67 research outputs found
The Impact of Lateral Gene Transfer in Chlamydia
Lateral gene transfer (LGT) facilitates many processes in bacterial ecology and pathogenesis, especially regarding pathogen evolution and the spread of antibiotic resistance across species. The obligate intracellular chlamydiae, which cause a range of diseases in humans and animals, were historically thought to be highly deficient in this process. However, research over the past few decades has demonstrated that this was not the case. The first reports of homologous recombination in the Chlamydiaceae family were published in the early 1990s. Later, the advent of whole-genome sequencing uncovered clear evidence for LGT in the evolution of the Chlamydiaceae, although the acquisition of tetracycline resistance in Chlamydia (C.) suis is the only recent instance of interphylum LGT. In contrast, genome and in vitro studies have shown that intraspecies DNA exchange occurs frequently and can even cross species barriers between closely related chlamydiae, such as between C. trachomatis, C. muridarum, and C. suis. Additionally, whole-genome analysis led to the identification of various DNA repair and recombination systems in C. trachomatis, but the exact machinery of DNA uptake and homologous recombination in the chlamydiae has yet to be fully elucidated. Here, we reviewed the current state of knowledge concerning LGT in Chlamydia by focusing on the effect of homologous recombination on the chlamydial genome, the recombination machinery, and its potential as a genetic tool for Chlamydia
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Virulence factors of Aeromonas salmonicida and their interaction with the salmonid host
Selected secreted and cellular virulence factors of Aeromonas
salmonicida were examined. A protocol was developed for the
separation of two secreted proteases (P1 and P2 protease), and
a trout erythrocyte specific hemolysin (T-lysin) from
supernatants of cultures of the bacterium. Distinctions between
the proteases were demonstrated using molecular weight
determinations, substrate specificities, sensitivity to chemical
protease inhibitor sensitivities, and polyacrylamide gel
electrophoresis using gels containing protease substrates (G-PAGE).
P1, but not P2, protease was detected in G-PAGE
analyses of protease from lesions of coho salmon (Oncorhynchus
kisutch) infected by injection. Other proteases of apparent host
origin were also detected in these assays. Analysis of the T-lysin
demonstrated that although the bacterium produced high
titers of the enzyme in vitro, no hemolytic activity was detected in vivo nor in cultures grown in salmonid sera. Subsequent
experiments demonstrated that salmonid sera possess an
inhibitor of hemolysis capable of protecting erythrocytes from
enzymatic or chemical lysis. The inhibitor was partially
purified using molecular sieve chromatography and preparative
isoelectric focusing. Analysis of P1 protease, P2 protease, and
T-lysin production was continued by examining their
production in the presence of salmonid sera and in the presence
of high concentrations of selected salts added to brain heart
infusion broth (BHI). The spectrum of proteases produced in
serum was similar to the spectrum produced in BHI. However,
a larger phenylmethylsufonyl fluoride sensitive fraction was
detected in supernatants from bacterial cells grown in serum.
Analysis of supernatants from the cultures grown in high salts
indicated that P1 protease and T-lysin production were
inhibited by these salts but P2 protease production was not.
Growth in high concentrations of magnesium salts also affected
the cellular morphology of the bacterium and this effect was
associated with the presence of an outer membrane protein
layer, the A layer.
Four monoclonal antibodies (Mabs) were produced with
specificity towards A. salmonicida lipopolysaccharide (LPS).
These Mabs were used to identify two distinct epitopes on LPS
and to show that the presence of each epitope varied among
different strains. The antibodies were also used to demonstrate
the difference in the host response of rabbits and rainbow trout
(Oncorhynchus mykiss) to A. salmonicida
Intrastrain and interstrain genetic variation within a paralogous gene family in Chlamydia pneumoniae
BACKGROUND: Chlamydia pneumoniae causes human respiratory diseases and has recently been associated with atherosclerosis. Analysis of the three recently published C. pneumoniae genomes has led to the identification of a new gene family (the Cpn 1054 family) that consists of 11 predicted genes and gene fragments. Each member encodes a polypeptide with a hydrophobic domain characteristic of proteins localized to the inclusion membrane. RESULTS: Comparative analysis of this gene family within the published genome sequences provided evidence that multiple levels of genetic variation are evident within this single collection of paralogous genes. Frameshift mutations are found that result in both truncated gene products and pseudogenes that vary among isolates. Several genes in this family contain polycytosine (polyC) tracts either upstream or within the terminal 5' end of the predicted coding sequence. The length of the polyC stretch varies between paralogous genes and within single genes in the three genomes. Sequence analysis of genomic DNA from a collection of 12 C. pneumoniae clinical isolates was used to determine the extent of the variation in the Cpn 1054 gene family. CONCLUSIONS: These studies demonstrate that sequence variability is present both among strains and within strains at several of the loci. In particular, changes in the length of the polyC tract associated with the different Cpn 1054 gene family members are common within each tested C. pneumoniae isolate. The variability identified within this newly described gene family may modulate either phase or antigenic variation and subsequent physiologic diversity within a C. pneumoniae population
