Evaluating methods to create protein functionalized catanionic vesicles.

Catanionic surfactant vesicles (SVs) composed of sodium dodecylbenzenesulfonate (SDBS) and cetyltrimethylammonium tosylate (CTAT) have potential applications as targeted drug delivery systems, vaccine platforms, and diagnostic tools. To facilitate these applications, we evaluated various methods to attach proteins to the surface of SDBS/CTAT vesicles. Acid phosphatase from wheat germ was used as a model protein. Acid phosphatase was successfully conjugated to vesicles enriched with a Triton-X 100 derivative containing an unsaturated ester. Enzymatic activity of acid phosphatase attached to vesicles was assessed using an acid phosphatase assay. Results from the acid phosphatase assay indicated that 15 ± 3% of the attached protein remained functional but the presence of vesicles interferes with the assay. DLS and zeta potential results correlated with the protein functionalization studies. Acid phosphatase functionalized vesicles had an average diameter of 175 ± 85 nm and an average zeta potential of -61 ± 5 mV in PBS. As a control, vesicles enriched with Triton-X 100 were prepared and analyzed by DLS and zeta potential measurements. Triton X-100 enriched vesicles had an average diameter of 140 ± 67 nm and an average zeta potential of -49 ± 2 mV in PBS. Functionalizing the surface of SVs with proteins may be a key step in developing vesicle-based technologies. For drug delivery, antibodies could be used as targeting molecules; for vaccine formulation, functionalizing the surface with spike proteins may produce novel vaccine platforms.

A New Vaccination Method Based on Phage NgoΦ6 and Its Phagemid Derivatives.

Phagemid particles based on the Neisseria gonorrhoeae filamentous phage NgoΦ6 were used as a vaccine delivery system. We demonstrate that the host proteins incorporated into/associated with these particles can be encoded by chromosomal genes of the host bacterium or from plasmids able to replicate as an autonomous entity in the phagemid host. Phagemid particles were prepared from three types of cells, namely, Salmonella enterica ser. Typhimurium [pBSKS::Φ6fm(ST)] containing phagemid genome as an autonomous plasmid, Haemophilus influenzae Rd containing phagemid [pBSKS::Φ6fm(Hin)] integrated into the chromosome, and S. enterica ser. Typhimurium [pMPMT6::Φ6fm(ST)] containing an additional plasmid, pE1 HCV, encoding the Hepatitis C virus envelope glycoprotein E1. Approximately 200 µg of purified phage particles was used to immunize rabbits. The phagemid particles prepared from these three strains all elicited a large amount of IgG antibodies that were able to recognize bacterial host cells and proteins, as determined by ELISA and FACS analysis. The amount of specific anti-S. enterica ser. Typhimurium, anti-H. influenzae, and anti-E1 HCV antibodies elicited by vaccination was 170 µg/ml for anti-Salmonella, 80 µg/ml for anti-H. influenzae, and 65 µg/ml for anti-E1 HCV. Taken in toto, these data suggest that classical phage display methods have underestimated the potential for filamentous phage as a novel immunogen delivery system.

In Vitro Analysis of Matched Isolates from Localized and Disseminated Gonococcal Infections Suggests That Opa Expression Impacts Clinical Outcome.

Gonorrhea is the second most common sexually transmitted infection, which is primarily localized but can be disseminated systemically. The mechanisms by which a localized infection becomes a disseminated infection are unknown. We used five pairs of Neisseria gonorrhoeae isolates from the cervix/urethra (localized) and the blood (disseminated) of patients with disseminated gonococcal infection to examine the mechanisms that confine gonococci to the genital tract or enable them to disseminate to the blood. Multilocus sequence analysis found that the local and disseminated isolates from the same patients were isogenic. When culturing in vitro, disseminated isolates aggregated significantly less and transmigrated across a polarized epithelial monolayer more efficiently than localized isolates. While localized cervical isolates transmigrated across epithelial monolayers inefficiently, those transmigrated bacteria self-aggregated less and transmigrated more than cervical isolates but comparably to disseminating isolates. The local cervical isolates recruited the host receptors of gonococcal Opa proteins carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) on epithelial cells. However, the transmigrated cervical isolate and the disseminated blood isolates recruit CEACAMs significantly less often. Our results collectively suggest that switching off the expression of CEACAM-binding Opa(s), which reduces self-aggregation, promotes gonococcal dissemination.

