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  • American Society for Microbiology  (11)
  • 1
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    Mutational Analysis of the Chlamydia muridarum Plasticity Zone
    American Society for Microbiology ; 2015
    In:  Infection and Immunity Vol. 83, No. 7 ( 2015-07), p. 2870-2881
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 83, No. 7 ( 2015-07), p. 2870-2881
    Abstract: Pathogenically diverse Chlamydia spp. can have surprisingly similar genomes. Chlamydia trachomatis isolates that cause trachoma, sexually transmitted genital tract infections (chlamydia), and invasive lymphogranuloma venereum (LGV) and the murine strain Chlamydia muridarum share 99% of their gene content. A region of high genomic diversity between Chlamydia spp. termed the plasticity zone (PZ) may encode niche-specific virulence determinants that dictate pathogenic diversity. We hypothesized that PZ genes might mediate the greater virulence and gamma interferon (IFN-γ) resistance of C. muridarum compared to C. trachomatis in the murine genital tract. To test this hypothesis, we isolated and characterized a series of C. muridarum PZ nonsense mutants. Strains with nonsense mutations in chlamydial cytotoxins, guaBA-add , and a phospholipase D homolog developed normally in cell culture. Two of the cytotoxin mutants were less cytotoxic than the wild type, suggesting that the cytotoxins may be functional. However, none of the PZ nonsense mutants exhibited increased IFN-γ sensitivity in cell culture or were profoundly attenuated in a murine genital tract infection model. Our results suggest that C. muridarum PZ genes are transcribed—and some may produce functional proteins—but are dispensable for infection of the murine genital tract.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.00106-15
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2015
    detail.hit.zdb_id: 1483247-1
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  • 2
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    Development of Surface Adhesion in Caulobacter crescentus
    American Society for Microbiology ; 2004
    In:  Journal of Bacteriology Vol. 186, No. 5 ( 2004-03), p. 1438-1447
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 186, No. 5 ( 2004-03), p. 1438-1447
    Abstract: Caulobacter crescentus has a dimorphic life cycle composed of a motile stage and a sessile stage. In the sessile stage, C. crescentus is often found tightly attached to a surface through its adhesive holdfast. In this study, we examined the contribution of growth and external structures to the attachment of C. crescentus to abiotic surfaces. We show that the holdfast is essential but not sufficient for optimal attachment. Rather, adhesion in C. crescentus is a complex developmental process. We found that the attachment of C. crescentus to surfaces is cell cycle regulated and that growth or energy or both are essential for this process. The initial stage of attachment occurs in swarmer cells and is facilitated by flagellar motility and pili. Our results suggest that strong attachment is mediated by the synthesis of a holdfast as the swarmer cell differentiates into a stalked cell.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.186.5.1438-1447.2004
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2004
    detail.hit.zdb_id: 1481988-0
    SSG: 12
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  • 3
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    Mutations in Sugar-Nucleotide Synthesis Genes Restore Holdfast Polysaccharide Anchoring to Caulobacter crescentus Holdfast Anchor Mutants
    American Society for Microbiology ; 2018
    In:  Journal of Bacteriology Vol. 200, No. 3 ( 2018-02)
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 200, No. 3 ( 2018-02)
