Pathogenic Diversity among<i>Chlamydia trachomatis</i>Ocular Strains in Nonhuman Primates Is Affected by Subtle Genomic Variations

Kari, László; Whitmire, William M.; Carlson, John H.; Crane, Deborah D.; Reveneau, Nathalie; Nelson, David E.; Mabey, David; Bailey, Robin L.; Holland, Martin J.; McClarty, Grant

doi:10.1086/525285

Cited by 103 publications

(120 citation statements)

References 40 publications

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“…We used these phenotypic and genetic markers of the trpBA operon to characterize the CTD trpB− mutant. Western blots using specific anti-TrpA and -TrpB antibodies were done with lysates of HeLa cells infected with CTD, CTD trpB−, and a C. trachomatis ocular strain A2497 (22) that has a frameshift mutation in trpA. As expected, the ocular strain A2497 expressed the 42-kDa TrpB but not the 26-kDa TrpA, whereas CTD expressed both polypeptides (Fig.…”

Section: Resultssupporting

confidence: 53%

Generation of targeted Chlamydia trachomatis null mutants

Kari

Goheen

Randall

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Chlamydia trachomatis is an obligate intracellular bacterial pathogen that infects hundreds of millions of individuals globally, causing blinding trachoma and sexually transmitted disease. More effective chlamydial control measures are needed, but progress toward this end has been severely hampered by the lack of a tenable chlamydial genetic system. Here, we describe a reversegenetic approach to create isogenic C. trachomatis mutants. C. trachomatis was subjected to low-level ethyl methanesulfonate mutagenesis to generate chlamydiae that contained less then one mutation per genome. Mutagenized organisms were expanded in small subpopulations that were screened for mutations by digesting denatured and reannealed PCR amplicons of the target gene with the mismatch specific endonuclease CEL I. Subpopulations with mutations were then sequenced for the target region and plaque-cloned if the desired mutation was detected. We demonstrate the utility of this approach by isolating a tryptophan synthase gene (trpB) null mutant that was otherwise isogenic to its parental clone as shown by de novo genome sequencing. The mutant was incapable of avoiding the anti-microbial effect of IFN-γ-induced tryptophan starvation. The ability to genetically manipulate chlamydiae is a major advancement that will enhance our understanding of chlamydial pathogenesis and accelerate the development of new anti-chlamydial therapeutic control measures. Additionally, this strategy could be applied to other medically important bacterial pathogens with no or difficult genetic systems.genetics | mutation screen

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Section: Resultssupporting

confidence: 53%

Generation of targeted Chlamydia trachomatis null mutants

Kari

Goheen

Randall

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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133

135

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“…In addition, IFN-␥ is unable to trigger C. trachomatis persistence in IDO1-deficient cells (34), confirming that the IDO1 gene is a key antichlamydial ISG. However, there is substantial variation in the IFN-␥ sensitivity of isolates within subgroups of tryptophan synthase-positive genital strains (19) and tryptophan synthase-negative trachoma strains (35). It has been reported that interferonstimulated p65 guanylate binding proteins (GBP) may contribute to IFN-␥-mediated control of C. trachomatis in HeLa cells (36) and macrophages (37), although more recent results indicate that human GBP1 (hGBP1) does not localize to C. trachomatis vacuoles and is dispensable for cell-autonomous control of this pathogen in both unprimed and IFN-␥-primed human cells (38).…”

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confidence: 99%

Beyond Tryptophan Synthase: Identification of Genes That Contribute to Chlamydia trachomatis Survival during Gamma Interferon-Induced Persistence and Reactivation

