Dual temporal transcription activation mechanisms control cesT expression in enteropathogenic Escherichia coli

Brouwers, Erin; Ma, Irene; Thomas, Nikhil A.

doi:10.1099/mic.0.059444-0

Cited by 6 publications

(6 citation statements)

References 62 publications

(74 reference statements)

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“…We generated EPEC strains that express (i) CesT Y152F, (ii) CesT Y153F and (iii) CesT Y152F, Y153F (non‐phosphorylatable at positions 152 and 153). Importantly, these strains maintain the normal chromosomal operon organization that is involved in dual transcriptional regulation of cesT mRNA expression (Brouwers et al ., ) and were found to produce the respective CesT variants (tyrosine to phenylalanine) at levels equivalent to wild type CesT as determined by immunoblotting with anti‐CesT antibodies (Fig. A).…”

Section: Resultsmentioning

confidence: 96%

“…The CesT protein sequence (Genbank accession number CAS11488.1) is highly conserved (96%–100% identity) across all serotypes and strains within publicly accessible databases. Located within the LEE‐PAI, the cesT allele is transcriptionally controlled by two differentially regulated promoter elements (Brouwers et al ., ). CesT homodimers have been shown to interact with at least 10 of the 21 annotated EPEC effectors (Tir, Map, EspF, EspH, EspG, EspZ, NleA, NleG, NleH1 and NleH2), which suggests a central role for this class 1B multicargo chaperone in the EPEC pathogenic strategy (Thomas et al ., ).…”

Section: Introductionmentioning

confidence: 97%

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Tandem tyrosine phosphosites in the Enteropathogenic Escherichia coli chaperone CesT are required for differential type III effector translocation and virulence

Runte

Jain

Getz

et al. 2018

Molecular Microbiology

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Enteropathogenic Escherichia coli (EPEC) use a type 3 secretion system (T3SS) for injection of effectors into host cells and intestinal colonization. Here, we demonstrate that the multicargo chaperone CesT has two strictly conserved tyrosine phosphosites, Y152 and Y153 that regulate differential effector secretion in EPEC. Conservative substitution of both tyrosine residues to phenylalanine strongly attenuated EPEC type 3 effector injection into host cells, and limited Tir effector mediated intimate adherence during infection. EPEC expressing a CesT Y152F variant were deficient for NleA effector expression and exhibited significantly reduced translocation of NleA into host cells during infection. Other effectors were observed to be dependent on CesT Y152 for maximal translocation efficiency. Unexpectedly, EPEC expressing a CesT Y153F variant exhibited significantly enhanced effector translocation of many CesT-interacting effectors, further implicating phosphosites Y152 and Y153 in CesT functionality. A mouse infection model of intestinal disease using Citrobacter rodentium revealed that CesT tyrosine substitution variants displayed delayed colonization and were more rapidly cleared from the intestine. These data demonstrate genetically separable functions for tandem tyrosine phosphosites within CesT. Therefore, CesT via its C-terminal tyrosine phosphosites, has relevant roles beyond typical type III secretion chaperones that interact and stabilize effector proteins.

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Section: Resultsmentioning

confidence: 96%

Section: Introductionmentioning

confidence: 97%

Tandem tyrosine phosphosites in the Enteropathogenic Escherichia coli chaperone CesT are required for differential type III effector translocation and virulence

Runte

Jain

Getz

et al. 2018

Molecular Microbiology

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“…We generated EPEC strains that express i) CesT Y152F, ii) CesT Y153F, and iii) CesT Y152F, Y153F (non-phosphorylatable at positons 152 and 153). Importantly, these strains maintain the normal chromosomal operon organization that is involved in dual transcriptional regulation of cesT mRNA expression (29) and were found to produce the respective CesT variants (tyrosine to phenylalanine) at levels equivalent to wild type CesT as determined by immunoblotting with anti-CesT antibodies (Fig 2A). Therefore, CesT tyrosine residues 152 and 153 are not essential for maintaining CesT steady state levels in EPEC.…”

Section: Resultsmentioning

confidence: 95%

“…The CesT protein sequence (Genbank accession number CAS11488.1) is highly conserved (96-100% identity) across all serotypes and strains within publicly accessible databases. Located within the LEE-PAI, the cesT allele is transcriptionally controlled by two differentially regulated promoter elements (29). CesT homodimers have been shown to interact with at least 10 of the 21 annotated EPEC effectors (Tir, Map, EspF, EspH, EspG, EspZ, NleA, NleG, NleH1, and NleH2), which suggests a central role for this class 1B multicargo chaperone in the EPEC pathogenic strategy (27).…”

