Factors affecting the adhesion and uptake of an oculo-genital strain of Chlamydia trachomatis to McCoy cells

Abstract We have studied adhesion and uptake of C. trachomatis serovar E in McCoy cells under various infection conditions. Adhesion and uptake of chlamydiae was completed about 3 h after the initiation of stationary infection at 37°C, but ingestion of cell membrane-attached organisms was finished within 0.5 h at 37°C. Reincubated chlamydiae, not attached after 3 h at 37°C, attached readily to fresh McCoy cell monolayers, but to a lesser extent than the original inoculum. Our results indicate that the lack of further attachment after 3 h incubation at 37°C under stationary infection conditions has complex causes, involving both host cell and parasite. Centrifugation did not affect the uptake of chlamydiae already bound to the cell membrane, suggesting that the uptake phase of C. trachomatis serovar E by McCoy cells is unaffected by centrifugation.     

HIV-1 attachment: another look

HIV-1 attachment to host cells is generally considered to take place via high-affinity binding between CD4 and gp120. However, the binding of virion-associated gp120 to cellular CD4 is often weak, and most cell types that are permissive for HIV-1 infection express little CD4. Thus, other interactions between the virion and the cell surface could dominate the attachment process.     

Primary Virus-Cell Interactions in the Immuno-fluorescence Assay of Venezuelan Equine Encephalomyelitis Virus

The conditions under which Venezuelan equine encephalomyelitis (VEE) virus attached to host cells markedly influenced the assay of virus by the fluorescent cell-counting technique. When virus inoculum was centrifuged onto McCoy cell monolayers, approximately 97% of virus was attached to cells within 10 min, in contrast to 34% after stationary incubation at 35 C for 2 hr. Maximal binding of virus occurred only in the presence of 0.1 to 0.15 m NaCl. This salt requirement, added to evidence of (p)H dependence and temperature independence of VEE virus attachment to cells, indicated that the initial union involved electrostatic forces. Virus penetration, measured by the insensitivity of virus-cell complexes to viral antiserum, was complete in 30 min at 35 C. The process was temperature-dependent and un-affected by the ionic content of medium. For assay of VEE virus by the fluorescent cell-counting technique, infected cells may be enumerated as early as 12 hr after infection of cell monolayers. The relationship between virus concentration and cell-infecting units was linear; the distribution of fluorescent cells was random. The virus assay was equivalent in sensitivity but more precise and rapid than that of intracerebral inoculation of mice.     

Control mechanisms governing the infectivity of Chlamydia trachomatis for HeLa cells: mechanisms of endocytosis

The mechanism by which Chlamydia trachomatis is endocytosed by host cells is unclear. Studies of the kinetics of chlamydial attachment and uptake in the susceptible HeLa 229 cell line showed that chlamydial endocytosis was rapid and saturable but limited by the slow rate of chlamydial attachment. To overcome this limitation and to investigate the mechanism of endocytosis, chlamydiae were centrifuged onto the host cell surface in the cold to promote attachment. Endocytosis of the adherent chlamydiae was initiated synchronously by rapid warming to 36 degrees C. Electron micrographs of chlamydial uptake 5 min after onset showed that chlamydial ingestion involves movement of the host cell membrane, leading to interiorization in tight, endocytic vacuoles which were not clathrin coated. Chlamydial ingestion was not inhibited by monodansylcadaverine or amantadine, inhibitors of receptor-mediated endocytosis and chlamydiae failed to displace [3H]sucrose from micropinocytic vesicles. Chlamydial endocytosis was markedly inhibited by cytochalasin D, an inhibitor of host cell microfilament function, and by vincristine or vinblastine, inhibitors of host cell microtubules. Hyperimmune rabbit antibody prevented the ingestion of adherent chlamydiae, suggesting that endocytosis requires the circumferential binding of chlamydial and host cell surface ligands. These findings were incompatible with the suggestion that chlamydiae enter cells by taking advantage of the classic mechanism of receptor-mediated endocytosis into clathrin-coated vesicles, used by the host cell for the internalization of beta-lipoprotein and other macromolecules, but were consistent with the hypothesis that chlamydiae enter cells by a microfilament-dependent zipper mechanism.     