    Abstract: Attachment is essential for microorganisms to establish interactions with both biotic and abiotic surfaces. Stable attachment of Caulobacter crescentus to surfaces requires an adhesive polysaccharide holdfast, but the exact composition of the holdfast is unknown. The holdfast is anchored to the cell envelope by outer membrane proteins HfaA, HfaB, and HfaD. H old f ast a nchor gene mutations result in holdfast shedding and reduced cell adherence. Translocation of HfaA and HfaD to the cell surface requires HfaB. The Wzx homolog HfsF is predicted to be a bacterial polysaccharide flippase. An hfsF deletion significantly reduced the amount of holdfast produced per cell and slightly reduced adherence. A Δ hfsF Δ hfaD double mutant was completely deficient in adherence. A suppressor screen that restored adhesion in the Δ hfsF Δ hfaD mutant identified mutations in three genes: wbqV , rfbB , and rmlA . Both WbqV and RfbB belong to a family of nucleoside-diphosphate epimerases, and RmlA has similarity to nucleotidyltransferases. The loss of wbqV or rfbB in the Δ hfsF Δ hfaD mutant reduced holdfast shedding but did not restore holdfast synthesis to parental levels. Loss of wbqV or rfbB did not restore adherence to a Δ hfsF mutant but did restore adherence and holdfast anchoring to a Δ hfaD mutant, confirming that suppression occurs through restoration of holdfast anchoring. The adherence and holdfast anchoring of a Δ hfaA ΔhfaD mutant could be restored by wbqV or rfbB mutation, but such mutations could not suppress these phenotypes in the Δ hfaB mutant. We hypothesize that HfaB plays an additional role in holdfast anchoring or helps to translocate an unknown factor that is important for holdfast anchoring. IMPORTANCE Biofilm formation results in increased resistance to both environmental stresses and antibiotics. Caulobacter crescentus requires an adhesive holdfast for permanent attachment and biofilm formation, but the exact mechanism of polysaccharide anchoring to the cell and the holdfast composition are unknown. Here we identify novel polysaccharide genes that affect holdfast anchoring to the cell. We identify a new role for the holdfast anchor protein HfaB. This work increases our specific knowledge of the polysaccharide adhesin involved in Caulobacter attachment and the general knowledge regarding production and anchoring of polysaccharide adhesins by bacteria. This work also explores the interactions between different polysaccharide biosynthesis and secretion systems in bacteria.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.00597-17
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2018
    detail.hit.zdb_id: 1481988-0
    SSG: 12
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  • 4
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    Characterization of the Caulobacter crescentus Holdfast Polysaccharide Biosynthesis Pathway Reveals Significant Redundancy in the Initiating Glycosyltransferase and Polymerase Steps
    American Society for Microbiology ; 2008
    In:  Journal of Bacteriology Vol. 190, No. 21 ( 2008-11), p. 7219-7231
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 190, No. 21 ( 2008-11), p. 7219-7231
    Abstract: Caulobacter crescentus cells adhere to surfaces by using an extremely strong polar adhesin called the holdfast. The polysaccharide component of the holdfast is comprised in part of oligomers of N -acetylglucosamine. The genes involved in the export of the holdfast polysaccharide and the anchoring of the holdfast to the cell were previously discovered. In this study, we identified a cluster of polysaccharide biosynthesis genes ( hfsEFGH ) directly adjacent to the holdfast polysaccharide export genes. Sequence analysis indicated that these genes are involved in the biosynthesis of the minimum repeat unit of the holdfast polysaccharide. HfsE is predicted to be a UDP-sugar lipid-carrier transferase, the glycosyltransferase that catalyzes the first step in polysaccharide biosynthesis. HfsF is predicted to be a flippase, HfsG is a glycosyltransferase, and HfsH is similar to a polysaccharide (chitin) deacetylase. In-frame