Identification of the Base-Pairing Requirements for Repression of hctA Translation by the Small RNA IhtA Leads to the Discovery of a New mRNA Target in Chlamydia trachomatis

Grieshaber, Nicole A.; Tattersall, J.; Liguori, Johella; Lipat, Joseph N.; Runac, Justin; Grieshaber, Scott S.

doi:10.1371/journal.pone.0116593

Cited by 12 publications

(16 citation statements)

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“…HctA is a small histone-like protein that is involved in the condensation of the chlamydial genome to form the compact nucleoid characteristic of the EB ( 20 ). The upstream promoter region as well as the first 10 codons of the open reading frame (ORF) of hctA were used to construct this reporter, as the regulation of HctA expression involves both the promoter and the IhtA binding site contained in the beginning of the ORF ( 21 ). Each reporter was transformed into C. trachomatis , generating the strains Ctr-ihtA prom-EGFP and Ctr-hctA prom-Clover (see Table S1 in the supplemental material).…”

Section: Resultsmentioning

confidence: 99%

Single-Inclusion Kinetics of Chlamydia trachomatis Development

et al. 2020

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The obligate intracellular bacterial pathogen Chlamydia trachomatis is reliant on a developmental cycle consisting of two cell forms, termed the elementary body (EB) and the reticulate body (RB). The EB is infectious and utilizes a type III secretion system and preformed effector proteins during invasion, but it does not replicate. The RB replicates in the host cell but is noninfectious. This developmental cycle is central to chlamydial pathogenesis. In this study, we developed mathematical models of the developmental cycle that account for potential factors influencing RB-to-EB cell type switching during infection. Our models predicted that two categories of regulatory signals for RB-to-EB development could be differentiated experimentally, an “intrinsic” cell-autonomous program inherent to each RB and an “extrinsic” environmental signal to which RBs respond. To experimentally differentiate between mechanisms, we tracked the expression of C. trachomatis development-specific promoters in individual inclusions using fluorescent reporters and live-cell imaging. These experiments indicated that EB production was not influenced by increased multiplicity of infection or by superinfection, suggesting the cycle follows an intrinsic program that is not directly controlled by environmental factors. Additionally, live-cell imaging revealed that EB development is a multistep process linked to RB growth rate and cell division. The formation of EBs followed a progression with expression from the euo and ihtA promoters evident in RBs, while expression from the promoter for hctA was apparent in early EBs/IBs. Finally, expression from the promoters for the true late genes, hctB, scc2, and tarp, was evident in the maturing EB. IMPORTANCE Chlamydia trachomatis is an obligate intracellular bacterium that can cause trachoma, cervicitis, urethritis, salpingitis, and pelvic inflammatory disease. To establish infection in host cells, Chlamydia must complete a multiple-cell-type developmental cycle. The developmental cycle consists of specialized cells, the EB cell, which mediates infection of new host cells, and the RB cell, which replicates and eventually produces more EB cells to mediate the next round of infection. By developing and testing mathematical models to discriminate between two competing hypotheses for the nature of the signal controlling RB-to-EB cell type switching, we demonstrate that RB-to-EB development follows a cell-autonomous program that does not respond to environmental cues. Additionally, we show that RB-to-EB development is a function of chlamydial growth and division. This study serves to further our understanding of the chlamydial developmental cycle that is central to the bacterium’s pathogenesis.

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Section: Resultsmentioning

confidence: 99%

Single-Inclusion Kinetics of Chlamydia trachomatis Development

et al. 2020

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“…In addition, they showed that the inhibition of HctA translation by lhtA is a conserved function across chlamydiae (33). Intriguingly, in addition to HctA, lhtA also regulates the expression of CTL0322 (CT066) a highly conserved chlamydia-specific hypothetical protein (41). Transcriptional studies indicate that the two primary plasmid antisense sRNAs, sRNA-2 and sRNA-7, encoded within pgp8 and pgp5, respectively, are highly expressed (32,42).…”

Section: Discussionmentioning

confidence: 99%

Plasmid Negative Regulation of CPAF Expression Is Pgp4 Independent and Restricted to InvasiveChlamydia trachomatisBiovars

