Synthesis of disulfide-bonded outer membrane proteins during the developmental cycle of Chlamydia psittaci and Chlamydia trachomatis.

The disulfide bond cross-linked major outer membrane protein (MOMP) of the extracellular elementary bodies (EBs) of Chlamydia psittaci was reduced to its monomeric form within 1 h of entry of EBs into host cells by a process which was inhibited by chloramphenicol, while monomeric forms of three cross-linked cysteine-rich proteins could not be detected in Sarkosyl outer membrane complexes at any time in either extracellular or intracellular forms of C. psittaci. Synthesis and incorporation of the MOMP into outer membrane complexes were detected early in the infection cycle (12 h postinfection), while synthesis and incorporation of the cysteine-rich proteins were not observed until reticulate bodies had begun to reorganize into EBs at 20 to 22 h postinfection. By 46 h postinfection, the intracellular population of C. psittaci consisted mainly of EBs, the outer membrane complexes of which were replete with monomeric MOMP and cross-linked cysteine-rich proteins. Upon lysis of infected cells at 46 h, the MOMP was rapidly cross-linked, and infectious EBs were released. The status of the MOMP of intracellular Chlamydia trachomatis was similar to the status of the MOMP of C. psittaci in that the MOMP was largely uncross-linked at 24 and 48 h postinfection, but formed interpeptide disulfide bonds when it was exposed to an extracellular environment late in the developmental cycle. In contrast to C. psittaci, only a fraction of the cross-linked MOMP of infecting EBs of C. trachomatis was reduced by 4 h postinfection, and reduction of the MOMP was not inhibited by chloramphenicol. Exposure of extracellular EBs of C. trachomatis and C. psittaci to dithiothreitol reduced the MOMP but failed to stimulate metabolic activities normally associated with reticulate bodies.     

Effects of chemically modified heparin on Chlamydia trachomatis serovar L2 infection of eukaryotic cells in culture

The mechanism and inhibitors of Chlamydia trachomatis serovar L2 infection of eukaryotic host cells were studied using a tissue culture model infection system. Potent inhibition of infectivity was observed when elementary bodies (EBs) were exposed to heparin or when HeLa 229 cells were treated with heparinase. No significant inhibition was seen the other way around. The same potent inhibition was observed when EBs were exposed to chemically 2-O-desulfated heparin (2-ODS heparin), which is composed of repeating disaccharide units of IdoA-GlcNS(6S), but not when exposed to chemically 6-ODS heparin or completely desulfated and N-resulfated heparin, which is composed of repeating disaccharide units of IdoA(2S)-GlcNS or IdoA-GlcNS, respectively. The inhibitory effects of 2-ODS heparin could be seen only with oligosaccharides longer than dodecasaccharides. The mutant Chinese hamster ovary (CHO) cell line 677, which is deficient in the biosynthesis of heparan sulfate, was less sensitive to C. trachomatis infection than were wild-type CHO cells. F-17 cells, deficient in 2-O-sulfation of heparan sulfate, had the same sensitivity to infection as wild-type CHO cells did. These data suggest that infection of host cells by EBS results from the specific binding of ligand molecules with affinity for heparin on the EB surface to heparan sulfate proteoglycans on the host cell surface. This binding may depend on host cell heparan sulfate chains that are 6-O-sulfated and longer than dodecasaccharides. The 2-ODS heparin oligosaccharides may be a potential agent for the prevention of C. trachomatis infection.     

Treatment of Chlamydia trachomatis with a small molecule inhibitor of the Yersinia type III secretion system disrupts progression of the chlamydial developmental cycle

The obligate intracellular bacterium Chlamydia trachomatis possesses a biphasic developmental cycle that is manifested by differentiation of infectious, metabolically inert elementary bodies (EBs) to larger, metabolically active reticulate bodies (RBs). The cycle is completed by asynchronous differentiation of dividing RBs back to a population of dormant EBs that can initiate further rounds of infection upon lysis of the host cell. Chlamydiae express a type III secretion system (T3SS) that is presumably employed to establish and maintain the permissive intracellular niche by secretion of anti-host proteins. We hypothesize that T3SS activity is essential for chlamydial development and pathogenesis. However, the lack of a genetic system has confounded efforts to establish any role of the T3SS. We therefore employed the small molecule Yersinia T3SS inhibitor N'-(3,5-dibromo-2-hydroxybenzylidene)-4-nitrobenzohydrazide, designated compound 1 (C1), to examine the interdependence of the chlamydial T3SS and development. C1 treatment inhibited C. trachomatis but not T4SS-expressing Coxiella burnetii development in a dose-dependent manner. Although chlamydiae remained viable and metabolically active, they failed to divide significantly and RB to EB differentiation was inhibited. These effects occurred in the absence of host cell cytotoxicity and were reversible by washing out C1. We further demonstrate that secretion of T3S substrates is perturbed in C1-treated chlamydial cultures. We have therefore provided evidence that C1 can inhibit C. trachomatis development and T3SS activity and present a model in which progression of the C. trachomatis developmental cycle requires a fully functional T3SS.     

