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Query: UNIPROT:P06889 (Mol)
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The metabolically inactive developmental form of Chlamydia trachomatis, the elementary body, contains two very basic DNA-binding proteins with homology to eukaryotic histone H1. One of these, Hc1, is relatively well characterized and induces DNA condensation in vitro, whereas the other, Hc2, is functionally virtually uncharacterized. In this study we describe the purification of Hc2, and a detailed comparative functional analysis of Hc2 and Hc1 is presented. By gel shift assays and electron microscopy, marked differences in the nucleic acid-binding properties of Hc2 and Hc1 were observed. Furthermore, Hc2 was found to strongly inhibit translation and transcription in vitro. Our results imply that DNA condensation is not the only function of Hc2.
Mol Microbiol 1996 Apr
PMID:Purification of recombinant Chlamydia trachomatis histone H1-like protein Hc2, and comparative functional analysis of Hc2 and Hc1. 873 29

The gene kdtA of Chlamydia pneumoniae strain TW-183, encoding the enzyme 3-deoxy-alpha-D-manno-octulosonic acid (Kdo) transferase of lipopolysaccharide biosynthesis, was cloned and sequenced. A single open reading frame of 1314 bp was identified, the deduced amino acid sequence of which revealed 69% similarity and 43% identity with KdtA of Chlamydia trachomatis and Chlamydia psittaci. The gene was expressed in the Gram-positive host Corynebacterium glutamicum and the primary gene product was characterized as a multifunctional glycosyltransferase. Cell-free extracts generated in vitro the genus-specific epitope of Chlamydia composed of the trisaccharide alphaKdo(2-8)alphaKdo(2-4)alphaKdo. The results show that a single polypeptide affords three different glycosidic bonds, which is in contradiction to the dogma of glycobiology: 'one enzyme - one glycosidic bond'.
Mol Microbiol 1995 Nov
PMID:Molecular cloning, sequence analysis, and functional characterization of the lipopolysaccharide biosynthetic gene kdtA encoding 3-deoxy-alpha-D-manno-octulosonic acid transferase of Chlamydia pneumoniae strain TW-183. 874 24

Chlamydia trachomatis is a nucleotide parasite, being entirely dependent on its host eukaryotic cell for a supply of ATP, GTP, and UTP. Chlamydiae are not, however, auxotrophic for CTP, as they are able both to transport CTP from the host and synthesize CTP de novo via a chlamydial CTP synthetase. This study addresses the developmental regulation of CTP synthetase over the course of the C. trachomatis life cycle. Given the distinct life stages of C. trachomatis, analysis of temporal changes in gene expression and regulation of protein activity is the key to unravelling the mechanism of pathogenesis of this bacterium. The results of immunodetection analysis indicate that CTP synthetase is present in C. trachomatis elementary bodies and reticulate bodies and that it is widespread in other chlamydial strains. Reverse transcriptase-polymerase chain reaction (RT-PCR) and metabolic labelling experiments show that CTP synthetase is transcribed and translated primarily during the mid- and late stages of the chlamydial growth cycle. In addition, C. trachomatis CTP synthetase was transcribed with the CTP utilizing enzyme CMP-2-keto-3-deoxy-octanoic acid synthetase (CMP-KDO synthetase) as part of a polycistronic mRNA. The co-expression of these two enzymes suggests a role for CTP synthetase in lipopolysaccharide biosynthesis, potentially channelling CTP directly to CMP-KDO synthetase. The ability of the intact operon to complement CTP synthetase and CMP-KDO deficiencies in mutant Escherichia coli strains indicates that both enzymes are efficiently translated from a single messenger RNA. Kinetic analysis revealed that the C. trachomatis CTP synthetase possessed co-operativity patterns typical of both prokaryotic and eukaryotic CTP synthetases. However, the K(m) of the enzyme for UTP was lower than that of E. coli CTP synthetase, presumably in response to the low intracellular concentration of this nucleotide in C. trachomatis.
Mol Microbiol 1996 Nov
PMID:Chlamydia trachomatis CTP synthetase: molecular characterization and developmental regulation of expression. 895 11