Attachment of Mycoplasma pulmonis to rat and mouse synovial cells cultured in vitro

The attachment of Mycoplasma pulmonis m53 organisms to mouse and rat synovial cells was examined by using the organisms and the synovial cells treated in various ways. M. pulmonis treated with trypsin attached to the synovial cells, but the organisms treated with pronase, formaldehyde, glutaraldehyde, or heat did not. These findings suggest that the sites for binding M. pulmonis to the mouse and rat synovial cells are of polypeptide nature. Treatment of M. pulmonis with sialic acid and treatment of the synovial cell sheets with neuraminidase did not affect the attachment. The synovial cell surface for receptors M. pulmonis organisms would be different from those on respiratory cells or erythrocytes for M. pneumoniae or M. gallisepticum. Even nonviable organisms and M. pulmonis membranes attached to the mouse or rat synovial cells. The nature of the receptor of mouse synovial cells would be different from that of rat cells, since rat cells were affected by treatment with formaldehyde or glutaraldehyde, but mouse cells were not.     

Mechanism of C. trachomatis attachment to eukaryotic host cells.

A novel trimolecular mechanism of microbial attachment to mammalian host cells was characterized for the obligate intracellular pathogen Chlamydia trachomatis. Using purified glycosaminoglycans (GAGs) and specific GAG lyases, we demonstrated that a heparan sulfate-like GAG present on the surface of chlamydia organisms is required for attachment to host cells. These observations were supported by inhibition of attachment following binding of heparan sulfate receptor analogs to chlamydiae and by demonstrating that chlamydiae synthesize a unique heparan sulfate-like GAG. Furthermore, exogenous heparan sulfate, as an adhesin analog, restored attachment and infectivity to organisms that had lost these attributes following treatment with heparan sulfate lyase. These data suggest that a GAG adhesin ligand mediates attachment by bridging mutual GAG receptors on the host cell surface and on the chlamydial outer membrane surface.     

Adhesion, penetration and intracellular replication of Legionella pneumophila: an in vitro model of pathogenesis

Legionella pneumophila attached to, penetrated and replicated within the three eukaryotic cell lines, MRC-5, HEp-2 and Vero. Multiplication occurred rapidly in these cells for an initial 48 h after inoculation and declined thereafter. Infected MRC-5 cell monolayers developed lytic-type cytopathic changes, with organisms being readily released. HEp-2 cells showed a more chronic infection, with slowly developing granular changes in the monolayers, and slow release of intracellular bacteria. In Vero cells, organisms were released rapidly along with a more progressively developing granular cytopathic effect in the monolayers. L. pneumophila was unable to grow in cell-free culture fluids. Uptake and intracellular development was similar for each cell type, and was initiated by 'bacteriopexis', a process in which the organisms bound via receptors and were surrounded by cellular microvilli which eventually fused, leading to bacterial engulfment. Replication of organisms in vacuoles within the cytoplasm of infected cells was confirmed by thorium labelling. These vacuoles were lined with ribosomes and, at the early stages of intracellular development, were found in close proximity to mitochondria, cytoplasmic filaments and banded enclosures. Ruthenium red staining showed that acid mucopolysaccharide capsular material was not present on these organisms during the attachment process or intracellular phase. Organism release was by lysis of the infected cells.     

