoclonal; Antigens, Bacterial; Bacterial Typing Techniques; Cholera; Disease Models, Animal; Flagella; Immunoglobulin Fab Fragments; Immunoglobulin G; Intestinal Mucosa; Lipid A; Mice; Movement; Vibrio cholerae

The causative agent of the life-threatening gastrointestinal infectious disease cholera is the Gram-negative, facultative human pathogen Vibrio cholerae. We recently started to investigate the potential of outer membrane vesicles (OMVs) derived from V. cholerae as an alternative approach for a vaccine candidate against cholera and successfully demonstrated the induction of a long-lasting, high-titer, protective immune response upon immunization with OMVs using the mouse model. In this study, we present immunization data using lipopolysaccharide (LPS)-modified OMVs derived from V. cholerae, which allowed us to improve and identify the major protective antigen of the vaccine candidate. Our results indicate that reduction of endotoxicity can be achieved without diminishing the immunogenic potential of the vaccine candidate by genetic modification of lipid A. Although the protective potential of anti-LPS antibodies has been suggested many times, this is the first comprehensive study that uses defined LPS mutants to characterize the LPS-directed immune response of a cholera vaccine candidate in more detail. Our results pinpoint the O antigen to be the essential immunogenic structure and provide a protective mechanism based on inhibition of motility, which prevents a successful colonization. In a detailed analysis using defined antisera, we can demonstrate that only anti-O antigen antibodies, but not antibodies directed against the major flagellar subunit FlaA or the most abundant outer membrane protein, OmpU, are capable of effectively blocking the motility by binding to the sheathed flagellum and provide protection in a passive immunization assay.

Topics: Adhesins, Bacterial; Animals; Animals, Newborn; Antibodies, Bacterial; Antibody Formation; Antibody Specificity; Cholera; Cholera Vaccines; Female; Fimbriae Proteins; Flagella; Humans; Lipid A; Macrophages; Mice; Mice, Inbred BALB C; O Antigens; Toxicity Tests; Vibrio cholerae

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.

Topics: Acyltransferases; Cell Membrane; Cholera; Drug Resistance, Bacterial; HEK293 Cells; Humans; Immunity, Innate; Lipid A; Lipopolysaccharides; Mass Spectrometry; O Antigens; Polymyxin B; Vibrio cholerae

The lipopolysaccharide (LPS) from Vibrio cholerae O139 was deacylated with KOH. The following structure of the oligosaccharide resulting from this treatment was established on the basis of monosaccharide and methylation analyses, 1H, 13C and 31P 1D and 2D NMR experiments and 1D analogues of 3D NOESY-TOCSY and 3D TOCSY-NOESY experiments. [formula: see text] 'C' is a beta-L-threo-hex-4-enuronopyranosyl residue. Hep is L-glycero-D-manno-heptose, QuiN is 2-amino-2,6-dideoxy-D-glucose, GlcN is 2-amino-2-deoxy-D-glucose, Glc is D-glucose, Fru is D-fructose, and Kdo is 3-deoxy-D-manno-2-octulosonic acid. All sugars are pyranoses except fructose which is furanosidic. The fructose residue was localised after deacylation of the LPS with anhydrous hydrazine, methylation, acid methanolysis, and remethylation using deuterated iodomethane. The elucidation of this structure allowed for a direct comparison to the previously determined structure for Vibrio cholerae O1 lipid A-core region. The two structures are almost identical, and, therefore, this study is consistent with the genetic data for the biogenesis of strain O139 from O1. Furthermore, the identification of a structural analogue to the capsular polysaccharide of O139 in the outer core of the LPS in conjunction with the identification of colitose as a constituent of the LPS, provides additional evidence that the O-antigen and capsular polysaccharide of this strain may share the same repeat unit.

Topics: Carbohydrate Conformation; Carbohydrate Sequence; Cholera; Electrophoresis, Polyacrylamide Gel; Lipid A; Lipopolysaccharides; Magnetic Resonance Spectroscopy; Mass Spectrometry; Molecular Sequence Data; Molecular Structure; Monosaccharides; Oligosaccharides; Serotyping; Vibrio cholerae