Phase Transition of Gonococci in Mammalian Cell Cultures

Neisseria gonorrhoeae was cultivated in mammalian cell cultures in an effort to determine if this environment will elicit a T4 --> T1 transition. Of four avirulent (T4) isolates tested, only one, H4, yielded T1 colonies. This change was consistently obtained in HeLa, WI-38, and MK2 cells, even when the multiplicity of the gonococcal infection was less than 1 per culture. Growth of the gonococci took place primarily on the surface of the cells, as demonstrated by light and electron microscopy, but occasional bacteria were undoubtedly intracellular. T1 colonies were seen at 24 h and were the major population at 48 h. This shift was favored by the presence of viable cells, since smaller yields of T1 were obtained when the cells were irradiated or heat inactivated. It was also favored by low pH, since T1 recovery was reduced when the buffering capacity of the medium was increased. Although the results suggest that T1 gonococci derived from H4 have a selective advantage over T4 in cell cultures, this is not true of all T1 and T4 colony types. F62 T4, which does not undergo a T4 --> T1 shift, propagated as well as T1 in HeLa cell cultures. The change in colony type of strain H4 to T1 was accompanied by formation of pili and by gain in capacity for deoxyribonucleic acid-mediated transformation. It is concluded that gonococci can undergo T4 --> T1 phase transition in mammalian cell cultures, but this property is not retained by all strains.     

Saturable attachment sites for type 3 mammalian reovirus on murine L cells and human HeLa cells

Attachment of [³⁵S]methionine-labelled mammalian type 3 reovirus to murine L cells and human HeLa cells was studied under equilibrium conditions. Cellular attachment sites could be completely saturated with ³⁵S-labelled reovirus, indicating that specific attachment sites for reovirus are present on the surface of these cells. We calculated that L cells possess about 86000–105000 attachment sites per cell while HeLa cells possess about 126000–147000 sites per cell for type 3 reovirus. Unlabelled reovirus was highly efficient in competing for attachment by ³⁵S-labelled reovirus to the saturable attachment sites of both L and HeLa cells, further indicating the specificity of the interaction. We also found that unlabelled reovirus competed equally well for both binding and internalization of ³⁵S-labelled reovirus into murine L cells, suggesting that the L cell attachment site may serve as a virus entry site. Phospholipase digestion of L cells had no effect on subsequent reovirus attachment, while treatment of L cells with moderate concentrations of bromelain (but not trypsin, proteinase K or pronase) and Vibrio cholerae neuraminidase reproducibly decreased subsequent reovirus attachment. These results and those of others (Epstein et al., 1984, Virology 133, 46–55) suggest that mammalian reoviruses attach to specific cell surface receptors on at least two species of mammalian cells to initiate the infectious cycle.     

Studies on Gonococcus Infection V. Observations on In Vitro Interactions of Gonococci and Human Neutrophils

The association of in vitro human leukocytes with pilated, type 2 Neisseria gonorrhoeae exceeds that for nonpilated, type 4 organisms but is less than that for nonpilated, type 4(*) gonococci. The two nonpilated forms of gonococci (types 4 and 4(*)) attach to tissue culture cells to a much lesser extent than do pilated, type 2 organisms of the same strain. Trypsin treatment of either pilated (type 2) or nonpilated (type 4(*)) gonococci markedly reduces the attachment-ingestion of these organisms with leukocytes, but the same trypsin treatment does not depilate the type 2 organisms nor visibly alter the morphology of their pili. Similar reductions in association with leukocytes are found if the gonococci are pretreated with chymotrypsin, heat, or glutaraldehyde. High levels of association between gonococci and leukocytes are reestablished if the trypsin or chymotrypsin-treated organisms are reincubated in protease-free medium. These data suggest that interactions between gonococci and human neutrophils are mediated through surface characteristics of the bacteria, different from those which influence attachment of the organisms to tissue culture cells. In the latter instance, pili appear to positively influence gonococcal attachment, whereas in the former a nonpilus bacterial cell wall nonpilus protein is probably the major determiner in the interaction between leukocytes and gonococci.     

Ultrastructural studies on the interaction between Salmonella typhimurium 395 M and HeLa cells.

