Invasion of human fallopian tube epithelium by Escherichia coli expressing combinations of a gonococcal porin, opacity-associated protein, and chimeric lipo-oligosaccharide.

The transepithelial migration of Escherichia coli that expressed all possible combinations of a plasmid-encoded gonococcal porin (Por), opacity-associated protein (Opa), and 3F11 lipo-oligosaccharide (LOS) epitope was investigated. Surface expression of Por mediated selective changes in E. coli antibiotic susceptibility, and coexpression of Opa and the 3F11 LOS epitope mediated bacterial clumping (P<.01). In the human fallopian tube organ-culture model, Opa-producing variants attached up to 44-fold better than control bacteria (P<.01), and Por-producing variants exceeded submucosal invasion of control bacteria by 500-fold (P<.01). Opa and Por each facilitated intracellular invasion 20-40-fold (P<.01). In dual expresser variants, the 3F11 LOS epitope markedly reduced attachment and invasion mediated by Opa or Por. The LOS inhibitory effect was curbed when all 3 factors were expressed, which suggests an additional interaction of the 3 factors at the bacterial surface. Por, Opa, and LOS play important roles in Neisseria gonorrhoeae trafficking across human fallopian tube epithelium.

Differential intracellular efficacies of ciprofloxacin and cefixime against Neisseria gonorrhoeae in human fallopian tube organ culture.

This study compared the abilities of ciprofloxacin and cefixime to kill intracellular Neisseria gonorrhoeae in a human fallopian tube organ culture assay. When invasion was inhibited by cytochalasin D, 0.996% of the tissue-associated gonococci survived ciprofloxacin exposure compared to 1.70% of gonococci exposed to cefixime (95% confidence interval for the ratio of the means, 0.267 to 1.30), indicating that the two antibiotics did not significantly differ in the ability to kill extracellular attached organisms. In the absence of cytochalasin D, 1.63% survived ciprofloxacin exposure while 9.76% survived cefixime treatment (95% confidence interval for the ratio of the means, 0.067 to 0.418). These results suggest that ciprofloxacin penetrated epithelial cells and killed intracellular gonococci better than did cefixime. Thus, at concentrations achievable in serum, ciprofloxacin was more effective in total gonococcal killing than cefixime in this human fallopian tube organ culture model.

Digital confocal microscopy allows measurement and three-dimensional multiple spectral reconstruction of Neisseria gonorrhoeae/epithelial cell interactions in the human fallopian tube organ culture model.

A strategy for measuring Neisseria gonorrhoeae attachment and invasion in the human Fallopian tube organ culture (FTOC) model via computerized image analysis (CIA) combined with "digital" confocal microscopy (DCM) was tested. DCM on serial image stacks of fluorescent latex beads reduced out-of-focus light propagation in the Z-axis (p < 0.005) and improved the shape factor of lateral three-dimensional reconstructions of the beads (p < 0.001). Sections of tissue infected for 44 hr with piliated, Opa+ gonococci were stained with fluorescein-labeled monoclonal anti-gonococcal antibodies, rhodamine-labeled phalloidin, and Hoechst 33342. Serial images collected at identical focal planes for each fluorochrome were subjected to DCM. Epithelial cytoplasmic regions of interest defined by rhodamine-stained actin were superimposed on the corresponding fluorescein-stained and Hoechst-stained images. Fluorescent objects defined by gray-scale threshold were measured by computerized image analysis using different border treatments to differentiate attached from intracellular gonococci or count cell nuclei. Compared with raw images, measurement of DCM images was less dependent on threshold choice (p < 0.05). DCM augments conventional microscopy in removing out-of-focus light from fluorescent images, in reconstructing three-dimensional images, and in quantitatively differentiating extracellular from intracellular gonococci in a natural target tissue.

Effect of attachment factors (pili plus Opa) on Neisseria gonorrhoeae invasion of human fallopian tube tissue in vitro: quantitation by computerized image analysis.

