Cell Wall Synthesis by Chlamydia psittaci Growing in L Cells

Biochemical events accompanying changes in structure and behavior of the cell walls of Chlamydia psittaci strain 6BC during its developmental cycle in L cells (mouse fibroblasts) were studied by measuring at short intervals the effect of d-cycloserine and penicillin G on incorporation of labeled intermediates into acid-insoluble fractions of infected L cells in which host incorporation had been inhibited by cycloheximide and into intact chlamydial cells and cell walls separated from the infected L cells. d-Cycloserine enhanced the incorporation of (14)C-l-alanine at all times in the developmental cycle, but the incorporation of (14)C-l-lysine was always inhibited. In parallel experiments, penicillin G had no effect on incorporation of any of these intermediates, but when infected L cells incorporated (14)C-l-alanine in the presence of penicillin G, the labeled alanine was released more rapidly in the subsequent absence of the antibiotic than in its continued presence. When either penicillin G or d-cycloserine was present throughout the developmental cycle, C. psittaci continued to synthesize deoxyribonucleic acid and protein, but at less than normal rates.     

Some aspects of the immune response of koalas (Phascolarctos cinereus) and in vitro neutralization of chlamydia psittaci (koala strains)

Abstract Western-blot analysis was used to study the reaction of koala antisera, two specific polyclonal antibodies and one monoclonal antibody, with chlamydial antigens in koalas infected with Chlamydia psittaci . The koala sera recognized four C. psittaci surface antigens, corresponding to the major outer membrane protein (39.5 kDa), 31 kDa protein, 18 kDa protein and lipopolysaccharide. The S25-23 LPS specific monoclonal antibody inhibited chlamydial infection (55–67%) with both koala strains (type I and type II). Both koala antiserum and rabbit polyclonal antibodies against either type of chlamydia significantly reduced the number of infected cells resulting from type II infections at a dilution of 1 in 20. Rabbit antiserum against type II was effective in neutralizing infection by type II elementary bodies, but was less effective against type I infection. In addition, no koala antiserum was effective in neutralizing type I infection.     

Oxygen-independent inhibition of intracellular Chlamydia psittaci growth by human monocytes and interferon-gamma-activated macrophages

We have demonstrated previously that Chlamydia psittaci grows well in human monocyte-derived macrophages, but to a limited extent in lymphokine-or interferon-gamma (IFN-gamma)-activated macrophages. In this investigation, freshly explanted human monocytes inhibited chlamydial inclusion formation by 85% as compared to macrophages, and the level of inhibition was similar to that exhibited by lymphokine-activated macrophages (79%). To determine whether the oxygen-dependent antimicrobial mechanisms of the mononuclear phagocyte were involved in the inhibition, cells were infected with C. psittaci in the presence of agents that either inhibit the respiratory burst (glucose deprivation) or diminish the effect of H2O2 (catalase). These treatments had no effect on the capacity of monocytes and lymphokine-activated macrophages to restrict chlamydial growth. In addition, monocytes and activated macrophages from an individual with chronic granulomatous disease suppressed chlamydial growth as effectively as normal cells. Oxidatively deficient HeLa and endothelial cells, once stimulated by lymphokine, also displayed normal levels of antichlamydial activity. The induction of this apparently oxygen-independent antichlamydial effect by lymphokine was completely neutralized by a monoclonal anti-IFN-gamma antibody, and could be achieved by treatment with recombinant (r)IFN-gamma alone. These results indicate that the primary antimicrobial mechanism of the human monocyte against C. psittaci is oxygen-independent, and that this response can be effectively stimulated in the macrophage by lymphokine (IFN-gamma).     

