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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.
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.
Chlamydia trachomatis is an important human pathogen. Research to develop a Chlamydia vaccine has focused on the major outer membrane protein (MOMP). Determinants of this protein elicit serovar-specific neutralizing antibodies which are thought to play a critical role in protective immunity. MOMP-specific antibody responses are highly variable in the polymorphic population. Genetic factors which might influence the MOMP-specific immune response are consequently of particular interest. The C. psittaci strain guinea pig inclusion conjunctivitis (GPIC) is a natural pathogen of the guinea pig that causes both ocular and genital tract infections that closely resemble those caused by C. trachomatis in humans. As such, it provides an excellent model for disease. In this report, we explore the influence of major histocompatibility complex-linked genes on the MOMP-specific antibody response in mice immunized with either whole GPIC elementary bodies or recombinant GPIC MOMP. Our results indicate that the MOMP-specific antibody response is major histocompatibility complex linked such that mice of the H-2d haplotype are high responders while mice of the H-2k haplotype are low responders. We demonstrate that MOMP-specific B cells are present in H-2k strains which are, however, deficient in MOMP-specific helper T cells. Although immunization of low-MOMP-responder strains with whole chlamydial elementary bodies induces high levels of immunoglobulin G antibody specific for Omp2, the cysteine-rich outer membrane protein, MOMP-specific B cells are unable to receive help from Omp2-specific T cells. The failure of intermolecular help from Omp2-specific T cells and related observations raise important issues regarding the processing and presentation of chlamydial antigens and the design of optimal subunit vaccines.
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.
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Chlamydia trachomatis were cultured from the conjunctiva of 8 newborn babies and 4 young adults' (age 20-30 years) affected with acute or therapy-resistant chronic conjunctivitis. The newborn had acute purulent conjunctivitis noticed 4 to 12 days after birth. Five of them had been treated with topical chloramphenicol without effect before the diagnosis was made. They responded well to topical tetracycline ointment, but 3 relapsed. Two developed chronic conjunctivitis and respiratory tract infection, one under long time topical chloramphenicol treatment. The adults had a chronic, in 3 unilateral, conjunctivitis with pronounced follicularly (3) or papillary (1) conjunctival hypertrophy. Different degrees of pseudoptosis, micropannus and corneal infiltrates (in 3) were noticed. In the tear fluid, the quantitative pipette method revealed inflammation shown by neutrophilic granulocytes and lymphocytes with an appreciable predominance of the former in the newborn, while less so in the adults. In the newborn the diagnosis was easily made by culture and by demonstrating inclusions bodies in Giemsa-stained conjunctival scrapings. In the adults the methods were less sensitive. A negative response to one culture does not preclude chlamydial infection.
The nucleotide sequence of nearly 6 kb of genomic DNA located immediately upstream of the omp3-omp2 operon of Chlamydia psittaci strain GPIC was obtained, revealing four significant open reading frames (ORFs), named ORF1, ORF2, ORF4 and ORF5. Searches for homologous sequences in the GenBank/EMBL databases have revealed that: (a) the open-ended ORF1 putatively encodes an homolog of RecJ of Escherichia coli, thought to be required for RecBCD-independent and conjugational recombination, and for UV repair; (b) the predicted translation product of ORF4 is highly homologous to the putative product of EUO, a previously described ORF of avian C. psittaci strain 6BC which is preferentially transcribed early during the life cycle; and (c) ORF5 putatively encodes an homolog of bacterial glutamyl-tRNA synthetases. This analysis establishes the genetic linkage of late (omp3-omp2) and of a proposed early (EUO) genes in Chlamydia.
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Adult inclusion conjunctivitis and neonatal inclusion conjunctivitis are important infectious diseases commonly encountered by ophthalmologists in industrialized countries. Patients with inclusion conjunctivitis are frequently complicated by genitourinary and epipharyngeal chlamydial infections. Thus, treatment of chlamydial conjunctivitis must include systemic antibiotics and not be limited to topical application of antibiotics to the eye. Effective anti-chlamydial agents include tetracyclines, macrolides, and some of the fluoroquinolones. Prolonged and frequent application of ophthalmic agents is vital for successful treatment of conjunctivitis.
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Responding to the pressure of large numbers of trachoma-inclusion conjunctivitis (TRIC)-lymphogranuloma venereum (LGV) isolates from field studies requiring serotyping, we have developed a simplified, less-precise method that utilizes cell culture-grown organisms to produce mouse antisera which is tested agaist prototype TRIC-LGV antigens in the micro-immunofluorescence test. Cell cultures with as few as 5 to 15% of cells showing inclusions produced adequate antibody in mice 4 days after single injection. Knowledge of the reaction of prototype antisera with the antigens has allowed typing of most isolates tested from the pattern of cross-reaction of their antiserum.
Trachoma inclusion conjunctivitis agent infections have a spectrum of clinical presentations which in their more chronic forms are often difficult to recognize. Patients with epithelial erosions of the upper cornea, episodic E.K.C. type subepithelial infiltrates, pannus or micropannus, and with or without lid scarring, merit conjunctival scrapings as an aid to diagnosis. Since the agents which produce these infections have a genital reservoir, as the prevalence of venereal diseases increases, the incidence of this type of keratoconjunctivitis will also increase. If the diagnosis is suspected treatment with systemic tetracycline is effective and relatively safe. Simultaneous treatment of sexual consorts of inclusion conjunctivitis patients and families of trachoma patients should decrease recurrences.