Phage infection of the obligate intracellular bacterium, Chlamydia psittaci strain guinea pig inclusion conjunctivitis

The infectious cycle of phiCPG1, a bacteriophage that infects the obligate intracellular pathogen, Chlamydia psittaci strain Guinea Pig Inclusion Conjunctivitis, was observed using transmission electron microscopy of phage-hyperinfected, Chlamydia-infected HeLa cells. Phage attachment to extracellular, metabolically dormant, infectious elementary bodies and cointernalisation are demonstrated. Following entry, phage infection takes place as soon as elementary bodies differentiate into metabolically active reticulate bodies. Phage-infected bacteria follow an altered developmental path whereby cell division is inhibited, producing abnormally large reticulate bodies, termed maxi-reticulate bodies, which do not mature to elementary bodies. These forms eventually lyse late in the chlamydial developmental cycle, releasing abundant phage progeny in the inclusion and, upon lysis of the inclusion membrane, into the cytosol of the host cell. Structural integrity of the hyperinfected HeLa cell is markedly compromised at late stages. Released phage particles attach avidly to the outer leaflet of the outer membranes of lysed and unlysed Chlamydiae at different stages of development, suggesting the presence of specific phage receptors in the outer membrane uniformly during the chlamydial developmental cycle. A mechanism for phage infection is proposed, whereby phage gains access to replicating chlamydiae by attaching to the infectious elementary body, subsequently subverting the chlamydial developmental cycle to its own replicative needs. The implications of phage infection in the context of chlamydial infection and disease are discussed.     

Trypanosoma cruzi: parasite-induced release of lysosomal enzymes by human polymorphonuclear leukocytes

The release of beta-glucuronidase and lysozyme from human polymorphonuclear leukocytes (PMN) engaged in phagocytosis and lysis of Trypanosoma cruzi epimastigotes was studied in the presence or absence of chagasic serum. Lysosomal enzyme release was enhanced when parasites were sensitized with serum from a chronic Chagas' patient, increased up to 3 hr of incubation at 28 C, and depended on the PMN:parasite ratio. The release of lysosomal enzymes was determined by the presence of 2 mM cyanide, 2 microM azide, 3 mM amobarbital, and 1 mM phenylbutazone. These drugs inhibited the killing of sensitized T. cruzi by interfering with the oxidative microbicidal mechanisms of PMN without affecting the uptake of the parasites. Lysosomal enzyme release occurred in the presence of cyanide and azide, indicating that in these cases the enzymatic release was unrelated to the killing of the parasites. Amobarbital and phenylbutazone, which stabilize PMN membranes, inhibited the release of beta-glucuronidase and lysozyme by PMN. The addition of 10 micrograms/ml of cytochalasin B inhibited the phagocytosis and killing of sensitized T. cruzi by PMN but increased the enzymatic release by effector cells. Since cytochalasin B did not affect the close contact between PMN and parasites, it appears that the enzymes released to the extracellular milieu were not toxic to noningested parasites. Furthermore, the lysosomal enzymes did not lyse bystander unsensitized parasites. Therefore, the release of lysosomal enzymes during the interaction of T. cruzi epimastigotes and PMN seems to be related to the triggering event of the phagocytic process and does not bear a cause-effect relationship with parasite death.     

Ultrastructural studies on the intracellular fate of Chlamydia psittaci (strain guinea pig inclusion conjunctivitis) and Chlamydia trachomatis (strain lymphogranuloma venereum 434): modulation of intracellular events and relationship with endocytic mechanism