Quantitative detection of Chlamydia spp. by fluorescent PCR in the LightCycler

Quantitative detection of intracellular bacteria of the genus Chlamydia by the standard cell culture method is cumbersome and operator dependent. As an alternative, we adapted hot-start PCR to the glass capillary quantitative PCR format of the LightCycler. The optimized PCR was consistently more efficient than commercially available pre-assembled PCRs. Detection by quantitative PCR of as few as single copies of DNA of Chlamydia spp. was accomplished by SYBR Green fluorescence of the dsDNA product and by fluorescence resonance energy transfer (FRET) hybridization probes. The PCRs were 15-fold more sensitive than the cell culture quantitative assay of C. psittaci B577 infectious stock. The number of chlamydial genomes detected by C. psittaci B577 FRET PCR correlated well with cell culture determination of inclusion forming units (IFUs) (r = 0.96, P < 0.0008). When infected tissue samples were analyzed by cell culture and PCR, the correlation coefficient between IFUs and chlamydial genomes was higher with C. psittaci B577 FRET PCR (r = 0.90, P < 0.0004) than with Chlamydia omp1 SYBR Green PCR (r = 0.85, P < 0.002).     

Interaction of L Cells and Chlamydia psittaci: Entry of the Parasite and Host Responses to Its Development

The entry and development of Chlamydia psittaci in the L cell was studied by using purified, infectious parasites at high multiplicity. Entry of the parasite was accomplished by an act of phagocytosis by the host which was independent of an adsorption stage but was temperature-dependent. Kinetic studies of phagocytosis performed with (14)C-amino acid-labeled, purified parasites indicated that the rate of phagocytosis was directly proportional to the multiplicity of inoculation. Electron microscopy of cells infected at high multiplicity with purified infectious C. psittaci showed that phagocytosed chlamydiae were segregated in a host phagocytic vacuole throughout their developmental cycle which consisted of the transition of infecting elementary bodies to reticulate bodies dividing by binary fission, followed by the reemergence of a population of elementary bodies. The process of the transition was examined and a proposed sequence of intermediate bodies is presented. In isopycnic gradients of fractionated, infected L cells, chlamydial phagocytic vacuoles were apparent as a dense band distinct from lysosome and mitochondrion peaks, as indicated by acid phosphatase and cytochrome oxidase activities. Chlamydiae inactivated by heat or neutralized by antiserum were phagocytosed and appeared in lysosomes within 12 hr after infection according to electron microscopy; however, chlamydiae which were continuously inhibited in their development by chloramphenicol were retained intact in the cell for 24 hr without lysosomal response. The possibility of a lysosomal inhibitor on the native parasite is discussed.     

Utilization of exogenous thymidine by Chlamydia psittaci growing in the thymidine kinase-containing and thymidine kinase-deficient L cells.

The incorporation of [3H]thymidine into the deoxyribonucleic acid (DNA) of Chlamydia psittaci (strain 6BC) growing in thymidine kinase (adenosine 5'-triphosphate-thymidine 5'-phosphotransferase, EC 1.7.1.21)-containing L cells, L(TK+), and thymidine kinase-deficient L cells, LM(TK-), was examined by autoradiography. Label was detected over C. psittaci inclusions in L(TK+) but not LM(TK-) cells. No evidence for a chlamydia-specific thymidine kinase activity in either L(TK+) or LM(TK-) cells was obtained. Entry of [3H]thymidine into the DNA of C. psittaci growing in L(TK+) cells was quantitated by measuring label in purified C. psittaci. It was 265 times less efficient than entry into infected host cell DNA. It is concluded that low levels of exogenous thymidine are incorporated into the DNA of C. psittaci and that this incorporation is dependent on a fully competent host thymidine kinase activity. Evidence also is presented that L cells possess at least two thymidine kinase activities, both of which are capable of supplying thymidylate precursors for nuclear DNA synthesis.     

