Immunization against chlamydial genital infection in guinea pigs with UV-inactivated and viable chlamydiae administered by different routes.

Female guinea pigs were immunized with viable or UV light-inactivated chlamydiae (agent of guinea pig inclusion conjunctivitis), belonging to the species Chlamydia psittaci, by intravenous, subcutaneous, oral, or ocular routes. All animals were then inoculated vaginally with viable chlamydiae to determine the extent of protection against challenge infection induced by the various regimens. The course of genital infection was significantly reduced in intensity in all groups of animals except the unimmunized controls and those animals immunized orally with inactivated antigen. Guinea pigs immunized with viable antigen were more likely to develop resistance to challenge infection and, in general, had a significantly greater degree of protection than animals immunized with inactivated antigen. No one route seemed superior in producing a protective response. Animals in all groups demonstrating protection developed serum and secretion immunoglobulin G antibody responses to chlamydiae. Lymphocyte proliferative reactions to chlamydial antigen were variable among groups. Immunoblot analysis of serum and secretions indicated a wide range of antibody specificities, but most protected animals produced antibodies to the major outer membrane protein, lipopolysaccharide, and the 61-kilodalton protein. No definitive associations could be made between the increased ability of immunization with viable organisms to produce resistance to challenge infection and a particular immune parameter. These data indicate that viable chlamydiae given by various routes are able to induce a strong immune response which can provide resistance against reinfection in some cases or at least reduce the degree of infection to a greater degree than inactivated antigen. However, complete resistance to genital tract infection may be difficult to obtain and alternate immunizations strategies may have to be developed.

Regulation of Chlamydia psittaci (strain guinea pig inclusion conjunctivitis) growth in McCoy cells by amino acid antagonism.

Chlamydiae have amino acid requirements for growth in tissue culture as defined by those amino acids whose individual omission from the growth medium prevents chlamydial multiplication. We have tested the hypothesis that this inhibition of growth arises as a result of antagonism between particular amino acids such that inhibition occurs when the concentration of one amino acid is reduced in the presence of the antagonist amino acid at high concentration. Using the Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC), in the presence of cycloheximide, the requirement for valine was abrogated by the simultaneous omission of isoleucine, that for phenylalanine by simultaneous omission of tryptophan and that for leucine by simultaneous omission of isoleucine plus valine. The antagonism shown between leucine and isoleucine plus valine appears to be unique among bacteria. In the absence of cycloheximide, GPIC had an additional need for tryptophan, tyrosine and isoleucine; these amino acid requirements were shown for both infected McCoy, HeLa and BHK cells. The results are consistent with a mechanism for regulation of parasite growth which depends on the balance of amino acid concentrations in the extracellular environment.

Requirements for ingestion of Chlamydia psittaci by mouse fibroblasts (L cells).

Ingestion of 14C-amino acid-labeled Chlamydia psittaci (6BC) by mouse fibroblasts (L cells) was inhibited when the host cells were incubated for 30 min at 37 degrees C in Earle salts containing 10 mug of crystalline trypsin per ml. Tryptic digestion also inhibited the ingestion of 1-mum polystrene latex beads. Trypsin-treated L cells almost completely recovered their ability to ingest chlamydiae after 4 h at 37 degrees C in medium 199 with 5% fetal calf serum. Cycloheximide (10 mug/ml) blocked this recovery. Heating 14C-amino acid-labeled C. psittaci for 3 min at 60 degrees C inhibited its ingestion by L cells, whereas inactivating it with ultraviolet light was without effect on the ingestion rate. These results show that efficient ingestion of C. psittaci by L cells involves trypsin-labile sites on the host and heat-sensitive sites on the parasite. The failure of excess unlabeled infectious C. psittaci to promote the ingestion of 14C-labeled heat-inactivated chlamydiae suggests that direct interaction between these two sites must occur for uptake to proceed normally.

Immediate toxicity of high multiplicities of Chlamydia psittaci for mouse fibroblasts (L cells).

One hour after suspensions of mouse fibroblasts (L cells) were exposed to 500 to 1,000 L-cell 50% infectious doses of Chlamydia psittaci (6BC), the L cells failed to attach to and spread out on solid substrates, phagocytosed polystyrene latex spheres at reduced rates, incorporated less [14C]isoleucine into protein, and had smaller soluble pools of nucleoside triphosphates. The infected L cells began to die at 8 h and were all dead by 20 h. Lower multiplicities of infection took correspondingly longer to kill the L cells. These effects of high multiplicities of C. psittaci on L cells will be referred to collectively as immediate toxicity. Similar effects were obtained with other strains of C. psittaci and C. trachomatis and with other cell lines. Ultraviolet-inactivated C. psittaci retained the ability to cause immediate toxicity, but heat-inactivated chlamydiae did not. C.psittaci cells had to be ingested by L cells before they were immediately toxic but, once they were phagocytosed, they did not need to multiply or to synthesize macromolecules in order to cause immediate injury to their hosts. Immediate toxicity was not the result of depression of energy metabolism, changes in the levels of intracellular cyclic nucleotides, or release of hydrolases from lysosomes. It was suggested that a lesion is produced in the plasma membrane of the L cell every time it ingests a chlamydial cell, that each act of ingestion produces an independent lesion, and that their injurious effects are additive. Thus, the more ingestion lesions there are, the faster the host cell dies. It was also suggested that induced phagocytosis, inhibition of lysosomal fusion, and death of mice and of cells in culture may all depend on a single activity of C. psittaci.