Previous observations on the highly infectious LGV strain 434 of Chlamydia trachomatis and the guinea pig inclusion conjunctivitis (GPIC) strain of C. psittaci (which requires centrifugation of inocula with host cell monolayers for maximum infectivity) indicated that infectivity differences were expressed, not at entry, but at an intracellular stage affecting multiplication. Centrifugation increased the potential of internalized chlamydiae to undergo productive infection. Here, analysis of the intracellular fate of chlamydiae by ultrastructural methods indicates that strain GPIC exhibits two patterns of behaviour depending on the mode of inoculation. Strain GPIC showed limited entry, with 47% of intracellular organisms becoming associated with thorotrast-labelled lysosomes, following static incubation with monolayers. In contrast, with centrifugation, entry was not limited and association with lysosomes was reduced to 12%; strain 434 behaved similarly but independently of the mode of inoculation. The different results for strain GPIC correlated with distinct entry mechanisms. Entry during static incubation was unimpaired either by treatment with cytochalasin D or by temperature reduction to 20 degrees C, suggesting that it was pinocytic. Entry during centrifugation was markedly impaired by both treatments, suggesting that it was phagocytic. The data lead to two novel conclusions: first, that chlamydiae can apparently enter cells by both pinocytic and phagocytic mechanisms; second, that the entry mechanism influences intracellular fate. It is suggested that entry mechanism is linked to selection of the vesicle membrane forming around the internalizing chlamydiae. This, in turn, may influence both intracellular translocation and subsequent inhibition or promotion of multiplication of the internalized parasite.     

Arrays of hemispheric surface projections on Chlamydia psittaci and Chlamydia trachomatis observed by scanning electron microscopy.

Scanning microscopy of two strains of Chlamydia psittaci and four strains of Chlamydia trachomatis representative of the wide diversity in origin and behavior of members of the genus revealed patches of regular arrays of hemispheric projections on the surfaces of elementary bodies of all six strains. These distinctive and perhaps unique surface structure are probably present in all populations of chlamydiae.     

Electron microscopy of the in vivo internalization of virulent Chlamydia psittaci 6BC strain.

The internalization of virulent Chlamydia psittaci 6BC particles by wandering mononuclear phagocytes in the peritoneal cavity of intraperitoneally inoculated mice occurred asynchronously, i.e., fragile reticulate bodies (RB) appeared to be more readily phagocytized than the rigid elementary bodies (EB). Early damage of mononuclear phagocytes occurred after internalization of chlamydiae. This was followed by a decreased uptake of particles, and may explain the relatively long persistence (up to 6 h after inoculation) of free, extracellular, "swollen", and RB-like particles. Internalized particles within phagolysosomes showed varying degrees of disintegration. The subsequent influx of polymorphonuclear phagocytes and monocytes into the inflammed peritoneal cavity may explain the rapid disappearance of chlamydiae and their antigens from the peritoneal fluid. The alteration in ultrastructure of peritoneal cells and chlamydial parasites during the inflammatory process are discussed.     

Phagocytosis by Trichomonas vaginalis: new insights

BACKGROUND INFORMATION: The parasitic protozoan Trichomonas vaginalis is the causative agent of trichomoniasis, a sexually transmitted disease. The phagocytic activity of this parasite has not been completely elucidated. In order to better understand the mechanisms of trichomonal phagocytosis, we have studied the in vitro capacity of T. vaginalis to phagocytose and degrade Saccharomyces cerevisiae cells. RESULTS AND CONCLUSIONS: To analyse the phagocytic ability and capacity, two isolates of T. vaginalis presenting different virulence grades were used. Complementary techniques, such as fluorescence microscopy, computer-based fluorescence analysis, scanning and transmission electron microscopy and the use of drugs that interfere with the actin microfilaments, were used in order to follow the behaviour of the actin cytoskeleton during phagocytosis of yeast cells by T. vaginalis. It was concluded that: (1) T. vaginalis changes its shape rapidly and engulfs the yeast cells, which are almost as large as the parasite; (2) long-term and fresh cultures are able to phagocytose, although the low-virulence strain JT demonstrated a lower activity when compared with the highly virulent T016 isolate; (3) the T016 strain exhibited an amoeboid morphology during the internalization of yeast cells in contrast with the JT strain; (4) attachment of yeast cells to the parasite occurs via the whole cell surface, including both anterior and recurrent flagella; (5) two forms of phagocytosis were observed: a 'sinking' process without any apparent participation of plasma membrane extensions and the classical phagocytosis where pseudopodia are extended toward the target cell; (6) the internalized S. cerevisiae are digested in lysosomes; (7) competitor sugars D-mannose or L-fucose inhibit the phagocytosis, and inhibition was 1.67 times higher in long-term cultured JT than that of the parasites from fresh isolate T016; (8) a thick layer of actin microfilaments was present underlying the plasma membrane, and especially in the pseudopodia and around the phagocytosed particles; (9) a dramatic change in the distribution pattern of fibrillar actin occurred during phagocytosis; (10) cytochalasin D depressed the phagocytosis; (11) a non-specific recognition and phagocytosis of yeast cells by T. vaginalis is mediated by a mannose receptor present on the parasite surface; (12) the phagocytic process may occur simultaneously during mitosis of the parasite.     