Cell death, BAX activation, and HMGB1 release during infection with Chlamydia

Infection by a number of Chlamydia species leads to resistance of the host cell to apoptosis, followed by induction of host-cell death. In a population of infected cells that displays protection against staurosporine-induced apoptosis among the adherent cells, we find that cells that had been recovered from the supernatant share characteristics of both apoptosis and necrosis, as assayed by the propidium iodide (PI)-annexin V double-labeling technique. Cell death was observed in both an epithelial cell line and primary fibroblasts, although the primary cells had a higher propensity to die through apoptosis than the immortalized cell line. Staurosporine-mediated activation of the pro-apoptotic BCL-2 family member, BAX, was inhibited in the epithelial cell line infected for 32 h with the lymphogranuloma venereum (LGV/L2) but not the murine pneumonitis (MoPn) strain of C. trachomatis, but inhibition of staurosporine-mediated BAX activation disappeared after 48 h of infection with the LGV/L2 strain. Conversely, infection with MoPn (C. muridarum) but not LGV/L2 led to BAX activation after 72 h, as previously reported for shorter (48 h) infection with the guinea pig inclusion conjunctivitis (GPIC) serovar of C. psittaci (C. caviae). These results suggest that the ability to inhibit staurosporine-mediated BAX activation or to activate BAX due to the infection itself may vary as a function of the chlamydial strain. Interestingly, both the epithelial cells and the fibroblasts also released high mobility group box 1 protein (HMGB1) during infection, although much less HMGB1 was released from fibroblasts, consistent with the higher level of apoptosis observed in the primary cells. HMGB1 is released preferentially by necrotic or permeabilized viable cells, but not apoptotic cells. In the extracellular space, HMGB1 promotes inflammation through interaction with specific cell-surface receptors. Higher levels of HMGB1 were also measured in the genital-tract secretions of mice infected vaginally with C. trachomatis, compared to uninfected controls. These results suggest that cells infected with Chlamydia release intracellular factors that may contribute to the inflammatory response observed in vivo.     

In vitro expression of factor-mediated cytotoxic activity generated during the immune response to Chlamydia in the mouse

Supernatant fluid (SF) prepared by mitogen incubation of spleen cells from A/J mice previously immunized against lethal challenge by the 6BC strain of Chlamydia psittaci was cytotoxic for mouse fibroblasts (L cells) infected with 6BC, as detected by the [3H]thymidine release assay and the trypan blue exclusion test. In contrast, SF prepared from spleen cells taken from unimmunized animals (controls) was not cytotoxic when added to infected L cells. No cytotoxicity was observed when SF was added to uninfected L cells. Maximal levels of cytotoxicity were observed only from cells infected with 6BC for at least 26 hr and exposed to SF for greater than 20 hr. Furthermore, the degree of cytotoxicity was dependent on both the dose of Chlamydia administered and the concentration of SF in the medium. We conclude that the capacity to secrete a spleen cell cytotoxic factor is an aspect of the immune response against the obligate intracellular prokaryotic pathogen Chlamydia. Our results indicate that SF-mediated cytotoxicity is induced subsequent to immunization with Chlamydia, and is significantly more pronounced against infected as opposed to uninfected L cells.     

Aspects of the epidemiology of Chlamydia psittaci infection in a population of koalas (Phascolarctos cinereus) in southeastern Queensland, Australia

A population of free-ranging koalas in southeastern Queensland was examined to determine the prevalence of Chlamydia psittaci infections. Although C. psittaci was isolated from 46 of 65 (71%) koalas studied, only six (9%) of these had clinical signs of disease. Most adult females (82%) had back or pouch young present even though 67% of them were infected. There were no significant correlations between age, sex or site of sampling (urogenital versus conjunctival tissues) and the isolation of C. psittaci. No other important bacterial or fungal pathogens were isolated. The complement fixation test had a sensitivity of 7% and a specificity of 94% in detecting chlamydial infections, suggesting that it is unsuitable for use as a screening test. Chlamydia psittaci infection within this population appeared to represent a generally well-balanced host-parasite relationship and few animals had clinical signs of disease. Only four of 27 (15%) healthy koalas infected with C. psittaci followed for 24 wk after sampling developed eye disease or "dirty tail." Two koalas with keratoconjunctivitis recovered without treatment during the study period. Additional factors, including the stresses imposed by loss of habitat, may act to produce overt disease in koalas with latent C. psittaci infections.     

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.     

