Novel Detection Strategy To Rapidly Evaluate the Efficacy of Antichlamydial Agents

Zhang, Yan; Xian, Yuqi; Gao, Leiqiong; Elaasar, Hiba; Wang, Yao; Tauhid, Lamiya; Zeng, Hua; Shen, Li

doi:10.1128/aac.02202-16

Cited by 6 publications

(9 citation statements)

References 51 publications

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“…Because Scc4 is highly toxic to E. coli, a tightly controlled strategy is desirable. We chose pBOMBP3 to make an Scc4 expression vector (36) because it contains (i) a tetracycline-inducible tetA promoter (P tetA ) that drives expression of the gene of interest in the presence of anhydrotetracycline hydrochloride (aTC) (41), (ii) a ␤-lactamase gene conferring ampicillin resistance that permits selection for the transformants, and (iii) a robust P ompA -gfp reporter that allows for the easy visualization of the growth behaviors of the transformants in live cells during the course of infection (42,43). We found that the native scc4 ribosome binding site (RBS) weakly functioned to direct initiation of Scc4 synthesis in E. coli (see Fig.…”

Section: Resultsmentioning

confidence: 99%

Context-Dependent Action of Scc4 Reinforces Control of the Type III Secretion System

et al. 2020

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ABSTRACT Chlamydia trachomatis Scc4 (formerly CT663) engages the transcription machinery and the pathogenic type III secretion system (T3SS). Both machines are required for Chlamydia infection. These requirements and the limited ability for genetic manipulation in Chlamydia have hampered dissection of Scc4’s contributions. Here, by developing bacterial systems that permit the controlled expression and stable maintenance of Scc4, we assess Scc4’s effects on chlamydial growth phenotype, secretion, and the patterns of T3SS gene expression. Expressing Scc4 in Escherichia coli lacking a T3SS injectisome causes a growth defect. This deficiency is rescued by overexpressing the β-subunit of RNA polymerase (RNAP) or by exploiting sigma 70 (σ70) (homologous to chlamydial σ66) mutants that strengthen the interaction between σ70 region 4 and the β-flap, confirming Scc4’s distinction as a module of RNAP holoenzyme capable of modulating transcription. Yersinia pestis expressing Scc4 sustains a functional T3SS, through which CopN secretion is boosted by cooption of Scc4 and Scc1. Finally, conditional expression of Scc4 in C. trachomatis results in fast expansion of the Chlamydia-containing vacuole and accelerated chlamydial development, coupled to selective up- or downregulation of gene expression from different T3SS genes. This work reveals, for the first time, the context-dependent action of Scc4 linking it to diverse protein networks in bacteria. It establishes that Scc4, when overexpressed, exerts incredible effects on chlamydial development by reinforcing control of the T3SS. IMPORTANCE The T3SS is a key virulence factor required for C. trachomatis infection. The control of the T3SS has not been well studied in this obligate intracellular pathogen. Here, we show that Scc4 plays a major role for precise control of the pathogenic T3SS at the levels of gene expression and effector secretion through genetically separable protein networks, allowing a fast adaptive mode of C. trachomatis development during infection in human epithelial cells.

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Section: Resultsmentioning

confidence: 99%

Context-Dependent Action of Scc4 Reinforces Control of the Type III Secretion System

et al. 2020

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“…Recently, other alternative approaches have been suggested for improving or automating C. trachomatis quantification or susceptibility testing. In particular, genetically modified C. trachomatis strains expressing Green Fluorescent Protein (GFP), in-silico analysis of Immuno-Spot data or modified plaque-forming assays [27][28][29][30][31] have been proposed. However, these approaches have not gained a lot of traction in the scientific community since, for example, the engineering of GFP-producing C. trachomatis is challenging and time-consuming [27][28][29][30][31], the ImmunoSpot imaging system shows a low resolving power, underestimating chlamydial quantification [30], and the plaque assay lack accuracy and reproducibility [31].…”