Identification and properties of chlamydial polypeptides that bind eucaryotic cell surface components.

An electroblotting technique was used to identify proteins of Chlamydia that bound surface-radioiodinated and Triton X-100-solubilized HeLa cell extracts. Two proteins, with apparent molecular masses of 18 and 32 kilodaltons (kDa), that bound HeLa cell surface components were identified on Chlamydia trachomatis L2 elementary bodies (EBs). Radioiodinated heparin, which disrupts chlamydial association with cultured cells, was also bound by these proteins. These two proteins were found on EBs but were absent or were present in reduced amounts on the noninfectious reticulate bodies. All C. trachomatis strains tested displayed two such proteins, although the apparent molecular weight of the larger protein varied with serotype in correlation with biotype and the disease that it caused. Two Chlamydia psittaci strains examined displayed only a single binding protein in the range of 17 to 19 kDa. All of the binding proteins stained intensely and distinctively on silver-stained sodium dodecyl sulfate-polyacrylamide gels and displayed an unusual sensitivity to reducing agents. The 32-kDa protein was not seen and did not bind 125I-labeled HeLa cell components if the EBs were solubilized in the presence of 2-mercaptoethanol. The 32-kDa protein was not affected by dithiothreitol, however. Similar to the effect of 2-mercaptoethanol, the 32-kDa protein was not visualized after treatment of EBs with the protease inhibitors tosyl-phenylalanine chloromethyl ketone (TPCK) or tosyl-lysine chloromethyl ketone (TLCK). TPCK and TLCK also abolished infectivity as did the alkylating agents N-ethylmaleimide and iodoacetamide, yet the latter two agents did not affect the appearance of the 32-kDa protein. These proteins were not detected in immunoblots with either rabbit antisera to C. trachomatis L2 EBs or by serum from a patient with lymphogranuloma venereum. The role of these proteins in the interaction of chlamydiae with host cells is not clear, but the binding of eucaryotic cell surface components and heparin, presence only during the infectious stage of the life cycle, variation between serotypes in correlation with disease, and sensitivity to reducing agents or protease inhibitors, collectively, suggest a role for these proteins in parasite-host interactions.     

Antibody-neutralizing activity against all urogenital Chlamydia trachomatis serovars in Chlamydia suis-infected pigs

It is known that neutralizing species-specific or serovar-specific antibodies are produced in response to chlamydial infection in humans and in some animal species. In a previous study, a strong in vitro neutralizing activity to Chlamydia suis in 80% of sera from C. suis-infected pigs had been observed. In view of the close relationship between C. suis and Chlamydia trachomatis, in the present study, the neutralizing activity against D-K C. trachomatis and C. suis purified elementary bodies (EBs) in sera collected from C. trachomatis-infected patients and C. suis-infected pigs was evaluated. A neutralizing activity of 50-70% was observed in the human sera against the homologous serovar and one to five heterologous C. trachomatis serovars. These sera were also able to neutralize C. suis EBs. The pig sera showed a strong neutralizing activity (70-100%) against C. suis EBs and all eight urogenital C. trachomatis serovars. These results suggested the presence of common immunogenic antigens in C. trachomatis and C. suis. Immunoblot analysis, performed to elucidate the target of this neutralizing activity, showed a clear reactivity in human and pig sera against two proteins of 150 and 40 kDa MW, when tested either with C. trachomatis or with C. suis EBs.     

The use of carboxyfluorescein diacetate succinimidyl ester to determine the site, duration and cell type responsible for antigen presentation in vivo

This report examines the use of 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) to determine the site, duration and cell type responsible for antigen presentation in vivo. Evidence that CFSE-labelled T cells can be used to determine where various types of antigens are presented, including auto-antigens, oral antigens and cell-associated foreign antigens, is provided. Using this technique, the length of time antigen is presented after acquisition by APC was measured. Finally, CFSE labelling was used to identify the origin of the APC responsible for different forms of antigen presentation.     