Section: Introductionmentioning

confidence: 99%

Tandem tyrosine residues in the EPEC multicargo chaperone CesT support differential type III effector translocation and early host colonization

Runte

Jain

Getz

et al. 2018

Preprint

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Enteropathogenic Escherichia coli (EPEC) are worldwide human enteric pathogens inflicting significant morbidity and causing large economic losses. A type 3 secretion system (T3SS) is critical for EPEC intestinal colonization, and injection of effectors into host cells contributes to cellular subversion and innate immune evasion. Here, we demonstrate that two strictly conserved C-terminal tyrosine residues, Y152 and Y153, within the multicargo T3SS chaperone CesT serve differential roles in regulating effector secretion in EPEC. Conservative substitution of both tyrosine residues to phenylalanine attenuated EPEC type 3 effector injection into host cells, and significantly limited Tir effector mediated intimate adherence, a key feature of attaching and effacing pathogenesis. Whereas CesT Y153 supported normal levels of Tir translocation, CesT Y152 was strictly required for the effector NleA to be expressed and subsequently translocated into host cells during infection. Other effectors were observed to be dependent on CesT Y152 for maximal translocation efficiency. Unexpectedly, EPEC expressing a CesT Y152, Y153F variant exhibited significantly enhanced effector translocation of many CesT-interacting effectors, further implicating Y152 in CesT functionality. A mouse infection model of EPEC intestinal disease using Citrobacter rodentium revealed that CesT tyrosine substitution variants displayed delayed colonization and were more rapidly cleared from the intestine. These data demonstrate genetically separable functions for strictly conserved tandem tyrosine residues within CesT. Tyrosine 152 of CesT is implicated in NleA expression, providing functional relevance for localized amino acid conservation. Therefore, CesT via its novel C-terminal domain, has relevant roles beyond typical T3SC that interact and stabilize effector proteins.

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“…Luciferase reporter assays were performed similar to a previous promoter study (Brouwers et al, 2012 ). End-point or multiple point assays were performed with Vp strains harboring lux plasmid constructs.…”

Section: Methodsmentioning

confidence: 99%

Transcriptional profiling of Vibrio parahaemolyticus exsA reveals a complex activation network for type III secretion

Liu

Thomas

2015

Front. Microbiol.

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Vibrio parahaemolyticus (Vp) is a marine halophilic bacterium that is commonly associated with oysters and shrimp. Human consumption of contaminated shellfish can result in Vp mediated gastroenteritis and severe diarrheal disease. Vp encodes two type 3 secretion systems (T3SS-1 and T3SS2) that have been functionally implicated in cytotoxicity and enterotoxicity respectively. In this study, we profiled protein secretion and temporal promoter activities associated with exsA and exsB gene expression. exsA is an AraC-like transcriptional activator that is critical for activating multiple operons that encode T3SS-1 genes, whereas exsB is thought to encode an outer membrane pilotin component for T3SS-1. The exsBA genetic locus has two predicted promoter elements. The predicted exsB and exsA promoters were individually cloned upstream of luxCDABE genes in reporter plasmid constructs allowing for in situ, real-time quantitative light emission measurements under many growth conditions. Low calcium growth conditions supported maximal exsB and exsA promoter activation. exsB promoter activity exhibited high basal activity and resulted in an exsBA co-transcript. Furthermore, a separate proximal exsA promoter showed initial low basal activity yet eventually exceeded that of exsB and reached maximal levels after 2.5 h corresponding to an entry into early log phase. exsA promoter activity was significantly higher at 30°C than 37°C, which also coincided with increased secretion levels of specific T3SS-1 effector proteins. Lastly, bioinformatic analyses identified a putative expanded ExsA binding motif for multiple transcriptional operons. These findings suggest a two wave model of Vp T3SS-I induction that integrates two distinct promoter elements and environmental signals into a complex ExsA activation framework.

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Dual temporal transcription activation mechanisms control cesT expression in enteropathogenic Escherichia coli

Cited by 6 publications

References 62 publications

Tandem tyrosine phosphosites in the Enteropathogenic Escherichia coli chaperone CesT are required for differential type III effector translocation and virulence

Tandem tyrosine phosphosites in the Enteropathogenic Escherichia coli chaperone CesT are required for differential type III effector translocation and virulence

Tandem tyrosine residues in the EPEC multicargo chaperone CesT support differential type III effector translocation and early host colonization

Transcriptional profiling of Vibrio parahaemolyticus exsA reveals a complex activation network for type III secretion

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