Section: Discussionmentioning

confidence: 99%

“…In particular, genetically modified C . trachomatis strains expressing Green Fluorescent Protein (GFP), in-silico analysis of ImmunoSpot data or modified plaque-forming assays [ 27 – 31 ] have been proposed. However, these approaches have not gained a lot of traction in the scientific community since, for example, the engineering of GFP-producing C .…”

Section: Discussionmentioning

confidence: 99%

“…However, these approaches have not gained a lot of traction in the scientific community since, for example, the engineering of GFP-producing C . trachomatis is challenging and time-consuming [ 27 – 31 ], the ImmunoSpot imaging system shows a low resolving power, underestimating chlamydial quantification [ 30 ], and the plaque assay lack accuracy and reproducibility [ 31 ].…”

Section: Discussionmentioning

confidence: 99%

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In-cell western assay as a high-throughput approach for Chlamydia trachomatis quantification and susceptibility testing to antimicrobials

Filardo¹,

Pietro²,

Pasqualetti³

et al. 2021

PLoS ONE

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Chlamydia trachomatis, the leading cause of bacterial sexually transmitted diseases in developed countries, with around 127 million new cases per year, is mainly responsible for urethritis and cervicitis in women, and urethritis and epididymitis in men. Most C. trachomatis infections remain asymptomatic (>50%) and, hence, untreated, leading to severe reproductive complications in both women and men, like infertility. Therefore, the detection of C. trachomatis as well as the antimicrobial susceptibility testing becomes a priority, and, along the years, several methods have been recommended, like cell culture and direct immunofluorescence (DFA) on cell cultures. Herein, we described the application of In-Cell Western assay (ICW) via Odyssey CLx as a fast, more accessible, and high-throughput platform for the quantification of C. trachomatis and the screening of anti-chlamydial drugs. As a first step, we set up a standard curve by infecting cell monolayers with 2-fold serial dilutions of C. trachomatis Elementary Body (EB) suspension. Then, different unknown C. trachomatis EB suspensions were quantified and the chlamydial susceptibility testing to erythromycin was performed, using the DFA as comparison. Our results showed a very high concordance between these two assays, as evidenced by the enumeration of chlamydial IFUs as well as the determination of erythromycin Minimum Inhibitory Concentration (MIC). In conclusion, the ICW assay may be a promising candidate as an accurate and accessible methodology for C. trachomatis antimicrobial susceptibility testing.

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“…We determined the MIC and minimum bactericidal concentration (MBC) values, as described previously (16,17), in cells infected with representative C. trachomatis strains, D/UW-3/Cx, L2/434/Bu, and the L2/P ompA -GFP expressing green fluorescence protein (GFP) (18,19). Sol at 0.004 g/ml inhibited and killed all C. trachomatis strains tested.…”

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confidence: 99%

Potency of Solithromycin against Fast- and Slow-Growing Chlamydial Organisms

Gao

Wang

Zeng

et al. 2018

Antimicrob Agents Chemother

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Evidence is provided that solithromycin is a bactericidal against not only fast-growing chlamydial organisms but also those slowed by gamma interferon (IFN-γ) At sublethal concentrations, Sol impedes homotypic fusion of-containing vacuoles and reduces secretion of the type III secretion (T3S) effector IncA. Sol may therefore represent a potential new clinical treatment for infections. Selective perturbation of the T3S system suggests a novel mode of antibacterial action for Sol that warrants further investigation.

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Novel Detection Strategy To Rapidly Evaluate the Efficacy of Antichlamydial Agents

Cited by 6 publications

References 51 publications

Context-Dependent Action of Scc4 Reinforces Control of the Type III Secretion System

Context-Dependent Action of Scc4 Reinforces Control of the Type III Secretion System

In-cell western assay as a high-throughput approach for Chlamydia trachomatis quantification and susceptibility testing to antimicrobials

Potency of Solithromycin against Fast- and Slow-Growing Chlamydial Organisms

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