scholarly journals Catalytically impaired TrpA subunit of tryptophan synthase from Chlamydia trachomatis is an allosteric regulator of TrpB

2021 ◽  
Author(s):  
Karolina Michalska ◽  
Samantha Wellington ◽  
Natalia Maltseva ◽  
Robert Jedrzejczak ◽  
Nelly Selem‐Mojica ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Tryptophan Synthase ◽  
Allosteric Regulator

scholarly journals Comparative Genomic Analysis of Chlamydia trachomatis Oculotropic and Genitotropic Strains

2005 ◽  
Vol 73 (10) ◽  
pp. 6407-6418 ◽  
Author(s):  
John H. Carlson ◽  
Stephen F. Porcella ◽  
Grant McClarty ◽  
Harlan D. Caldwell
Keyword(s):  
Chlamydia Trachomatis ◽  
Protein A ◽  
Transmitted Disease ◽  
Genomic Analysis ◽  
Hla Class I ◽  
Comparative Genomic ◽  
Toxin Gene ◽  
Snp Analysis ◽  
Tryptophan Synthase ◽  
T Cell Epitopes

ABSTRACT Chlamydia trachomatis infection is an important cause of preventable blindness and sexually transmitted disease (STD) in humans. C. trachomatis exists as multiple serovariants that exhibit distinct organotropism for the eye or urogenital tract. We previously reported tissue-tropic correlations with the presence or absence of a functional tryptophan synthase and a putative GTPase-inactivating domain of the chlamydial toxin gene. This suggested that these genes may be the primary factors responsible for chlamydial disease organotropism. To test this hypothesis, the genome of an oculotropic trachoma isolate (A/HAR-13) was sequenced and compared to the genome of a genitotropic (D/UW-3) isolate. Remarkably, the genomes share 99.6% identity, supporting the conclusion that a functional tryptophan synthase enzyme and toxin might be the principal virulence factors underlying disease organotropism. Tarp (translocated actin-recruiting phosphoprotein) was identified to have variable numbers of repeat units within the N and C portions of the protein. A correlation exists between lymphogranuloma venereum serovars and the number of N-terminal repeats. Single-nucleotide polymorphism (SNP) analysis between the two genomes highlighted the minimal genetic variation. A disproportionate number of SNPs were observed within some members of the polymorphic membrane protein (pmp) autotransporter gene family that corresponded to predicted T-cell epitopes that bind HLA class I and II alleles. These results implicate Pmps as novel immune targets, which could advance future chlamydial vaccine strategies. Lastly, a novel target for PCR diagnostics was discovered that can discriminate between ocular and genital strains. This discovery will enhance epidemiological investigations in nations where both trachoma and chlamydial STD are endemic.

scholarly journals Tryptophan Operon Diversity Reveals Evolutionary Trends among Geographically Disparate Chlamydia trachomatis Ocular and Urogenital Strains Affecting Tryptophan Repressor and Synthase Function

mBio ◽  
2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Sankhya Bommana ◽  
Naraporn Somboonna ◽  
Gracie Richards ◽  
Maryam Tarazkar ◽  
Deborah Dean
Keyword(s):  
Chlamydia Trachomatis ◽  
Selective Pressure ◽  
Intracellular Survival ◽  
Tryptophan Synthase ◽  
Evolutionary Mechanism ◽  
Content Type ◽  
Sexually Transmitted ◽  
Host Pathogen ◽  
Tryptophan Operon ◽  
Tryptophan Repressor

