A Novel C-Terminal Region within the Multicargo Type III Secretion Chaperone CesT Contributes to Effector Secretion

Ramu, Thangadurai; Prasad, Madhulika Esther; Connors, Erica; Mishra, Amit V.; Thomassin, Jenny‐Lee; LeBlanc, Jason J.; Rainey, Jan K.; Thomas, Nikhil A.

doi:10.1128/jb.01967-12

Cited by 20 publications

(48 citation statements)

References 68 publications

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“…Many pathogenic bacteria use T3SS to rapidly inject effector proteins into host cells during infection. Effector proteins are synthesised in the bacterial cytosol and are trafficked across the bacterial inner and outer membranes and eventually across the host cell membrane (Ramu et al, 2013) to the cellular cytoplasm, where they modulate a variety host cells functions for the benefit of the pathogen (Adam et al, 2012). T3SS is known as one of the most important virulence factors in E. piscicida; it facilitates the survival and replication of E. piscicida in host cells (Okuda et al, 2006;Srinivasa, Lim, & Leung, 2001;Tan, Zheng, Tung, Rosenshine, & Leung, 2005).…”

Section: Discussionmentioning

confidence: 99%

Novel T3SS effector EseK in Edwardsiella piscicida is chaperoned by EscH and EscS to express virulence

Cao

Yang

Qin

et al. 2017

Cellular Microbiology

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Bacterium usually utilises type III secretion systems (T3SS) to deliver effectors directly into host cells with the aids of chaperones. Hence, it is very important to identify bacterial T3SS effectors and chaperones for better understanding of host-pathogen interactions. Edwardsiella piscicida is an invasive enteric bacterium, which infects a wide range of hosts from fish to human. Given E. piscicida encodes a functional T3SS to promote infection, very few T3SS effectors and chaperones have been identified in this bacterium so far. Here, we reported that EseK is a new T3SS effector protein translocated by E. piscicida. Bioinformatic analysis indicated that escH and escS encode two putative class I T3SS chaperones. Further investigation indicated that EscH and EscS can enhance the secretion and translocation of EseK. EscH directly binds EseK through undetermined binding domains, whereas EscS binds EseK via its N-terminal α-helix. We also found that EseK has an N-terminal chaperone-binding domain, which binds EscH and EscS to form a ternary complex. Zebrafish infection experiments showed that EseK and its chaperones EscH and EscS are necessary for bacterial colonisation in zebrafish. This work identified a new T3SS effector, EseK, and its two T3SS chaperones, EscH and EscS, in E. piscicida, which enriches our knowledge of bacterial T3SS effector-chaperone interaction and contributes to our understanding of bacterial pathogenesis.

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Section: Discussionmentioning

confidence: 99%

Novel T3SS effector EseK in Edwardsiella piscicida is chaperoned by EscH and EscS to express virulence

Cao

Yang

Qin

et al. 2017

Cellular Microbiology

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“…Furthermore, a C-terminal tail of CesAB was critical for the EscV interaction. It is tempting to speculate the same is true for CesT, especially in context of our previous observations relating to C-terminal tyrosine residues in CesT (24). Interestingly, FlgN, a Salmonella type III export flagellar chaperone, requires a highly conserved C-terminal Tyr122 residue to interact with FlhA (its EscV homologue) to support flagellar assembly (45).…”

Section: Discussionmentioning

confidence: 60%

“…The C-terminal ~20 amino acids are located outside the dimerization interface, and the last 11 amino acids were not resolved within protein crystals, suggesting it might be a disordered terminal region. Moreover, this terminal region of CesT and specific amino acid residues within it, are important for effector secretion (24) (Fig 1A). CesT is highly conserved over its entire 156 amino acid sequence, displaying 96-100% identity for over 300 NCBI database entries (Dataset S1).…”

Section: Resultsmentioning

confidence: 99%

“…Low calcium sensing by EPEC has been implicated in transitioning from translocon to effector protein secretion (20). The first secreted, or hierarchical effector, is the t ranslocated i ntimin r eceptor (Tir) (23, 24), although the mechanism behind this hierarchy remains elusive. Once Tir is translocated and localized within the host cytoplasmic membrane, it acts as a high affinity surface exposed receptor for intimin which is embedded within the outer membrane of EHEC/EPEC (25, 26).…”

Section: Introductionmentioning

confidence: 99%

“…CesT has been shown to interact with EscN, a hexameric ATPase that is required for T3SS function (31, 32). A genetic screen to assess CesT function identified many CesT C-terminal substitution variants that were defective for effector secretion (24). Specifically, some CesT variants only supported Tir secretion and not other effectors, thus implicating the C-terminal region of CesT in coordinating effector secretion.…”

Section: Introductionmentioning

confidence: 99%

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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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Binding specificity of type three secretion system effector NleH2 to multi‐cargo chaperone CesT and their phosphorylation

et al. 2021

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Gram-negative pathogens like Enteropathogenic Escherichia coli (EPEC) utilize the type three secretion system (T3SS) to translocate various effector proteins that are needed to "hijack" the host system for pathogenic survival. Specialized T3SS chaperones inside bacterial cells stabilize these effector proteins and facilitate their translocation. CesT is a unique multi-cargo chaperone that interacts with and translocates ~10 different effector proteins. Here, we report the specific interaction between CesT and its key effector, NleH2, and explore the potential role of NleH2 as a kinase for CesT phosphorylation. First, we identified the chaperone-binding domain (CBD; 19-97aa) of NleH2, and mapped the specific interaction sites for both CesT and NleH2. The N-and Cterminal residues of the CBD interact with the dimeric interface of CesT. Further, we compared the CesT binding to NleH2, to that of another key effector Tir and with the global carbon regulator CsrA. Notably, the effectors have the binding regions at the β-sheet core and dimer interface of CesT, whereas the CsrA regulator interacts predominantly through the C-terminal region, which is found ~17 Å away from the effectors-binding sites. Next, we showed that NleH2 remains an active kinase even as a complex with CesT and is responsible for its autophosphorylation as well as phosphorylation of CesT at Tyr153. Collectively, our findings enhance the understanding of the role of multi-cargo chaperone CesT in orchestrating effector translocation through T3SS.

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A Novel C-Terminal Region within the Multicargo Type III Secretion Chaperone CesT Contributes to Effector Secretion

Cited by 20 publications

References 68 publications

Novel T3SS effector EseK in Edwardsiella piscicida is chaperoned by EscH and EscS to express virulence

Novel T3SS effector EseK in Edwardsiella piscicida is chaperoned by EscH and EscS to express virulence

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

Binding specificity of type three secretion system effector NleH2 to multi‐cargo chaperone CesT and their phosphorylation

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