Gonococcal invasion into epithelial cells depends on both cell polarity and ezrin.

Neisseria gonorrhoeae (GC) establishes infection in women from the cervix, lined with heterogeneous epithelial cells from non-polarized stratified at the ectocervix to polarized columnar at the endocervix. We have previously shown that GC differentially colonize and transmigrate across the ecto and endocervical epithelia. However, whether and how GC invade into heterogeneous cervical epithelial cells is unknown. This study examined GC entry of epithelial cells with various properties, using human cervical tissue explant and non-polarized/polarized epithelial cell line models. While adhering to non-polarized and polarized epithelial cells at similar levels, GC invaded into non-polarized more efficiently than polarized epithelial cells. The enhanced GC invasion in non-polarized epithelial cells was associated with increased ezrin phosphorylation, F-actin and ezrin recruitment to GC adherent sites, and the elongation of GC-associated microvilli. Inhibition of ezrin phosphorylation inhibited F-actin and ezrin recruitment and microvilli elongation, leading to a reduction in GC invasion. The reduced GC invasion in polarized epithelial cells was associated with non-muscle myosin II-mediated F-actin disassembly and microvilli denudation at GC adherence sites. Surprisingly, intraepithelial GC were only detected inside epithelial cells shedding from the cervix by immunofluorescence microscopy, but not significantly in the ectocervical and the endocervical regions. We observed similar ezrin and F-actin recruitment in exfoliated cervical epithelial cells but not in those that remained in the ectocervical epithelium, as the luminal layer of ectocervical epithelial cells expressed ten-fold lower levels of ezrin than those beneath. However, GC inoculation induced F-actin reduction and myosin recruitment in the endocervix, similar to what was seen in polarized epithelial cells. Collectively, our results suggest that while GC invade non-polarized epithelial cells through ezrin-driven microvilli elongation, the apical polarization of ezrin and F-actin inhibits GC entry into polarized epithelial cells.

Adaptation of Neisseria gonorrhoeae to the Female Reproductive Tract.

Gonorrhea, caused by Neisseria gonorrhoeae, is a common sexually transmitted infection and an urgent public health problem. Humans are the exclusive host, and the genital tract with heterogeneous epithelia is the primary niche of this bacterium, creating unique challenges for understanding its pathogenesis. The cervical tissue explant model that we have developed enabled us to show that the properties of the epithelial cells in the female reproductive tract are the main factors driving gonococcal adaptation. Gonococcal variants that colonize strongly and penetrate poorly, thereby causing asymptomatic infection, survive better in the cervix. Gonococci adapt to different epithelial cell types by varying their surfaces and modulating distinct epithelial cell-cell adhesion complexes through manipulation of host cell signaling. These findings provide critical new insights on the mechanisms by which N. gonorrhoeae adapts to the human mucosal surface and causes asymptomatic infection.

Extraction of Membrane Components from Neisseria gonorrhoeae Using Catanionic Surfactant Vesicles: A New Approach for the Study of Bacterial Surface Molecules.

Identification of antigens is important for vaccine production. We tested extraction protocols using cetyltrimethylammonium tosylate (CTAT) and sodium dodecylbenzenesulfonate (SDBS) to formulate surfactant vesicles (SVs) containing components from Neisseria gonorrhoeae. Carbohydrate and protein assays demonstrated that protein and carbohydrates were incorporated into the vesicle leaflet. Depending on the extraction protocol utilized, 100-400 µg of protein/mL of SVs solution was obtained. Gel electrophoresis followed by silver staining demonstrated that SV extracts contained lipooligosaccharide and a subset of bacterial proteins and lipoproteins. Western blotting and mass spectral analysis indicated that the majority of the proteins were derived from the outer membrane. Mass spectrometric and bioinformatics analysis of SVs identified 29 membrane proteins, including porin and opacity-associated protein. Proteins embedded in the SVs leaflet could be degraded by the addition of trypsin or proteinase K. Our data showed that the incorporation of CTAT and SDBS into vesicles eliminated their toxicity as measured by a THP-1 killing assay. Incorporation of gonococcal cell surface components into SVs reduced toxicity as compared to the whole cell extracts, as measured by cytokine induction, while retaining the immunogenicity. This process constitutes a general method for extracting bacterial surface components and identification of antigens that might be included in vaccines.