The ultrastructure of a new species of Chlamydia--Chlamydia pneumoniae]

The dynamic study of a new Chlamydia species, C. pneumoniae (strain TWAR, isolate TW-480), inoculated into the monolayer culture of cells L-929 was made 24, 48, 72 and 96 hours after inoculation. When compared with C. trachomatis and C. psittaci, C. pneumoniae were found to stand between these two species with respect to the morphology of their intracytoplasmic microcolonies (inclusions): they were round, almost bubble-like, but more densely packed with chlamydiae, surrounded by an undulate membrane, preserving its integrity until the late stages of their development cycle. In cells L-929 C. pneumoniae had a typical development cycle accompanied by the formation of vegetative and spore-like cells, reticular and elementary bodies, as well as intermediate cells, though this process was slower than in C. trachomatis and C. psittaci. Besides normal elementary bodies, many altered ones were formed in the process of the development of C. pneumoniae in cells L-929. Most of these alterations were similar to the process of bacterial L-transformation and could be regarded as the manifestation of chlamydial pathology related to the adaptation to new host cells.     

Effect of Penicillin on the Multiplication of Meningopneumonitis Organisms (Chlamydia psittaci)

Although formation of infectious particles of meningopneumonitis organism in L cells was completely inhibited by 1 or more units of penicillin per ml, multiplication of reticulate bodies was observed, by light microscopy, in the presence of 200 units of penicillin per ml in stained smears of infected cells. When reticulate bodies were purified from cultures containing penicillin after 18, 30, and 45 hr of incubation, continuously increasing yields were obtained. When penicillin was added to infected cultures 0 to 15 hr after infection, no increase in infectivity was observed at 40 hr, but when antibiotic was added between 20 and 35 hr, partial synthesis of infectious particles was observed at 40 hr. On the other hand, removal of penicillin from an infected culture before 15 hr after infection did not affect the final yields of infectivity when assayed at 40 hr, but elimination of penicillin after 20 hr resulted in a decrease in infectivity. In suspensions of (32)P-labeled purified reticulate bodies grown in cultures containing penicillin and harvested 18 and 40 hr after infection, the (32)P distributions obtained by acid fractionation were similar to those of reticulate bodies from penicillin-free cultures. Cell membranes of reticulate bodies were also prepared from 40-hr cultures with penicillin. The size and shape of purified membranes, as seen by electron microscopy, and their amino acid compositions were similar to membranes prepared from reticulate bodies grown without penicillin, except that very small structures were observed in membranes from cultures containing penicillin. These results indicated that penicillin does not inhibit reproduction of reticulate bodies and formation of their cell membranes, but does inhibit the formation of elementary body cell envelopes.     

Pyrimidine metabolism by intracellular Chlamydia psittaci.