hfsG and hfsH deletion mutants resulted in severe deficiencies both in surface adhesion and in binding to the holdfast-specific lectin wheat germ agglutinin. In contrast, hfsE and hfsF mutants exhibited nearly wild-type levels of adhesion and holdfast synthesis. We identified three paralogs to hfsE , two of which are redundant to hfsE for holdfast synthesis. We also identified a redundant paralog to the hfsC gene, encoding the putative polysaccharide polymerase, and present evidence that the hfsE and hfsC paralogs, together with the hfs genes, are absolutely required for proper holdfast synthesis.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.01003-08
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2008
    detail.hit.zdb_id: 1481988-0
    SSG: 12
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  • 5
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    Genetic Screen in Chlamydia muridarum Reveals Role for an Interferon-Induced Host Cell Death Program in Antimicrobial Inclusion Rupture
    American Society for Microbiology ; 2019
    In:  mBio Vol. 10, No. 2 ( 2019-04-30)
    Details
    In: mBio, American Society for Microbiology, Vol. 10, No. 2 ( 2019-04-30)
    Abstract: Interferon-regulated immune defenses protect mammals from pathogenically diverse obligate intracellular bacterial pathogens of the genus Chlamydia . Interferon gamma (IFN-γ) is especially important in controlling the virulence of Chlamydia species and thus impacts the modeling of human chlamydial infection and disease in mice. How IFN-γ contributes to cell-autonomous defenses against Chlamydia species and how these pathogens evade IFN-γ-mediated immunity in their natural hosts are not well understood. We conducted a genetic screen which identified 31 IFN-γ - s ensitive (Igs) mutants of the mouse model pathogen Chlamydia muridarum . Genetic suppressor analysis and lateral gene transfer were used to map the phenotype of one of these mutants, Igs4, to a missense mutation in a putative chlamydial inclusion membrane protein, TC0574. We observed the lytic destruction of Igs4-occupied inclusions and accompanying host cell death in response to IFN-γ priming or various proapoptotic stimuli. However, Igs4 was insensitive to IFN-γ-regulated cell-autonomous defenses previously implicated in anti- Chlamydia trachomatis host defense in mice. Igs4 inclusion integrity was restored by caspase inhibitors, indicating that the IFN-γ-mediated destruction of Igs4 inclusions is dependent upon the function of caspases or related prodeath cysteine proteases. We further demonstrated that the Igs4 mutant is immune restricted in an IFN-γ-dependent manner in a mouse infection model, thereby implicating IFN-γ-mediated inclusion destruction and host cell death as potent in vivo host defense mechanisms to which wild-type C. muridarum is resistant. Overall, our results suggest that C. muridarum evolved resistance mechanisms to counter IFN-γ-elicited programmed cell death and the associated destruction of intravacuolar pathogens. IMPORTANCE Multiple obligatory intracellular bacteria in the genus Chlamydia are important pathogens. In humans, strains of C. trachomatis cause trachoma, chlamydia, and lymphogranuloma venereum. These diseases are all associated with extended courses of infection and reinfection that likely reflect the ability of chlamydiae to evade various aspects of host immune responses. Interferon-stimulated genes, driven in part by the cytokine interferon gamma, restrict the host range of various Chlamydia species, but how these pathogens evade interferon-stimulated genes in their definitive host is poorly understood. Various Chlamydia species can inhibit death of their host cells and may have evolved this strategy to evade prodeath signals elicited by host immune responses. We present evidence that chlamydia-induced programmed cell death resistance evolved to counter interferon- and immune-mediated killing of Chlamydia -infected cells.
