Nontypeable Haemophilus influenzae Lipooligosaccharide Expresses a Terminal Ketodeoxyoctanoate In Vivo, Which Can Be Used as a Target for Bactericidal Antibody.

Nontypeable Haemophilus influenzae (NTHi) is an important pathogen in individuals of all ages. The lipooligosaccharide (LOS) of NTHi has evolved a complex structure that can be attributed to a multiplicity of glycosyltransferases, the random switching of glycosyltransferase gene expression via phase variation, and the complex structure of its core region with multiple glycoform branch points. This article adds to that complexity by describing a multifunctional enzyme (LsgB) which optimally functions when the species is grown on a solid surface and which can add either a ketodeoxyoctanoate (KDO) or an N-acetylneuramic acid (Neu5Ac) moiety to a terminal N-acetyllactosamine structure of LOS. Our studies show that expression of lsgB is reduced four- to sixfold when NTHi is grown in broth. The substrate that the enzyme utilizes is dependent upon the concentration of free Neu5Ac (between 1 and 10 µg/ml) in the environment. In environments in which Neu5Ac is below that level, the enzyme utilizes endogenous CMP-KDO as the substrate. Our studies show that during in vivo growth in an NTHi biofilm, the KDO moiety is expressed by the organism. Monoclonal antibody 6E4, which binds KDO, is bactericidal for NTHi strains that express the KDO epitope at high levels. In a survey of 33 NTHi strains isolated from healthy and diseased individuals, the antibody was bactericidal (>90% kill) for 12 strains (36%). These studies open up the possibility of using a KDO-based glycoconjugate vaccine as part of a multicomponent vaccine against NTHi.IMPORTANCE Nontypeable Haemophilus influenzae is an important pathogen in middle ear infections in children, sinusitis in adults, and acute bronchitis in individuals with chronic obstructive lung disease. The organism is very well adapted to the human host environment, and this has hindered successful development of an effective vaccine. In this article, we describe a mechanism by which the bacteria decorates its surface lipooligosaccharide with a sugar unique to Gram-negative bacteria, ketodeoxyoctanoate (KDO). This sugar decoration is present during active infection and we have shown that an antibody directed against this sugar can result in killing of the organism. These data demonstrate that the lipooligosaccharide ketodeoxyoctanoate epitope may be a novel NTHi-specific candidate vaccine antigen.

Perturbing the acetylation status of the Type IV pilus retraction motor, PilT, reduces Neisseria gonorrhoeae viability.

Post-translational acetylation is a common protein modification in bacteria. It was recently reported that Neisseria gonorrhoeae acetylates the Type IV pilus retraction motor, PilT. Here, we show recombinant PilT can be acetylated in vitro and acetylation does not affect PilT ultrastructure. To investigate the function of PilT acetylation, we mutated an acetylated lysine, K117, to mimic its acetylated or unacetylated forms. These mutations were not tolerated by wild-type N. gonorrhoeae, but they were tolerated by N. gonorrhoeae carrying an inducible pilE when grown without inducer. We identified additional mutations in pilT and pilU that suppress the lethality of K117 mutations. To investigate the link between PilE and PilT acetylation, we found the lack of PilE decreases PilT acetylation levels and increases the amount of PilT associated with the inner membrane. Finally, we found no difference between wild-type and mutant cells in transformation efficiency, suggesting neither mutation inhibits Type IV pilus retraction. Mutant cells, however, form microcolonies morphologically distinct from wt cells. We conclude that interfering with the acetylation status of PilTK117 greatly reduces N. gonorrhoeae viability, and mutations in pilT, pilU and pilE can overcome this lethality. We discuss the implications of these findings in the context of Type IV pilus retraction regulation.

Characterization of Inner and Outer Membrane Proteins from Francisella tularensis Strains LVS and Schu S4 and Identification of Potential Subunit Vaccine Candidates.