Patton

Chen

Yang

et al. 2018

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Chlamydia trachomatis is an obligate intracellular bacterial pathogen that causes blinding trachoma and sexually transmitted disease. C. trachomatis isolates are classified into 2 biovars-lymphogranuloma venereum (LGV) and trachomawhich are distinguished biologically by their natural host cell infection tropism.LGV biovars infect macrophages and are invasive, whereas trachoma biovars infect oculourogenital epithelial cells and are noninvasive. The C. trachomatis plasmid is an important virulence factor in the pathogenesis of these infections. Central to its pathogenic role is the transcriptional regulatory function of the plasmid protein Pgp4, which regulates the expression of plasmid and chromosomal virulence genes. As many gene regulatory functions are post-transcriptional, we employed a comparative proteomic study of cells infected with plasmid-cured C. trachomatis serovars A and D (trachoma biovar), a L2 serovar (LGV biovar), and the L2 serovar transformed with a plasmid containing a nonsense mutation in pgp4 to more completely elucidate the effects of the plasmid on chlamydial infection biology. Our results show that the Pgp4-dependent elevations in the levels of Pgp3 and a conserved core set of chromosomally encoded proteins are remarkably similar for serovars within both C. trachomatis biovars. Conversely, we found a plasmid-dependent, Pgp4-independent, negative regulation in the expression of the chlamydial protease-like activity factor (CPAF) for the L2 serovar but not the A and D serovars. The molecular mechanism of plasmiddependent negative regulation of CPAF expression in the LGV serovar is not understood but is likely important to understanding its macrophage infection tropism and invasive infection nature. IMPORTANCEThe Chlamydia trachomatis plasmid is an important virulence factor in the pathogenesis of chlamydial infection. It is known that plasmid protein 4 (Pgp4) functions in the transcriptional regulation of the plasmid virulence protein 3 (Pgp3) and multiple chromosomal loci of unknown function. Since many gene regulatory functions can be post-transcriptional, we undertook a comparative proteomic analysis to better understand the plasmid's role in chlamydial and host protein expression. We report that Pgp4 is a potent and specific master positive regulator of a common core of plasmid and chromosomal virulence genes shared by multiple C. trachomatis serovars. Notably, we show that the plasmid is a negative regulator of the expression of the chlamydial virulence factor CPAF. The plasmid regulation of CPAF is independent of Pgp4 and restricted to a C. trachomatis macrophage-tropic strain. These findings are important because they define a previously unknown role for the plasmid in the pathophysiology of invasive chlamydial infection.

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“…For example, Grieshaber et al recently reported that the noncoding RNA (ncRNA) IhtA interferes with the translation of the chlamydial histone-like protein HctA and CTL0322 (49). HctA functions similarly to a eukaryotic histone and mediates condensation of chromatin during the RB-to-EB transition (50)(51)(52).…”

Section: Discussionmentioning

confidence: 99%

“…Like HctA, CTL0322 is predicted to be a small, highly basic protein, and the C. trachomatis serovar D homolog of CTL0322 is nucleotropic in Hep2 cells (53). Greishaber proposed that, similarly to HctA, CTL0322 might bind DNA and mediate the RB-to-EB transition (49). Supporting this hypothesis, HS11, which contains CTL0322 M51I , has a TS defect that initiates between 18 and 24 hpi, corresponding to the beginning of the RBto-EB transition in these strains of C. trachomatis.…”

Section: Discussionmentioning

confidence: 99%

Interrogating Genes That Mediate Chlamydia trachomatis Survival in Cell Culture Using Conditional Mutants and Recombination

et al. 2016

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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. Temperatureshift 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. IMPORTANCEStudy 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. Chlamydiae are obligate intracellular pathogens that share a characteristic biphasic developmental cycle where these organisms alternate between extracellular, infectious elementary body (EB) and intracellular, replicative reticulate body (RB) forms (1). Multiple species of the family Chlamydiaceae, including Chlamydia trachomatis, C. pneumoniae, and C. psittaci, are common or incidental pathogens of humans (2). Despite their medical importance, a lack of tools for genetic manipulation of these organisms was a major impediment in chlamydial research until recently (3, 4).Only ϳ60% of Chlamydiaceae genes share significant ...

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Identification of the Base-Pairing Requirements for Repression of hctA Translation by the Small RNA IhtA Leads to the Discovery of a New mRNA Target in Chlamydia trachomatis

Cited by 12 publications

References 47 publications

Single-Inclusion Kinetics of Chlamydia trachomatis Development

Single-Inclusion Kinetics of Chlamydia trachomatis Development

Plasmid Negative Regulation of CPAF Expression Is Pgp4 Independent and Restricted to InvasiveChlamydia trachomatisBiovars

Interrogating Genes That Mediate Chlamydia trachomatis Survival in Cell Culture Using Conditional Mutants and Recombination

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