The use of signature sequences in different proteins to determine the relative branching order of bacterial divisions: evidence that Fibrobacter diverged at a similar time to Chlamydia and the Cytophaga-Flavobacterium-Bacteroides division.
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The phylogenetic placement of the rumen bacterium Fibrobacter succinogenes was determined using a signature sequence approach that allows determination of the relative branching order of the major divisions among Bacteria [Gupta, R. S. (2000) FEMS Microbiol Rev 24, 367-402]. For this purpose, segments of the Hsp60 (groEL), Hsp70 (dnaK), CTP synthase and alanyl-tRNA synthetase genes, which are known to contain signature sequences that are useful for phylogenetic deterministic purposes, were cloned. Using degenerate oligonucleotide primers for highly conserved regions in these proteins, 1.4 kb, 0.75 kb, 401 bp and 171 bp fragments of the Hsp70, Hsp60, CTP synthase and alanyl-tRNA synthetase genes respectively were amplified by PCR, and these fragments were cloned and sequenced. These primers, because of their high degree of conservation, could also be used for cloning these genes from other bacterial species. The Hsp70 homologues from different Gram-negative bacteria contain a 21-23 aa insert that is not found in any Gram-positive bacteria. The presence of this insert in the F. succinogenes Hsp70 supports its placement within the Gram-negative group of bacteria. A conserved insert in F. succinogenes Hsp60 that is commonly present in all bacterial species, except various Gram-positive bacteria, Deinococcus-Thermus groups and green non-sulphur bacteria, provides evidence that F. succinogenes does not belong to these taxa. A particularly useful signature consisting of a 4 aa insert is found in Ala-tRNA synthetase. This insert is present in all proteobacterial homologues as well as in homologues from species belonging to the Chlamydia and Cytophaga-Flavobacterium- Bacteroides (CFB) groups, but it is not found in homologues from any other groups of bacteria. The presence of this insert in F. succinogenes Ala-tRNA synthetase provides evidence that this species is related to these groups. However, two other signatures in CTP synthase and Hsp70 proteins, that are distinctive of the proteobacterial species, are not present in the F. succinogenes homologues. These results provide evidence that F. succinogenes does not belong to the proteobacterial division and thus should be placed in a similar position as the Chlamydia and CFB groups of species. (+info)
Mechanisms of evolution in Rickettsia conorii and R. prowazekii.
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Rickettsia conorii is an obligate intracellular bacterium that causes Mediterranean spotted fever in humans. We determined the 1,268,755-nucleotide complete genome sequence of R. conorii, containing 1374 open reading frames. This genome exhibits 804 of the 834 genes of the previously determined R. prowazekii genome plus 552 supplementary open reading frames and a 10-fold increase in the number of repetitive elements. Despite these differences, the two genomes exhibit a nearly perfect colinearity that allowed the clear identification of different stages of gene alterations with gene remnants and 37 genes split in 105 fragments, of which 59 are transcribed. A 38-kilobase sequence inversion was dated shortly after the divergence of the genus. (+info)
Recombination in the ompA gene but not the omcB gene of Chlamydia contributes to serovar-specific differences in tissue tropism, immune surveillance, and persistence of the organism.
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Sequences of the major outer membrane protein (MOMP) gene (ompA) and the outer membrane complex B protein gene (omcB) from Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci were analyzed for evidence of intragenic recombination and for linkage equilibrium. The Sawyer runs test, compatibility matrices, and index of association analyses provided substantial evidence that there has been a history of intragenic recombination at ompA including one instance of interspecies recombination between the C. trachomatis mouse pneumonitis strain and the C. pneumoniae horse N16 strain. Although none of these methods detected intragenic recombination within omcB, differences in divergence reported in earlier studies suggested that there has been intergenic recombination involving omcB, and the analyses presented in this study are consistent with this. For C. trachomatis, index-of-association analyses suggested a higher degree of recombination for C class than for B class strains and a higher degree of recombination in the downstream half of ompA. In concordance with these findings, many significant breakpoints were found in variable segments 3 and 4 of MOMP for the recombinant strains D/B120, G/UW-57, E/Bour, and LGV-98 identified in this study. We provide examples of how genetic diversity generated by repeated recombination in these regions may be associated with evasion of immune surveillance, serovar-specific differences in tissue tropism, and persistence. (+info)
In vitro and in vivo activities of sitafloxacin against Chlamydia spp.
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The in vitro and in vivo antichlamydial activity of sitafloxacin was investigated. The MICs and minimal chlamydiacidal concentrations of sitafloxacin for various species of chlamydia ranged from 0.031 to 0.125 microg/ml. Sitafloxacin had an excellent therapeutic effect on experimental Chlamydia psittaci pneumonia and was more potent than tosufloxacin, ofloxacin, and ciproflxacin, although slightly less potent than sparfloxacin. (+info)
Utilization of L-cell nucleoside triphosphates by Chlamydia psittaci for ribonucleic acid synthesis.