The interaction of Salmonella typhimurium 395 MS and its rough Rd-mutant 395 MR10 with HeLa cells was studied by transmission and scanning electron microscopy. The bacteria attached to central as well as more marginal positions of the HeLa cell surface. Bacteria associated preferentially to HeLa cells with a relatively low number of microvilli, in which they often were entangled. Bacteria attached to the cell border were sometimes surrounded by membrane folds, possibly as a response to their attachment. Infected cells had longer and more slender microvilli compared with noninfected cells. Some parts of the attached bacteria were in close contact with the HeLa cell membrane, whereas other parts were separated from the latter by a gap. Bacteria adhered preferentially to microvilli without obvious membrane damage. Most of the intracellular bacteria were surrounded by a membrane, often appearing as a vacuole, which sometimes contained more than one bacterium. Intracellular bacteria seemed to be morphologically intact. We propose that S. typhimurium enter HeLa cells by a process of phagocytosis.     

The effect of tendon surface treatment on cell attachment for potential enhancement of tendon graft healing: An ex vivo model

For both tendon allografts and autografts, the surface, initially optimized for gliding, may not be ideal to facilitate tissue integration for graft healing to host tendon or bone. As a prelude to studying tendon–bone integration, we investigated the effect of surface treatments with trypsin or mechanical abrasion on cell attachment to the tendon surface in a canine ex vivo intrasynovial tendon tissue culture model. Intrasynovial tendon allograft surfaces were seeded with cells after the following treatments: (1) no treatment, (2) mechanical abrasion, (3) trypsin, and (4) abrasion and trypsin. The area covered by cells was determined using confocal laser microscopy at one and two weeks. Results were compared to untreated extrasynovial tendon. Additional tendons were characterized with scanning electron microscopy. Tendons with trypsin treatment had significantly more surface coverage with cells than the other groups, after both one and two weeks of culture. In terms of the cellular shape and size, cells on tendons with trypsin treatment spread more and were more polygonal in shape, whereas tendons with mechanical abrasion with/without trypsin treatment contained smaller, more spindle-like cells. Surface roughening can affect cell behavior with topographical stimulation. Trypsin surface digestion exposes a mesh-like structure on the tendon surface, which could enhance cell adherence and, possibly, tendon/bone healing.     

Adherence of staphylococcus aureus to influenza A virus-infected Madin-Darby canine kidney cell cultures.

Madin-Darby canine kidney cells infected with influenza A virus (strains PR8, FM1, Jap 305, and Tex 1) were tested with nine strains of Staphylococcus aureus and group B Streptococcus type Ic to determine whether mammalian cells become susceptible to bacterial adherence as a result of virus infection. Bacterial adherence to virus-infected cells varied depending on the virus strain and on the strain of bacteria tested. A quantitative radioassay was developed to study the parameters of adherence. Attachment of 3H-labeled S. aureus grown in chemically defined or biologically complex medium to FM1 virus-infected cells was significantly increased (P less than 0.0005) compared with attachment to control cells. Adherence coincided with the appearance of hemadsorption, which is a marker of the presence of virus-induced glycoproteins on the cell surface. Adherence was temperature dependent, increased with a decrease in hydrogen ion concentration, and was not affected by the presence of K+, Mg2+, or Ca2+. Adherence was blocked when 3H-labeled S. aureus was pretreated with trypsin but not when cells were pretreated with neuraminidase.     

Specific nature of Trichomonas vaginalis parasitism of host cell surfaces.