ABSTRACT The obligate intracellular pathogen Chlamydia trachomatis (Ct) is the leading cause of bacterial sexually transmitted infections and blindness globally. To date, Ct urogenital strains are considered tryptophan prototrophs, utilizing indole for tryptophan synthesis within a closed-conformation tetramer comprised of two α (TrpA)- and two β (TrpB)-subunits. In contrast, ocular strains are auxotrophs due to mutations in TrpA, relying on host tryptophan pools for survival. It has been speculated that there is strong selective pressure for urogenital strains to maintain a functional operon. Here, we performed genetic, phylogenetic, and novel functional modeling analyses of 595 geographically diverse Ct ocular, urethral, vaginal, and rectal strains with complete operon sequences. We found that ocular and urogenital, but not lymphogranuloma venereum, TrpA-coding sequences were under positive selection. However, vaginal and urethral strains exhibited greater nucleotide diversity and a higher ratio of nonsynonymous to synonymous substitutions [Pi(a)/Pi(s)] than ocular strains, suggesting a more rapid evolution of beneficial mutations. We also identified nonsynonymous amino acid changes for an ocular isolate with a urogenital backbone in the intergenic region between TrpR and TrpB at the exact binding site for YtgR—the only known iron-dependent transcription factor in Chlamydia—indicating that selective pressure has disabled the response to fluctuating iron levels. In silico effects on protein stability, ligand-binding affinity, and tryptophan repressor (TrpR) affinity for single-stranded DNA (ssDNA) measured by calculating free energy changes (ΔΔG) between Ct reference and mutant tryptophan operon proteins were also analyzed. We found that tryptophan synthase function was likely suboptimal compared to other bacterial tryptophan prototrophs and that a diversity of urogenital strain mutations rendered the synthase nonfunctional or inefficient. The novel mutations identified here affected active sites in an orthosteric manner but also hindered α- and β-subunit allosteric interactions from distant sites, reducing efficiency of the tryptophan synthase. Importantly, strains with mutant proteins were inclined toward energy conservation by exhibiting an altered affinity for their respective ligands compared to reference strains, indicating greater fitness. This is not surprising as l-tryptophan is one of the most energetically costly amino acids to synthesize. Mutations in the tryptophan repressor gene (trpR) among urogenital strains were similarly detrimental to function. Our findings indicate that urogenital strains are evolving more rapidly than previously recognized with mutations that impact tryptophan operon function in a manner that is energetically beneficial, providing a novel host-pathogen evolutionary mechanism for intracellular survival. IMPORTANCE Chlamydia trachomatis (Ct) is a major global public health concern causing sexually transmitted and ocular infections affecting over 130 million and 260 million people, respectively. Sequelae include infertility, preterm birth, ectopic pregnancy, and blindness. Ct relies on available host tryptophan pools and/or substrates to synthesize tryptophan to survive. Urogenital strains synthesize tryptophan from indole using their intact tryptophan synthase (TS). Ocular strains contain a trpA frameshift mutation that encodes a truncated TrpA with loss of TS function. We found that TS function is likely suboptimal compared to other tryptophan prototrophs and that urogenital stains contain diverse mutations that render TS nonfunctional/inefficient, evolve more rapidly than previously recognized, and impact operon function in a manner that is energetically beneficial, providing an alternative host-pathogen evolutionary mechanism for intracellular survival. Our research has broad scientific appeal since our approach can be applied to other bacteria that may explain evolution/survival in host-pathogen interactions.

scholarly journals Ammonia generation by tryptophan synthase drives a key genetic difference between genital and ocular Chlamydia trachomatis isolates

2019 ◽  
Vol 116 (25) ◽  
pp. 12468-12477 ◽  
Author(s):  
Shardulendra P. Sherchand ◽  
Ashok Aiyar
Keyword(s):  
Chlamydia Trachomatis ◽  
Genetic Difference ◽  
Bacterial Species ◽  
Serum Levels ◽  
High Serum ◽  
Striking Difference ◽  
Tryptophan Synthase ◽  
Indole Derivatives ◽  
Infected Cells ◽  
Inactivating Mutations