The soluble form of human respiratory syncytial virus attachment protein differs from the membrane-bound form in its oligomeric state but is still capable of binding to cell surface proteoglycans

The soluble (Gs) and membrane-bound (Gm) forms of human respiratory syncytial virus (HRSV) attachment protein were purified by immunoaffinity chromatography from cultures of HEp-2 cells infected with vaccinia virus recombinants expressing either protein. Sucrose gradient centrifugation indicated that Gs, which is secreted into the culture medium, remains monomeric, whereas Gm is an oligomer, probably a homotetramer. Nevertheless, Gs was capable of binding to the surface of cells in vitro, as assessed by a flow cytometry-based binding assay. The attachment of Gs to cells was inhibited by previous heparinase treatment of living cells, and Gs did not bind to CHO cell mutants defective in proteoglycan biosynthesis. Thus, Gs, as previously reported for the G protein of intact virions, binds to glycosaminoglycans presented at the cell surface as proteoglycans. Deletion of a previously reported heparin binding domain from Gs protein substantially inhibited its ability to bind to cells, but the remaining level of binding was still sensitive to heparinase treatment, suggesting that other regions of the Gs molecule may contribute to attachment to proteoglycans. The significance of these results for HRSV infection is discussed.     

Separation and partial characterization of a type-specific antigen from Chlamydia trachomatis.

Type-specific antigens of Chlamydia trachomatis have been demonstrated by the mouse toxicity prevention test and a variety of immunofluorescent techniques. In addition, biologic activity has been associated with these antigens in terms of type-specific immunity to trachoma infections. This report is the first to describe the detection of a soluble type-specific antigen of C. trachomatis and its separation from those antigens that cross-react among different immunotypes. Test antigens were prepared by labeling the surface components of purified, yolk sac grown organisms with a radioiodinated intermediate (Bolton-Hunter reagent, 125I). The organisms were solubilized with Triton X-100 and gel filtered through Sepharose 6B. All fractions were then tested in radioimmunoassay for binding with rabbit antisera raised against solubilized immunogens prepared from homologous and heterologous strain organisms propagated in BHK-21 cells. A fraction demonstrating homologous binding only was used in subsequent modified procedures for the preparation of quantities of type-specific antigen sufficient for analysis. The antigen appears to be a heat labile, cell surface protein associated with apparent immunogenic activity during the course of actual chlamydial eye infection.     

Physical and metabolic requirements for early interaction of poliovirus and human rhinovirus with HeLa cells.

Attachment, "tight binding' and eclipse of radioactive poliovirus 2 (P2) and human rhinovirus 2 (HRV 2) were investigated. The activation energy for attachment of both HRV2 and P2 was about 13 kcal/mol. HRV2 differed from P2 in two respects: the Arrhenius plot for attachment of HRV2 showed a break at 15 to 19 degrees C when the cells were first treated several hours at 0 degrees C, and attachment of HRV2 was inhibited by treatment of cells with metabolic poisons able to reduce cellular ATP by more than 90%. Tight binding was determined by isolation of a specific P2-membrane complex or by loss of EDTA dissociability of HRV2. Tight binding of both viruses was slowed by 0.01 M iodoacetamide but not by 0.02 M F-; F- plus 0.002 M CN- slowed tight binding of HRV2 but not of P2. Eclipse, the irreversible alteration of parental virions, was detected by isolation of cell-associated subviral particles or by loss of cell-associated infectious virus. Eclipse of both viruses is slowed by iodoacetamide or F-. It seems likely that the early steps of infection with picornaviruses may be sensitive to alterations in the cell membrane produced by metabolic inhibitors or by treatment at low temperature.     