The adherence of Trichomonas vaginalis NYH 286 to host cells was evaluated by using monolayer cultures of HeLa and HEp-2 epithelial cells and human fibroblast cell lines. Saturation of sites on HeLa cells was achieved, yielding a maximal T. vaginalis NYH 286-to-cell ratio of two. The ability of radiolabeled NYH 286 to compete with unlabeled trichomonads for attachment and the time, temperature, and pH-dependent nature of host cell parasitism reinforced the idea of specific parasite-cell associations. Other trichomonal isolates (JH31A, RU375, and JHHR) were also found to adhere to cell monolayers, albeit to different degrees, and all isolates produced maximal contact-dependent HeLa cell cytotoxicity. The avirulent trichomonad, Trichomonas tenax, did not adhere to cell monolayers and did not cause host cell damage. Interestingly, parasite cytadherence was greater with HeLa and HEp-2 epithelial cells than with fibroblast cells. In addition, cytotoxicity with fibroblast cells never exceeded 20% of the level of cell killing observed for epithelial cells. Elucidation of properties of the pathogenic human trichomonads that allowed for host cell surface parasitism was also attempted. Treatment of motile T. vaginalis NYH 286 with trypsin diminished cell parasitism. Incubation of trypsinized organisms in growth medium allowed for regeneration of trichomonal adherence, and cycloheximide inhibited the regeneration of attachment. Organisms poisoned with metronidazole or iodoacetate failed to attach to host cells, and adherent trichomonads exposed to metronidazole or iodoacetate were readily released from parasitized cells. Coincubation experiments with polycationic proteins and sugars and pretreatment of parasites or cells with neuraminidase or periodate had no effect on host cell parasitism. Colchicine and cytochalasin B, however, did produce some inhibition of adherence to HeLa cells. The data suggest that metabolizing T. vaginalis adheres to host cells via parasite surface proteins in a specific receptor-ligand fashion. Furthermore, parasitism of epithelial cells appears to render this cell type more susceptible than fibroblast cell types to contact-dependent cytotoxicity.     

Surface properties of Yersinia species and epithelial cell interactions in vitro by a method measuring total associated, attached and intracellular bacteria

A procedure was developed for enumeration of total associated, attached and intracellular bacteria after interaction of Yersinia spp. with epithelial cells in vitro. Isogenic cultures of Y. enterocolitica grown at 25 degrees C had greater affinity for epithelial cells (Henle, HeLa and Vero) than for polystyrene, and they invaded the cells. Y. kristenseni and Y. intermedia showed less attachment to either surface and were non-invasive. The degree of attachment to cells and invasion by Y. enterocolitica was related to number of bacteria added and interaction time, whereas attachment to polystyrene occurred rapidly and did not change. Y. enterocolitica was more hydrophobic when grown at 35 degrees C than at 25 degrees C according to partitioning in a biphasic dextran-polyethylene glycol system, and attached strongly to both polystyrene and epithelial-cell monolayers. Y. kristenseni grown at 25 degrees C was also hydrophobic but did not have the same attachment properties. Y. kristenseni and Y. intermedia showed slightly reduced electrostatic interactions with the anion exchangers DEAE-Sepharose and DEAE-Trisacryl. Attachment of Y. enterocolitica to epithelial cells probably involves non-specific surface properties that are not entirely explicable by hydrophobic and electrostatic interactions, whereas invasion of epithelial cells appears to resemble "receptor-mediated endocytosis".     

Attachment of Moraxella catarrhalis occurs to the positively charged domains of pharyngeal epithelial cells

Attachment of bacteria to host cells is the initial step in the pathogenesis of infection. Several factors, such as hydrophobicity, surface electric charge, and van der Waals force, are considered to be responsible for the attachment step. However, it is not clear why bacteria and epithelial cells, both of which possess a negative surface charge, do not repel one another. In the present study, we used Moraxella catarrhalis and pharyngeal epithelial cells to study the surface charges of structures involved in the attachment. By atomic force microscopy (AFM) equipped with surface potential spectroscopy, it was found that the cell surface microplicae have a positive charge of 30.1+/-3.6 mV (mean+/-SE). The depressions between the microplicae have a negative surface charge of 43.5+/-4.0 mV. Using cationic ferritin and electron microscopy (EM) we confirmed that the depressions between the microplicae have a negative charge. By AFM and by using cationic ferritin with EM, it was found that the net surface charge of the bacterial cells is negative. By both AFM and EM, it was found that the bacterial cells attach to the microplicae of the pharyngeal epithelial cell. Our work confirmed the general belief that both kinds of cells do have a net negative charge. We conclude that there are positively and negatively charged domains on the surface of human pharyngeal epithelial cells. M. catarrhalis evidently attaches to the positively charged domain (i.e. microplicae) of pharyngeal epithelial cells.     

Cell behaviour on polymer surfaces grafted with non-ionic and ionic monomers.