Neisseria gonorrhoeae infects the heterogeneous epithelia of the human cervix using distinct mechanisms.

Sexually transmitted infections are a critical public health issue. However, the mechanisms underlying sexually transmitted infections in women and the link between the infection mechanism and the wide range of clinical outcomes remain elusive due to a lack of research models mimicking human infection in vivo. We established a human cervical tissue explant model to mimic local Neisseria gonorrhoeae (GC) infections. We found that GC preferentially colonize the ectocervix by activating integrin-ß1, which inhibits epithelial shedding. GC selectively penetrate into the squamocolumnar junction (TZ) and endocervical epithelia by inducing ß-catenin phosphorylation, which leads to E-cadherin junction disassembly. Epithelial cells in various cervical regions differentially express carcinoembryonic antigen-related cell adhesion molecules (CEACAMs), the host receptor for GC opacity-associated proteins (OpaCEA). Relatively high levels were detected on the luminal membrane of ecto/endocervical epithelial cells but very low levels intracellularly in TZ epithelial cells. CEACAM-OpaCEA interaction increased ecto/endocervical colonization and reduced endocervical penetration by increasing integrin-ß1 activation and inhibiting ß-catenin phosphorylation respectively, through CEACAM downstream signaling. Thus, the intrinsic properties of cervical epithelial cells and phase-variation of bacterial surface molecules both play a role in controlling GC infection mechanisms and infectivity, preferential colonization or penetration, potentially leading to asymptomatic or symptomatic infection.

Quantitative Examination of Antibiotic Susceptibility of Neisseria gonorrhoeae Aggregates Using ATP-utilization Commercial Assays and Live/Dead Staining.

The emergence of antibiotic resistant Neisseria gonorrhoeae (GC) is a worldwide health threat and highlights the need to identify individuals who fail treatment. This Gram-negative bacterium causes gonorrhea exclusively in humans. During infection, it is able to form aggregates and/or biofilms. The minimum inhibitory concentration (MIC) test is used for to determine susceptibility to antibiotics and to define appropriate treatment. However, the mechanism of the eradication in vivo and its relationship to laboratory results are not known. A method that examines how GC aggregation affects antibiotic susceptibility and shows the relationship between aggregate size and antibiotic susceptibility was developed. When GC aggregate, they are more resistant to antibiotic killing, with bacteria in the center surviving ceftriaxone treatment better than those in the periphery. The data indicate that N. gonorrhoeae aggregation can reduce its susceptibility to ceftriaxone, which is not reflected using the standard agar plate-based MIC methods. The method used in this study will allow researchers to test bacterial susceptibility under clinically relevant conditions.

Neisseria gonorrhoeae Aggregation Reduces Its Ceftriaxone Susceptibility.

Antibiotic resistance in Neisseria gonorrhoeae (GC) has become an emerging threat worldwide and heightens the need for monitoring treatment failures. N. gonorrhoeae, a gram-negative bacterium responsible for gonorrhea, infects humans exclusively and can form aggregates during infection. While minimal inhibitory concentration (MIC) tests are often used for determining antibiotic resistance development and treatment, the knowledge of the true MIC in individual patients and how it relates to this laboratory measure is not known. We examined the effect of aggregation on GC antibiotic susceptibility and the relationship between bacterial aggregate size and their antibiotic susceptibility. Aggregated GC have a higher survival rate when treated with ceftriaxone than non-aggregated GC, with bacteria in the core of the aggregates surviving the treatment. GC lacking opacity-associated protein or pili, or expressing a truncated lipooligosaccharide, three surface molecules that mediate GC-GC interactions, reduce both aggregation and ceftriaxone survival. This study demonstrates that the aggregation of N. gonorrhoeae can reduce the susceptibility to antibiotics, and suggests that antibiotic utilization can select for GC surface molecules that promote aggregation which in turn drive pathogen evolution. Inhibiting aggregation may be a potential way of increasing the efficacy of ceftriaxone treatment, consequently reducing treatment failure.

Immunofluorescence Analysis of Human Endocervical Tissue Explants Infected with Neisseria gonorrhoeae.