Effect of Ionizing Irradiation on Susceptibility of McCoy Cell Cultures to Chlamydia trachomatis

The effect of graded doses of irradiation (cobalt-60) on the morphology of McCoy cells was analyzed, and 4,000 to 5,000 r was selected as a satisfactory dose for production of giant cells. The susceptibility of radiation-induced giant cells to chlamydial infection was compared with that of nonirradiated cells by using three strains of Chlamydia trachomatis and one of C. psittaci. Monolayers of giant cells were more susceptible than normal McCoy cells as indicated by (i) greater numbers of inclusions (four- to eightfold) per unit area of monolayer, (ii) larger inclusions (fourfold greater in area), (iii) higher infective titers (1 log or more greater) of harvested cells, and (iv) greater ease of promoting a second cycle of growth. Graded doses of irradiation were applied also to mouse fibroblast (L) cells, and a similar increase in susceptibility to chlamydial infection was noted. It is concluded that giant cells produced by irradiation possess advantages over nonirradiated cells in culture for growth of Chlamydia.     

Antichlamydial activity of ofloxacin

The in vitro activity of ofloxacin, a new pyridone-carboxylic acid, against 11 strains of Chlamydia trachomatis and six strains of Chlamydia psittaci was determined. All test strains of both species were inhibited by 0.39 micrograms of ofloxacin per ml. The antichlamydial activity of ofloxacin was comparable to that of doxycycline and four- to eightfold less than that of minocycline. The results of this susceptibility test, coupled with those of previous pharmacokinetic analyses of ofloxacin, warrant further evaluation of its clinical usefulness against chlamydial infections.     

Specificity of the microimmunofluorescence assay for the serodiagnosis of Chlamydia pneumoniae infections.

Chlamydia pneumoniae infections are mostly confirmed using an indirect microimmunofluorescence test for which potential cross-reactions between antigens from different chlamydial species are not well documented. Using this assay, 928 sera (507 subjects) submitted for Chlamydia pneumoniae serology were tested for specific IgM and IgG to this bacteria using the TW-183 antigen. IgM and IgG reactivities to Chlamydia trachomatis serotypes C, D, E, and L2 and Chlamydia psittaci strain 6BC antigens were also tested. A sample was interpreted as positive only when evenly fluorescent elementary bodies were observed. Twenty-five subjects (4.9%) showed serological evidence of recent Chlamydia pneumoniae infection (IgM positive and (or) IgG seroconversion); 11 of them also showed serological evidence of recent infection with at least one other chlamydial species. Specificity was 50 and 63% for IgM and IgG detection, respectively. These results suggest that mixed or temporally related infections might occur, or, more likely, that some Chlamydia pneumoniae IgM or IgG reactivities might be due to heterotypic antibodies.     

Effect of Infection with the Meningopneumonitis Agent on Deoxyribonucleic Acid and Protein Synthesis by Its L-Cell Host

Cycloheximide, which had already been shown to inhibit protein synthesis in Earle's L cells (mouse fibroblasts) without having any effect on the multiplication or protein synthesis in Chlamydia psittaci (strain meningopneumonitis) infecting these host cells, also caused greater than 90% inhibition of deoxyribonucleic acid (DNA) synthesis in L cells after a 3-hr exposure to the drug. L cells infected with the meningopneumonitis agent and treated with cycloheximide were used to follow meningopneumonitis-specific DNA synthesis during intracellular growth of the parasite. The rate at which labeled precursors were incorporated into parasite DNA doubled every 2 hr. The effect of meningopneumonitis infection on L-cell DNA and protein synthesis was investigated in logarithmically growing and in stationary-phase (nondividing) populations of L cells. Host-specific DNA and protein synthesis appeared to be inhibited in infected L cells when compared with logarithmically growing control cells, whereas no inhibition was apparent when the comparison was made with stationary-phase control cells. The maximal amount of protein and DNA synthesis that occurred in meningopneumonitis-infected L cells was equal to the amount of DNA and protein synthesized in logarithmically growing, uninfected L cells. A possible explanation of these results is given.     

Immunoelectron microscopy of Chlamydia psittaci with monoclonal antibodies.