    Type of Medium: Online Resource
    ISSN: 2161-2129 , 2150-7511
    URL: Article
    DOI: 10.1128/mBio.00385-19
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2019
    detail.hit.zdb_id: 2557172-2
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  • 6
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    A Genital Infection-Attenuated Chlamydia muridarum Mutant Infects the Gastrointestinal Tract and Protects against Genital Tract Challenge
    American Society for Microbiology ; 2020
    In:  mBio Vol. 11, No. 6 ( 2020-12-22)
    Details
    In: mBio, American Society for Microbiology, Vol. 11, No. 6 ( 2020-12-22)
    Abstract: Chlamydia spp. productively infect mucosal epithelial cells of multiple anatomical sites, including the conjunctiva, lungs, gastrointestinal (GI) tract, and urogenital tract. We, and others, previously established that chlamydial GI tropism is mediated by distinct chromosomal and plasmid factors. In this study, we describe a genital infection-attenuated Chlamydia muridarum mutant (GIAM-1) that is profoundly and specifically attenuated in the murine genital tract. GIAM-1 infected the murine GI tract similarly to wild-type (WT) Chlamydia muridarum but did not productively infect the lower genital tract of female mice, ascend to infect the upper genital tract, or cause hydrosalpinx. However, GI infection of mice with GIAM-1 elicited a transmucosal immune response that protected against subsequent genital challenge with WT Chlamydia muridarum . Collectively, our results demonstrate that chlamydia mutants that are profoundly attenuated for specific organ tissues can be derived and demonstrate that live-attenuated vaccine strains that infect the GI tract, but do not elicit genital tract disease, could be used to protect against chlamydia genital tract infection and disease. IMPORTANCE Chlamydia is the most common sexually transmitted bacterial infection in the United States. Most chlamydia genital infections resolve without serious consequences; however, untreated infection in women can cause pelvic inflammatory disease and infertility. Antibiotics are very effective in treating chlamydia, but most genital infections in both men and women are asymptomatic and go undiagnosed. Therefore, there is a critical need for an effective vaccine. In this work, we show that a mutant chlamydia strain, having substantially reduced virulence for genital infection, colonizes the gastrointestinal tract and produces robust immunity to genital challenge with fully virulent wild-type chlamydia. These results are an important advance in understanding chlamydial virulence and provide compelling evidence that safe and effective live-attenuated chlamydia vaccines may be feasible.
    Type of Medium: Online Resource
    ISSN: 2161-2129 , 2150-7511
    URL: Article
    DOI: 10.1128/mBio.02770-20
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2020
    detail.hit.zdb_id: 2557172-2
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  • 7
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    A Member of an Ancient Family of Bacterial Amino Acids Transporters Contributes to Chlamydia Nutritional Virulence and Immune Evasion
    American Society for Microbiology ; 2023
    In:  Infection and Immunity Vol. 91, No. 3 ( 2023-03-15)
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 91, No. 3 ( 2023-03-15)
    Abstract: Many obligate intracellular bacteria, including members of the genus Chlamydia , cannot synthesize a variety of amino acids de novo and acquire these from host cells via largely unknown mechanisms. Previously, we determined that a missense mutation in ctl0225 , a conserved Chlamydia open reading frame of unknown function, mediated sensitivity to interferon gamma. Here, we show evidence that CTL0225 is a member of the SnatA family of neutral amino acid transporters that contributes to the import of several amino acids into Chlamydia cells. Further, we show that CTL0225 orthologs from two other distantly related obligate intracellular pathogens ( Coxiella burnetii and Buchnera aphidicola ) are sufficient to import valine into Escherichia coli . We also show that chlamydia infection and interferon exposure have opposing effects on amino acid metabolism, potentially explaining the relationship between CTL0225 and interferon sensitivity. Overall, we show that phylogenetically diverse intracellular pathogens use an ancient family of amino acid transporters to acquire host amino acids and provide another example of how nutritional virulence and immune evasion can be linked in obligate intracellular pathogens.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/iai.00483-22
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2023
    detail.hit.zdb_id: 1483247-1
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  • 8
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    Evidence of Uncultivated Bacteria in the Adult Female Bladder
    American Society for Microbiology ; 2012
    In:  Journal of Clinical Microbiology Vol. 50, No. 4 ( 2012-04), p. 1376-1383
    Details
    In: Journal of Clinical Microbiology, American Society for Microbiology, Vol. 50, No. 4 ( 2012-04), p. 1376-1383
    Abstract: Clinical urine specimens are usually considered to be sterile when they do not yield uropathogens using standard clinical cultivation procedures. Our aim was to test if the adult female bladder might contain bacteria that are not identified by these routine procedures. An additional aim was to identify and recommend the appropriate urine collection method for the study of bacterial communities in the female bladder. Consenting participants who were free of known urinary tract infection provided urine samples by voided, transurethral, and/or suprapubic collection methods. The presence of bacteria in these samples was assessed by bacterial culture, light microscopy, and 16S rRNA gene sequencing. Bacteria that are not or cannot be routinely cultivated (hereinafter called uncultivated bacteria) were common in voided urine, urine collected by transurethral catheter (TUC), and urine collected by suprapubic aspirate (SPA), regardless of whether the subjects had urinary symptoms. Voided urine samples contained mixtures of urinary and genital tract bacteria. Communities identified in parallel urine samples collected by TUC and SPA were similar. Uncultivated bacteria are clearly present in the bladders of some women. It remains unclear if these bacteria are viable and/or if their presence is relevant to idiopathic urinary tract conditions.