Francisella tularensis is the causative agent of tularemia and a potential bioterrorism agent. In the present study, we isolated, identified, and quantified the proteins present in the membranes of the virulent type A strain, Schu S4, and the attenuated type B strain, LVS (live vaccine strain). Spectral counting of mass spectrometric data showed enrichment for membrane proteins in both strains. Mice vaccinated with whole LVS membranes encapsulated in poly (lactic-co-glycolic acid) (PLGA) nanoparticles containing the adjuvant polyinosinic-polycytidylic acid [poly(I·C)] showed significant protection against a challenge with LVS compared to the results seen with naive mice or mice vaccinated with either membranes or poly(I·C) alone. The PLGA-encapsulated Schu S4 membranes with poly(I·C) alone did not significantly protect mice from a lethal intraperitoneal challenge with Schu S4; however, this vaccination strategy provided protection from LVS challenge. Mice that received the encapsulated Schu S4 membranes followed by a booster of LVS bacteria showed significant protection with respect to a lethal Schu S4 challenge compared to control mice. Western blot analyses of the sera from the Schu S4-vaccinated mice that received an LVS booster showed four immunoreactive bands. One of these bands from the corresponding one-dimensional (1D) SDS-PAGE experiment represented capsule. The remaining bands were excised, digested with trypsin, and analyzed using mass spectrometry. The most abundant proteins present in these immunoreactive samples were an outer membrane OmpA-like protein, FopA; the type IV pilus fiber building block protein; a hypothetical membrane protein; and lipoproteins LpnA and Lpp3. These proteins should serve as potential targets for future recombinant protein vaccination studies.IMPORTANCE The low infectious dose, the high potential mortality/morbidity rates, and the ability to be disseminated as an aerosol make Francisella tularensis a potential agent for bioterrorism. These characteristics led the Centers for Disease Control (CDC) to classify F. tularensis as a Tier 1 pathogen. Currently, there is no vaccine approved for general use in the United States.

Identification and characterization of AckA-dependent protein acetylation in Neisseria gonorrhoeae.

Neisseria gonorrhoeae, the causative agent of gonorrhea, has a number of factors known to contribute to pathogenesis; however, a full understanding of these processes and their regulation has proven to be elusive. Post-translational modifications (PTMs) of bacterial proteins are now recognized as one mechanism of protein regulation. In the present study, Western blot analyses, with an anti-acetyl-lysine antibody, indicated that a large number of gonococcal proteins are post-translationally modified. Previous work has shown that Nε-lysine acetylation can occur non-enzymatically with acetyl-phosphate (AcP) as the acetyl donor. In the current study, an acetate kinase mutant (1291ackA), which accumulates AcP, was generated in N. gonorrhoeae. Broth cultures of N. gonorrhoeae 1291wt and 1291ackA were grown, proteins extracted and digested, and peptides containing acetylated-lysines (K-acetyl) were affinity-enriched from both strains. Mass spectrometric analyses of these samples identified a total of 2686 unique acetylation sites. Label-free relative quantitation of the K-acetyl peptides derived from the ackA and wild-type (wt) strains demonstrated that 109 acetylation sites had an ackA/wt ratio>2 and p-values <0.05 in at least 2/3 of the biological replicates and were designated as "AckA-dependent". Regulated K-acetyl sites were found in ribosomal proteins, central metabolism proteins, iron acquisition and regulation proteins, pilus assembly and regulation proteins, and a two-component response regulator. Since AckA is part of a metabolic pathway, comparative growth studies of the ackA mutant and wt strains were performed. The mutant showed a growth defect under aerobic conditions, an inability to grow anaerobically, and a defect in biofilm maturation. In conclusion, the current study identified AckA-dependent acetylation sites in N. gonorrhoeae and determined that these sites are found in a diverse group of proteins. This work lays the foundation for future studies focusing on specific acetylation sites that may have relevance in gonococcal pathogenesis and metabolism.

Comparative Analyses of the Lipooligosaccharides from Nontypeable Haemophilus influenzae and Haemophilus haemolyticus Show Differences in Sialic Acid and Phosphorylcholine Modifications.