A striking difference between genital and ocular clinical isolates of Chlamydia trachomatis is that only the former express a functional tryptophan synthase and therefore can synthesize tryptophan by indole salvage. Ocular isolates uniformly cannot use indole due to inactivating mutations within tryptophan synthase, indicating a selection against maintaining this enzyme in the ocular environment. Here, we demonstrate that this selection occurs in two steps. First, specific indole derivatives, produced by the human gut microbiome and present in serum, rapidly induce expression of C. trachomatis tryptophan synthase, even under conditions of tryptophan sufficiency. We demonstrate that these indole derivatives function by acting as de-repressors of C. trachomatis TrpR. Second, trp operon de-repression is profoundly deleterious when infected cells are in an indole-deficient environment, because in the absence of indole, tryptophan synthase deaminates serine to pyruvate and ammonia. We have used biochemical and genetic approaches to demonstrate that expression of wild-type tryptophan synthase is required for the bactericidal production of ammonia. Pertinently, although these indole derivatives de-repress the trpRBA operon of C. trachomatis strains with trpA or trpB mutations, no ammonia is produced, and no deleterious effects are observed. Our studies demonstrate that tryptophan synthase can catalyze the ammonia-generating β-elimination reaction within any live bacterium. Our results also likely explain previous observations demonstrating that the same indole derivatives inhibit the growth of other pathogenic bacterial species, and why high serum levels of these indole derivatives are favorable for the prognosis of diseased conditions associated with bacterial dysbiosis.

scholarly journals Polymorphisms in Chlamydia trachomatis tryptophan synthase genes differentiate between genital and ocular isolates

2003 ◽  
Vol 111 (11) ◽  
pp. 1757-1769 ◽  
Author(s):  
Harlan D. Caldwell ◽  
Heidi Wood ◽  
Debbie Crane ◽  
Robin Bailey ◽  
Robert B. Jones ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Tryptophan Synthase

scholarly journals Molecular Mechanism of Tryptophan-Dependent Transcriptional Regulation in Chlamydia trachomatis

2006 ◽  
Vol 188 (12) ◽  
pp. 4236-4243 ◽  
Author(s):  
Johnny C. Akers ◽  
Ming Tan
Keyword(s):  
Chlamydia Trachomatis ◽  
Transcriptional Repression ◽  
Defense Mechanisms ◽  
Biosynthetic Pathway ◽  
In Vitro Transcription ◽  
Tryptophan Metabolism ◽  
Gram Negative Bacteria ◽  
Tryptophan Synthase ◽  
The Face

ABSTRACT Tryptophan is an essential amino acid that is required for normal development in Chlamydia species, and tryptophan metabolism has been implicated in chlamydial persistence and tissue tropism. The ability to synthesize tryptophan is not universal among the Chlamydiaceae, but species that have a predicted tryptophan biosynthetic pathway also encode an ortholog of TrpR, a regulator of tryptophan metabolism in many gram-negative bacteria. We show that in Chlamydia trachomatis serovar D, TrpR regulates its own gene and trpB and trpA, the genes for the two subunits of tryptophan synthase. These three genes form an operon that is transcribed by the major form of chlamydial RNA polymerase. TrpR acts as a tryptophan-dependent aporepressor that binds specifically to operator sequences upstream of the trpRBA operon. We also found that TrpR repressed in vitro transcription of trpRBA in a promoter-specific manner, and the level of repression was dependent upon the concentrations of TrpR and tryptophan. Our findings provide a mechanism for chlamydiae to sense changes in tryptophan levels and to respond by modulating expression of the tryptophan biosynthesis genes, and we present a unified model that shows how C. trachomatis can combine transcriptional repression and attenuation to regulate intrachlamydial tryptophan levels. In the face of host defense mechanisms that limit tryptophan availability from the infected cell, the ability to maintain homeostatic control of intrachlamydial tryptophan levels is likely to play an important role in chlamydial pathogenesis.

scholarly journals Clinical Persistence of Chlamydia trachomatis Sexually Transmitted Strains Involves Novel Mutations in the Functional αββα Tetramer of the Tryptophan Synthase Operon

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Naraporn Somboonna ◽  
Noa Ziklo ◽  
Thomas E. Ferrin ◽  
Jung Hyuk Suh ◽  
Deborah Dean
Keyword(s):  
Amino Acid ◽  
Chlamydia Trachomatis ◽  
Essential Amino Acid ◽  
Tryptophan Synthase ◽  
Content Type ◽  
Sexually Transmitted ◽  
Host Pathogen ◽  
Ifn Γ