Following exposure to corona discharge, a polyethylene film was graft polymerized with different water-soluble monomers such as acrylamide (non-ionic), acrylic acid (anionic), 2-acrylamide-2-methyl propane sulphonic acid (anionic), styrene sulphonic acid sodium salt (anionic) and N,N-dimethylaminopropyl acrylamide (cationic). Attachment and proliferation of HeLa S3 cells were studied for grafted surfaces with different zeta potentials and contact angles. The polyethylene surface graft polymerized with styrene sulphonic acid sodium salt exhibited high cell attachment and protein adsorption, whereas the cells did not adhere to the 2-acrylamide-2-methyl propane sulphonic acid graft-polymerized surface, although both surfaces had high negative zeta potentials. Graft polymerization of acrylamide reduced the zeta potential of surface close to zero and rejected the cell attachment. The polyethylene surface became highly cell-adhesive through graft polymerization of the cationic N,N-dimethylaminopropyl acrylamide monomer, but too much grafting killed the attaching cells. Once the cells attached to a surface without being killed, they could proliferate at the same growth rate, whatever their surface zeta potential.     

Differential effect of trypsin on infectivity of Chlamydia trachomatis: loss of infectivity requires cleavage of major outer membrane protein variable domains II and IV.

The initial interaction of chlamydiae with host cells is not well understood. Chlamydial cell surface components that function in attachment are key virulence factors, and their identification is critical for understanding the pathogenic strategies of this very successful parasite. We used trypsin proteolysis of chlamydiae to define surface components that function in chlamydia-host cell interactions. We found that trypsin had a differential effect on the infectivity of Chlamydia trachomatis serovars B and L2 for HeLa 229 cells. Trypsin treatment resulted in a significant loss of attachment and infectivity of serovar B but had no effect on the infectivity of serovar L2. Fluorograms of chlamydiae metabolically labeled with 14C-amino acids and treated with trypsin showed that the major outer membrane protein (MOMP) of both serovars was cleaved. Evidence for two trypsin cleavage sites was found for the serovar B MOMP. One cleavage site was located between lysine 145 and valine 146 in variable domain (VD) II of the protein. The second site was located between lysine 309 and threonine 310 in VD IV. In contrast, the serovar L2 MOMP was cleaved only at lysine 309 in VD IV. These results suggest a functional role for MOMP in chlamydial attachment and implicate VDs II and IV of MOMP in this interaction.     

The use of mild trypsinization conditions in the detachment of endothelial cells to promote subsequent endothelialization on synthetic surfaces

A necessary condition for endothelialization of small diameter grafts is rapid and firm adhesion of endothelial cells upon exposure to flow. To retain integrins on the cell surface, we assessed the effects of trypsin concentration, the duration of trypsin incubation, and trypsin neutralization methods on endothelial cell adhesion. Human umbilical vein endothelial cells which were detached using 0.025% trypsin for 5 min and seeded onto glass pretreated with fibronectin had close to 100% cell retention when shear stresses as high as 200 dyn/cm2 were applied for 2 min. An equivalent level of cell retention was observed on fibronectin coated Teflon-AF for shear stresses up to 60 dyn/cm2 applied for 4h. Using 0.025% trypsin, initial cell spreading and cell surface alpha5beta1 integrins were increased relative to cells treated with 0.5% trypsin. After 1h of attachment, focal adhesions formed when low trypsin concentrations were used but were less evident with high trypsin concentrations. These results showed that low trypsin concentrations produced faster spreading, a higher number of intact integrins, and rapid focal adhesion formation.     

Wg3h细胞系转染PSV_2-neo质粒前后生长特性及表面超微结构改变的研究

本文对转染PSV_2-neo质粒后的Wg3h细胞系(Wg3h-neo)在长期传代中的生长特性和表面超微结构与母系Wg3h细胞进行了比较研究。结果发现:转染后Wg3h细胞的DNA合成及生长速度明显高于母系Wg3h细胞,生长饱和密度增大。在软琼脂培养中不形成集落,接种在裸鼠不长肿瘤。在扫描电镜下,细胞表面的微绒毛较母系Wg3h细胞丰富。Southern印迹杂交实验证明PSV_2-neo质粒已整合到宿主细胞的基因组中。转染后Wg3h细胞的生长特性和表面超微结构均发生了某些转化特征。     

