A 7.5 kbp cryptic plasmid is found in almost all isolates of Chlamydia trachomatis. Real-time PCR assays, using TaqMan chemistry, were set up to quantify accurately both the chlamydial plasmid and the single copy, chromosomal omcB gene in the infectious, elementary bodies (EBs) of C. trachomatis L1 440. Plasmid copy number was also determined in the EBs of six other lymphogranuloma venereum (LGV) isolates (serovars L1-L3), ten trachoma isolates (serovars A-C) and nine urogenital isolates (serovars D-J). The results indicated an average plasmid copy number of 4.0+/-0.8 (mean+/-95 % confidence interval) plasmids per chromosome. During the chlamydial developmental cycle, up to 7.6 plasmids per chromosome were detected, indicating an increased plasmid copy number in the actively replicating reticulate bodies. Attempts to eliminate the plasmid from strain L1 440 using the plasmid-curing agents ethidium bromide, acridine orange or imipramine/novobiocin led to a paradoxical increase in plasmid copy number. It is speculated that the stress induced by chemical curing agents may stimulate the activity of plasmid-encoded replication (Rep) proteins. In contrast to C. trachomatis, only a single isolate of Chlamydophila pneumoniae bears a plasmid. C. pneumoniae strain N16 supports a 7.4 kbp plasmid in which ORF1, encoding one of the putative Rep proteins, is disrupted by a deletion and split into two smaller ORFs. Similar assay techniques revealed 1.3+/-0.2 plasmids per chromosome (mean+/-95 % confidence interval) in EBs of this strain. These findings are in agreement with the hypothesis that the ORF1-encoded protein is involved in, but not essential for, plasmid replication and control of copy number.

ABSTRACT Chlamydiaphage Chp2 is a member of the family Microviridae , of which bacteriophage φX174 is the type species. Although grouped in the same family, the relationship between the Microviridae coliphages and the Chp2-like viruses, which infect obligate intracellular parasitic bacteria, is quite distant, with major differences in structural protein content and scaffolding protein dependence. To investigate the morphogenesis of Chp2, large particles were isolated from infected Chlamydophila abortus by equilibrium and rate zonal sedimentation. A monoclonal antibody that recognizes only assembled viral coat proteins was used in these detection assays. Thus, the detected particles represent virions and/or postcapsid formation assembly intermediates. Two distinct particle types were detected, differing in both protein and DNA content. Filled particles lacked VP3, the putative internal scaffolding protein, whereas empty particles contained this protein. These results indicate that VP3 is a scaffolding protein and that the isolated VP3-containing particles most likely represent Chp2 procapsids.

The host range of phiCPAR39 is limited to four Chlamydophila species: C. abortus, C. caviae, C. pecorum, and C. pneumoniae. Chp3 (a newly discovered bacteriophage isolated from C. pecorum) shares three of these hosts (C. abortus, C. caviae, and C. pecorum) but can additionally infect Chlamydophila felis. The ability to support replication was directly correlated with the binding properties of the respective bacteriophages with their host species. Binding studies also show that phiCPAR39 and Chp3 use different host receptors to infect the same host cells: cell binding is sensitive to proteinase K treatment, confirming that the chlamydiaphage receptors are proteinaceous in nature.

ABSTRACT A number of bacteriophages belonging to the Microviridae have been described infecting chlamydiae. Phylogenetic studies divide the Chlamydiaceae into two distinct genera, Chlamydia and Chlamydophila , containing three and six different species, respectively. In this work we investigated the biological properties and host range of the recently described bacteriophage Chp2 that was originally discovered in Chlamydophila abortus . The obligate intracellular development cycle of chlamydiae has precluded the development of quantitative approaches to assay bacteriophage infectivity. Thus, we prepared hybridomas secreting monoclonal antibodies (monoclonal antibodies 40 and 55) that were specific for Chp2. We demonstrated that Chp2 binds both C. abortus elementary bodies and reticulate bodies in an enzyme-linked immunosorbent assay. Monoclonal antibodies 40 and 55 also detected bacteriophage Chp2 antigens in chlamydia-infected eukaryotic cells. We used these monoclonal antibodies to monitor the ability of Chp2 to infect all nine species of chlamydiae. Chp2 does not infect members of the genus Chlamydia ( C. trachomatis, C. suis , or C. muridarum ). Chp2 can infect C. abortus, C. felis , and C. pecorum but is unable to infect other members of this genus, including C. caviae and C. pneumoniae , despite the fact that these chlamydial species support the replication of very closely related bacteriophages.