Colonization and penetration of the epithelium is the infection-initiating route of mucosal pathogens. The epithelium counteracts infection by eliciting host cell responses while maintaining the mucosal barrier function. The obligate human sexually transmitted bacterium Neisseria gonorrhoeae, or gonococcus (GC) infects the female reproductive tract primarily from the endocervical epithelium. Due to lack of an infection model that mimics all aspects of human infections in the female reproductive tract, GC pathogenesis is poorly understood. This protocol takes advantage of the viability and functional integrity of human cervical tissues propagated in culture to generate an ex vivo infection model. This tissue model maintains the nature of the infection target and environment without any manipulation such as immortalization of epithelial cells by viruses. Using immunofluorescence microscopy, the interaction of GC with the endocervical epithelium was analyzed.

Catanionic Surfactant Vesicles as a New Platform for probing Glycan-Protein Interactions.

Glycomics lags substantially behind proteomics and genomics in its ability to decipher and synthesize complex glycans. The slow progress in deciphering glycan interactions at a molecular level is in large part due to the absence of a functional system to express, on a large scale, carbohydrates of known structure, in the context of a biologically relevant assay system. Here we describe the characterization of a glycan-functionalized catanionic surfactant vesicles (CVs) as a platform for glycan synthesis, and to demonstrate that the resulting glycan-functionalized CVs can serve as a scaffold for the interrogation of protein-glycan interactions. We demonstrate that N. gonorrhoeae lipooligosaccharide (LOS) glycosyltransferase LgtE, an enzyme that catalyzes the addition of galactose onto a terminal glucose found on LOS can be used to biochemically modify LOS or glucose functionalized CVs. CVs were characterized by differential lectin binding using flow cytometry. LgtE activity was measured on whole cells and LOS functionalized vesicles and found to have approximately the same biochemical properties. We further demonstrate that CVs can be ink-jet printed. This paper presents proof-of-concept that glycan-functionalized catanionic vesicles can be used to create a high-specificity and high-throughput glycan array that will allow for the investigation of a variety of protein-glycan interactions.

Using a Concept Inventory to Reveal Student Thinking Associated with Common Misconceptions about Antibiotic Resistance.

Misconceptions, also known as alternate conceptions, about key concepts often hinder the ability of students to learn new knowledge. Concept inventories (CIs) are designed to assess students' understanding of key concepts, especially those prone to misconceptions. Two-tiered CIs include prompts that ask students to explain the logic behind their answer choice. Such two-tiered CIs afford an opportunity for faculty to explore the student thinking behind the common misconceptions represented by their choice of a distractor. In this study, we specifically sought to probe the misconceptions that students hold prior to beginning an introductory microbiology course (i.e., preconceptions). Faculty-learning communities at two research-intensive universities used the validated Host-Pathogen Interaction Concept Inventory (HPI-CI) to reveal student preconceptions. Our method of deep analysis involved communal review and discussion of students' explanations for their CI answer choice. This approach provided insight valuable for curriculum development. Here the process is illustrated using one question from the HPI-CI related to the important topic of antibiotic resistance. The frequencies with which students chose particular multiple-choice responses for this question were highly correlated between institutions, implying common underlying misconceptions. Examination of student explanations using our analysis approach, coupled with group discussions within and between institutions, revealed patterns in student thinking to the participating faculty. Similar application of a two-tiered concept inventory by general microbiology instructors, either individually or in groups, at other institutions will allow them to better understand student thinking related to key concepts in their curriculum.

Neisseria gonorrhoeae infects the human endocervix by activating non-muscle myosin II-mediated epithelial exfoliation.