A Sphingomonas bacterium interacting with epithelial cells

Bacteria of the genus Sphingomonas are environmental organisms that have recently been implicated in a variety of community-acquired and nosocomial infections. During studies on bacteria-cell interactions, we incurred a microorganism contaminating our HeLa cell culture, possibly from water utilized for reagent preparation; this bacterium appeared to tightly adhere to cell monolayers and to survive, with only limited growth rate, which did not seem to alter cells as far as shape, growth rate or survival were concerned. The contaminating organism was isolated and partially characterized by morphological, genetic, and biochemical assays. Mechanisms of cell interaction and entry into epithelial cells were investigated by electron microscopy, immunofluorescence, and biochemical inhibitors. Morphological and biochemical features indicated that the microorganism belonged to the genus Sphingomonas. Electron microscopy showed that contact between the Sphingomonas bacterium and epithelial cells leads to a dramatic alteration of the cell surface, with formation of numerous microvillar extensions plus membrane ruffling. Confocal microscopy and the use of inhibitors showed that actin microfilaments were involved during attachment and entry into HeLa cells. Macropinosome formation and an inhibitory effect by amiloride indicate that internalization occurs in part via a macropinocytosis mechanism. Moreover, cholesterol distribution at the site of bacterial binding suggests that Sphingomonas bacteria could use the lipid rafts as initial binding sites.     

Modulation of Candida albicans attachment to human epithelial cells by bacteria and carbohydrates.

The effects of carbohydrates (mannose and dextrose). Escherichia coli 07KL. and Klebsiella pneumoniae on Candida albicans attachment to epithelial cells was studied. Dextrose had no effect on yeast attachment to epithelial cells. Conversely, mannose significantly decreased both yeast and piliated bacterial attachment (E. coli 07KL, heavily piliated K. pneumoniae) whereas having no effect on nonpiliated K. pneumoniae attachment to epithelial cells. The number of yeasts attaching to epithelial cells was enhanced by preincubation of epithelial cells with piliated strains of bacteria, whereas preincubation with nonpiliated strains of bacteria had no effect on yeast attachment. Scanning electron microscopy showed that piliated bacteria and yeasts were juxtaposed on the epithelial cell surface. These data suggest that certain piliated strains of bacteria can enhance C. albicans attachment to epithelial cells and that type 1 pili of bacteria can be a factor in the enhanced attachment of C. albicans to epithelial cells.     

Role of Pili in the Virulence of Neisseria gonorrhoeae

Gonococci of the colonial types that are associated with virulence, types 1 and 2, have pili that enable the bacteria both to attach in vitro to human epithelial cells and to resist phagocytosis by polymorphonuclear leukocytes. These piliated gonococci also agglutinate various mammalian and chicken erythrocytes. Gonococci of an avirulent colonial type, i.e., type 4, have no pili and neither attach to epithelial cells or erythrocytes nor resist phagocytosis. Like the type 4 bacteria, mechanically or enzymatically (trypsin) depiliated type 1 gonococci failed to attach to epithelial cells and erythrocytes and were susceptible to phagocytosis. Pili of types 1 and 2 gonococci were antigenically similar. Both type 1 gonococci and pili isolated from them induced in rabbits antibody that (i) precipitated gonococcal pili in immunodiffusion, (ii) reacted with piliated gonococci as tested by indirect immunofluorescent analysis, (iii) inhibited attachment of piliated gonococci to both human epithelial cells and erythrocytes, and (iv) opsonized piliated gonococci.     

Attachment of Cryptosporidium parvum sporozoites to MDCK cells in vitro.

The initial attachment of Cryptosporidium parvum sporozoites to host cells in vivo may be a critical event in the pathogenesis of this infection. The molecular basis of attachment and the conditions influencing this host-parasite interaction have not been studied systematically. Therefore, we have developed a sporozoite attachment model by using paraformaldehyde-fixed Madin-Darby canine kidney (MDCK) cells. Attachment of sporozoites to fixed MDCK cells was quantitated by indirect immunofluorescence and confirmed by transmission electron microscopy. Attachment in this system was time, temperature (37 degrees C), and pH (7.2 to 7.6) dependent. Dose-response studies demonstrated that the attachment of sporozoites to fixed MDCK cells was a saturable process. Attachment was enhanced in the presence of 10 mM manganese, 1 mM calcium, and 1 to 10 mM zinc. Attachment of sporozoites to MDCK cells was inhibited in a dose-dependent manner by polyclonal anti-Cryptosporidium antisera and by purified immunoglobulin G (IgG). This model will be useful for the study of parasite and host cell molecules involved in the initial interaction of C. parvum sporozoites with their target cell.     