Haemophilus haemolyticus and nontypeable Haemophilus influenzae (NTHi) are closely related upper airway commensal bacteria that are difficult to distinguish phenotypically. NTHi causes upper and lower airway tract infections in individuals with compromised airways, while H. haemolyticus rarely causes such infections. The lipooligosaccharide (LOS) is an outer membrane component of both species and plays a role in NTHi pathogenesis. In this study, comparative analyses of the LOS structures and corresponding biosynthesis genes were performed. Mass spectrometric and immunochemical analyses showed that NTHi LOS contained terminal sialic acid more frequently and to a higher extent than H. haemolyticus LOS did. Genomic analyses of 10 strains demonstrated that H. haemolyticus lacked the sialyltransferase genes lic3A and lic3B (9/10) and siaA (10/10), but all strains contained the sialic acid uptake genes siaP and siaT (10/10). However, isothermal titration calorimetry analyses of SiaP from two H. haemolyticus strains showed a 3.4- to 7.3-fold lower affinity for sialic acid compared to that of NTHi SiaP. Additionally, mass spectrometric and immunochemical analyses showed that the LOS from H. haemolyticus contained phosphorylcholine (ChoP) less frequently than the LOS from NTHi strains. These differences observed in the levels of sialic acid and ChoP incorporation in the LOS structures from H. haemolyticus and NTHi may explain some of the differences in their propensities to cause disease.

Phosphoethanolamine Transferase LptA in Haemophilus ducreyi Modifies Lipid A and Contributes to Human Defensin Resistance In Vitro.

Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, ß-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. The triple mutant was significantly more susceptible to both α- and ß-defensins; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin LL-37 may be more important than defensin resistance to H. ducreyi pathogenesis.

Francisella tularensis Schu S4 lipopolysaccharide core sugar and O-antigen mutants are attenuated in a mouse model of tularemia.

The virulence factors mediating Francisella pathogenesis are being investigated, with an emphasis on understanding how the organism evades innate immunity mechanisms. Francisella tularensis produces a lipopolysaccharide (LPS) that is essentially inert and a polysaccharide capsule that helps the organism to evade detection by components of innate immunity. Using an F. tularensis Schu S4 mutant library, we identified strains that are disrupted for capsule and O-antigen production. These serum-sensitive strains lack both capsule production and O-antigen laddering. Analysis of the predicted protein sequences for the disrupted genes (FTT1236 and FTT1238c) revealed similarity to those for waa (rfa) biosynthetic genes in other bacteria. Mass spectrometry further revealed that these proteins are involved in LPS core sugar biosynthesis and the ligation of O antigen to the LPS core sugars. The 50% lethal dose (LD50) values of these strains are increased 100- to 1,000-fold for mice. Histopathology revealed that the immune response to the F. tularensis mutant strains was significantly different from that observed with wild-type-infected mice. The lung tissue from mutant-infected mice had widespread necrotic debris, but the spleens lacked necrosis and displayed neutrophilia. In contrast, the lungs of wild-type-infected mice had nominal necrosis, but the spleens had widespread necrosis. These data indicate that murine death caused by wild-type strains occurs by a mechanism different from that by which the mutant strains kill mice. Mice immunized with these mutant strains displayed >10-fold protective effects against virulent type A F. tularensis challenge.

Comparative analyses of proteins from Haemophilus influenzae biofilm and planktonic populations using metabolic labeling and mass spectrometry.

BACKGROUND: Non-typeable H. influenzae (NTHi) is a nasopharyngeal commensal that can become an opportunistic pathogen causing infections such as otitis media, pneumonia, and bronchitis. NTHi is known to form biofilms. Resistance of bacterial biofilms to clearance by host defense mechanisms and antibiotic treatments is well-established. In the current study, we used stable isotope labeling by amino acids in cell culture (SILAC) to compare the proteomic profiles of NTHi biofilm and planktonic organisms. Duplicate continuous-flow growth chambers containing defined media with either "light" (L) isoleucine or "heavy" (H) (13)C6-labeled isoleucine were used to grow planktonic (L) and biofilm (H) samples, respectively. Bacteria were removed from the chambers, mixed based on weight, and protein extracts were generated. Liquid chromatography-mass spectrometry (LC-MS) was performed on the tryptic peptides and 814 unique proteins were identified with 99% confidence. RESULTS: Comparisons of the NTHi biofilm to planktonic samples demonstrated that 127 proteins showed differential expression with p-values ≤0.05. Pathway analysis demonstrated that proteins involved in energy metabolism, protein synthesis, and purine, pyrimidine, nucleoside, and nucleotide processes showed a general trend of downregulation in the biofilm compared to planktonic organisms. Conversely, proteins involved in transcription, DNA metabolism, and fatty acid and phospholipid metabolism showed a general trend of upregulation under biofilm conditions. Selected reaction monitoring (SRM)-MS was used to validate a subset of these proteins; among these were aerobic respiration control protein ArcA, NAD nucleotidase and heme-binding protein A. CONCLUSIONS: The present proteomic study indicates that the NTHi biofilm exists in a semi-dormant state with decreased energy metabolism and protein synthesis yet is still capable of managing oxidative stress and in acquiring necessary cofactors important for biofilm survival.

