The Edwardsiella piscicida Type III Translocon Protein EseC Inhibits Biofilm Formation by Sequestering EseE

Abstract

The type III secretion system (T3SS) is one of the most important virulence factors of the fish pathogen Edwardsiella piscicida. It contains three translocon proteins, EseB, EseC, and EseD, required for translocation of effector proteins into host cells. We have previously shown that EseB forms filamentous appendages on the surface of E. piscicida, and these filamentous structures mediate bacterial cell-cell interactions promoting autoaggregation and biofilm formation. In the present study, we show that EseC, but not EseD, inhibits the autoaggregation and biofilm formation of E. piscicida. At 18 h postsubculture, a ΔeseC strain developed strong autoaggregation and mature biofilm formation, accompanied by enhanced formation of EseB filamentous appendages. This is in contrast to the weak autoaggregation and immature biofilm formation seen in the E. piscicida wild-type strain. EseE, a protein that directly binds to EseC and also positively regulates the transcription of the escC-eseE operon, was liberated and showed increased levels in the absence of EseC. This led to augmented transcription of the escC-eseE operon, thereby increasing the steady-state protein levels of intracellular EseB, EseD, and EseE, as well as biofilm formation. Notably, the levels of intracellular EseB and EseD produced by the ΔeseE and ΔeseC ΔeseE strains were similar but remarkably lower than those produced by the wild-type strain at 18 h postsubculture. Taken together, we have shown that the translocon protein EseC inhibits biofilm formation through sequestering EseE, a positive regulator of the escC-eseE operon. IMPORTANCE Edwardsiella piscicida, previously known as Edwardsiella tarda, is a Gram-negative intracellular pathogen that mainly infects fish. The type III secretion system (T3SS) plays a pivotal role in its pathogenesis. The T3SS translocon protein EseB is required for the assembly of filamentous appendages on the surface of E. piscicida. The interactions between the appendages facilitate autoaggregation and biofilm formation. In this study, we explored the role of the other two translocon proteins, EseC and EseD, in biofilm formation. We have demonstrated that EseC, but not EseD, inhibits the autoaggregation and biofilm formation of E. piscicida, providing new insights into the regulatory mechanism involved in E. piscicida biofilm formation.

HutZ is required for biofilm formation and contributes to the pathogenicity of Edwardsiella piscicida

“…Among these factors, some inhibit biofilm formation. For example, the type III translocon protein EseC inhibits biofilm formation by sequestering the regulator EseE [54], and an rpoS sigma factor mutant displayed markedly increased biofilm formation [55]. Deletion of the ugd gene, which encodes UDP-glucose dehydrogenase, enhanced autoaggregation and biofilm formation [56].…”

Bacteria autoaggregation: how and why bacteria stick together

“…These dual function adhesins are important in the initiation, growth, maintenance and disassembly of auto-aggregates (microcolonies) within infection niches. Further examples of surface organelles involved in autoaggregation are the Vibrio cholerae DNA-uptake pili [ 15 ], curli [ 16 ], Edwardsiella piscida EseB filaments [ 17 ] and the Vibro vulnificicus Tad pili [ 18 ].…”

Proteus faecis: a putative pathogen of the freshwater Yangtze finless porpoise

“…The ∆eseB strain and the ∆eseC strain were included as the negative control and positive control, respectively. The EseB lament-mediated bacterial cell-cell interaction prompts bio lm formation, however, EseC inhibits bio lm formation through sequestering EseE, a positive regulator for EseB (Gao et al 2015;Liu et al, 2019). Since bio lms act as a primary barrier to protect bacteria from the detrimental effects of antibiotics, cytokines, and complement (Wakimoto et al 2004), our results suggest P. faecis could evade host defenses and survive in vivo through bio lm formation.…”

“…The ∆eseB and ∆eseC strains were included as the negative control and positive control, respectively. They both are derived from Edwardsiella piscicida PPD130/91 (Ling et al 2000; Liu et al, 2019;Shao et al 2015). The culture supernatants were removed carefully at 24 h post-subculture, and the coverslips were gently rinsed three times with prewarmed PBS, and the bacteria attached to the coverslips were xed in 4% paraformaldehyde and stained with 0.1% crystal violet for 30 min.…”