    Type of Medium: Online Resource
    ISSN: 0095-1137 , 1098-660X
    URL: Article
    DOI: 10.1128/JCM.05852-11
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2012
    detail.hit.zdb_id: 1498353-9
    SSG: 12
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  • 9
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    Chlamydia muridarum Genital and Gastrointestinal Infection Tropism Is Mediated by Distinct Chromosomal Factors
    American Society for Microbiology ; 2018
    In:  Infection and Immunity Vol. 86, No. 7 ( 2018-07)
    Details
    In: Infection and Immunity, American Society for Microbiology, Vol. 86, No. 7 ( 2018-07)
    Abstract: Some members of the genus Chlamydia , including the human pathogen Chlamydia trachomatis , infect multiple tissues, including the genital and gastrointestinal (GI) tracts. However, it is unknown if bacterial targeting to these sites is mediated by multifunctional or distinct chlamydial factors. We previously showed that disruption of individual large clostridial toxin homologs encoded within the Chlamydia muridarum plasticity zone were not critical for murine genital tract infection. Here, we assessed whether cytotoxin genes contribute to C. muridarum GI tropism. Infectivity and shedding of wild-type (WT) C. muridarum and three mutants containing nonsense mutations in different cytotoxin genes, tc0437 , tc0438 , and tc0439 , were compared in mouse genital and GI infection models. One mutant, which had a nonsense mutation in tc0439 , was highly attenuated for GI infection and had a GI 50% infectious dose (ID 50 ) that was 1,000 times greater than that of the WT. GI inoculation with this mutant failed to elicit anti-chlamydial antibodies or to protect against subsequent genital tract infection. Genome sequencing of the tc0439 mutant revealed additional chromosomal mutations, and phenotyping of additional mutants suggested that the GI attenuation might be linked to a nonsense mutation in tc0600 . The molecular mechanism underlying this dramatic difference in tissue-tropic virulence is not fully understood. However, isolation of these mutants demonstrates that distinct chlamydial chromosomal factors mediate chlamydial tissue tropism and provides a basis for vaccine initiatives to isolate chlamydia strains that are attenuated for genital infection but retain the ability to colonize the GI tract and elicit protective immune responses.
    Type of Medium: Online Resource
    ISSN: 0019-9567 , 1098-5522
    URL: Article
    DOI: 10.1128/IAI.00141-18
    RVK:
    XA 10000
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2018
    detail.hit.zdb_id: 1483247-1
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  • 10
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    Interrogating Genes That Mediate Chlamydia trachomatis Survival in Cell Culture Using Conditional Mutants and Recombination
    American Society for Microbiology ; 2016
    In:  Journal of Bacteriology Vol. 198, No. 15 ( 2016-08), p. 2131-2139
    Details
    In: Journal of Bacteriology, American Society for Microbiology, Vol. 198, No. 15 ( 2016-08), p. 2131-2139
    Abstract: 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.
    Type of Medium: Online Resource
    ISSN: 0021-9193 , 1098-5530
    URL: Article
    DOI: 10.1128/JB.00161-16
    Language: English
    Publisher: American Society for Microbiology
    Publication Date: 2016
    detail.hit.zdb_id: 1481988-0
    SSG: 12
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