Proteomic analysis of Neisseria gonorrhoeae biofilms shows shift to anaerobic respiration and changes in nutrient transport and outermembrane proteins.

Neisseria gonorrhoeae, the causative agent of gonorrhea, can form biofilms in vitro and in vivo. In biofilms, the organism is more resistant to antibiotic treatment and can serve as a reservoir for chronic infection. We have used stable isotope labeling by amino acids in cell culture (SILAC) to compare protein expression in biofilm and planktonic organisms. Two parallel populations of N. gonorrhoeae strain 1291, which is an arginine auxotroph, were grown for 48 h in continuous-flow chambers over glass, one supplemented with (13)C(6)-arginine for planktonic organisms and the other with unlabeled arginine for biofilm growth. The biofilm and planktonic cells were harvested and lysed separately, and fractionated into three sequential protein extracts. Corresponding heavy (H) planktonic and light (L) biofilm protein extracts were mixed and separated by 1D SDS-PAGE gels, and samples were extensively analyzed by liquid chromatography-mass spectrometry. Overall, 757 proteins were identified, and 152 unique proteins met a 1.5-fold cutoff threshold for differential expression with p-values <0.05. Comparing biofilm to planktonic organisms, this set included 73 upregulated and 54 downregulated proteins. Nearly a third of the upregulated proteins were involved in energy metabolism, with cell envelope proteins making up the next largest group. Of the downregulated proteins, the largest groups were involved in protein synthesis and energy metabolism. These proteomics results were compared with our previously reported results from transcriptional profiling of gonococcal biofilms using microarrays. Nitrite reductase and cytochrome c peroxidase, key enzymes required for anaerobic growth, were detected as highly upregulated in both the proteomic and transcriptomic datasets. These and other protein expression changes observed in the present study were consistent with a shift to anaerobic respiration in gonococcal biofilms, although changes in membrane proteins not explicitly related to this shift may have other functions.

O-antigen and core carbohydrate of Vibrio fischeri lipopolysaccharide: composition and analysis of their role in Euprymna scolopes light organ colonization.

Vibrio fischeri exists in a symbiotic relationship with the Hawaiian bobtail squid, Euprymna scolopes, where the squid provides a home for the bacteria, and the bacteria in turn provide camouflage that helps protect the squid from night-time predators. Like other gram-negative organisms, V. fischeri expresses lipopolysaccharide (LPS) on its cell surface. The structure of the O-antigen and the core components of the LPS and their possible role in colonization of the squid have not previously been determined. In these studies, an O-antigen ligase mutant, waaL, was utilized to determine the structures of these LPS components and their roles in colonization of the squid. WaaL ligates the O-antigen to the core of the LPS; thus, LPS from waaL mutants lacks O-antigen. Our results show that the V. fischeri waaL mutant has a motility defect, is significantly delayed in colonization, and is unable to compete with the wild-type strain in co-colonization assays. Comparative analyses of the LPS from the wild-type and waaL strains showed that the V. fischeri LPS has a single O-antigen repeat composed of yersiniose, 8-epi-legionaminic acid, and N-acetylfucosamine. In addition, the LPS from the waaL strain showed that the core structure consists of L-glycero-D-manno-heptose, D-glycero-D-manno-heptose, glucose, 3-deoxy-D-manno-octulosonic acid, N-acetylgalactosamine, 8-epi-legionaminic acid, phosphate, and phosphoethanolamine. These studies indicate that the unusual V. fischeri O-antigen sugars play a role in the early phases of bacterial colonization of the squid.

Identification, characterization and immunogenicity of an O-antigen capsular polysaccharide of Francisella tularensis.