ABSTRACT Comparisons of the proteome of abortifacient Chlamydia psittaci isolates from sheep by two-dimensional gel electrophoresis identified a novel abundant protein with a molecular mass of 61.4 kDa and an isoelectric point of 6.41. C-terminal sequence analysis of this protein yielded a short peptide sequence that had an identical match to the viral coat protein (VP1) of the avian chlamydiaphage Chp1. Electron microscope studies revealed the presence of a 25-nm-diameter bacteriophage (Chp2) with no apparent spike structures. Thin sections of chlamydia-infected cells showed that Chp2 particles were located to membranous structures surrounding reticulate bodies (RBs), suggesting that Chp2 is cytopathic for ovine C. psittaci RBs. Chp2 double-stranded circular replicative-form DNA was purified and used as a template for DNA sequence analysis. The Chp2 genome is 4,567 bp and encodes up to eight open reading frames (ORFs); it is similar in overall organization to the Chp1 genome. Seven of the ORFs (1 to 5, 7, and 8) have sequence homologies with Chp1. However, ORF 6 has a different spatial location and no cognate partner within the Chp1 genome. Chlamydiaphages have three viral structural proteins, VP1, VP2, and VP3, encoded by ORFs 1 to 3, respectively. Amino acid residues in the φX174 procapsid known to mediate interactions between the viral coat protein and internal scaffolding proteins are conserved in the Chp2 VP1 and VP3 proteins. We suggest that VP3 performs a scaffolding-like function but has evolved into a structural protein.

Infection of the endometrium by Neisseria gonorrhoeae is a pivotal stage in the development of pelvic inflammatory disease in women. An ex vivo model of cultures of primary human endometrial cells was developed to study gonococcal–host cell interactions. To facilitate these studies, gonococci were transformed with a hybrid shuttle vector containing the gfp gene from Aequoria victoria, encoding the green fluorescent protein (GFP), to produce intrinsically fluorescent bacteria. The model demonstrated that both pili and Opa proteins were important for both mediating gonococcal interactions with endometrial cells and inducing the secretion of pro‐inflammatory cytokines and chemokines. Pil+ gonococci showed high levels of adherence and invasion, regardless of Opa expression, which was associated with increased secretion of IL‐8 chemokine and reduced secretion of IL‐6 cytokine. Gonococcal challenge also caused increased secretion of TNF‐α cytokine, but this did not correlate with expression of pili or Opa, suggesting that release of components from non‐adherent bacteria may be involved in TNF‐α induction. Thus, the use of cultured primary endometrial cells, together with gonococci expressing green fluorescent protein, has the potential to extend significantly our knowledge, at the molecular level, of the role of this important human pathogen in the immunobiology of pelvic inflammatory disease.

One of the critical developmental events during the unique intracellular life cycle of Chlamydiae is their differentiation from a metabolically active, replicative form or reticulate body (RB) to an infectious extracellular form of the organism (elementary body or EB). This process is characterized by the expression of two extraordinarily cysteine-rich envelope proteins of molecular masses 9 kDa and 60 kDa. We describe the molecular cloning and sequence determination of the 9 kDa cysteine-rich proteins (CrPs) of C. pneumoniae and C. psittaci. Comparison of these 9 kDa CrP amino acid sequences with those of C. trachomatis showed regions of structural variation and conservation. Transcription of the 9 kDa CrP genes occurred as both a monocistronic message and as a bicistronic message which included the 60 kDa CrP gene. Transcription of the 9 kDa and 60 kDa CrP genes was tightly linked to the chlamydial growth cycle with synthesis of their mRNAs and consequent translation of the 60 kDa CrPs occurring as RBs differentiated to form EBs. The maximal rate of transcription occurred late in the growth cycle from a single but highly conserved promoter which had close similarity with the Escherichia coli consensus promoter sequences. A stem and loop structure which could be involved in regulating translation of mRNA occurred in all three species between the transcriptional start point and the ribosome binding site. Although transcription is initiated from a single promoter in all three chlamydial species, transcriptional termination points for the monocistronic and bicistronic mRNAs differ in both number and position.