A model of neutralization of Chlamydia trachomatis based on antibody and host cell aggregation on the elementary body surface

Humoral immunity is that aspect of specific immunity that is mediated by B lymphocytes and involves the neutralizing of pathogens by means of antibodies attaching to the pathogen's binding sites. Antibodies bind to and block ligand sites on the pathogen which prevents these sites from attaching to target cell receptors and so cell entry is inhibited. Many studies investigate the role of humoral immunity for protection against chlamydial challenge and they have shown that neutralization of the chlamydial body requires a large number of attached antibodies. Steric hindrance greatly influences the number of available sites that may be bound, reducing relative occupancy well below 100%. We model steric effects of antibody Fab fragment attachment indicating that they must be taken into consideration to accurately model valency, the number of available binding sites. We derive a partial differential equation for the number of antibody Fabs and host cell receptors that are aggregated to extracellular chlamydial elementary bodies. We consider steric effects in describing the size distribution of aggregates. Our theory is in good agreement with Monte Carlo simulations of binding. We use our theoretical prediction for the valency in a model for the in-host population dynamics of a chlamydial infection and we fit our model to experimental data.     

Endocytosis of chlamydiae by McCoy cells: measurement and effects of centrifugation

Abstract Chlamydia trachomatis strain 434 and C. psittaci strain guinea pig inclusion conjunctivitis (GPIC) were compared for entry into McCoy cells and expression of productive infection (inclusion body formation). Entry was measured as the difference between extracellular cell-associated organisms, determined directly after fluorescence staining of live cells, and total cell-associated organisms (intracellular and extracellular); the latter were evaluated from radioactivity measurement and known particle: radioactivity ratios for stock radiolabelled suspensions. Under inoculation conditions of natural (spontaneous) infection, 69–82% of cell-associated organisms of both strains were internalised and entry was not enhanced by centrifugation of inocula with monolayers. For 434, inclusion bodies were seen in 10–20% of cells containing organisms and numbers were little influenced by mode of infection. For GPIC, productive infection initiated by centrifugation was comparable with that of 434 but some 15-fold reduced in spontaneous infection. The results suggest that unproductive infection by GPIC occurs, not because of defective entry, but from inhibition at an intracellular step which is circumvented when infection is initiated by centrifugation.     

Bacterial adhesion to hydroxyl- and methyl-terminated alkanethiol self-assembled monolayers.

The attachment of bacteria to solid surfaces is influenced by substratum chemistry, but to determine the mechanistic basis of this relationship, homogeneous, well-defined substrata are required. Self-assembled monolayers (SAMs) were constructed from alkanethiols to produce a range of substrata with different exposed functional groups, i.e., methyl and hydroxyl groups and a series of mixtures of the two. Percentages of hydroxyl groups in the SAMs and substratum wettability were measured by X-ray photoelectron spectroscopy and contact angles of water and hexadecane, respectively. SAMs exhibited various substratum compositions and wettabilities, ranging from hydrophilic, hydroxyl-terminated monolayers to hydrophobic, methyl-terminated monolayers. The kinetics of attachment of an estuarine bacterium to these surfaces in a laminar flow chamber were measured over periods of 120 min. The initial rate of net adhesion, the number of cells attached after 120 min, the percentage of attached cells that adsorbed or desorbed between successive measurements, and the residence times of attached cells were quantified by phase-contrast microscopy and digital image processing. The greatest numbers of attached cells occurred on hydrophobic surfaces, because (i) the initial rates of adhesion and the mean numbers of cells that attached after 120 min increased with the methyl content of the SAM and the contact angle of water and (ii) the percentage of cells that desorbed between successive measurements (ca. 2 min) decreased with increasing substratum hydrophobicity. With all surfaces, 60 to 80% of the cells that desorbed during the 120-min exposure period had residence times of less than 10 min, suggesting that establishment of firm adhesion occurred quickly on all of the test surfaces.     

Association and uptake studies of a Chlamydia trachomatis lymphogranuloma venereum strain by eukaryotic cells

We have studied association and uptake of Chlamydia trachomatis serovar L1 under various infection conditions. Chlamydiae attached to a greater extent to McCoy cells than to HeLa cells, although the number of inclusions formed in the latter was the same or higher. The amount of internalised chlamydiae was similar in the 2 cell types. Centrifugation of McCoy cell-attached chlamydiae did not affect the uptake of this serovar. However, if the inoculum was centrifuged to the cell surface and then incubated at 37°C, there was a pronounced increase in the relative amount of ingested chlamydiae in comparison with stationary infection. Chlamydiae were attached to and internalised insubconfluent McCoy cell monolayers as efficiently as in confluent layers. If the monolayers were sparse or very sparse, there was a great reduction of associated chlamydiae. Our results indicate that the host cell binding for chlamydiae vary with cell type, cell density, and mode of infection.     