Chlamydiae are obligate intracellular bacteria that replicate within a non-acidified vacuole called an inclusion. Chlamydia psittaci (strain GPIC) produces a 39 kDa protein (IncA) that is localized to the inclusion membrane. While IncA is present as a single 39 kDa species in purified reticulate bodies, two additional higher M(r) forms are found in C. psittaci-infected cells. This finding suggested that IncA may be post-translationally modified in the host cell. Here we present evidence that IncA is a serine/threonine phosphoprotein that is phosphorylated by host cell enzymes. This conclusion is supported by the following experimental findings: (i) treatment of infected cells with inhibitors of host cell phosphatases or kinases altered the electrophoretic migration pattern of IncA; (ii) treatment with calf intestinal alkaline phosphatase eliminated the multiple-banding pattern of IncA, leaving only the protein band with the lowest relative molecular weight; and (iii) radioimmunoprecipitation of lysates of [32P]-orthophosphate-labelled infected HeLa cells with anti-IncA antisera demonstrated that the two highest M(r) IncA bands were phosphorylated. A vaccinia-virus recombinant expressing incA was used to determine if HeLa cells can phosphorylate IncA in the absence of a chlamydial background. IncA in lysates of these cells migrated identically to that seen in C. psittaci-infected cells, indicating the host cell was responsible for the phosphorylation of the protein. Microinjection of fluorescently labelled anti-IncA antibodies into C. psittaci-infected HeLa cells resulted in immunostaining of the outer face of the inclusion membrane. Collectively, these results demonstrate that IncA is phosphorylated by the host cell, and regions of IncA are exposed at the cytoplasmic face of the inclusion.
Mol Microbiol 1997 Apr
PMID:Chlamydia psittaci IncA is phosphorylated by the host cell and is exposed on the cytoplasmic face of the developing inclusion. 914 Sep 78

In a comparison of commercial ligase chain reaction (LCR; Abbott) and polymerase chain reaction (PCR; Roche) assays, measuring plasmid genes of Chlamydia trachomatis, some specimens were found to be negative by either or both assays but positive in traditional culture or antigen detection tests. Of 767 women, 35 were found to be infected by cervical or urine testing. Twenty three specimens from 16 women may have contained inhibitors in six cervical swabs (CS) and 15 first void urines (FVU). By performing dilution and 'spiking' experiments on five FVU, inhibitors of PCR, LCR or both, which disappeared by dilution, were demonstrated. Confirmatory assays were used which amplified segments of the major outer membrane gene by PCR or LCR. When comparisons of assays were made on a single specimen type, the sensitivities of the amplification assays, compared to an expanded reference standard, were as follows: on CS, PCR was 93.8% (30/32) and LCR was 96.9% (31/32); on FVU, PCR was 76.6% (23/30) and LCR was 93.3% (28/30). When a combined calculation was made to determine the ability of the assays to detect patients infected in the cervix or urethra by testing FVU, the sensitivities dropped to 71.4% (25/35) for PCR and 80.0% (28/35) for LCR: CS sensitivity was 88.6% (31/35) for both amplified tests. There were two CS and five FVU false-positives by PCR which reduced to one CS and three FVU in the combined analysis. There were no false-positives by LCR. Inhibitors and low levels of chlamydial plasmid nucleic acids may have contributed to lower than expected sensitivities, suggesting a possible need for internal positive controls, especially for PCR, when testing urine. More studies with multiple sampling and more than one amplification assay are needed to confirm these findings and to identify and remove inhibitors of amplification assays.
Mol Cell Probes 1997 Aug
PMID:Urinary inhibitors of polymerase chain reaction and ligase chain reaction and testing of multiple specimens may contribute to lower assay sensitivities for diagnosing Chlamydia trachomatis infected women. 928 9

Four genes of Chlamydia psittaci strain guinea pig inclusion conjunctivitis (GPIC), whose predicted products are highly homologous to structural and regulatory components of a contact-dependent or type III secretion apparatus, were isolated. Related to genes present in several animal and plant bacterial pathogens, these genes may represent a section of a previously undetected chromosomal virulence locus analogous to several recently described virulence-associated type III secretion loci. The existence of contact-dependent secretion in Chlamydia strongly suggests that these bacteria use pathogenic mechanisms that are similar to those of other intracellular bacterial pathogens. Unlike other intracellular bacteria, however, chlamydiae are metabolically inactive extracellularly and only become capable of global protein synthesis several hours after infection. This implies that chlamydial contact-dependent secretion is only active from within, uniquely after the bacteria have been internalized by eukaryotic cells. The possible role(s) of this pathway in chlamydial pathogenesis are discussed.
Mol Microbiol 1997 Jul
PMID:Type III secretion genes identify a putative virulence locus of Chlamydia. 928 47