Capsular polysaccharides are important factors in bacterial pathogenesis and have been the target of a number of successful vaccines. Francisella tularensis has been considered to express a capsular antigen but none has been isolated or characterized. We have developed a monoclonal antibody, 11B7, which recognizes the capsular polysaccharide of F. tularensis migrating on Western blot as a diffuse band between 100 kDa and 250 kDa. The capsule stains poorly on SDS-PAGE with silver stain but can be visualized using ProQ Emerald glycoprotein stain. The capsule appears to be highly conserved among strains of F. tularensis as antibody 11B7 bound to the capsule of 14 of 14 F. tularensis type A and B strains on Western blot. The capsular material can be isolated essentially free of LPS, is phenol and proteinase K resistant, ethanol precipitable and does not dissociate in sodium dodecyl sulfate. Immunoelectron microscopy with colloidal gold demonstrates 11B7 circumferentially staining the surface of F. tularensis which is typical of a polysaccharide capsule. Mass spectrometry, compositional analysis and NMR indicate that the capsule is composed of a polymer of the tetrasaccharide repeat, 4)-alpha-D-GalNAcAN-(1->4)-alpha-D-GalNAcAN-(1->3)-beta-D-QuiNAc-(1->2)-beta-D-Qui4NFm-(1-, which is identical to the previously described F. tularensis O-antigen subunit. This indicates that the F. tularensis capsule can be classified as an O-antigen capsular polysaccharide. Our studies indicate that F. tularensis O-antigen glycosyltransferase mutants do not make a capsule. An F. tularensis acyltransferase and an O-antigen polymerase mutant had no evidence of an O-antigen but expressed a capsular antigen. Passive immunization of BALB/c mice with 75 microg of 11B7 protected against a 150 fold lethal challenge of F. tularensis LVS. Active immunization of BALB/c mice with 10 microg of capsule showed a similar level of protection. These studies demonstrate that F. tularensis produces an O-antigen capsule that may be the basis of a future vaccine.

Proposed second class of Haemophilus ducreyi strains show altered protein and lipooligosaccharide profiles.

Haemophilus ducreyi is the etiologic agent of chancroid, a sexually transmitted genital ulcer disease. Previously we have shown that the protein profiles and lipooligosaccharide (LOS) structures from various strains of H. ducreyi are generally well conserved. Previous studies have demonstrated that at least one strain, 33921, has a variant protein profile and LOS structure. In this study, both the whole cell lysate and the membrane proteins from strain 33921 were further examined and compared to the prototypical strain 35000HP by 2-DE and by the 16-BAC (benzyldimethyl-n-hexadecylammonium chloride)/SDS-PAGE two-detergent system, respectively. These comparisons demonstrated that a number of the proteins that could be identified from both strains had altered positions on the gels, both in their apparent molecular weight and pI values. Strain 33921 has been suggested to be a member of a second class of strains, termed class II strains. In this study, the proteomic profiles and the LOS structures from the five potential class II strains were examined and found to be similar to strain 33921.

Identification of genes involved in the expression of atypical lipooligosaccharide structures from a second class of Haemophilus ducreyi.

Haemophilus ducreyi is a gram-negative bacterium that is the causative agent of chancroid. Strain 35000HP has been well characterized and is representative of the majority of H. ducreyi strains. Strain 35000HP produces a lipooligosaccharide (LOS) that contains D-glycero-D-manno-heptose in the main oligosaccharide chain extension; the lbgB gene has been shown to encode the DD-heptosyltransferase. The lbgB gene is found in a gene cluster together with the lbgA gene, which encodes for the galactosyltransferase I. These two genes are flanked by two housekeeping genes, rpmE and xthA, encoding the ribosomal protein L31 and the exonuclease III, respectively. Recently, a second group of H. ducreyi strains have been identified. Strain 33921, a representative of the class II strains, produces an LOS that lacks DD-heptose in the oligosaccharide portion of its LOS. To better understand the biosynthesis of the DD-heptose-deficient 33921 LOS, we cloned and sequenced the corresponding lbgAB genomic region from strain 33921. Similar to strain 35000HP, the 33921 genome contains xthA and rpmE. However, between these two genes we identified genes encoding two putative glycosyltransferases that were not highly homologous to the 35000HP lbgAB genes. In this study, we demonstrate that the product of one of these genes encodes a galactosyltransferase. In addition, dot blot hybridization determined that 3 of 35 strains tested had the atypical transferases present, as did 4 strains characterized as class II strains by other criterion. These data indicate that the lbgAB genes can serve as one indicator of the classification of H. ducreyi strains.