Giardia lamblia attachment force is insensitive to surface treatments

Giardia lamblia cell populations show 90% detachment from glass under normal forces of 2.43+/-0.33 nN applied by centrifugation. Detachment forces were not significantly different for cells attached to positively charged, hydrophobic, or inert surfaces than for cells attached to plain glass. The insensitivity of attachment force to surface treatment is consistent with a suction-based mechanism of attachment.     

Host modification of chlamydiae: differential infectivity for cell monolayers of chlamydiae grown in eggs and monolayers.

Cell monolayer-grown chlamydiae (CGO) differed from egg-grown organisms (EGO) in their increased spontaneous infectivity relative to centrifuge-assisted infectivity for monolayers. For each population spontaneous: centrifuge-assisted infectivity ratios were constant over a wide dose range. Spontaneous infection increased linearly with time and could not be exhausted from either population by prolonged adsorption; there was no change in infectivity ratios in residual supernatants. Further, one passage of EGO through monolayers gave CGO with stable infectivity properties not increased by further cell passage yet reverting on a single passage in eggs. Spontaneous infection of monolayers with EGO gave progeny with the same infectivity ratios as monolayers infected with EGO by centrifugation. The change in properties following EGO infection of monolayers occurred prior to natural release from cells. We conclude that EGO and CGO are two phenotypically distinct, homogeneous populations. The two infection modes are not properties of subpopulations within EGO and CGO. The relationship of these observations on chlamydiae to other possible host-imposed phenomena is considered.     

The synergy peptide PHSRN and the adhesion peptide RGD mediate cell adhesion through a common mechanism

This work reports on the role of the synergy peptide PHSRN in mediating the adhesion of cells. The attachment of baby hamster kidney cells and 3T3 Swiss fibroblasts to model substrates presenting either GRGDS or PHSRN was evaluated using self-assembled monolayers of alkanethiolates on gold presenting the peptide ligands mixed with tri(ethylene glycol) groups. These substrates permit rigorous control over the structures and densities of peptide ligands and at the same time prevent nonspecific interactions with adherent cells. Both cell types attached efficiently to monolayers presenting either the RGD or the PHSRN peptide but not to monolayers presenting scrambled peptide GRDGS or HRPSN. Cell attachment was comparable on substrates presenting either peptide ligand but less efficient than on substrates presenting the protein fibronectin. The degree of cell spreading, however, was substantially higher on substrates presenting RGD relative to PHSRN. Staining of 3T3 fibroblasts with anti-vinculin and phalloidin revealed clear cytoskeletal filaments and focal adhesions for cells attached by way of either RGD or PHSRN. Inhibition experiments showed that the attachment of 3T3 fibroblasts to monolayers presenting RGD could be inhibited completely by a soluble RGD peptide and partially by a soluble PHSRN peptide. IMR 90 fibroblast attachment to monolayers presenting PHSRN could be inhibited with anti-integrin alpha(5) or anti-integrin beta(1) antibody. This work demonstrates unambiguously that PHSRN alone can support the attachment of cells and that the RGD and PHSRN bind competitively to the integrin receptors.     