Colonization and disruption of the epithelium is a major infection mechanism of mucosal pathogens. The epithelium counteracts infection by exfoliating damaged cells while maintaining the mucosal barrier function. The sexually transmitted bacterium Neisseria gonorrhoeae (GC) infects the female reproductive tract primarily from the endocervix, causing gonorrhea. However, the mechanism by which GC overcome the mucosal barrier remains elusive. Using a new human tissue model, we demonstrate that GC can penetrate into the human endocervix by inducing the exfoliation of columnar epithelial cells. We found that GC colonization causes endocervical epithelial cells to shed. The shedding results from the disassembly of the apical junctions that seal the epithelial barrier. Apical junction disruption and epithelial exfoliation increase GC penetration into the endocervical epithelium without reducing bacterial adherence to and invasion into epithelial cells. Both epithelial exfoliation and junction disruption require the activation and accumulation of non-muscle myosin II (NMII) at the apical surface and GC adherent sites. GC inoculation activates NMII by elevating the levels of the cytoplasmic Ca2+ and NMII regulatory light chain phosphorylation. Piliation of GC promotes, but the expression of a GC opacity-associated protein variant, OpaH that binds to the host surface proteins CEACAMs, inhibits GC-induced NMII activation and reorganization and Ca2+ flux. The inhibitory effects of OpaH lead to reductions in junction disruption, epithelial exfoliation, and GC penetration. Therefore, GC phase variation can modulate infection in the human endocervix by manipulating the activity of NMII and epithelial exfoliation.

Innate immune response to lipooligosaccharide: pivotal regulator of the pathobiology of invasive Neisseria meningitidis infections.

Infections due to Neisseria meningitidis afflict more than one million people worldwide annually and cause death or disability in many survivors. The clinical course of invasive infections has been well studied, but our understanding of the cause of differences in patient outcomes has been limited because these are dependent on multiple factors including the response of the host, characteristics of the bacteria and interactions between the host and the bacteria. The meningococcus is a highly inflammatory organism, and the lipooligosaccharide (LOS) on the outer membrane is the most potent inflammatory molecule it expresses due to the interactions of the lipid A moiety of LOS with receptors of the innate immune system. We previously reported that increased phosphorylation of hexaacylated neisserial lipid A is correlated with greater inflammatory potential. Here we postulate that variability in lipid A phosphorylation can tip the balance of innate immune responses towards homeostatic tolerance or proinflammatory signaling that affects adaptive immune responses, causing disease with meningitis only, or septicemia with or without meningitis, respectively. Furthermore, we propose that studies of the relationship between bacterial virulence and gene expression should consider whether genetic variation could affect properties of biosynthetic enzymes resulting in LOS structural differences that alter disease pathobiology.

nagZ Triggers Gonococcal Biofilm Disassembly.

Bacterial-bacterial interactions play a critical role in promoting biofilm formation. Here we show that NagZ, a protein associated with peptidoglycan recycling, has moonlighting activity that allows it to modulate biofilm accumulation by Neisseria gonorrhoeae. We characterize the biochemical properties of NagZ and demonstrate its ability to function as a dispersing agent for biofilms formed on abiotic surfaces. We extend these observations to cell culture and tissue explant models and show that in nagZ mutants, the biofilms formed in cell culture and on human tissues contain significantly more biomass than those formed by a wild-type strain. Our results demonstrate that an enzyme thought to be restricted to peptidoglycan recycling is able to disperse preformed biofilms.

Oral Immunization of Rabbits with S. enterica Typhimurium Expressing Neisseria gonorrhoeae Filamentous Phage Φ6 Induces Bactericidal Antibodies Against N. gonorrhoeae.

All Neisseria gonorrhoeae strains whose DNA sequences have been determined possess filamentous phage DNA sequences. To ascertain if phage encoded proteins could form the basis of a gonococcal vaccine, rabbits were orally infected with S. enterica Typhimurium strain χ3987 harboring phagemid NgoΦ6 fm. The elicited sera contained large quantities of anti-phage IgG and IgA antibodies that bound to the surface of N. gonorrhoeae cells, as shown by indirect fluorescent analysis and flow cytometry. The elicited sera was able to bind to several phage proteins. The sera also had bactericidal activity. These data demonstrate that N. gonorrhoeae filamentous phage can induce antibodies with anti-gonococcal activity and that phage proteins may be a candidate for vaccine development.

Lipooligosaccharide Structures of Invasive and Carrier Isolates of Neisseria meningitidis Are Correlated with Pathogenicity and Carriage.

