Use of green fluorescent protein (GFP) to study the invasion pathways of Edwardsiella tarda in in vivo and in vitro fish models

Ling, Sharon Hee Ming; Wang, X. H.; Xie, Linfeng; Lim, Tit Meng; Leung, Ka Yin

doi:10.1099/00221287-146-1-7

Cited by 174 publications

(126 citation statements)

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“…The bacterial strains and plasmids used in this study are described in Table 1. E. tarda PPD130/91 (22) and its derived strains were grown in tryptic soy broth (TSB; BD Biosciences) at 25°C and Escherichia coli strains in Luria-Bertani broth (LB; BD Biosciences) at 37°C. To induce the expression of T3SS proteins, E. tarda strains were grown at 25°C in Dulbecco modified Eagle medium (DMEM; Invitrogen) under 5% (vol/vol) CO 2 atmosphere.…”

Section: Methodsmentioning

confidence: 99%

EseE of Edwardsiella tarda Augments Secretion of Translocon Protein EseC and Expression of the escC - eseE Operon

et al. 2016

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Edwardsiella tarda is an important Gram-negative pathogen that employs a type III secretion system (T3SS) to deliver effectors into host cells to facilitate bacterial survival and replication. These effectors are translocated into host cells through a translocon complex composed of three secreted proteins, namely, EseB, EseC, and EseD. The secretion of EseB and EseD requires a chaperone protein called EscC, whereas the secretion of EseC requires the chaperone EscA. In this study, we identified a novel protein (EseE) that also regulates the secretion of EseC. An eseE deletion mutant secreted much less EseC into supernatants, accompanied by increased EseC levels within bacterial cells. We also demonstrated that EseE interacted directly with EseC in a pulldown assay. Interestingly, EseC, EseE, and EscA were able to form a ternary complex, as revealed by pulldown and gel filtration assays. Of particular importance, the deletion of eseE resulted in decreased levels of EseB and EseD proteins in both the bacterial pellet and supernatant fraction. Furthermore, real-time PCR assays showed that EseE positively regulated the transcription of the translocon operon escC-eseE, comprising escC, eseB, escA, eseC, eseD, and eseE. These effects of EseE on the translocon components/operon appeared to have a functional consequence, since the ⌬eseE strain was outcompeted by wild-type E. tarda in a mixed infection in blue gourami fish. Collectively, our results demonstrate that EseE not only functions as a chaperone for EseC but also acts as a positive regulator controlling the expression of the translocon operon escC-eseE, thus contributing to the pathogenesis of E. tarda in fish.T he type III secretion system (T3SS) is a contact-dependent translocation system. It forms a syringe-like structure spanning the inner and outer membranes of bacteria and induces pore formation on host cells (1). Through these pores, an array of bacterial proteins (effectors) are delivered into host cells, where they manipulate host cell signaling pathways to promote bacterial survival (2).Edwardsiella tarda is a Gram-negative intracellular pathogen that can cause hemorrhagic septicemia in fish (3), as well as gastroand extraintestinal infections in humans (4, 5). The T3SS is well known to be one of the most important virulence factors in E. tarda (6, 7). It facilitates the survival and replication of E. tarda in host cells (6-9). E. tarda T3SS contains 34 open reading frames (ORFs), which encode the apparatus, chaperones, effectors, and regulators (6, 10). The expression of E. tarda T3SS is controlled by many factors, such as the two-component system esrA-esrB (6) and esrC (11) inside the T3SS gene cluster, as well as phoP-phoQ (12) and phoR-phoB (13) located outside the T3SS gene cluster. Intriguingly, our group recently showed that a type III secretion system-secreted protein (EscE) also functions as a regulator controlling the injection of effectors and secretion of translocators (14).EseB, EseC, and EseD, secreted by the E. tarda T3SS, are homologous ...

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Section: Methodsmentioning

confidence: 99%

EseE of Edwardsiella tarda Augments Secretion of Translocon Protein EseC and Expression of the escC - eseE Operon

et al. 2016

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“…The pathogenesis of Ed. tarda is multifactorial, including factors that enable the bacteria to invade non-phagocytic cells (Kourany et al, 1977;Ling et al, 2000), survive in phagocytes (Srinivasa Rao et al, 2001, and produce virulence factors such as haemolysins (Janda & Abbott, 1993;Chen et al, 1996) and catalases (Srinivasa Rao et al, 2003). Recently, two important protein secretion systems, a type III secretion system (T3SS) (Tan et al, 2005;Zheng et al, 2007) and a type VI secretion system (T6SS) (Zheng & Leung, 2007), have been demonstrated to be virulence-associated.…”

Section: Introductionmentioning

confidence: 99%

Investigation of EscA as a chaperone for the Edwardsiella tarda type III secretion system putative translocon component EseC