The host cell cytoskeleton is known to play a vital role in the life cycles of several pathogenic intracellular microorganisms by providing the basis for a successful invasion and by promoting movement of the pathogen once inside the host cell cytoplasm. McCoy cells infected with Chlamydia trachomatis serovars E or L2 revealed, by indirect immunofluorescence microscopy, collocation of microtubules and Chlamydia-containing vesicles during the process of migration from the host cell surface to a perinuclear location. The vast majority of microtubule-associated Chlamydia vesicles also collocated with tyrosine-phosphorylated McCoy cell proteins. After migration, the Chlamydia-containing vesicles were positioned exactly at the centre of the microtubule network, indicating a microtubule-dependent mode of chlamydial redistribution. Inhibition of host cell dynein, a microtubule-dependent motor protein known to be involved in directed vesicle transport along microtubules, was observed to have a pronounced effect on C. trachomatis infectivity. Furthermore, dynein was found to collocate with perinuclear aggregates of C. trachomatis E and L2 but not C. pneumoniae VR-1310, indicating a marked difference in the cytoskeletal requirements for C. trachomatis and C. pneumoniae during early infection events. In support of this view, C. pneumoniae VR-1310 was shown to induce much less tyrosine phosphorylation of HeLa cell proteins during uptake than that seen for C. trachomatis.
Mol Microbiol 1997 Aug
PMID:Chlamydia trachomatis utilizes the host cell microtubule network during early events of infection. 930 7

Following binding and internalization into the host cell cytoplasm, elementary bodies (EB) of the obligate intracellular bacterium Chlamydia trachomatis undergo a developmental process resulting in production of reticulate bodies (RB), the vegetative growth form of the organism. EB are metabolically inactive, but EB to RB transformation requires bacterial protein synthesis. Using HeLa cells infected with EB of C. trachomatis serovar C, we examined the time of first appearance of transcripts from several genes whose products are required for assembly and function of the chlamydial protein synthetic system. We monitored appearance of chlamydial RNAs using reverse transcription-polymerase chain reaction assays targeting primary transcripts from the bacterial rRNA operons, and mRNAs encoding the glycyl tRNA synthetase and the ribosomal proteins S5 and L5. Transcripts from the proximal rRNA promoters, and those from the r-protein and tRNA synthetase genes, are detectable as early as 4 h after EB-host binding; transcripts from distal rRNA promoters do not appear until 6 h post-infection. Thus, expression of bacterial genes whose products are required for protein synthesis begins earlier in chlamydial EB to RB development than previously thought.
Mol Gen Genet 1997 Aug
PMID:Genes required for assembly and function of the protein synthetic system in Chlamydia trachomatis are expressed early in elementary to reticulate body transformation. 932 68

A simple assay format was developed for the direct detection of C. trachomatis rRNA utilizing ligation of recombinant MDV-1 probe RNA fragments hybridized to 23S rRNA after capture and release from a solid support. Assay background (equivalent to 10(4) targets) was suppressed by blocking sequences in the 5' MDV reporter probe fragment complementary to the 3' fragment by prehybridization of a DNA oligonucleotide. A pair of reporter fragments bearing a deletion within the region, obtained by a hydrid-selection-amplification protocol, yielded a low level of assay background which was reduced to < 2% with a blocker directed against the remaining pairing sequence. This probe set showed a sensitivity of 10(3) molecules of 23S rRNA (> 95% responding) and could detect a single elementary body (EB) of Chlamydia trachomatis or 1-10 EB added to a clinical matrix of pooled negative human cervical swab samples. The time of first appearance of amplification products by real-time fluorescence detection showed a linear response to log increases in the target level over a 10(5)-fold range, permitting the determination of target level within an order of magnitude. The assay showed approximately 10(9)-fold discrimination over Chlamydia pneumonae (TWAR) rRNA. High levels of cultured C. albicans, E. coli, S. aureus, or N. gonorrhoeae had no detectable effect on assay background or the ability to detect a single elementary body.
Mol Cell Probes 1997 Dec
PMID:Rapid and sensitive detection of Chlamydia trachomatis using a ligatable binary RNA probe and Q beta replicase. 950 Aug 10

Infections of the eye and genital tract with the bacterium Chlamydia trachomatis are a major cause of morbidity worldwide and are costly to treat. Development of a vaccine capable of protecting against infection or severe disease presents special challenges but would be the most effective long-term option for control of chlamydial disease. Progress has been made in understanding protective and pathological immune mechanisms in these infections, and a number of potential vaccine candidates have been developed.
Mol Med Today 1998 Apr
PMID:Vaccines against Chlamydia: approaches and progress. 957 58


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