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.

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.

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.

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.

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.

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.

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.

Neisseria gonorrhoeae breaches the apical junction of polarized epithelial cells for transmigration by activating EGFR.

Neisseria gonorrhoeae initiates infection at the apical surface of columnar endocervical epithelial cells in the female reproductive tract. These cells provide a physical barrier against pathogens by forming continuous apical junctional complexes between neighbouring cells. This study examines the interaction of gonococci (GC) with polarized epithelial cells. We show that viable GC preferentially localize at the apical side of the cell-cell junction in polarized endometrial and colonic epithelial cells, HEC-1-B and T84. In GC-infected cells, continuous apical junctional complexes are disrupted, and the junction-associated protein ß-catenin is redistributed from the apical junction to the cytoplasm and to GC adherent sites; however, overall cellular levels remain unchanged. This redistribution of junctional proteins is associated with a decrease in the 'fence' function of the apical junction but not its 'gate' function. Disruption of the apical junction by removing calcium increases GC transmigration across the epithelial monolayer. GC inoculation induces the phosphorylation of both epidermal growth factor receptor (EGFR) and ß-catenin, while inhibition of EGFR kinase activity significantly reduces both GC-induced ß-catenin redistribution and GC transmigration. Therefore, the gonococcus is capable of weakening the apical junction and polarity of epithelial cells by activating EGFR, which facilitates GC transmigration across the epithelium.

Construction and characterization of a derivative of Neisseria gonorrhoeae strain MS11 devoid of all opa genes.

To better understand the role of Opa in gonococcal infections, we created and characterized a derivative of MS11 (MS11Δopa) that had the coding sequence for all 11 Opa proteins deleted. The MS11Δopa bacterium lost the ability to bind to purified lipooligosaccharide (LOS). While nonpiliated MS11Δopa and nonpiliated Opa-expressing MS11 cells grew at the same rate, nonpiliated MS11Δopa cells rarely formed clumps of more than four bacteria when grown in broth with vigorous shaking. Using flow cytometry analysis, we demonstrated that MS11Δopa produced a homogeneous population of bacteria that failed to bind monoclonal antibody (MAb) 4B12, a MAb specific for Opa. Opa-expressing MS11 cells consisted of two predominant populations, where ∼85% bound MAb 4B12 to a significant level and the other population bound little if any MAb. Approximately 90% of bacteria isolated from a phenotypically Opa-negative colony (a colony that does not refract light) failed to bind MAb 4B12; the remaining 10% bound MAb to various degrees. Piliated MS11Δopa cells formed dispersed microcolonies on ME180 cells which were visually distinct from those of piliated Opa-expressing MS11 cells. When Opa expression was reintroduced into MS11Δopa, the adherence ability of the strain recovered to wild-type levels. These data indicate that Opa contributes to both bacterium-bacterium and bacterium-host cell interactions.

Lack of lipid A pyrophosphorylation and functional lptA reduces inflammation by Neisseria commensals.

The interaction of the immune system with Neisseria commensals remains poorly understood. We have previously shown that phosphoethanolamine on the lipid A portion of lipooligosaccharide (LOS) plays an important role in Toll-like receptor 4 (TLR4) signaling. For pathogenic Neisseria, phosphoethanolamine is added to lipid A by the phosphoethanolamine transferase specific for lipid A, which is encoded by lptA. Here, we report that Southern hybridizations and bioinformatics analyses of genomic sequences from all eight commensal Neisseria species confirmed that lptA was absent in 15 of 17 strains examined but was present in N. lactamica. Mass spectrometry of lipid A and intact LOS revealed the lack of both pyrophosphorylation and phosphoethanolaminylation in lipid A of commensal species lacking lptA. Inflammatory signaling in human THP-1 monocytic cells was much greater with pathogenic than with commensal Neisseria strains that lacked lptA, and greater sensitivity to polymyxin B was consistent with the absence of phosphoethanolamine. Unlike the other commensals, whole bacteria of two N. lactamica commensal strains had low inflammatory potential, whereas their lipid A had high-level pyrophosphorylation and phosphoethanolaminylation and induced high-level inflammatory signaling, supporting previous studies indicating that this species uses mechanisms other than altering lipid A to support commensalism. A meningococcal lptA deletion mutant had reduced inflammatory potential, further illustrating the importance of lipid A pyrophosphorylation and phosphoethanolaminylation in the bioactivity of LOS. Overall, our results indicate that lack of pyrophosphorylation and phosphoethanolaminylation of lipid A contributes to the immune privilege of most commensal Neisseria strains by reducing the inflammatory potential of LOS.

Neisseria gonorrhoeae-induced transactivation of EGFR enhances gonococcal invasion.

Neisseria gonorrhoeae, the causative agent of the sexually transmitted infection gonorrhoea, adheres to and invades into genital epithelial cells. Here, we investigate host components that are used by the bacteria for their entry into epithelial cells. We found that gonococcal microcolony formation on the surface of HEC-1-B cells disrupted the polarized, basolateral distribution of both epidermal growth factor receptor (EGFR) and ErbB2, a related family member, and induced their accumulation under the microcolonies at the apical membrane. Gonococcal infection increased EGFR and ErbB2 phosphorylation. The EGFR kinase inhibitor, AG1478, reduced gonococcal invasion by 80%, but had no effect on adherence or the recruitment of EGFR and ErbB2 to the microcolonies. Gonococcal inoculation upregulated the mRNA levels of several ligands of EGFR. Prevention of EGFR ligand shedding by blocking matrix metalloproteinase activation reduced gonococcal invasion without altering their adherence, while the addition of the EGFR ligand, HB-EGF, was able to restore invasion to 66% of control levels. These data indicate that N. gonorrhoeae modulates the activity and cellular distribution of host EGFR, facilitating their invasion. EGFR activation does not appear to be due to direct gonococcal binding to EGFR, but instead by its transactivation by gonococcal induced increases in EGFR ligands.