Pyrimidine metabolism was studied in the obligate intracellular bacterium Chlamydia psittaci AA Mp in the wild type and a variety of mutant host cell lines with well-defined mutations affecting pyrimidine metabolism. C. psittaci AA Mp cannot synthesize pyrimidines de novo, as assessed by its inability to incorporate aspartic acid into nucleic acid pyrimidines. In addition, the parasite cannot take UTP, CTP, or dCTP from the host cell, nor can it salvage exogenously supplied uridine, cytidine, or deoxycytidine. The primary source of pyrimidine nucleotides is via the salvage of uracil by a uracil phosphoribosyltransferase. Uracil phosphoribosyltransferase activity was detected in crude extracts prepared from highly purified C. psittaci AA Mp reticulate bodies. The presence of CTP synthetase and ribonucleotide reductase is implicated from the incorporation of uracil into nucleic acid cytosine and deoxycytidine. Deoxyuridine was used by the parasite only after cleavage to uracil. C. psittaci AA Mp grew poorly in mutant host cell lines auxotrophic for thymidine. Furthermore, the parasite could not synthesize thymidine nucleotides de novo. C. psittaci AA Mp could take TTP directly from the host cell. In addition, the parasite could incorporate exogenous thymidine and thymine into DNA. Thymidine kinase activity and thymidine-cleaving activity were detected in C. psittaci AA Mp reticulate body extract. Thus, thymidine salvage was totally independent of other pyrimidine salvage.     

Schistosoma mansoni modulation of phagocytosis in Biomphalaria glabrata

Both short-term (3 hr) exposure of Biomphalaria glabrata snails (M-line and 13-16-R1) to Schistosoma mansoni (PR1) miracidia and in vitro incubation of parasite sporocysts with host hemolymph components altered host phagocytic ability. Hemocytes obtained from susceptible (M-line) snails that had been exposed to parasite miracidia for 3 hr showed reduced levels of phagocytosis of yeast cells in vitro compared to hemocytes from unexposed individuals. Incubation of whole hemolymph with sporocysts in vitro also reduced yeast phagocytosis in this susceptible strain. In contrast, resistant (13-16-R1) hemocytes showed increased levels of yeast phagocytosis after in vitro incubation with the parasite, and the opsonic properties of 13-16-R1 plasma were greater after exposure of snails to miracidia. These strain-specific effects of S. mansoni on host hemocyte phagocytosis and plasma opsonization were seen only when both plasma and hemocytes were present at the time of exposure to the parasite.     

Phagocytosis of Plasmodium chabaudi: modulation by immune serum and antigen in vitro

The phagocytosis of free Plasmodium chabaudi parasite by resident peritoneal macrophages of mouse was studied in an in vitro system. The effect of antimalarial antiserum (HIS) was assessed by preincubation of parasite macrophages and both parasite and macrophages with HIS prior to use in phagocytic assays. Highest phagocytic index was obtained with HIS pretreated parasites. The two activities viz. opsonic (parasite dependent) and cytophilic (macrophage dependent) were noted to operate independent of each other. The phagocytosis promoting activity was found to be complement dependent. The receptor site for binding of HIS opsonized but not medium opsonized parasite on the surface of macrophages was blocked by pretreatment of these cells with HIS-soluble antigen combination.     

Adenine nucleotide and lysine transport in Chlamydia psittaci.

Isolated reticulate bodies of Chlamydia psittaci were found to transport ATP and ADP by an ATP-ADP exchange mechanism. ATP uptake activity was not detected in elementary bodies. The apparent Km of transport for both ATP and ADP was approximately 5 microM, and the calculated Vmax for both was about 1 nmol of nucleotide transported per min per mg of protein. ADP competitively inhibited ATP transport with a Ki of 4.5 microM. Other nucleotides tested had no effect on the uptake of ATP. A magnesium-dependent, oligomycin-sensitive ATPase (ATP phosphohydrolase, EC 3.6.1.3) was associated with reticulate bodies, and most of the transported ATP was hydrolyzed to ADP, which was exchanged for additional, extracellular nucleotide. Some ADP was hydrolyzed to AMP, which exited the cells slowly. Lysine was transported against the electrochemical gradient by reticulate bodies in the presence of ATP. Oligomycin and carbonyl cyanide p-trifluoromethoxyphenylhydrazone inhibited ATP-dependent lysine transport. Lysine exited reticulate bodies when the reticulate bodies were incubated in the presence of ADP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, or a reduced concentration of ATP. The results support the concept that chlamydiae are energy parasites which are capable of drawing upon the adenine nucleotides of their hosts, hydrolyzing ATP, and establishing an energized membrane.     