et al. 2016

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cChlamydia 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-␥)-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-␥-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. Chlamydia trachomatis has a characteristic biphasic developmental cycle in cell culture (1). The two dominant chlamydial forms are elementary bodies (EBs) and reticulate bodies (RBs) (1). EBs represent the infectious, nonreplicative form capable of invading host cells (1). Following entry, EBs differentiate into RBs that replicate inside parasitophorous vacuoles termed inclusions (1). Maturing RBs then redifferentiate back to EBs and exit the host cell via lysis or extrusion (2). Morphologically aberrant RBs have been observed in clinical specimens (3). This abnormal developmental cycle can be recapitulated in a laboratory setting by exposing C. trachomatis to various stressors such as host cytokines (4-6), excess amino acids (7), iron deficiency (8, 9), virus coinfections (10), and antibiotics (11, 12) (reviewed in reference 13). Under these conditions, normal C. trachomatis RBs can transition into persistent forms that do not divide, are enlarged, and have aberrant morphology (13). Upon removal of the persistence-inducing stressor, RBs transition back into normal RBs and continue normal development (13). Aberrant RB morphology is a shared feature of multiple C. trachomatis persistence models and may reflect activation of a common stress response program (13). Thus, characterizing how C. trachomatis enters, survives, and reactivates from persistence could reveal new insights into chlamydial pathogenesis.The Th1 cytokine interferon gamma (IFN-␥) can induce dif...

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“…The genomic sequences of different ocular, urogenital, and LGV strains exhibit Ͼ98% identity and a high degree of synteny (12,25,29,31,50,53,55,59,60). Therefore, the determinants of the different types of infection (invasive or noninvasive) and tissue tropism (eyes, genitals, and lymph nodes) must rely on the few genes present in some strains but not in others and on nucleotide differences which may lead either to proteins with disease group-specific amino acids or to differential gene expression.…”

mentioning

confidence: 99%

“…Our studies suggest that a subset of Inc proteins quences of the inc, pmp, and housekeeping genes analyzed were from the available genomes of 51 C. trachomatis strains (12,25,29,31,50,53,55,59,60). The strains and corresponding genome accession numbers are listed in Table S1 in the supplemental material.…”

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confidence: 99%

Polymorphisms in Inc Proteins and Differential Expression ofincGenes among Chlamydia trachomatis Strains Correlate with Invasiveness and Tropism of Lymphogranuloma Venereum Isolates

et al. 2012

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Chlamydia trachomatis is a human bacterial pathogen that multiplies only within an intracellular membrane-bound vacuole, the inclusion. C. trachomatis includes ocular and urogenital strains, usually causing infections restricted to epithelial cells of the conjunctiva and genital mucosa, respectively, and lymphogranuloma venereum (LGV) strains, which can infect macrophages and spread into lymph nodes. However, C. trachomatis genomes display >98% identity at the DNA level. In this work, we studied whether C. trachomatis Inc proteins, which have a bilobed hydrophobic domain that may mediate their insertion in the inclusion membrane, could be a factor determining these different types of infection and tropisms. Analyses of polymorphisms and phylogeny of 48 Inc proteins from 51 strains encompassing the three disease groups showed significant amino acid differences that were mainly due to variations between Inc proteins from LGV and ocular or urogenital isolates. Studies of the evolutionary dynamics of inc genes suggested that 10 of them are likely under positive selection and indicated that most nonsilent mutations are LGV specific. Additionally, real-time quantitative PCR analyses in prototype and clinical strains covering the three disease groups identified three inc genes with LGV-specific expression. We determined the transcriptional start sites of these genes and found LGV-specific nucleotides within their promoters. Thus, subtle variations in the amino acids of a subset of Inc proteins and in the expression of inc genes may contribute to the unique tropism and invasiveness of C. trachomatis LGV strains.

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Pathogenic Diversity amongChlamydia trachomatisOcular Strains in Nonhuman Primates Is Affected by Subtle Genomic Variations

Cited by 103 publications

References 40 publications

Generation of targeted Chlamydia trachomatis null mutants

Generation of targeted Chlamydia trachomatis null mutants

Beyond Tryptophan Synthase: Identification of Genes That Contribute to Chlamydia trachomatis Survival during Gamma Interferon-Induced Persistence and Reactivation

Polymorphisms in Inc Proteins and Differential Expression ofincGenes among Chlamydia trachomatis Strains Correlate with Invasiveness and Tropism of Lymphogranuloma Venereum Isolates

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