An immunoelectron microscopic study was performed to determine the distribution of antigenic components on particles of Chlamydia psittaci and infected cells using a number of monoclonal antibodies (MAbs). Of three anti-lipopolysaccharide (LPS) antibodies (4D5, A2 and 4G5), two antibodies (4D5 and A2) reacted with the surface of reticulate bodies (RBs) but not with that of elementary bodies (EBs). The other antibody (4G5) reacted with both EBs and RBs. Examination of infected cells in thin sections revealed that 4D5 and A2 combined with the membranes of both EBs and RBs. These results indicate that each LPS epitope localized at a different position in the chlamydial membrane. Most MAbs directed to protein antigens reacted on the surface of both EBs and RBs though 3E9 specific for the 90 kDa and 50 kDa protein components combined with RBs only.     

The Chlamydia trachomatis IncA protein is required for homotypic vesicle fusion

Chlamydiae replicate within an intracellular vacuole, termed an inclusion, that is non-fusogenic with vesicles of the endosomal or lysosomal compartments. Instead, the inclusion appears to intersect an exocytic pathway from which chlamydiae intercept sphingomyelin en route from the Golgi apparatus to the plasma membrane. Chlamydial protein synthesis is required to establish this interaction. In an effort to identify those chlamydial proteins controlling vesicle fusion, we have prepared polyclonal antibodies against several Chlamydia trachomatis inclusion membrane proteins. Microinjection of polyclonal antibodies against three C. trachomatis inclusion membrane proteins, IncA, F and G, into the cytosol of cells infected with C. trachomatis demonstrates reactivity with antigens on the cytoplasmic face of the inclusion membrane, without apparent inhibition of chlamydial multiplication. Microinjection of antibodies against the C. trachomatis IncA protein, however, results in the development of an aberrant multilobed inclusion structure remarkably similar to that of C. psittaci GPIC. These results suggest that the C. trachomatis IncA protein is involved in homotypic vesicle fusion and/or septation of the inclusion membrane that is believed to accompany bacterial cell division in C. psittaci . This proposal is corroborated by the expression of C. trachomatis and C. psittaci IncA in a yeast two-hybrid system to demonstrate C. trachomatis , but not C. psittaci , IncA interactions. Despite the inhibition of homotypic fusion of C. trachomatis inclusions, fusion of sphingomyelin-containing vesicles with the inclusion was not suppressed.     

Serological profiling with Chlamycheck, a commercial multiplex recombinant antigen Western blot assay of chlamydial infections

A new chlamydial test system, the Chlamycheck assay, which uses 4 purified recombinant antigens of Chlamydia trachomatis and Chlamydophila pneumoniae and one antigen of Chlamydophila psittaci, has been developed and commercialized. We investigated the reactivities of the recombinant antigens with sera from a group of 30 patients with acute Chlamydia trachomatis infection, 88 patients consulting for sexually transmitted infections, and 46 patients with serological evidence of Chlamydophila pneumoniae infection. The results obtained from human and infected mouse sera suggest that Chlamycheck serology against multiple proteins may provide additional useful information that is not available by conventional whole elementary body microimmunofluorescence or single-antigen enzyme-linked immunosorbent assay serology. Specific serological profiles were associated with acute versus past Chlamydia trachomatis infection or with Chlamydia trachomatis primo-infection versus infection in a Chlamydophila pneumoniae history context.     

A secondary structure motif predictive of protein localization to the chlamydial inclusion membrane