Real-time measurements of membrane surface dynamics on macrophages and the phagocytosis of Leishmania parasites

Defocusing microscopy was used for real-time observation and quantification of membrane surface dynamics in murine bone marrow macrophages. Small random membrane fluctuations (SRMF), possibly metabolic driven, were detected uniformly over all membrane surface. Morphological and dynamical parameters of ruffles, such as shape, dimensions, and velocity of propagation, were analyzed. Optical tweezers were used to promote phagocytosis of single Leishmania amazonensis amastigotes by selected macrophages. Analysis of ruffling activity on the macrophages before and during phagocytosis of the parasites indicated that increased ruffling response near forming phagosomes, most likely induced by the parasite, accelerates phagocytosis. The effects of temperature decrease on the dynamics of membrane surface fluctuations and on the phagocytosis of parasites were used to determine the overall activation energies involved in these processes. The values obtained support the existence of strong correlation between membrane motility and phagocytic capacity.     

Localization by immunoelectron microscopy of antigens of Chlamydia psittaci suitable for diagnosis or vaccine development

Two different antigens of serotype 1 Chlamydia psittaci were localized using three immunoelectron microscopy techniques: non-embedding, pre-embedding and post-embedding. The antigens had previously been described as being of potential use in diagnosis (80–90 kDa protein region) and vaccine development (110 kDa protein). The results show a direct relationship between the protective capacity of the antigens and their surface localization on the elementary bodies, which are the infectious form of Chlamydia. The 80–90 kDa protein region is located on the surface of reticulate bodies but not of elementary bodies, where it was located periplasmically, while the 110 kDa protein occurs on the surface of both elementary and reticulate bodies.     

Low-nutrient induction of abnormal chlamydial development: A novel component of chlamydial pathogenesis?

Abstract The intracellular development of chlamydiae in McCoy cells incubated in Eagle's minimal essential medium lacking all 13 amino acids was examined both by fluorescence and electron microscopy and by infectivity titration. Aberrant development occurred in almost all inclusions of strains of Chlamydia trachomatis and C. psittaci with the production of abnormal forms which differed in size, shape and internal structure from normal reticulate and elementary body forms. Detailed analysis of the response of C. trachomatis L2 strain 434 to graded reductions in amino acid level showed that infectivity was reduced and morphological abnormality increased as amino acid concentrations were lowered from 33 to 0% of amino acids present in minimal essential medium. Reversion of inclusions to normal and reappearance of infectious forms occured on restoration of amino acids and further incubation. It is suggested that aberrant development may account for the presence in vivo of non-cultivable chlamydiae and that such development can arise via tryptophan deprivation mediated by local release of interferon gamma.     

Effector mechanism of host resistance in murine giardiasis: specific IgG and IgA cell-mediated toxicity

The role of specific serum and milk anti-Giardia muris antibodies in mediation of host-effector responses to this enteric pathogen is unknown. We have investigated antibody-dependent cell-parasite interactions, potentially important as mediators of protection against infection at the mucosal surface. Elicited mouse peritoneal neutrophils and macrophages were incubated with G. muris trophozoites in the presence of either serum or milk antibodies, and their adherence and phagocytosis of the parasites were assessed. The percentage of trophozoites with adherent neutrophils increased significantly in the presence of heat-inactivated immune rabbit serum (93.5% +/- 6.5) and immune mouse milk (54.4% +/- 11.3) and their purified IgG (35.2% +/- 9.7) and secretory IgA fractions (48.0% +/- 12.3) when compared with incubation in RPMI-10% FCS (21.7% +/- 13.9). Similarly, macrophage adherence to trophozoites increased from 49.7% +/- 14.3 in medium alone to respective values of 92.8% +/- 7.1 in immune rabbit serum and 77.3% +/- 11.0 in immune milk. Phagocytosis of parasites by macrophages also was enhanced after incubation in immune rabbit serum (48.0% +/- 4.0) and immune mouse milk (35.0% +/- 5.0) when compared with the percentage of trophozoites ingested when cells and parasites were incubated in RPMI-10% FCS (3.3% +/- 3.0). Transmission electron microscopy showed ingestion of parasites by neutrophils or macrophages after 15 min of incubation. Morphologic evidence of intracellular parasite injury was observed at 6 hr. A decrease in parasite infectivity also resulted when trophozoites were incubated with neutrophils or macrophages and a source of antibodies, and intragastrically fed to weanling mice. These observations show that both antitrophozoite IgG, secretory IgA, and mouse phagocytic cells interact in vitro to promote parasite clearance. Because both the humoral and cellular components of this system are found intraluminally in the small intestine and in milk, they may represent a biologically relevant protective response against giardiasis.     

