23 research outputs found
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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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Essential Role for the Response Regulator PmrA in Coxiella burnetii Type 4B Secretion and Colonization of Mammalian Host Cells
Successful host cell colonization by the Q fever pathogen, Coxiella burnetii, requires translocation of effector proteins into
the host cytosol by a Dot/Icm type 4B secretion system (T4BSS). In Legionella pneumophila, the two-component system (TCS)
PmrAB regulates the Dot/Icm T4BSS and several additional physiological processes associated with pathogenesis. Because PmrA
consensus regulatory elements are associated with some dot/icm and substrate genes, a similar role for PmrA in regulation of the
C. burnetii T4BSS has been proposed. Here, we constructed a C. burnetii pmrA deletion mutant to directly probe PmrA-mediated
gene regulation. Compared to wild-type bacteria, C. burnetii ΔpmrA exhibited severe intracellular growth defects that coincided
with failed secretion of effector proteins. Luciferase gene reporter assays demonstrated PmrA-dependent expression of 5 of
7 dot/icm operons and 9 of 11 effector-encoding genes with a predicted upstream PmrA regulatory element. Mutational analysis
verified consensus sequence nucleotides required for PmrA-directed transcription. RNA sequencing and whole bacterial cell
mass spectrometry of wild-type C. burnetii and the ΔpmrA mutant uncovered new components of the PmrA regulon, including
several genes lacking PmrA motifs that encoded Dot/Icm substrates. Collectively, our results indicate that the PmrAB TCS is a
critical virulence factor that regulates C. burnetii Dot/Icm secretion. The presence of PmrA-responsive genes lacking PmrA regulatory
elements also suggests that the PmrAB TCS controls expression of regulatory systems associated with the production of
additional C. burnetii proteins involved in host cell parasitism
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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
Peptidoglycan Production by an Insect-Bacterial Mosaic
Peptidoglycan (PG) is a defining feature of bacteria, involved in cell division, shape, and integrity. We previously reported that several genes related to PG biosynthesis were horizontally transferred from bacteria to the nuclear genome of mealybugs. Mealybugs are notable for containing a nested bacteria-within-bacterium endosymbiotic structure in specialized insect cells, where one bacterium, Moranella, lives in the cytoplasm of another bacterium, Tremblaya. Here we show that horizontally transferred genes on the mealybug genome work together with genes retained on the Moranella genome to produce a PG layer exclusively at the Moranella cell periphery. Furthermore, we show that an insect protein encoded by a horizontally transferred gene of bacterial origin is transported into the Moranella cytoplasm. These results provide a striking parallel to the genetic and biochemical mosaicism found in organelles, and prove that multiple horizontally transferred genes can become integrated into a functional pathway distributed between animal and bacterial endosymbiont genomes
Peptidoglycan Production by an Insect-Bacterial Mosaic
Peptidoglycan (PG) is a defining feature of bacteria, involved in cell division, shape, and integrity. We previously reported that several genes related to PG biosynthesis were horizontally transferred from bacteria to the nuclear genome of mealybugs. Mealybugs are notable for containing a nested bacteria-within-bacterium endosymbiotic structure in specialized insect cells, where one bacterium, Moranella, lives in the cytoplasm of another bacterium, Tremblaya. Here we show that horizontally transferred genes on the mealybug genome work together with genes retained on the Moranella genome to produce a PG layer exclusively at the Moranella cell periphery. Furthermore, we show that an insect protein encoded by a horizontally transferred gene of bacterial origin is transported into the Moranella cytoplasm. These results provide a striking parallel to the genetic and biochemical mosaicism found in organelles, and prove that multiple horizontally transferred genes can become integrated into a functional pathway distributed between animal and bacterial endosymbiont genomes
Antibiotic resistance in Chlamydiae
Abstract There are few documented reports of antibiotic resistance in Chlamydia and no examples of natural and stable antibiotic resistance in strains collected from humans. While there are several reports of clinical isolates exhibiting resistance to antibiotics, these strains either lost their resistance phenotype in vitro, or lost viability altogether. Differences in procedures for chlamydial culture in the laboratory, low recovery rates of clinical isolates and the unknown significance of heterotypic resistance observed in culture may interfere with the recognition and interpretation of antibiotic resistance. Although antibiotic resistance has not emerged in chlamydiae pathogenic to humans, several lines of evidence suggest they are capable of expressing significant resistant phenotypes. The adept ability of chlamydiae to evolve to antibiotic resistance in vitro is demonstrated by contemporary examples of mutagenesis, recombination and genetic transformation. The isolation of tetracycline-resistant Chlamydia suis strains from pigs also emphasizes their adaptive ability to acquire antibiotic resistance genes when exposed to significant selective