An early event in the herpes simplex virus type-2 replication cycle is sufficient to induce Chlamydia trachomatis persistence

Epidemiological studies have demonstrated that co-infections of herpes simplex virus type 2 (HSV-2) and Chlamydia trachomatis occur in vivo. Data from a tissue culture model of C. trachomatis/HSV-2 co-infection indicate that viral co-infection stimulates the formation of persistent chlamydiae. Transmission electron microscopic (TEM) analyses demonstrated that in both HeLa and HEC-1B cells, co-infection caused developing chlamydiae to exhibit swollen, aberrantly shaped reticulate bodies (RBs), characteristically observed in persistence. Additionally, HSV-2 co-infection suppressed production of infectious chlamydial elementary bodies (EBs) in both host cell types. Co-infection with HSV type 1 (HSV-1) produced similar morphologic alterations and abrogated infectious EB production. These data indicate that virus-induced chlamydial persistence was neither host cell- nor virus strain-specific. Purification of crude HSV-2 stocks demonstrated that viral particles were required for coinfection-induced chlamydial persistence to occur. Finally, co-infection with either UV-inactivated, replication-incompetent virus or replication-competent HSV-2 in the presence of cyclohexamide reduced chlamydial infectivity without altering chlamydial genomic DNA accumulation. These data demonstrate that productive viral replication is not required for the induction of chlamydial persistence and suggest that HSV attachment and entry can provide the necessary stimulus to alter C. trachomatis development.     

Evidence that CT694 is a novel Chlamydia trachomatis T3S substrate capable of functioning during invasion or early cycle development

Chlamydia trachomatis is an obligate intracellular parasite, occupies a membrane-bound vacuole throughout development and is capable of manipulating the eukaryotic host by translocating effector molecules via a type III secretion system (T3SS). The infectious chlamydial elementary body (EB) is metabolically inactive yet possesses a functional T3S apparatus capable of translocating effector proteins into the host cell to facilitate invasion and other early cycle events. We present evidence here that the C. trachomatis protein CT694 represents an early cycle-associated effector protein. CT694 is secreted by the Yersinia T3SS and immunodetection studies of infected HeLa cultures indicate that CT694-specific signal accumulates directly adjacent to, but not completely overlapping with EBs during invasion. Yeast two-hybrid analyses revealed an interaction of CT694 with the repeat region and C-terminus of human AHNAK. Immunolocalization studies of CT694 ectopically expressed in HeLa cells were consistent with an interaction with endogenous AHNAK. Additionally, expression of CT694 in HeLa cells resulted in alterations in the detection of stress fibres that correlated with the ability of CT694 to interact with AHNAK. These data indicate that CT694 is a novel T3S-dependent substrate unique to C. trachomatis , and that its interaction with host proteins such as AHNAK may be important for aspects of invasion or development particular to this species.     

Chlamydia trachomatis type III secretion: evidence for a functional apparatus during early-cycle development

The obligate intracellular bacterium Chlamydia trachomatis occupies a parasitophorous vacuole termed an inclusion. During its intracellular developmental cycle, C. trachomatis maintains this intracellular niche, presumably by expressing a type III secretion system, which deploys a set of host cell-interactive proteins including inclusion membrane-localized proteins termed Incs. Some Incs are expressed and secreted by 2 h (early cycle) after infection, whereas the expression of type III-specific genes is not detectable until 6-12 h (mid-cycle). To resolve this paradox, we investigated the presence of a type III apparatus on elementary bodies (EBs) that might function early in infection. We demonstrate the existence of the type III secretory apparatus by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) and immunoblot analyses of purified EB extracts. Immunoblots using polyclonal antibodies specific for the core apparatus component CdsJ identified this protein in both EB and reticulate body (RB) extracts. Furthermore, CdsJ-specific signals were detected by immunoblot of whole infected-culture extracts and by indirect immunofluorescence of infected monolayers at times before the detection of cdsJ-specific message. Finally, expression of IncC, expressed by 2 h after infection during C. trachomatis infections, in Yersinia pseudotuberculosis resulted in its secretion via the Yersinia type III apparatus. Based on these data, we propose a model in which type III secretion pores are present on EBs and mediate secretion of early Incs and possible additional effectors. Mid-cycle expression of type III genes would then replenish secretion apparatus on vegetative RBs and serve as a source of secretion pores for subsequently formed EBs.     