Stable isotope metabolic labeling of Neisseria meningitidis lipooligosaccharide.

The lipooligosaccharide (LOS) of a Neisseria meningitidis acetate auxotroph was metabolically labeled with either [2-13C]-sodium acetate or [1,2-13C2]-sodium acetate. In this study, we demonstrated that this label was efficiently incorporated into both the lipid A acyl moieties and the two N-acetylglucosamines present in the oligosaccharide branch of the LOS. The development of this efficient labeling protocol should prove useful in future structural studies analyzing the interactions between LOS and host proteins.

Identification of a novel sialic acid transporter in Haemophilus ducreyi.

Haemophilus ducreyi, the causative agent of chancroid, produces a lipooligosaccharide (LOS) which terminates in N-acetyllactosamine. This glycoform can be further extended by the addition of a single sialic acid residue to the terminal galactose moiety. H. ducreyi does not synthesize sialic acid, which must be acquired from the host during infection or from the culture medium when the bacteria are grown in vitro. However, H. ducreyi does not have genes that are highly homologous to the genes encoding known bacterial sialic acid transporters. In this study, we identified the sialic acid transporter by screening strains in a library of random transposon mutants for those mutants that were unable to add sialic acid to N-acetyllactosamine-containing LOS. Mutants that reacted with the monoclonal antibody 3F11, which recognizes the terminal lactosamine structure, and lacked reactivity with the lectin Maackia amurensis agglutinin, which recognizes alpha2,3-linked sialic acid, were further characterized to demonstrate that they produced a N-acetyllactosamine-containing LOS by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis and mass spectrometric analyses. The genes interrupted in these mutants were mapped to a four-gene cluster with similarity to genes encoding bacterial ABC transporters. Uptake assays using radiolabeled sialic acid confirmed that the mutants were unable to transport sialic acid. This study is the first report of bacteria using an ABC transporter for sialic acid uptake.

Biochemical and functional characterization of membrane blebs purified from Neisseria meningitidis serogroup B.

Studies with purified aggregates of endotoxin have revealed the importance of lipopolysaccharide-binding protein (LBP)-dependent extraction and transfer of individual endotoxin molecules to CD14 in Toll-like receptor 4 (TLR4)-dependent cell activation. Endotoxin is normally embedded in the outer membrane of intact Gram-negative bacteria and shed membrane vesicles ("blebs"). However, the ability of LBP and CD14 to efficiently promote TLR4-dependent cell activation by membrane-associated endotoxin has not been studied extensively. In this study, we used an acetate auxotroph of Neisseria meningitidis serogroup B to facilitate metabolic labeling of bacterial endotoxin and compared interactions of purified endotoxin aggregates and of membrane-associated endotoxin with LBP, CD14, and endotoxin-responsive cells. The endotoxin, phospholipid, and protein composition of the recovered blebs indicate that the blebs derive from the bacterial outer membrane. Proteomic analysis revealed an unusual enrichment in highly cationic (pI > 9) proteins. Both purified endotoxin aggregates and blebs activate monocytes and endothelial cells in a LBP-, CD14-, and TLR4/MD-2-dependent fashion, but the blebs were 3-10-fold less potent when normalized for the amount of endotoxin added. Differences in potency correlated with differences in efficiency of LBP-dependent delivery to and extraction of endotoxin by CD14. Both membrane phospholipids and endotoxin are extracted by LBP/soluble CD14 (sCD14) treatment, but only endotoxin.sCD14 reacts with MD-2 and activates cells. These findings indicate that the proinflammatory potency of endotoxin may be regulated not only by the intrinsic structural properties of endotoxin but also by its association with neighboring molecules in the outer membrane.

The msbB mutant of Neisseria meningitidis strain NMB has a defect in lipooligosaccharide assembly and transport to the outer membrane.