pressure. Keywords antibiotic resistance; Chlamydia; heterotypic resistance; persistence; phenotypic resistance; sexually transmitted infection; trachoma recombination; transformation Chlamydiae are a successful group of obligate intracellular pathogens that cause serious diseases in a wide range of hosts (Box 1). Chlamydial infection of cells is initiated by an infectious, but metabolically inactive, elementary body (EB) that subsequently differentiates into a metabolically active, but noninfectious, reticulate body (RB). All chlamydial development occurs within a membrane bound vacuole termed the inclusion NIH Public Access Genital Sexually transmitted infections caused by C. trachomatis are the most prevalent bacterial cause of sexually transmitted infections worldwide, and around 92 million men and women are estimated to be infecte
Noncanonical Inhibition of mTORC1 by Coxiella burnetii Promotes Replication within a Phagolysosome-Like Vacuole
Coxiella burnetii is an intracellular pathogenic bacterium that replicates within a lysosomal vacuole. Biogenesis of the Coxiella-containing vacuole (CCV) requires effector proteins delivered into the host cell cytosol by the type 4B secretion system (T4BSS). Modifications to lysosomal physiology required for pathogen replication within the CCV are poorly understood. Mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) is a master kinase that regulates lysosome structure and function. Nutrient deprivation inhibits mTORC1, which promotes cell catabolism in the form of accelerated autophagy and increased lysosome biosynthesis. Here, we report that C. burnetii growth is enhanced by T4BSS-dependent inhibition of mTORC1 that does not activate autophagy. Canonical inhibition of mTORC1 by starvation or inhibitor treatment that induces autophagic flux does not benefit C. burnetii growth. Furthermore, hyperactivation of mTORC1 impairs bacterial replication. These findings indicate that C. burnetii inhibition of mTORC1 without accelerated autophagy promotes bacterial growth.The Q fever agent Coxiella burnetii is a Gram-negative bacterium that invades macrophages and replicates inside a specialized lysosomal vacuole. The pathogen employs a type 4B secretion system (T4BSS) to deliver effector proteins into the host cell that modify the Coxiella-containing vacuole (CCV) into a replication-permissive niche. Mature CCVs are massive degradative organelles that acquire lysosomal proteins. Inhibition of mammalian (or mechanistic) target of rapamycin complex 1 (mTORC1) kinase by nutrient deprivation promotes autophagy and lysosome fusion, as well as activation of the transcription factors TFE3 and TFEB (TFE3/B), which upregulates expression of lysosomal genes. Here, we report that C. burnetii inhibits mTORC1 as evidenced by impaired localization of mTORC1 to endolysosomal membranes and decreased phosphorylation of elF4E-binding protein 1 (4E-BP1) and S6 kinase 1 in infected cells. Infected cells exhibit increased amounts of autophagy-related proteins protein 1A/1B-light chain 3 (LC3) and p62 as well as of activated TFE3. However, C. burnetii did not accelerate autophagy or block autophagic flux triggered by cell starvation. Activation of autophagy or transcription by TFE3/B increased CCV expansion without enhancing bacterial replication. By contrast, knockdown of tuberous sclerosis complex 1 (TSC1) or TSC2, which hyperactivates mTORC1, impaired CCV expansion and bacterial replication. Together, these data demonstrate that specific inhibition of mTORC1 by C. burnetii, but not amplified cell catabolism via autophagy, is required for optimal pathogen replication. These data reveal a complex interplay between lysosomal function and host cell metabolism that regulates C. burnetii intracellular growth
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KronmillerBrentCGRBEssentialRoleResponse_DatasetS2.xlsx
Successful host cell colonization by the Q fever pathogen, Coxiella burnetii, requires translocation of effector proteins into
the host cytosol by a Dot/Icm type 4B secretion system (T4BSS). In Legionella pneumophila, the two-component system (TCS)
PmrAB regulates the Dot/Icm T4BSS and several additional physiological processes associated with pathogenesis. Because PmrA
consensus regulatory elements are associated with some dot/icm and substrate genes, a similar role for PmrA in regulation of the
C. burnetii T4BSS has been proposed. Here, we constructed a C. burnetii pmrA deletion mutant to directly probe PmrA-mediated
gene regulation. Compared to wild-type bacteria, C. burnetii ΔpmrA exhibited severe intracellular growth defects that coincided
with failed secretion of effector proteins. Luciferase gene reporter assays demonstrated PmrA-dependent expression of 5 of
7 dot/icm operons and 9 of 11 effector-encoding genes with a predicted upstream PmrA regulatory element. Mutational analysis
verified consensus sequence nucleotides required for PmrA-directed transcription. RNA sequencing and whole bacterial cell
mass spectrometry of wild-type C. burnetii and the ΔpmrA mutant uncovered new components of the PmrA regulon, including
several genes lacking PmrA motifs that encoded Dot/Icm substrates. Collectively, our results indicate that the PmrAB TCS is a
critical virulence factor that regulates C. burnetii Dot/Icm secretion. The presence of PmrA-responsive genes lacking PmrA regulatory
elements also suggests that the PmrAB TCS controls expression of regulatory systems associated with the production of
additional C. burnetii proteins involved in host cell parasitism