The Chlamydia outer membrane protein OmcB is required for adhesion and exhibits biovar-specific differences in glycosaminoglycan binding

Chlamydia pneumoniae, an obligate intracellular human pathogen, causes a number of respiratory diseases. We explored the role of the conserved OmcB protein in C. pneumoniae infections, using yeast display technology. (i) Yeast cells presenting OmcB were found to adhere to human epithelial cells. (ii) Pre-incubation of OmcB yeast cells with heparin, but not other glycosaminoglycans (GAGs), abrogated adhesion. (iii) Pre-treatment of the target cells with heparinase inhibited adherence, and GAG-deficient CHO cell lines failed to bind OmcB yeast. (iv) A heparin-binding motif present near the N-terminus of OmcB is required for host cell binding. (v) Pre-treatment of chlamydial elementary bodies (EBs) with anti-OmcB antibody or pre-incubation of target cells with recombinant OmcB protein reduced infectivity upon challenge with C. pneumoniae. (vi) Adhesion of fluorescently labelled EBs to epithelial or endothelial cells was abrogated by prior addition of heparin or OmcB protein. Thus, C. pneumoniae OmcB is an adhesin that binds heparan sulphate-like GAGs. OmcB from Chlamydia trachomatis serovar L1 also adheres to human cells in a heparin-dependent way, unlike its counterpart from serovar E. We show that a single position in the OmcB sequence determines heparin dependence/independence, and variations there may reflect differences between the two serovars in cell tropism and disease pattern.     

Disulfide-mediated interactions of the chlamydial major outer membrane protein: role in the differentiation of chlamydiae?

The effects of exogenous reducing agents on a number of biological properties of purified Chlamydia trachomatis LGV-434 and Chlamydia psittaci meningopneumonitis elementary bodies (EBs) have been examined in an attempt to identify in vitro correlates of early events in the differentiation of the infectious EB to the replicative cell type, the reticulate body (RB). Treatment of EBs with dithiothreitol elicited a number of changes normally associated with differentiation to the RB. EBs in the presence of 10 mM dithiothreitol displayed enhanced rates of [14C]glutamate oxidation, reduced infectivity, and decreased osmotic stability, and their Machiavello staining properties changed to those characteristic of the RB. A true differentiation of EB to RB did not take place under these conditions, since EBs treated in this manner and examined by transmission electron microscopy did not demonstrate increased size or decreased electron density as do isolated RBs. Additional studies were initiated to identify the macromolecules involved in this process. With polyacrylamide gel electrophoresis and immunoblotting procedures with monoclonal and polyclonal monospecific antibodies, the chlamydial major outer membrane protein was found to be the predominant component that varied under reducing versus nonreducing conditions. Furthermore, the extent of disulfide-mediated cross-linking of the major outer membrane protein varied between the infective and replicative forms of the C. trachomatis LGV-434 life cycle. Implications of disulfide interactions in the life cycle of chlamydiae are discussed.     

Development of a transformation system for Chlamydia trachomatis: restoration of glycogen biosynthesis by acquisition of a plasmid shuttle vector

Chlamydia trachomatis remains one of the few major human pathogens for which there is no transformation system. C. trachomatis has a unique obligate intracellular developmental cycle. The extracellular infectious elementary body (EB) is an infectious, electron-dense structure that, following host cell infection, differentiates into a non-infectious replicative form known as a reticulate body (RB). Host cells infected by C. trachomatis that are treated with penicillin are not lysed because this antibiotic prevents the maturation of RBs into EBs. Instead the RBs fail to divide although DNA replication continues. We have exploited these observations to develop a transformation protocol based on expression of β-lactamase that utilizes rescue from the penicillin-induced phenotype. We constructed a vector which carries both the chlamydial endogenous plasmid and an E.coli plasmid origin of replication so that it can shuttle between these two bacterial recipients. The vector, when introduced into C. trachomatis L2 under selection conditions, cures the endogenous chlamydial plasmid. We have shown that foreign promoters operate in vivo in C. trachomatis and that active β-lactamase and chloramphenicol acetyl transferase are expressed. To demonstrate the technology we have isolated chlamydial transformants that express the green fluorescent protein (GFP). As proof of principle, we have shown that manipulation of chlamydial biochemistry is possible by transformation of a plasmid-free C. trachomatis recipient strain. The acquisition of the plasmid restores the ability of the plasmid-free C. trachomatis to synthesise and accumulate glycogen within inclusions. These findings pave the way for a comprehensive genetic study on chlamydial gene function that has hitherto not been possible. Application of this technology avoids the use of therapeutic antibiotics and therefore the procedures do not require high level containment and will allow the analysis of genome function by complementation.     