Lysosomes of Toxoplasma gondii and Their Possible Relation to the Host-Cell Penetration of Toxoplasma Parasites

Lysosome-like structures of Toxoplasma gondii were observed by means of vital staining with acridine orange and by the Gomori technique. These structures were found scattered over the cytoplasm but were often located at one end of the parasite. In comparison with parasites of the inoculum used for infecting HeLa cell cultures, the toxoplasma which had penetrated the HeLa cells revealed a markedly lower percentage of parasites showing lysosomal staining. After the penetration, the number of parasites with demonstrable lysosomes increased successively and, at the time for release of newly formed parasites (at 24 hr), the majority of the parasites demonstrated lysosome-like bodies in the cytoplasm. The observations are discussed with special reference to the mechanism of host-cell penetration.     

The effects of macrophage source on the mechanism of phagocytosis and intracellular survival of Leishmania

Leishmania spp. protozoa are obligate intracellular parasites that replicate in macrophages during mammalian infection. Efficient phagocytosis and survival in macrophages are important determinants of parasite virulence. Macrophage lines differ dramatically in their ability to sustain intracellular Leishmania infantum chagasi (Lic). We report that the U937 monocytic cell line supported the intracellular replication and cell-to-cell spread of Lic during 72?h after parasite addition, whereas primary human monocyte-derived macrophages (MDMs) did not. Electron microscopy and live cell imaging illustrated that Lic promastigotes anchored to MDMs via their anterior ends and were engulfed through symmetrical pseudopods. In contrast, U937 cells bound Lic in diverse orientations, and extended membrane lamellae to reorient and internalize parasites through coiling phagocytosis. Lic associated tightly with the parasitophorous vacuole (PV) membrane in both cell types. PVs fused with LAMP-1-expressing compartments 24?h after phagocytosis by MDMs, whereas U937 cell PVs remained LAMP-1 negative. The expression of one phagocytic receptor (CR3) was higher in MDMs than U937 cells, leading us to speculate that parasite uptake proceeds through dissimilar pathways between these cells. We hypothesize that the mechanism of phagocytosis differs between primary versus immortalized human macrophage cells, with corresponding differences in the subsequent intracellular fate of the parasite.     

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.     

Purification of Chlamydia trachomatis lymphogranuloma venereum elementary bodies and their interaction with HeLa cells

A procedure has been developed to yield infectious elementary bodies of the lymphogranuloma venereum strains LGV 434 and 404 of Chlamydia trachomatis, labelled during intracellular growth in HeLa 229 cells. The final preparation, obtained after velocity sedimentation of a polycarbonate membrane-filtered sample through a sucrose gradient, is free of host proteins and, more importantly, of chlamydial reticulate bodies. Using such purified preparations, it was found that the association of LGV 434 elementary bodies with HeLa 229 cultures was unaffected by the pretreatment of the host cells with a variety of lectins or with neuraminidases from Clostridium perfringens and Vibrio cholerae. The association was inhibited by dextran sulphate and by mild trypsin treatment of HeLa cultures. Treatment of purified elementary bodies with trypsin, chymotrypsin, neuraminidases and a variety of carbohydrates and lectins did not produce any change in the rate of association with HeLa cultures. Heat-inactivated elementary bodies were significantly less able to associate with the host cells.