A deletion-insertion mutation in msbB, a gene that encodes a lipid A acyltransferase, was introduced into encapsulated Neisseria meningitidis serogroup B strain NMB and an acapsular mutant of the same strain. These mutants were designated NMBA11K3 and NMBA11K3cap-, respectively. Neither lipooligosaccharide (LOS) nor lipid A could be isolated from NMBA11K3 although a number of techniques were tried, but both were easily extracted from NMBA11K3cap-. Immunoelectron microscopy using monoclonal antibody (MAb) 6B4, which recognizes the terminal Galbeta1-4GlcNAc of LOS, demonstrated that NMB, NMBcap-, and NMBA11K3cap- expressed LOS circumferentially, while MAb 6B4 did not bind to the surface of NMBA11K3. However, cytoplasmic staining of NMBA11K3 with MAb 6B4 was a consistent observation. Mass-spectrometric analyses demonstrated that the relative amounts of the lipid A-specific C12:0 3-OH and C14:0 3-OH present in the membrane preparations (MP) from NMBA11K3 were substantially decreased (25- and 23-fold, respectively) compared to the amount in MP from its parent strain, NMB. Western blot analyses of MP from NMBA11K3 demonstrated that the levels of porin in the outer membrane of NMBA11K3 were also substantially decreased. These studies suggest that the lipid A acylation defect in encapsulated NMBA11K3 influences the assembly of the lipid A and consequently the incorporation of porin in the outer membrane.

Immortalization of human urethral epithelial cells: a model for the study of the pathogenesis of and the inflammatory cytokine response to Neisseria gonorrhoeae infection.

The primary human urethral epithelial cells developed by our laboratory have been immortalized by transduction with a retroviral vector expressing the human papillomavirus E6E7 oncogenes. Analysis of telomerase expression and comparison to that in primary cells revealed detectable levels in the transduced human urethral epithelial cells. Immortalized urethral cells could be passaged over 20 times. Immunofluorescence microscopy studies showed that the immortalized cells were phenotypically similar and responded to gonococcal infection similarly to primary cells. Specifically, positive cytokeratin staining showed that the immortalized cells are keratinocytes; cell surface levels of human asialoglycoprotein receptor increase following gonococcal infection, and, like the primary cells, the immortalized urethral epithelial cells are CD14 negative. Using enzyme-linked immunosorbent assay, we found that interleukin-6 (IL-6) and IL-8 levels in primary urethral epithelial cell supernatants increase after challenge with N. gonorrhoeae. Likewise, the immortalized urethral epithelial cells produced higher levels of IL-6 and IL-8 cytokines in response to gonococcal infection. Cells challenged with a gonococcal lipid A msbB mutant produced reduced IL-6 and IL-8 levels when compared to the parent strain. Additionally, these data suggest that the 1291 msbB lipooligosaccharide may suppress cytokine induction.

Intracellular survival of Neisseria gonorrhoeae in male urethral epithelial cells: importance of a hexaacyl lipid A.

Neisseria gonorrhoeae is a strict human pathogen that invades and colonizes the urogenital tracts of males and females. Lipooligosaccharide (LOS) has been shown to play a role in gonococcal pathogenesis. The acyl transferase MsbB is involved in the biosynthesis of the lipid A portion of the LOS. In order to determine the role of an intact lipid A structure on the pathogenesis of N. gonorrhoeae, the msbB gene was cloned and sequenced, a deletion and insertion mutation was introduced into N. gonorrhoeae, and the mutant strain was designated 1291A11K3. Mass spectrometric analyses of 1291A11K3 LOS determined that this mutation resulted in a pentaacyl rather than a hexaacyl lipid A structure. These analyses also demonstrated an increase in the phosphorylation of lipid A and an increase in length of the oligosaccharide of a minor species of the msbB LOS. The interactions of this mutant with male urethral epithelial cells (uec) were examined. Transmission and scanning electron microscopy studies indicated that the msbB mutants formed close associations with and were internalized by the uec at levels similar to those of the parent strain. Gentamicin survival assays performed with 1291A11K3 and 1291 bacteria demonstrated that there was no difference in the abilities of the two strains to adhere to uec; however, significantly fewer 1291A11K3 bacteria than parent strain bacteria were recovered from gentamicin-treated uec. These studies suggest that the lipid A modification in the N. gonorrhoeae msbB mutant may render it more susceptible to innate intracellular killing mechanisms when internalized by uec.