Chlamydia trachomatis has penicillin-binding proteins but not detectable muramic acid.

Chlamydia trachomatis LGV-434 was grown in HeLa 229 cells. Benzylpenicillin completely inhibited the formation of infectious elementary bodies (EBs) at a concentration of 19 pmol/ml or higher and produced abnormally large reticulate bodies (RBs) in the inclusions at 30 pmol/ml or higher. The possible targets for penicillin in C. trachomatis were three penicillin-binding proteins (PBPs) which were identified in the Sarkosyl-soluble fractions of both RBs and EBs. The apparent subunit molecular weights were 88,000 (PBP 1), 61,000 (BPB 2), and 36,000 (PBP 3). The 50% binding concentrations of [3H]penicillin for PBPs 1 to 3 in EBs and RBs were between 7 and 70 pmol/ml. Such high susceptibility to penicillin was shown by an organism that did not have detectable muramic acid (less than 0.02% by weight) in preparations of either whole cells or sodium dodecyl sulfate-insoluble residues.     

Immunoelectron microscopic localization of chlamydial lipopolysaccharide (LPS) in McCoy cells inoculated with Chlamydia trachomatis.

Interactions between Chlamydia trachomatis, host cells, and the immune system are believed to involve lipopolysaccharide (LPS). We used immunogold techniques to study the distribution of chlamydial LPS in cultured cells infected with C. trachomatis LGV-L1. McCoy cells inoculated with C. trachomatis were cultured and then fixed and embedded in situ with acrylic resins. Sections were immunolabeled with a protein A-gold method using antisera to the genus-specific, periodate-sensitive epitope on chlamydial LPS. Pre-embedding immunogold labeling on permeabilized cells was also done. By post-embedding methods, labeling for LPS was equally abundant over the outer membranes of elementary (EB) and reticulate bodies (RB). By post-embedding labeling, the sub-surface side of the EB outer membrane was more heavily labeled than the surface side. By pre-embedding labeling, LPS was found to be less abundant on the surface of EBs than RBs. Labeling for LPS was found over apparent lysosomes in McCoy cells and over electron-dense blebs on or near the surface of the plasma membranes of McCoy cells. These results indicate that the concentration of LPS in chlamydial membranes is constant during development but that with development its location changes from being mostly cell-surface to sub-surface. These results show that the post-embedding immunogold technique can be a useful approach for the cell culture-based study of chlamydial LPS.     

High-yield culture and purification of Chlamydiaceae bacteria

Research on intracellular bacteria of the family Chlamydiaceae, and the diseases they cause, requires large amounts of infectious elementary bodies (EB). We describe an approach that maximizes the generation of Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydia abortus, or Chlamydia pecorum EBs in several replication cycles over approximately 10 days or more in a saturated equilibrium monolayer cell culture system. Buffalo Green Monkey Kidney (BGMK) cells, Human Epidermoid Carcinoma-2 (HEp-2) cells, or mouse McCoy cells were tested. BGMK cells best supported C. pneumoniae replication when cultivated in Iscove's Modified Dulbecco's Medium. From day 1 to day 9 after inoculation, C. pneumoniae genomes per ml culture medium increased from 10(5.1) to 10(8.6) in BGMK, from 10(5.6) to 10(8.1) in HEp-2, and remained at 10(5.2) in McCoy cell cultures. Three-month pre-inoculation maintenance of BGMK cells in different culture media did not influence C. pneumoniae yields. Inoculation at multiplicities of infection (MOI) of 10 or higher and supplementation of the cell culture medium on day 7 after inoculation with 0.1% glucose enhanced C. pneumoniae EB yields in harvested cell culture medium. For purification, EBs in medium were concentrated by sedimentation, followed by low-speed centrifugation for removal of host cell nuclei, and by step-gradient centrifugation of the supernatant in a 30% RenoCal-76-50% sucrose step-gradient. Extensive sonication increased yield and infectivity of chlamydial EB. The combined method typically produced from 1000 ml infected BGMK culture medium 10 ml homogeneous, single-cell, highly infectious EB stock containing approximately 5x10(11) C. pneumoniae genomes equivalent to 4-5x10(11) inclusion forming units.     

In situ studies on incorporation of nucleic acid precursors into Chlamydia trachomatis DNA.

