Phenotypic diversity of type III secretion system activity in enteropathogenic Escherichia coli clinical isolates

Abstract

Introduction. Enteropathogenic Escherichia coli (EPEC) strains pose a significant threat as a leading cause of severe childhood diarrhoea in developing nations. EPEC pathogenicity relies on the type III secretion system (T3SS) encoded by the locus of enterocyte effacement (LEE), facilitating the secretion and translocation of bacterial effector proteins.Gap Statement. While the regulatory roles of PerC (plasmid-encoded regulator) and GrlA (global regulator of LEE-activator) in ler expression and LEE gene activation are well-documented in the EPEC prototype strain E2348/69, understanding the variability in LEE gene expression control mechanisms among clinical EPEC isolates remains an area requiring further investigation.Aim. This study aims to explore the diversity in LEE gene expression control mechanisms among clinical EPEC isolates through a comparative analysis of secretion profiles under defined growth conditions favouring either PerC- or GrlA-mediated activation of LEE expression.Methodology. We compared T3SS-dependent secretion patterns and promoter expression in both typical EPEC (tEPEC) and atypical EPEC (aEPEC) clinical isolates under growth conditions favouring either PerC- or GrlA-mediated activation of LEE expression. Additionally, we conducted promoter reporter activity assays, quantitative real-time PCR and Western blot experiments to assess gene expression activity.Results. Significant differences in T3SS-dependent secretion were observed among tEPEC and aEPEC strains, independent of LEE sequence variations or T3SS gene functionality. Notably, a clinical tEPEC isolate exhibited increased secretion levels under repressive growth conditions and in the absence of both PerC and GrlA, implicating an alternative mechanism in the activation of Ler (LEE-encoded regulator) expression.Conclusion. Our findings indicate that uncharacterized LEE regulatory mechanisms contribute to phenotypic diversity among clinical EPEC isolates, though their impact on clinical outcomes remains unknown. This challenges the conventional understanding based on reference strains and highlights the need to investigate beyond established models to comprehensively elucidate EPEC pathogenesis.

Keywords: GrlA; Ler; PerC; clinical isolates; enteropathogenic Escherichia coli; locus of enterocyte effacement; pathogenesis; phenotypic diversity; regulatory mechanisms; type III secretion system.

Fig. 1.. The protein secretion profiles of 17 representative tEPEC and aEPEC clinical isolates demonstrate the diversity within EPEC. Bacteria were cultured under various conditions: shaken DMEM (a), static DMEM in a 5% CO₂ atmosphere (b), and shaken LB medium (c), all at 37 °C. Secreted proteins were precipitated from culture supernatants collected at the stationary phase using 10% TCA and resolved by 15% SDS-PAGE. Total cell extracts were subjected to Western blot analysis using anti-EscJ (LEE2) and anti-GroEL antibodies as loading controls. The strains were categorized into three groups: Group 1 (lanes 1 to 8), Group 2 (lanes 9 to 11), and Group 3 (lanes 12* to 17). The experiment was replicated at least three times, yielding similar results. The symbol ‘+’ indicates tEPEC strains carrying the pEAF plasmid.

Fig. 2.. Constitutive expression of GrlA and PerC and elimination of GrlR restored T3S and LEE expression in the non-secreting tEPEC strain D3309. tEPEC strain D3309 transformants carrying plasmids pMPM-K3 (empty vector), pK3-Ler_E2348, pK3-GrlA_E2348, pK3-PerC_E2348 or pK3-GrlR_E2348 (a and b) or WT and its Δler, ΔgrlA and ΔgrlR isogenic mutants (c and d) were grown in shaken DMEM or static DMEM + CO₂. Culture supernatants and bacterial pellets were collected at an OD₆₀₀ of approximately 1.0. Secreted proteins were precipitated using 10% TCA, resolved by 15% SDS-PAGE and stained with Coomassie brilliant blue. Western blot analysis of total cell extracts from the same strains was conducted using α-GroEL as a loading control (middle panels) and α-EscJ to detect the non-secreted LEE-encoded T3SS component (bottom panels). The experiment was replicated at least three times, yielding similar results.

Fig. 3.. The secreted protein profile of the hypersecretory tEPEC D3158 strain is maintained even in the absence of GrlA and PerC. tEPEC strains D3158 and E2348/69 and their corresponding isogenic mutants, Δler, ΔgrlA, ΔperC, ΔgrlR, ΔperC ΔgrlA and ΔgrlR/A, were grown in LB medium with shaking (a), DMEM with shaking (b) and static DMEM under a 5% CO₂ atmosphere (c), at 37 °C. Culture supernatants and bacterial pellets were collected at an OD₆₀₀ of approximately 1.0. Secreted proteins were precipitated using 10% TCA, resolved by 15% SDS-PAGE and stained with Coomassie brilliant blue (upper panels). Western blot analysis of total cell extracts from the same strains using α-GroEL (loading control, middle panels) and α-EscJ (non-secreted LEE-encoded T3SS component, bottom panels). The experiment was replicated at least three times, yielding similar results.

Fig. 4.. The transcriptional activity of the sepZ11-cat fusion in tEPEC D3158 remains active in the absence of the ler-specific activators GrlA and PerC, but it remains dependent on Ler. tEPEC strains D3158 (a) and E2348/69 (b), along with their corresponding isogenic mutants (Δler, ΔgrlA, ΔperC, ΔgrlR, ΔperCΔgrlA and ΔgrlR/A), carrying a plasmid with the sepZ11-cat fusion, were cultured in LB medium with shaking, shaken DMEM and static DMEM under 5% CO₂ at 37 °C. CAT-specific activity was determined from samples collected at an OD₆₀₀ of ∼1. The data represent the average of three independent experiments conducted in duplicate.

Fig. 5.. tEPEC strain D3158 expresses a functional GrlR protein that lacks the last five amino acids compared to that of strain E2348/69. Alignment of the amino acid sequences of GrlR from strains E2348/69 and D3158 (a). Secreted protein profiles of wild-type E2348/69 and D3158, along with their ∆grlR isogenic mutants, transformed with plasmids pMPM-K3 (empty vector), pK3-GrlR_E2348 and pK3-GrlR_D3158 (b and c, upper panels). Culture supernatants and bacterial pellets were collected at an OD₆₀₀ of approximately 1.0. Secreted proteins were precipitated using 10% TCA, resolved by 15% SDS-PAGE and stained with Coomassie brilliant blue (upper panels). Western blot analysis of total cell extracts from the same strains using α-GroEL (loading control), α-EscJ (non-secreted LEE-encoded T3SS component) and α-GrlR antibodies (b and c, lower panels). The experiment was conducted three times independently, yielding similar results.