Pili (P) and opacity-associated proteins (Opa) facilitate Neisseria gonorrhoeae attachment to human fallopian tube epithelium. Subsequent effects on invasion are unproven. Computerized image analysis was used to study the effects of attachment factors on invasion by comparing a P+Opa+ variant to a P-Opa- variant of strain R10 in the fallopian tube organ culture model. Gonococci in sections of infected fallopian tube tissue were identified with FITC-labelled monoclonal anti-gonococcal antibodies. Nomarski DIC microscopy was used to establish anatomic boundaries that excluded extracellular gonococci from invasion measurements. The area of intracellular fluorescence served as an index of gonococcal invasion. With conservative criteria to exclude extracellular gonococci, the per cent of the intracellular area occupied by fluorescent P+Opa+ gonococci was 18% compared to 4.7% for the P-Opa- variant (P < 0.001). Data suggest that P+Opa+ organisms invaded deeper than P-Opa- microbes over the same time period (P = 0.029). Intra-observer variation in invasion measurements was not significant (P > or = 0.85), and inter-observer correlation was high (correlation coefficient = 0.96). Computerized image analysis is a rapid, reliable means of quantifying gonococcal invasion of fallopian tube epithelium. We conclude that gonococcal attachment factors can facilitate events which enhance gonococcal invasion of fallopian tube epithelium.

Local induction of tumor necrosis factor as a molecular mechanism of mucosal damage by gonococci.

Tumor necrosis factor (TNF) is an endogenously produced cytokine that plays a critical role in mediating septic shock and multi-organ failure, but previous studies of the role TNF in disease have not examined its role in mucosal disease processes. In an experimental model of acute gonococcal salpingitis, gonococcal infection of human fallopian tube mucosa resulted in increased mucosal production of TNF. Recombinant human TNF-alpha damaged fallopian tube mucosa in a dose-response manner and produced epithelial damage with the same ultrastructural features as those observed in gonococcal infection. Blocking production of TNF during gonococcal infection diminished the extent of damage to fallopian tube mucosa. In addition to mediating systemic disease, such as septic shock, TNF is also produced locally, and can play a critical role in mediating mucosal disease processes, such as acute gonococcal salpingitis.

Effect of human chorionic gonadotropin (hCG) on Neisseria gonorrhoeae invasion of and IgA secretion by human fallopian tube mucosa.

The possible effect of human chorionic gonadotropin (hCG) on the mucosal immune response and susceptibility of the fallopian tube mucosa to invasion by Neisseria gonorrhoeae (gonococci) was investigated in the fallopian tube organ culture (FTOC) model. Immunohistochemical and radioreceptor assay techniques showed specific high affinity binding of hCG in vitro to the apices of non-ciliated fallopian tube cells (Kd approximately 10(-9) M). Continuous exposure of the FTOC mucosa to hCG during infection with gonococci resulted in a marked increase (6- to 15-fold) in IgA secretion and significantly reduced gonococcal invasion (invasion score range 0.7 to 1.75) compared to infected control tissue which was not exposed to hCG (invasion score range 2.9 to 4.95, P less than or equal to 0.01). By contrast, exposure of the mucosa to hCG during the 24 h preceding gonococcal infection followed by the removal of hCG from the system at the time of infection resulted in enhanced gonococcal invasion (invasion score range 7.95 to 9.7, P less than 0.001). We conclude that hCG can modulate the mucosal immune response and susceptibility of fallopian tube epithelium to gonococcal invasion.

Antimicrobial interference with bacterial mechanisms of pathogenicity: effect of sub-MIC azithromycin on gonococcal piliation and attachment to human epithelial cells.

The effects of subinhibitory concentrations of azithromycin (CP-62,993) on the piliation and attachment properties of Neisseria gonorrhoeae were examined. Subinhibitory concentrations of azithromycin significantly reduced the percentage of gonococci that expressed assembled pili on their surfaces by decreasing pilin subunit synthesis and substantially decreased gonococcal adherence to human mucosal cells.