The 60 kDa cysteine-rich proteins (CrPs) of Chlamydia are developmentally regulated outer envelope proteins synthesized late in the chlamydial growth cycle. These proteins, found only on the extracellular infectious elementary bodies, elicit major antibody responses in chlamydial infection. We have cloned and expressed in Escherichia coli the complete 60 kDa CrP genes from Chlamydia trachomatis, C. psittaci and C. pneumoniae. The recombinant products were expressed as either 'native' proteins or as fusions with the bacteriophage T7 gene 10 protein. Electron microscopy showed that recombinant proteins were produced as insoluble inclusions within the E. coli host cells. The recombinant 60 kDa CrPs were purified and used to raise high titre polyclonal antisera. In immunoblot analysis these antisera reacted with the 60 kDa CrPs from purified elementary bodies of all three chlamydial species in a genus-specific manner. Further molecular analysis allowed the genus-specific cross-reacting epitopes to be localized by using overlapping synthetic peptides covering the C. trachomatis 60 kDa CrP. Immunogold labelling experiments using purified infectious elementary bodies from the three chlamydial species indicated that the 60 kDa CrPs are not surface accessible to antibody binding.

Respiratory syncytial (RS) virus continues to cause serious human respiratory disease and no prophylactic vaccine is yet available. Serum antibodies to RS virus fusion protein (F) that have the appropriate specificities and activities could confer protection against severe RS virus infections. To explore human serum antibody responses to RS virus F we first characterised four epitopes on F and then measured the concentrations of human serum antibodies to these sites for 389 sera. Individuals varied in serum antibody concentration to the epitopes. The distribution patterns of the concentrations of antibodies reactive to each epitope were different. Antigenic variation of F at these epitopes in Southampton RS virus isolates was examined by immunofluorescence. The F proteins from different isolates varied within and between RS virus subtypes which co-circulated in the outbreak of winter 1985-1986. Variations in F detected by immunofluorescence were consistent with differences between the strains' susceptibilities to monoclonal antibody antiviral action.

The major outer-membrane protein (MOMP) of Chlamydia trachomatis is the focus of attention for chlamydial vaccine design, particularly those serovar- and subspecies-specific epitopes which provoke neutralizing immune responses. Selected surface-exposed B-cell epitopes of MOMP, incorporating B-subspecies specificities, were expressed as fusions with LamB, an inducible outer-membrane transport protein of Escherichia coli. These recombinant chlamydial-LamB proteins were correctly transported to the outer membrane of both E. coli and an aro A mutant of Salmonella typhimurium. The immunogenicity of the constructs was investigated in a mouse model of chlamydial salpingitis. After oral immunization, recombinant S. typhimurium were recovered from the livers of mice for up to two weeks, and a serum IgG response was induced both to the Salmonella and to the inserted chlamydial epitopes. By contrast, intravenous inoculation was ineffective. Although these LamB fusions proved only weakly immunogenic, this approach should be useful for investigating the ability of attenuated S. typhimurium vaccines incorporating chlamydial epitopes to stimulate protective mucosal immunity in the mouse model of chlamydial salpingitis.

The major outer-membrane protein (MOMP) of Chlamydia trachomatis is a promising candidate antigen for chlamydial vaccine development. We have sequenced the MOMP genes for a serovar A and a serovar B isolate and have compared these new sequences with those already reported. Intra-serovar changes in the inferred amino acid sequences of the surface-exposed variable segments known to be responsible for binding of neutralizing antibody were observed. Nevertheless, epitope mapping with solid-phase peptides showed that these intra-serovar changes did not affect the binding of serovar- and subspecies-specific, potentially protective antibodies. Variable segment 1 of C. trachomatis serovar A contained two adjacent antibody-binding sites, one of which was C-subspecies specific while the other was serovar A specific. Therefore the subspecies binding site for C-complex organisms is in variable segment 1, whilst that for B-complex organisms is in variable segment 4. This work shows that MOMP sequences are relatively stable within the serovar categorization for isolates taken decades apart from different continents. Within a given serovar, however, limited interchange of functionally related amino acids may occur without impairing the binding of serovar-specific antibody.