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.

Neisseria gonorrhoeae FA1090 carries genes encoding two classes of Vsr endonucleases.

A very short patch repair system prevents mutations resulting from deamination of 5-methylcytosine to thymine. The Vsr endonuclease is the key enzyme of this system, providing sequence specificity. We identified two genes encoding Vsr endonucleases V.NgoAXIII and V.NgoAXIV from Neisseria gonorrhoeae FA1090 based on DNA sequence similarity to genes encoding Vsr endonucleases from other bacteria. After expression of the gonococcal genes in Escherichia coli, the proteins were biochemically characterized and the endonucleolytic activities and specificities of V.NgoAXIII and V.NgoAXIV were determined. V.NgoAXIII was found to be multispecific and to recognize T:G mismatches in every nucleotide context tested, whereas V.NgoAXIV recognized T:G mismatches in the following sequences: GTGG, CTGG, GTGC, ATGC, and CTGC. Alanine mutagenesis of conserved residues showed that Asp50 and His68 of V.NgoAXIII and Asp51 and His69 of V.NgoAXIV are essential for hydrolytic activity. Glu25, His64, and Asp97 of V.NgoAXIV and Glu24, Asp63, and Asp97 of V.NgoAXIII are important but not crucial for the activity of V.NgoAXIII and V.NgoAXIV. However, Glu24 and Asp63 are also important for the specificity of V.NgoAXIII. On the basis of our results concerning features of Vsr endonucleases expressed by N. gonorrhoeae FA1090, we postulate that at least two types of Vsr endonucleases can be distinguished.

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.

Structural characterization of an oligosaccharide made by Neisseria sicca.

Neisseria sicca 4320 expresses two carbohydrate-containing components with sodium dodecyl sulfate-polyacrylamide gel electrophoresis mobilities that resemble those of lipooligosaccharide and lipopolysaccharide. Using matrix-assisted laser desorption ionization--time of flight and electrospray ionization mass spectrometry, we characterized a disaccharide carbohydrate repeating unit expressed by this strain. Gas chromatography identified the sugars composing the unit as rhamnose and N-acetyl-D-glucosamine. Glycosidase digestion confirmed the identity of the nonreducing terminal sugar of the disaccharide and established its beta-anomeric configuration. Mass spectrometry analysis and lectin binding were used to verify the linkages within the disaccharide repeat. The results revealed that the disaccharide repeat is [-4) beta-L-rhamnose (1-3) beta-N-acetyl-D-glucosamine (1-] with an N-acetyl-D-glucosamine nonreducing terminus. This work is the first structural characterization of a molecule that possesses rhamnose in the genus Neisseria.

Use of nfsB, encoding nitroreductase, as a reporter gene to determine the mutational spectrum of spontaneous mutations in Neisseria gonorrhoeae.

BACKGROUND: Organisms that are sensitive to nitrofurantoin express a nitroreductase. Since bacterial resistance to this compound results primarily from mutations in the gene encoding nitroreductase, the resulting loss of function of nitroreductase results in a selectable phenotype; resistance to nitrofurantoin. We exploited this direct selection for mutation to study the frequency at which spontaneous mutations arise (transitions and transversions, insertions and deletions). RESULTS: A nitroreductase- encoding gene was identified in the N. gonorrhoeae FA1090 genome by using a bioinformatic search with the deduced amino acid sequence derived from the Escherichia coli nitroreductase gene, nfsB. Cell extracts from N. gonorrhoeae were shown to possess nitroreductase activity, and activity was shown to be the result of NfsB. Spontaneous nitrofurantoin-resistant mutants arose at a frequency of approximatelty 3 x 10(-6) - 8 x 10(-8) among the various strains tested. The nfsB sequence was amplified from various nitrofurantoin-resistant mutants, and the nature of the mutations determined. Transition, transversion, insertion and deletion mutations were all readily detectable with this reporter gene. CONCLUSION: We found that nfsB is a useful reporter gene for measuring spontaneous mutation frequencies. Furthermore, we found that mutations were more likely to arise in homopolymeric runs rather than as base substitutions.

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.

Quantification of bacterial internalization by host cells using a beta-lactamase reporter strain: Neisseria gonorrhoeae invasion into cervical epithelial cells requires bacterial viability.

Neisseria gonorrhoeae can invade into cervical epithelial cells to overcome this host defense barrier. We developed a beta-lactamase reporter system that allowed us to quantify at the single cell level if a host cell internalized a viable or nonviable microorganism. We autodisplayed beta-lactamase on the surface of FA1090 [FA1090Phi(bla-iga')] and demonstrated by confocal fluorescence microscopy and flow cytometry that FA1090Phi(bla-iga') cleaved the beta-lactamase substrate CCF2-AM loaded into host cells only when gonococci were internalized by these host cells. While FA1090Phi(bla-iga') adhered to almost all ME180 cells, viable N. gonorrhoeae were internalized by only a subset of cells during infection. Nonviable gonococci adhered to, but were not internalized by ME180 cells, and failed to recruit F-actin to sites of adherent bacteria. Overall, we show that epithelial cell invasion is a dynamic process that requires viable N. gonorrhoeae. We demonstrate the advantages of the beta-lactamase reporter system over the gentamicin protection assay in quantifying bacterial invasion. The reporter system that we have developed can be adapted to studying the internalization of any bacterial species into any host cell.