The use of neutrophils, macrophages and organ cultures to assess the penetration of human cells by antimicrobials.

Recent studies of pathogenic mechanisms of bacteria have revealed that in addition to traditional intracellular parasites such as Brucella spp. and Listeria, many other human pathogens reside inside cells during disease processes. Thus, studies of how well antimicrobials are delivered to and perform within phagocytic or endocytic vacuoles have become of increasing importance. Whereas studies of penetration of PMN neutrophils and macrophages have indicated whether antimicrobials penetrate the cytoplasm of human cells, studies in organ cultures can reveal whether antimicrobials enter phagosomes or endocytic vacuoles where the bacteria actually reside. Such information is probably more predictive of antimicrobial efficacy in naturally occurring intracellular infections than are data from studies with PMN's or macrophages.

Parasite-directed endocytosis.

Following the attachment of gonococci to human fallopian tube mucosa in organ culture, the gonococci are endocytosed by specialized low columnar epithelial cells, are transported to the base of the epithelial cells, and are subsequently exocytosed into the subepithelial tissues. This transepithelial transport process by which "invasion" of the host occurs appears to be dependent on microbial factors is designated parasite-directed endocytosis to distinguish it from host-directed endocytosis by cells such as macrophages that eventually degrade the parasites. "Invasion" of the host by a number of human pathogens--bacteria (e.g., Neisseria meningitidis, Haemophilus influenzae, and Listeria monocytogenes), viruses, or protozoa--appears to be accomplished by parasite-directed endocytosis.

Microbial invasion: a covert activity?

In contrast to nonpathogenic microorganisms that exist happily in biofilms on various organic and inorganic surfaces, many pathogenic microbes have the additional ability to invade host tissues by inducing their own endocytosis and transport across normally protective barriers. This phenomenon, designated "parasite-directed endocytosis," has been observed with a variety of surfaces (intestinal, genital, nasopharyngeal, and tracheal epithelium) as well as in endothelial cells. The mechanisms involved in invasion may involve a single factor as described for some species of Yersinia, or may require multiple factors as observed in Shigellae. For the majority of pathogens, the molecular mechanisms of invasion are not well understood (e.g., Neisseria gonorrhoeae). Because parasite-directed endocytosis is reminiscent of receptor-mediated endocytosis, it is quite possible that some pathogens engage in biologic mimicry by producing a molecule that resembles a natural host ligand, for which there is a host cell receptor. Such a masquerade may allow some microbes to enter the host's inner sanctum covertly in a manner analogous to the Trojan horse, rather than overtly by destroying the mucosa and entering host tissues directly. Whereas this hypothesis is speculative at present, bacteria that produce molecules resembling insulin, calmodulin, and chorionic gonadotropin have been described.

Erysipelothrix rhusiopathiae endocarditis: microbiologic, epidemiologic, and clinical features of an occupational disease.

Ninety percent of the 49 reported cases of serious Erysipelothrix rhusiopathiae infection have been episodes of presumed or proved endocarditis. E. rhusiopathiae endocarditis correlates highly with occupation (farming, animal exposure), affects more males than females, exhibits a peculiar aortic valve tropism, displays a characteristic erysipeloid cutaneous lesion (in 40% of cases), and is associated with significant mortality (overall rate, 38%). Comparison with other unusual gram-positive rods causing endocarditis shows that E. rhusiopathiae resembles Listeria monocytogenes and Lactobacillus species in its propensity to involve structurally damaged but native left-sided valves. Unlike diphtheroid endocarditis, E. rhusiopathiae endocarditis has not involved prosthetic valves and is not associated with intravenous drug abuse, as is Bacillus species endocarditis. E. rhusiopathiae is exquisitely susceptible to penicillin but resistant to vancomycin. Since vancomycin is often employed in empiric therapy for presumed endocarditis, prompt microbiologic differentiation of E. rhusiopathiae from other gram-positive organisms is necessary to avoid delays in the initiation of appropriate antibiotic therapy.