Wáng

Mao

et al. 2009

Microbiology

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Edwardsiella tarda is an important Gram-negative enteric pathogen affecting both animals and humans. It possesses a type III secretion system (T3SS) essential for pathogenesis. EseB, EseC and EseD have been shown to form a translocon complex after secretion, while EscC functions as a T3SS chaperone for EseB and EseD. In this paper we identify EscA, a protein required for accumulation and proper secretion of another translocon component, EseC. The escA gene is located upstream of eseC and the EscA protein has the characteristics of T3SS chaperones. Cell fractionation experiments indicated that EscA is located in the cytoplasm and on the cytoplasmic membrane. Mutation with in-frame deletion of escA greatly decreased the secretion of EseC, while complementation of escA restored the wild-type secretion phenotype. The stabilization and accumulation of EseC in the cytoplasm were also affected in the absence of EscA. Mutation of escA did not affect the transcription of eseC but reduced the accumulation level of EseC as measured by using an EseC-LacZ fusion protein in Ed. tarda. Co-purification and coimmunoprecipitation studies demonstrated a specific interaction between EscA and EseC. Further analysis showed that residues 31-137 of EseC are required for EseC-EscA interaction. Mutation of EseC residues 31-137 reduced the secretion and accumulation of EseC in Ed. tarda. Finally, infection experiments showed that mutations of EscA and residues 31-137 of EseC increased the LD 50 by approximately 10-fold in blue gourami fish. These results indicated that EscA functions as a specific chaperone for EseC and contributes to the virulence of Ed. tarda.

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“…It has been isolated from a variety of animals, including fish, birds, mammals, and reptiles (12,27,31,46,47), and environmental water (32,50). Several potential virulence properties have been suggested to contribute to the pathogenesis of E. tarda, namely, the production of dermatotoxin (45) and hemolysin (14) and the ability to resist phagocyte-mediated destruction and to invade epithelial cells (19,22,43). However, little is known about the pathogenic mechanism of E. tarda, and the causes of disease occurrence are still elusive.…”

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confidence: 99%

Retention of Virulence in a Viable but Nonculturable Edwardsiella tarda Isolate

Chen

Zhang

et al. 2007

Appl Environ Microbiol

100

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Edwardsiella tarda is pathogen of fish and other animals. The aim of this study was to investigate the viable but nonculturable (VBNC) state and virulence retention of this bacterium. Edwardsiella tarda CW7 was cultured in sterilized aged seawater at 4°C. Total cell counts remained constant throughout the 28-day period by acridine orange direct counting, while plate counts declined to undetectable levels (<0.1 CFU/ml) within 28 days by plate counting. The direct viable counts, on the other hand, declined to ca. 10 9 CFU/ml active cells and remained fairly constant at this level by direct viable counting. These results indicated that a large population of cells existed in a viable but nonculturable state. VBNC E. tarda CW7 could resuscitate in experimental chick embryos and in the presence of nutrition with a temperature upshift. The resuscitative times were 6 days and 8 days, respectively. The morphological changes of VBNC, normal, and resuscitative E. tarda CW7 cells were studied with a scanning electron microscope. The results showed that when the cells entered into the VBNC state, they gradually changed in shape from short rods to coccoid and decreased in size, but the resuscitative cells did not show any obvious differences from the normal cells. The VBNC and the resuscitative E. tarda CW7 cells were intraperitoneally inoculated into turbot separately, and the fish inoculated with the resuscitative cells died within 7 days, which suggested that VBNC E. tarda CW7 might retain pathogenicity.Edwardsiella tarda, an enteric gram-negative bacterium of the Enterobacteriaceae, is the causative agent of the systemic disease edwardsiellosis in freshwater and marine fish worldwide. The bacterium causes septicemia with extensive skin lesions and affects internal organs, such as the liver, kidney, spleen, and muscle (33, 52). It has been isolated from a variety of animals, including fish, birds, mammals, and reptiles (12,27,31,46,47), and environmental water (32, 50). Several potential virulence properties have been suggested to contribute to the pathogenesis of E. tarda, namely, the production of dermatotoxin (45) and hemolysin (14) and the ability to resist phagocyte-mediated destruction and to invade epithelial cells (19,22,43). However, little is known about the pathogenic mechanism of E. tarda, and the causes of disease occurrence are still elusive.Many bacteria have developed strategies for metamorphosis into more or less sophisticated survival forms in response to harsh environmental conditions, such as a temperature change, high salinity, or nutrient deprivation (4, 5). The formation of viable but nonculturable (VBNC) cells of bacteria has been proposed as a strategy to survive adverse conditions (10). The first clear evidence of the existence of the VBNC state in pathogens was provided by Xu et al. It has been demonstrated that VBNC cells are active in metabolism (16,20,53) and are able to recover from their dormant state, becoming metabolically active and fully culturable (26,40,48). Some pathogens in the VBNC ...

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Use of green fluorescent protein (GFP) to study the invasion pathways of Edwardsiella tarda in in vivo and in vitro fish models

Cited by 174 publications

References 40 publications

EseE of Edwardsiella tarda Augments Secretion of Translocon Protein EseC and Expression of the escC - eseE Operon

EseE of Edwardsiella tarda Augments Secretion of Translocon Protein EseC and Expression of the escC - eseE Operon

Investigation of EscA as a chaperone for the Edwardsiella tarda type III secretion system putative translocon component EseC

Retention of Virulence in a Viable but Nonculturable Edwardsiella tarda Isolate

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