The degree of phosphorylation and phosphoethanolaminylation of lipid A on neisserial lipooligosaccharide (LOS), a major cell-surface antigen, can be correlated with inflammatory potential and the ability to induce immune tolerance in vitro. On the oligosaccharide of the LOS, the presence of phosphoethanolamine and sialic acid substituents can be correlated with in vitro serum resistance. In this study, we analyzed the structure of the LOS from 40 invasive isolates and 25 isolates from carriers of Neisseria meningitidis without disease. Invasive strains were classified as groups 1-3 that caused meningitis, septicemia without meningitis, and septicemia with meningitis, respectively. Intact LOS was analyzed by high resolution matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Prominent peaks for lipid A fragment ions with three phosphates and one phosphoethanolamine were detected in all LOS analyzed. LOS from groups 2 and 3 had less abundant ions for highly phosphorylated lipid A forms and induced less TNF-α in THP-1 monocytic cells compared with LOS from group 1. Lipid A from all invasive strains was hexaacylated, whereas lipid A of 6/25 carrier strains was pentaacylated. There were fewer O-acetyl groups and more phosphoethanolamine and sialic acid substitutions on the oligosaccharide from invasive compared with carrier isolates. Bioinformatic and genomic analysis of LOS biosynthetic genes indicated significant skewing to specific alleles, dependent on the disease outcome. Our results suggest that variable LOS structures have multifaceted effects on homeostatic innate immune responses that have critical impact on the pathophysiology of meningococcal infections.

Expression of Opacity Proteins Interferes with the Transmigration of Neisseria gonorrhoeae across Polarized Epithelial Cells.

Neisseria gonorrhoeae (GC) establishes infection at the mucosal surface of the human genital tract, most of which is lined with polarized epithelial cells. GC can cause localized as well as disseminated infections, leading to various complications. GC constantly change their surface structures via phase and antigenic variation, which has been implicated as a means for GC to establish infection at various anatomic locations of male and female genital tracks. However, the exact contribution of each surface molecule to bacterial infectivity remains elusive due to their phase variation. Using a GC derivative that is genetically devoid of all opa genes (MS11∆Opa), this study shows that Opa expression interferes with GC transmigration across polarized human epithelial cells. MS11∆Opa transmigrates across polarized epithelial cells much faster and to a greater extent than MS11Opa+, while adhering at a similar level as MS11Opa+. When MS11Opa+, able to phase vary Opa expression, was inoculated, only those bacteria that turn off Opa expression transmigrate across the polarized epithelial monolayer. Similar to bacteria alone or co-cultured with non-polarized epithelial cells, MS11∆Opa fails to form large microcolonies at the apical surface of polarized epithelial cells. Apical inoculation of MS11Opa+, but not MS11∆Opa, induces the recruitment of the Opa host-cell receptor carcinoembryonic antigen-related cell adhesion molecules (CEACAMs) to the apical junction and the vicinity of bacterial adherent sites. Our results suggest that Opa expression limits gonococcal ability to invade into subepithelial tissues by forming tight interactions with neighboring bacteria and by inducing CEACAM redistribution to cell junctions.

Neisseria gonorrhoeae filamentous phage NgoΦ6 is capable of infecting a variety of Gram-negative bacteria.

We constructed a phagemid consisting of the whole genome of the Neisseria gonorrhoeae bacteriophage NgoΦ6 cloned into a pBluescript plasmid derivative lacking the f1 origin of replication (named pBS::Φ6). Escherichia coli cells harboring pBS::Φ6 were able to produce a biologically active phagemid, NgoΦ6fm, capable of infecting, integrating its DNA into the chromosome of, and producing progeny phagemids in, a variety of taxonomically distant Gram-negative bacteria, including E. coli, Haemophilus influenzae, Neisseria sicca, Pseudomonas sp., and Paracoccus methylutens. A derivative of pBS::Φ6 lacking the phage orf7 gene, a positional homolog of filamentous phage proteins that mediate the interaction between the phage and the bacterial pilus, was capable of producing phagemid particles that were able to infect E. coli, Haemophilus influenzae, N. sicca, Pseudomonas sp., and Paracoccus methylutens, indicating that NgoΦ6 infects cells of these species using a mechanism that does not involve the Orf7 gene product and that NgoΦ6 initiates infection through a novel process in these species. We further demonstrate that the establishment of the lysogenic state does not require an active phage integrase. Since phagemid particles were capable of infecting diverse hosts, this indicates that NgoΦ6 is the first broad-host-range filamentous bacteriophage described.