Cytokinesis is blocked in mammalian cells transfected with Chlamydia trachomatis gene CT223
<p>Abstract</p> <p>Background</p> <p>The chlamydiae alter many aspects of host cell biology, including the division process, but the molecular biology of these alterations remains poorly characterized. Chlamydial inclusion membrane proteins (Incs) are likely candidates for direct interactions with host cell cytosolic proteins, as they are secreted to the inclusion membrane and exposed to the cytosol. The <it>inc </it>gene <it>CT223 </it>is one of a sequential set of orfs that encode or are predicted to encode Inc proteins. CT223p is localized to the inclusion membrane in all tested <it>C. trachomatis </it>serovars.</p> <p>Results</p> <p>A plasmid transfection approach was used to examine the function of the product of <it>CT223 </it>and other Inc proteins within uninfected mammalian cells. Fluorescence microscopy was used to demonstrate that <it>CT223</it>, and, to a lesser extent, adjacent <it>inc </it>genes, are capable of blocking host cell cytokinesis and facilitating centromere supranumeracy defects seen by others in chlamydiae-infected cells. Both phenotypes were associated with transfection of plasmids encoding the carboxy-terminal tail of CT223p, a region of the protein that is likely exposed to the cytosol in infected cells.</p> <p>Conclusion</p> <p>These studies suggest that certain Inc proteins block cytokinesis in <it>C. trachomatis</it>-infected cells. These results are consistent with the work of others showing chlamydial inhibition of host cell cytokinesis.</p
Beyond Tryptophan Synthase: Identification of Genes That Contribute to Chlamydia trachomatis Survival during Gamma Interferon-Induced Persistence and Reactivation
Chlamydia trachomatis can enter a viable but nonculturable state in vitro termed persistence. A common feature of C. trachomatis persistence models is that reticulate bodies fail to divide and make few infectious progeny until the persistence-inducing stressor is removed. One model of persistence that has relevance to human disease involves tryptophan limitation mediated by the host enzyme indoleamine 2,3-dioxygenase, which converts l-tryptophan to N-formylkynurenine. Genital C. trachomatis strains can counter tryptophan limitation because they encode a tryptophan-synthesizing enzyme. Tryptophan synthase is the only enzyme that has been confirmed to play a role in interferon gamma (IFN-γ)-induced persistence, although profound changes in chlamydial physiology and gene expression occur in the presence of persistence-inducing stressors. Thus, we screened a population of mutagenized C. trachomatis strains for mutants that failed to reactivate from IFN-γ-induced persistence. Six mutants were identified, and the mutations linked to the persistence phenotype in three of these were successfully mapped. One mutant had a missense mutation in tryptophan synthase; however, this mutant behaved differently from previously described synthase null mutants. Two hypothetical genes of unknown function, ctl0225 and ctl0694, were also identified and may be involved in amino acid transport and DNA damage repair, respectively. Our results indicate that C. trachomatis utilizes functionally diverse genes to mediate survival during and reactivation from persistence in HeLa cells
Interrogating Genes That Mediate Chlamydia trachomatis Survival in Cell Culture Using Conditional Mutants and Recombination
Intracellular bacterial pathogens in the family Chlamydiaceae are causes of human blindness, sexually transmitted disease, and pneumonia. Genetic dissection of the mechanisms of chlamydial pathogenicity has been hindered by multiple limitations, including the inability to inactivate genes that would prevent the production of elementary bodies. Many genes are also Chlamydia-specific genes, and chlamydial genomes have undergone extensive reductive evolution, so functions often cannot be inferred from homologs in other organisms. Conditional mutants have been used to study essential genes of many microorganisms, so we screened a library of 4,184 ethyl methanesulfonate-mutagenized Chlamydia trachomatis isolates for temperature-sensitive (TS) mutants that developed normally at physiological temperature (37°C) but not at nonphysiological temperatures. Heat-sensitive TS mutants were identified at a high frequency, while cold-sensitive mutants were less common. Twelve TS mutants were mapped using a novel markerless recombination approach, PCR, and genome sequencing. TS alleles of genes that play essential roles in other bacteria and chlamydia-specific open reading frames (ORFs) of unknown function were identified. Temperature-shift assays determined that phenotypes of the mutants manifested at distinct points in the developmental cycle. Genome sequencing of a larger population of TS mutants also revealed that the screen had not reached saturation. In summary, we describe the first approach for studying essential chlamydial genes and broadly applicable strategies for genetic mapping in Chlamydia spp. and mutants that both define checkpoints and provide insights into the biology of the chlamydial developmental cycle.