Evidence of Haemophilus ducreyi adherence to and cytotoxin destruction of human epithelial cells

The adherence of ten different Haemophilus ducreyi strains to cultured human epithelial cells and the subsequent destruction of these cells was investigated in vitro using H Ep-2 and HeLa cells. Bacterial adherence was measured with two assays, one employing viable bacteria and the other radiolabeled bacteria. In addition, the capacity of H. ducreyi to invade/penetrate the H Ep-2 cells was examined. Differential interference contrast and transmission electron microscopy techniques were also used. In both cell lines, all ten strains of H. ducreyi manifested substantial adherence (the rates being 4-20% of the inoculum), irrespective of whether the bacteria were cultivated on solid or liquid media. Bacterial adherence reached a peak after about 2-3 h of incubation, though it was already manifest after only 15 min, a finding suggesting constitutive rather than inducible properties of H. ducreyi adhesins to be involved. The adherence capacity was diminished, but not totally abolished, when bacteria were heat-treated at 100°C for 30 min, indicating the adhesins to be fairly stable. On the other hand, treatment of H Ep-2 cells with methanol, glutaraldehyde and emetine dichloride significantly reduced the adherence, indicating viable eukaryotic cells with native surface structures to be involved in bacterial adherence. This capacity of H. ducreyi to adhere to H Ep-2 cells was confirmed both by electron microscopy and by differential interference microscopy. Some adherent bacteria were also capable of penetrating epithelial cells, as observed with an invasion assay and confirmed by transmission electron microscopy. Further incubation of the cell monolayers with the ten strains resulted in the cell-death and total damage of monolayers for seven cytotoxin-producing strains, indicating cytotoxin action to be responsible for the destruction of the monolayer. All strains manifested capacity to survive and multiply on the cell monolayer. We propose the first step in the pathogenesis of chancroid to be the adherence of bacteria to epithelial cells, followed by the action of cytotoxin and further bacterial proliferation. This sequence of events is suggested to result in the production of genital ulcers by H. ducreyi organisms.     

The 120 kDa outer membrane protein of Ehrlichia chaffeensis: preferential expression on dense-core cells and gene expression in Escherichia coli associated with attachment and entry

The immunodominant 120 kDa protein (p120) of Ehrlichia chaffeensis was demonstrated to be exposed on the surface of purified whole ehrlichial cells examined by immunoelectron microscopy with a rabbit antibody against a portion of the domain containing tandem repeat units. In the intracellular location, the 120 kDa protein was detected by immunoelectron microscopy in the outer membrane of the cell wall of dense-core forms of the ehrlichiae in infected canine macrophage-like cells and as a component of the intramorular fibrillary matrix. No 120 kDa protein was detected in the cell wall of ehrlichial reticulate cells. Recombinant Escherichia coli with a plasmid containing the entire 120 kDa protein gene, but no bacteria with non-recombinant plasmid, attached to the surface of HeLa cells as visualized by electron microscopy. Some of the recombinant 120 kDa protein expressing E. coli invaded the HeLa cells as determined by gentamicin protection assays and by intravacuolar localization ultrastructurally.     

Control of cell attachment on pH-responsive chitosan surface by precise adjustment of medium pH

The purpose of this study is to demonstrate pH-responsive chitosan is able to control cell behavior in response to small changes in environmental pH, which is at useful pH suitable for recovering cultured cells without additional enzymatic treatment and extensive washing steps. HeLa cells attached and spread well on chitosan at pH 6.99 and 7.20. When the pH was increased to 7.65, over 90% of cells would rapidly detached from chitosan surface within 1 h. Similarly, fibronectin adsorbed on chitosan at pH 7.20 also rapidly desorbed after increasing the medium pH. Most importantly and interestingly, medium pH adjustment could be facilitated by altering environment pCO₂. It was found over 80% of HeLa cells could be recovered from chitosan surface within 1 h and the viability of detached cells was more than 95% by transferring the culture plate from incubator to atmospheric condition. Additionally, chitosan substrate could effectively control attachment/detachment of various types of cells including cell lines HaCaT, H1299, NIH-3T3, and primary corneal fibroblasts, indicating the technology described here is easily reproducible and should be promising for controlling rapid fibronectin adsorption/desorption and cell attachment/detachment for tissue engineering applications.