ABSTRACT Clinical persistence of Chlamydia trachomatis (Ct) sexually transmitted infections (STIs) is a major public health concern. In vitro persistence is known to develop through interferon gamma (IFN-γ) induction of indoleamine 2,3-dioxygenase (IDO), which catabolizes tryptophan, an essential amino acid for Ct replication. The organism can recover from persistence by synthesizing tryptophan from indole, a substrate for the enzyme tryptophan synthase. The majority of Ct strains, except for reference strain B/TW-5/OT, contain an operon comprised of α and β subunits that encode TrpA and TrpB, respectively, and form a functional αββα tetramer. However, trpA mutations in ocular Ct strains, which are responsible for the blinding eye disease known as trachoma, abrogate tryptophan synthesis from indole. We examined serial urogenital samples from a woman who had recurrent Ct infections over 4 years despite antibiotic treatment. The Ct isolates from each infection episode were genome sequenced and analyzed for phenotypic, structural, and functional characteristics. All isolates contained identical mutations in trpA and developed aberrant bodies within intracellular inclusions, visualized by transmission electron microscopy, even when supplemented with indole following IFN-γ treatment. Each isolate displayed an altered αββα structure, could not synthesize tryptophan from indole, and had significantly lower trpBA expression but higher intracellular tryptophan levels compared with those of reference Ct strain F/IC-Cal3. Our data indicate that emergent mutations in the tryptophan operon, which were previously thought to be restricted only to ocular Ct strains, likely resulted in in vivo persistence in the described patient and represents a novel host-pathogen adaptive strategy for survival. IMPORTANCE Chlamydia trachomatis (Ct) is the most common sexually transmitted bacterium with more than 131 million cases occurring annually worldwide. Ct infections are often asymptomatic, persisting for many years despite treatment. In vitro recovery from persistence occurs when indole is utilized by the organism’s tryptophan synthase to synthesize tryptophan, an essential amino acid for replication. Ocular but not urogenital Ct strains contain mutations in the synthase that abrogate tryptophan synthesis. Here, we discovered that the genomes of serial isolates from a woman with recurrent, treated Ct STIs over many years were identical with a novel synthase mutation. This likely allowed long-term in vivo persistence where active infection resumed only when tryptophan became available. Our findings indicate an emerging adaptive host-pathogen evolutionary strategy for survival in the urogenital tract that will prompt the field to further explore chlamydial persistence, evaluate the genetics of mutant Ct strains and fitness within the host, and their implications for disease pathogenesis.

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

2016 ◽  
Vol 84 (10) ◽  
pp. 2791-2801 ◽  
Author(s):  
Matthew K. Muramatsu ◽  
Julie A. Brothwell ◽  
Barry D. Stein ◽  
Timothy E. Putman ◽  
Daniel D. Rockey ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Tryptophan Synthase ◽  
Content Type ◽  
Damage Repair ◽  
Nonculturable State ◽  
Ifn Γ ◽  
Functionally Diverse ◽  
Hypothetical Genes ◽  
Viable But Nonculturable State

Chlamydia trachomatiscan enter a viable but nonculturable statein vitrotermed persistence. A common feature ofC. trachomatispersistence 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 convertsl-tryptophan toN-formylkynurenine. GenitalC. trachomatisstrains 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 mutagenizedC. trachomatisstrains 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,ctl0225andctl0694, were also identified and may be involved in amino acid transport and DNA damage repair, respectively. Our results indicate thatC. trachomatisutilizes functionally diverse genes to mediate survival during and reactivation from persistence in HeLa cells.

scholarly journals Regulation of tryptophan synthase gene expression in Chlamydia trachomatis

2003 ◽  
Vol 49 (5) ◽  
pp. 1347-1359 ◽  
Author(s):  
Heidi Wood ◽  
Christine Fehlner-Gardner ◽  
Jody Berry ◽  
Elizabeth Fischer ◽  
Bonnie Graham ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chlamydia Trachomatis ◽  
Tryptophan Synthase

Genetic differences in the Chlamydia trachomatis tryptophan synthase α-subunit can explain variations in serovar pathogenesis

2000 ◽  
Vol 2 (6) ◽  
pp. 581-592 ◽  
Author(s):  
Allan C. Shaw ◽  
Gunna Christiansen ◽  
Peter Roepstorff ◽  
Svend Birkelund
Keyword(s):  
Chlamydia Trachomatis ◽  
Genetic Differences ◽  
Tryptophan Synthase ◽  
Α Subunit
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