IMPORTANCE:
Study of the pathogenesis of Chlamydia spp. has historically been hampered by a lack of genetic tools. Although there has been recent progress in chlamydial genetics, the existing approaches have limitations for the study of the genes that mediate growth of these organisms in cell culture. We used a genetic screen to identify conditional Chlamydia mutants and then mapped these alleles using a broadly applicable recombination strategy. Phenotypes of the mutants provide fundamental insights into unexplored areas of chlamydial pathogenesis and intracellular biology. Finally, the reagents and approaches we describe are powerful resources for the investigation of these organisms
Unraveling the basic biology and clinical significance of the chlamydial plasmid
New evidence indicates that the conserved plasmid shared among Chlamydial species may be key for understanding and vaccinating against these pathogenic bacteria
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Genomic and phenotypic characterization of in vitro-generated Chlamydia trachomatis recombinants
Background: Pre-genomic and post-genomic studies demonstrate that chlamydiae actively recombine in vitro and
in vivo, although the molecular and cellular biology of this process is not well understood. In this study, we
determined the genome sequence of twelve Chlamydia trachomatis recombinants that were generated in vitro
under antibiotic selection. These strains were used to explore the process of recombination in Chlamydia spp.,
including analysis of candidate recombination hotspots, and to correlate known C. trachomatis in vitro phenotypes
with parental phenotypes and genotypes.
Results: Each of the 190 examined recombination events was the product of homologous recombination, and no
candidate targeting motifs were identified at recombination sites. There was a single deletion event in one
recombinant progeny that resulted in the removal of 17.1 kilobases between two rRNA operons. There was no
evidence for preference for any specific region of the chromosome for recombination, and analyses of a total of
over 200 individual recombination events do not provide any support for recombination hotspots in vitro. Two
measurable phenotypes were analyzed in these studies. First, the efficiency of attachment to host cells in the
absence of centrifugation was examined, and this property segregated to regions of the chromosome that carry
the polymorphic membrane protein (Pmp) genes. Second, the formation of secondary inclusions within cells varied
among recombinant progeny, but this did not cleanly segregate to specific regions of the chromosome.
Conclusions: These experiments examined the process of recombination in C. trachomatis and identified tools that
can be used to associate phenotype with genotype in recombinant progeny. There were no data supporting the
hypothesis that particular nucleotide sequences are preferentially used for recombination in vitro. Selected
phenotypes can be segregated by analysis of recombination, and this technology may be useful in preliminary
analysis of the relationship of genetic variation to phenotypic variation in the chlamydiae.Keywords: Attachment, Chlamydia, Secondary inclusions, Recombination, Hotspo
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Enhanced Direct Major Histocompatibility Complex Class I Self-Antigen Presentation Induced by Chlamydia Infection
The direct major histocompatibility complex (MHC) class I antigen presentation pathway ensures intracellular peptides are displayed at the cellular surface for recognition of infected or transformed cells by CD8⁺ cytotoxic T lymphocytes. Chlamydia spp. are obligate intracellular bacteria and, as such, should be targeted by CD8⁺ T cells. It is likely that Chlamydia spp. have evolved mechanisms to avoid the CD8⁺ killer T cell responses by interfering with MHC class I antigen presentation. Using a model system of self-peptide presentation which allows for posttranslational control of the model protein's stability, we tested the ability of various Chlamydia species to alter direct MHC class I antigen presentation. Infection of the JY lymphoblastoid cell line limited the accumulation of a model host protein and increased presentation of the model-protein-derived peptides. Enhanced self-peptide presentation was detected only when presentation was restricted to defective ribosomal products, or DRiPs, and total MHC class I levels remained unaltered. Skewed antigen presentation was dependent on a bacterial synthesized component, as evidenced by reversal of the observed phenotype upon preventing bacterial transcription, translation, and the inhibition of bacterial lipooligosaccharide synthesis. These data suggest that Chlamydia spp. have evolved to alter the host antigen presentation machinery to favor presentation of defective and rapidly degraded forms of self-antigen, possibly as a mechanism to diminish the presentation of peptides derived from bacterial proteins
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The Broad-Spectrum Antiviral Compound ST-669 Restricts Chlamydial Inclusion Development and Bacterial Growth and Localizes to Host Cell Lipid Droplets within Treated Cells
Novel broad-spectrum antimicrobials are a critical component of a strategy for combating antibiotic-resistant pathogens. In this
study, we explored the activity of the broad-spectrum antiviral compound ST-669 for activity against different intracellular bacteria
and began a characterization of its mechanism of antimicrobial action. ST-669 inhibits the growth of three different species
of chlamydia and the intracellular bacterium Coxiella burnetii in Vero and HeLa cells but not in McCoy (murine) cells. The antichlamydial
and anti-C. burnetii activity spectrum was consistent with those observed for tested viruses, suggesting a common
mechanism of action. Cycloheximide treatment in the presence of ST-669 abrogated the inhibitory effect, demonstrating that
eukaryotic protein synthesis is required for tested activity. Immunofluorescence microscopy demonstrated that different chlamydiae
grow atypically in the presence of ST-669, in a manner that suggests the compound affects inclusion formation and organization.
Microscopic analysis of cells treated with a fluorescent derivative of ST-669 demonstrated that the compound localized
to host cell lipid droplets but not to other organelles or the host cytosol. These results demonstrate that ST-669 affects intracellular
growth in a host-cell-dependent manner and interrupts proper development of chlamydial inclusions, possibly through a
lipid droplet-dependent process
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