RNA was extracted at various times from cells infected with Chlamydia trachomatis serovar L1. Northern-blot analysis showed that transcription of the CrP gene encoding the 60-kDa cysteine-rich outer membrane protein (CrP) produces a temporally controlled polycistronic mRNA. Primer extension analysis indicated the presence of tandem promoters separated by 66 nt with transcriptional start points (tsp) located 577 and 643 nt upstream from the start codon of the mature 60-kDa CrP. Nucleotide (nt) sequencing of this region revealed a small open reading frame (SORF) with coding potential for an 88-amino acid protein containing 13 cysteine residues. This SORF is transcribed as both a polycistronic 2300-nt mRNA together with the CrP gene, and as a separate 480-nt mRNA. Analysis of the upstream sequences, around the tsp for these mRNAs, revealed the presence of three inverted repeat structures that might act as binding domain(s) for a regulatory protein.

The complete nucleotide sequence encoding the major outer membrane protein (MOMP) of Chlamydia psittaci strain A22/M, responsible for enzootic abortion of ewes (EAE), has been determined. An 800bp EcoRI/XbaI fragment containing a portion of the MOMP coding sequence from C. trachomatis serovar L1 was used to probe a λL47.1 genomic library constructed from DNA obtained from C. psittaci EAE A22/M. The recombinant L47.1/EA1 was selected and contained the entire C. psittaci MOMP gene within a 7.5 kb BamHI fragment. The DNA sequence revealed an open reading frame encoding 402 amino acids, including a 22 amino acid signal peptide, which exhibited 17/22 conservation with the signal peptide of C. trachomatis MOMP. The calculated molecular mass of the C. psittaci MOMP was 43 kDa. A comparison of the MOMP genes of C. psittaci and C. trachomatis revealed only 34% nucleotide sequence homology, but 65% amino acid homology.

Antigenic and structural variation in the major nucleocapsid protein, VPN41, from different strains of respiratory syncytial (RS) virus was observed using a combination of monoclonal antibodies and two-dimensional peptide mapping. Limited trypsin treatment of intact nucleocapsids produced two peptide fragments Mr 27K and Mr 14K. Two monoclonal antibodies, N1 and N2, reactive with primary sequence epitopes located on intact nucleocapsids also reacted with either the 27K fragment (N2) or the 14K fragment (N1). Competitive radioimmunoassay studies using N1 and N2 antibodies revealed two discrete antigenic groups among the seven human strains of RS virus examined. A bovine strain of RS virus, although antigenically similar to the human strain of RS virus, was placed in a separate group. Two-dimensional peptide mapping of 125I-labelled VPN41 purified by SDS-PAGE revealed extensive structural homology between all strains. However, several unique tryptic/chymotryptic peptides supported the grouping obtained with the monoclonal antibodies.

Variants of Neisseria gonorrhoeae P9 possessing a, gamma or delta pili were shown to vary in their toxicity and virulence for human epithelial cells. Studies with antisera raised against purified pili showed that attachment and virulence were reduced to a significant degree in the presence of antisera to homologous pili. Heterologous antisera, while capable of agglutinating whole organisms, were largely ineffective in reducing attachment and cytotoxicity. Using an enzyme-linked immunosorbent assay (ELISA) system, the cross-reactivity between pili and heterologous antisera was estimated to be no more than 10-20%.

The invasive properties of nine variants of Neisseria gonorrhoeae strain P9 known to vary in their surface composition have been investigated. Relative virulence was evaluated by their cytotoxic effect on Chang epithelial cell monolayers. Piliated variants P9-2 (with alpha pili) and P9-20 (with beta pili plus protein II) showed increased ability to kill the target cells compared with the prototype P9-1 (lacking pili and additional outer membrane proteins). Two nonpiliated variants, P9-11 (with proteins IIa and IId) and P9-19 (with proteins II and IIc), were also relatively more virulent compared with P9-1. Enhanced attachment was exhibited by both piliated and some nonpiliated variants: beta-piliated P9-20 (with protein II; molecular weight, 29,000) and nonpiliated P9-16 (with protein IIb; molecular weight, 28,000) were the most effective in adherence to the target monolayers.

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Isoelectric focusing showed that Neisseria gonorrhoeae has an overall negative surface charge. Chemical modification of protein amino or carboxyl groups changed the surface charge and thereby altered the ability of the organisms to attach to human amnion cells grown in tissue culture. Attachment of modified and unmodified N. gonorrhoeae was increased by the presence of pili only when the bacteria bore a negative surface charge. Thus an important factor in the pathogenesis of gonorrhoea may be the ability of pili to facilitate attachment of N. gonorrhoeae by overcoming the initial electrostatic repulsive barrier which exists between it and the host cell.