Identification of MEK- and phosphoinositide 3-kinase-dependent signalling as essential events during Chlamydia pneumoniae invasion of HEp2 cells

The ability of Chlamydia pneumoniae to survive and cause disease is predicated on efficient invasion of cellular hosts. While it is recognized that chlamydial determinants are important for mediating attachment and uptake into non-phagocytic cells, little is known about the bacterial ligands and cellular receptors that facilitate invasion or host cell signal transduction pathways implicated in this process. We used transmission and scanning electron microscopy to demonstrate that attachment of bacteria to host cells induced the appearance of microvilli on host cell membranes. Invasion occurred 30-120 min after cell contact with the subsequent loss of membrane microvilli. Using an epithelial cell infection model, C. pneumoniae invasion caused a rapid and sustained increase in MEK-dependent phosphorylation and activation of ERK1/2, followed by PI 3-kinase-dependent phosphorylation and activation of Akt. Tyrosine phosphorylation of focal adhesion kinase (FAK) preceded its appearance in a complex with the p85 subunit of PI 3-kinase during chlamydial invasion and isoform-specific tyrosine phosphorylation of the docking protein Shc also occurred at the time of attachment and entry of bacteria. Chlamydia entry but not attachment could be abrogated with specific inhibitors of MEK, PI 3-kinase and actin polymerization, demonstrating the importance of these signalling pathways and an intact actin cytoskeleton for C. pneumoniae invasion. These results suggest that activation of specific cell signalling pathways is an essential strategy used by C. pneumoniae to invade epithelial cells.     

Differential cell behaviour on polylysine and gelatin in primary culture of mouse mammary adenocarcinoma.

Cells dissociated from spontaneous and transplanted tumours of C3HJax mammary gland have been cultured on polylysine and gelatin substrates. The isolated cells proliferated to form monolayers with high degree of organoid structure as indicated by formation of alveolar cavities. Differences were observed in the cell attachment, growth pattern, number and size of alveolar cavities, cells which lined the cavity and cell morphology on polylysine and gelatin substrates as compared to conventional cell culture plastic surface. On polylysine more than 90% cells attached rapidly, within 15-45 min after plating, with or without serum and formed confluent monolayers marked by presence of large and small alveolar cavities. Multiple interacting cell types took part in organization of the cavity. Cells lining the cavity constantly proliferated and rearranged to expand it. On gelatin, 60-70% cells attached over a period of 6-24 hr in presence of serum and formed confluent monolayers dominated by small alveolar cavities. Cells forming the cavities were epithelial in nature and cavities once formed did not increase in size. Upon subculture, the cell morphology on these substrates was strikingly different. On polylysine, the predominant cell type had numerous irregular microvilli whereas on gelatin, cells had smoother boundaries with a few stunted cytoplasmic extensions. The cell attachment on conventional surface was low, 40-50%. When seeded at high cell density, formation of alveolar cavities was suppressed and at low cell density, cultures were marked by contact inhibition of cells and failure to attain confluence. These results suggest differential behaviour and interaction of mammary tumour epithelium with the substrates used.     

The invasion of HeLa cells by Salmonella typhimurium: reversible and irreversible bacterial attachment and the role of bacterial motility

The interactions which brought about the invasion of HeLa cells by Salmonella typhimurium consisted of a sequence of three phases. Initially, the motility of the bacteria facilitated their contact with the HeLa cells whereupon the bacteria became attached in a reversible manner (i.e. the bacteria could be removed readily by washing the HeLa cell monolayers with Hanks' Balanced Salt solution). The binding forces responsible for reversible attachment were probably the weak long-range forces of the secondary minimum level of attractive interactions between the bacterium and the HeLa cell. Reversible attachment was a necessary interlude before the bacteria became irreversibly attached to the surfaces of the HeLa cells (i.e. the bacteria were no longer removed by the washing procedure that removed the reversibly attached salmonellae). Irreversible attachment was prevented in solutions of low ionic strength; the forces responsible were probably those of the primary minimum generated between the HeLa cell and a bacterial adhesion which was capable of acting over only short distances between the reversibly attached bacterium and the HeLa cell (i.e. probably less than 15 nm). Only irreversibly attached bacteria proceeded to the third phase and were internalized by the HeLa cells.