Chlamydiae are obligate intracellular bacteria that are dependent on eukaryotic host cells for ribonucleoside triphosphates. The purpose of the present study was to determine whether Chlamydia trachomatis obtains deoxyribonucleotides from the host cell. The study was aided by the finding that host and parasite DNA synthesis activity could be distinguished by their differing sensitivities to aphidicolin and norfloxacin. Results from isotope incorporation experiments indicated that any nucleobase or ribonucleoside that could serve as a precursor for host DNA synthesis could also be utilized by C. trachomatis for DNA replication. C. trachomatis utilized only those precursors which the host cell converted to the nucleotide level. Pyrimidine deoxyribonucleotides were efficient precursors for host DNA synthesis; however, they were not used by C. trachomatis. On the other hand, purine deoxyribonucleosides are rapidly catabolized by host cells, it is necessary to regulate their metabolism to determine whether they serve as direct precursors for C. trachomatis DNA synthesis. This was partially achieved by using a hypoxanthine-guanine phosphoribosyltransferase-negative cell line and using deoxycoformycin and 8-aminoguanosine as inhibitors of (deoxy)adenosine deaminase and purine nucleoside phosphorylase, respectively. The results indicated that purine deoxyribonucleosides are efficiently utilized for host cell DNA synthesis even if degradation pathways are inhibited and salvage to ribonucleotides is minimized. In sharp contrast, the purine deoxyribonucleosides were utilized by C. trachomatis as precursors for DNA synthesis only when host catabolic pathways and salvage reactions were intact. High-pressure liquid chromatographic analysis of nucleotide pools extracted from host cells pulsed with radiolabeled precursors suggests that infected cells transport and phosphorylate all deoxynucleosides as effectively as mock-infected control cultures. In aggregate, these results show that chlamydiae do not take up deoxyribonucleotides from the host cells.     

Chlamydia trachomatis Mip-like protein

A 27 kDa Chlamydia trachomatis Mip-like protein with homology of a 175-amino-acid C-terminal fragment to the surface-exposed Legionella pneumophila mip-gene product has previously been described. In this paper the entire chlamydia Mip-like sequence of C. trachomatis serovar L2 (lymphogranuloma venereum (LGV) biovar) is presented. The sequence shows high similarity to the legionella Mip protein and its C-terminal region, like that of the legionella Mip, has high amino acid similarity to eukaryotic and prokaryotic FK506-binding proteins. The chlamydial mip-like gene was detected by polymerase chain reaction (PCR) in other C. trachomatis serovars and by sequencing of the mip-like genes of serovars B and E (trachoma biovar) was shown to be highly conserved within the two major biovars of C. trachomatis. Monoclonal and polyclonal antibodies raised against the recombinant Mip-like protein failed to demonstrate surface-exposed epitopes on infectious elementary bodies or reproductive reticulate body forms either by immunofluorescence or immuno-gold electron microscopy. However, a complement-dependent inhibition of up to 91% of infectivity for cell cultures was observed with antibodies to the N-terminal fragment of the Mip-like protein suggesting that antibody-accessible epitopes are present on infectious EBs.     

Chlamydia trachomatis utilizes the host cell microtubule network during early events of infection

The host cell cytoskeleton is known to play a vital role in the life cycles of several pathogenic intracellular microorganisms by providing the basis for a successful invasion and by promoting movement of the pathogen once inside the host cell cytoplasm. McCoy cells infected with Chlamydia trachomatis serovars E or L2 revealed, by indirect immunofluorescence microscopy, collocation of microtubules and Chlamydia -containing vesicles during the process of migration from the host cell surface to a perinuclear location. The vast majority of microtubule-associated Chlamydia vesicles also collocated with tyrosine-phosphorylated McCoy cell proteins. After migration, the Chlamydia -containing vesicles were positioned exactly at the centre of the microtubule network, indicating a microtubule-dependent mode of chlamydial redistribution. Inhibition of host cell dynein, a microtubule-dependent motor protein known to be involved in directed vesicle transport along microtubules, was observed to have a pronounced effect on C. trachomatis infectivity. Furthermore, dynein was found to collocate with perinuclear aggregates of C. trachomatis E and L2 but not C. pneumoniae VR-1310, indicating a marked difference in the cytoskeletal requirements for C. trachomatis and C. pneumoniae during early infection events. In support of this view, C. pneumoniae VR-1310 was shown to induce much less tyrosine phosphorylation of HeLa cell proteins during uptake than that seen for C. trachomatis .