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Long-term, 32-P-labeled L cells were infected with the obligately intracellular parasite Chlamydia psittaci (strain 6 BC). At 20 h postinfection, [3-H]uridine was added, and the infected cells were sampled at intervals for incorporation of the labels into the uridine triphosphate (UTP) and cytidine triphosphate (CTP) pools of the host L cell and the uridine monophosphate (UMP) and cytidine monophosphate (CMP) in 16S ribosomal ribonucleic acid (RNA) of the parasite. The specific activity of the nucleotides was calculated from the ratio of 3-H to 32-P counts in the nucleotides. The rate of approach to equilibrium labeling of UTP and CTP in L-cell pools and UMP and CMP in 16S RNA from the exogenous uridine label was determined from the increase in the ratios of the specific activities of CTP to UTP and CMP to UMP with time. The rate of approach to equilibrium CMP:UMP labeling of the 16S RNA of C. psittaci was consistent with the rate predicted from the kinetics of labeling of the CTP and UTP pools of the host L cell. In analogous experiments, the rate of approach to equilibrium guanosine monophosphate:adenosine monophosphate labeling of 16S RNA from an exogenous [14-C]adenine label was consistent with the rate predicted from the kinetics of labeling of the purine nucleoside triphosphate pool of the host cell. These results support the concept that members of the genus Chlamydia owe their obligate intracellular mode of reproduction to a requirement for energy intermediates which is fulfilled by the host cell. In addition, evidence was obtained that the total acid-soluble purine nucleoside triphosphate pool of L cells accurately represents the precursors of L-cell 18S ribosomal RNA. (+info)
Ultrastructural cytochemical evidence for the activation of lysosomes in the cytocidal effect of Chlamydia psittaci.
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The cytopathic effect of the polyarthritis strain of Chlamydia psittaci was studied in cultured bovine fetal spleen cells and found to be mediated by the release of lysosomal enzymes into the host cytoplasm during the late stages of chlamydial development. Ultrastructural cytochemical analysis and cell fractionation studies of infected cells revealed a close relationship between the stage of chlamydial development, fine structural features of the host, and localization of lysosomal enzyme activities. After adsorption, chlamydiae entered the host cells by endocytosis. The endocytic vacuoles containing individual chlamydiae and later the inclusion vacuoles containing the different chlamydial developmental forms were always free from lysosomal enzyme activity. Even after extensive multiplication of chlamydiae, lysosomal enzymes remained localized within lysosomes or their precursors in the host cell. Coincident with the process of chlamydial maturation, lysosomal enzymes were released into the host cytoplasm and were always associated with disintegration of host cell constituents and lysis. The chlamydiae appeared to be protected from this lysosomal enzyme activity by the inclusion membrane. After release from the inclusion, elementary bodies maintained their fine structural features, whereas all other chlamydial developmental forms lost their ultrasturctural integrity. (+info)
CADD, a Chlamydia protein that interacts with death receptors.
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We report here the identification of a bacterial protein capable of interacting with mammalian death receptors in vitro and in vivo. The protein is encoded in the genome of Chlamydia trachomatis and has homologues in other Chlamydia species. This protein, which we refer to as "Chlamydia protein associating with death domains" (CADD), induces apoptosis in a variety of mammalian cell lines when expressed by transient gene transfection. Apoptosis induction can be blocked by Caspase inhibitors, indicating that CADD triggers cell death by engaging the host apoptotic machinery. CADD interacts with death domains of tumor necrosis factor (TNF) family receptors TNFR1, Fas, DR4, and DR5 but not with the respective downstream adaptors. In infected epithelial cells, CADD is expressed late in the infectious cycle of C. trachomatis and co-localizes with Fas in the proximity of the inclusion body. The results suggest a role for CADD modulating the apoptosis pathways of cells infected, revealing a new mechanism of host-pathogen interaction. (+info)
The quantity of nitric oxide released by macrophages regulates Chlamydia-induced disease.
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Intracellular bacteria of the genus Chlamydia cause numerous typically chronic diseases, frequently with debilitating sequelae. Genetic determinants of disease susceptibility after infection with Chlamydia bacteria are unknown. C57BL/6 mice develop severe pneumonia and poor immunity against Chlamydia after moderate respiratory infection whereas BALB/c mice are protected from disease and develop vigorous Th1 immunity. Here we show that infected C57BL/6 macrophages release more NO synthesized by NO synthase 2 (NOS2) than BALB/c macrophages and have lower mRNA concentrations of arginase II, a competitor of NOS2 for the common substrate, l-arginine. Reduction, but not elimination, of NO production by incomplete inhibition of NOS2 abolishes susceptibility of C57BL/6 mice to Chlamydia-induced disease. Thus, the quantity of NO released by infected macrophages is the effector mechanism that regulates between pathogenic and protective responses to chlamydial infection, and genes controlling NO production determine susceptibility to chlamydial disease. (+info)