Chlamydiae are obligate intracellular pathogens that spend their entire growth phase sequestered in a membrane-bound vacuole called an inclusion. A set of chlamydial proteins, labelled Inc proteins, has been identified in the inclusion membrane (IM). The predicted IncA, IncB and IncC amino acid sequences share very limited similarity, but a common hydrophobicity motif is present within each Inc protein. In an effort to identify a relatively complete catalogue of Chlamydia trachomatis proteins present in the IM of infected cells, we have screened the genome for open reading frames encoding this structural motif. Hydropathy plot analysis was used to screen each translated open reading frame in the C. trachomatis genome database. Forty-six candidate IM proteins (C-lncs) that satisfied the criteria of containing a bilobed hydrophobic domain of at least 50 amino acids were identified. The genome of Chlamydia pneumoniae encodes a larger collection of C-lnc proteins, and only approximately half of the C-lncs are encoded within both genomes. In order to confirm the hydropathy plot screening method as a valid predictor of C-lncs, antisera and/or monoclonal antibodies were prepared against six of the C. trachomatis C-lncs. Immunofluorescence microscopy of C. trachomatis-infected cells probed with these antibodies showed that five out of six C-lncs are present in the chlamydial IM. Antisera were also produced against C. pneumoniae p186, a protein sharing identity with Chlamydia psittaci lncA and carrying a similar bilobed hydrophobic domain. These antisera labelled the inclusion membrane in C. pneumoniae infected cells, confirming that proteins sharing the unique secondary structural characteristic also localize to the inclusion membrane of C. pneumoniae. Sera from patients with high-titre antibodies to C. trachomatis were examined for reactivity with each tested C-lnc protein. Three out of six tested C-lncs were recognized by a majority of these patient sera. Collectively, these studies identify and characterize novel proteins localized to the chlamydial IM and demonstrate the existence of a potential secondary structural targeting motif for localization of chlamydial proteins to this unique intracellular environment.     

Sequence analysis and lipid modification of the cysteine-rich envelope proteins of Chlamydia psittaci 6BC.

The envelopes of elementary bodies of Chlamydia spp. consist largely of disulfide-cross-linked major outer membrane protein (MOMP) and two cysteine-rich proteins (CRPs). The MOMP gene of Chlamydia psittaci 6BC has been sequenced previously, and the genes encoding the small and large CRPs from this strain were cloned and sequenced in this study. The CRP genes were found to be tandemly arranged on the chlamydial chromosome but could be independently expressed in Escherichia coli. The deduced 87-amino-acid sequence of the small-CRP gene (envA) contains 15 cysteine residues, a potential signal peptide, and a potential signal peptidase II-lipid modification site. Hydropathy plot and conformation analysis of the small-CRP amino acid sequence indicated that the protein was unlikely to be associated with a membrane. However, the small CRP was specifically labeled in host cells incubated with [3H]palmitic acid and may therefore be associated with a membrane through a covalently attached lipid portion of the molecule. The deduced 557-amino-acid sequence of the large-CRP gene (envB) contains 37 cysteine residues and a single putative signal peptidase I cleavage site. In one recombinant clone the large CRP appeared to be posttranslationally cleaved at two sites, forming a doublet in a manner similar to the large-CRP doublet made in native C. psittaci 6BC. Comparison of the deduced amino acid sequences of the CRPs from chlamydial strains indicated that the small CRP is moderately conserved, with 54% identity between C. psittaci 6BC and Chlamydia trachomatis, and the large CRP is highly conserved, with 71% identity between C. psittaci and C. trachomatis and 85% identity between C. psittaci 6BC and Chlamydia pneumoniae. The positions of the cysteine residues in both CRPs are highly conserved in Chlamydia spp. From the number of cysteine residues in the MOMP and the CRPs and the relative incorporation of [35S]cysteine into these proteins, it was calculated that the molar ratio of C. psittaci 6BC elementary body envelope proteins is about one large-CRP molecule to two small-CRP molecules to five MOMP molecules.     

Differential amino acid utilization by Chlamydia psittaci (strain guinea pig inclusion conjunctivitis) and its regulatory effect on chlamydial growth

The effect of omission of individual amino acids from growth medium on the multiplication of Chlamydia psittaci (strain guinea pig inclusion conjunctivitis) in cycloheximide-treated McCoy cells has been examined. Marked differences were observed in the amounts of particular amino acids required for normal chlamydial multiplication: omission of either leucine, phenylalanine or valine completely inhibited multiplication, whereas absence of any one of another 10 amino acids had no effect on numbers of cells infected. Threshold concentrations of 80, 80 and approx. 8 nmol ml-1 for leucine, valine and phenylalanine, respectively, were needed for normal chlamydial multiplication. These requirements could not be related either to unusually high content in the whole organism, to degradation in the medium, or, from studies with leucine, to deficient association of leucine with host cells. Leucine deprivation at late stages of the developmental cycle also appeared to regulate multiplication. Possible mechanisms responsible for these effects are discussed.