Identification and Characterization of EvpQ, a Novel T6SS Effector Encoded on a Mobile Genetic Element in Edwardsiella piscicida

Abstract

In this study, a hypothetical protein (ORF02740) secreted by Edwardsiella piscicida was identified. We renamed the ORF02740 protein as EvpQ, which is encoded by a mobile genetic element (MGE) in E. piscicida genome. The evpQ gene is spaced by 513 genes from type VI secretion system (T6SS) gene cluster. Low GC content, three tRNA, and three transposase genes nearby evpQ define this MGE that evpQ localizes as a genomic island. Sequence analysis reveals that EvpQ shares a conserved domain of C70 family cysteine protease and shares 23.91% identity with T3SS effector AvrRpt2 of phytopathogenic Erwinia amylovora. Instead, EvpQ of E. piscicida is proved to be secreted at a T6SS-dependent manner, and it can be translocated into host cells. EvpQ is thereof a novel T6SS effector. Significantly decreased competitive index of ΔevpQ strain in blue gourami fish (0.53 ± 0.27 in head kidney and 0.44 ± 0.19 in spleen) indicates that EvpQ contributes to the pathogenesis of E. piscicida. At 8-, 18-, and 24-h post-subculture into DMEM, the transcription of evpQ was found to be negatively regulated by Fur and positively regulated by EsrC, and the steady-state protein levels of EvpQ are negatively controlled by RpoS. Our study lays a foundation for further understanding the pathogenic role of T6SS in edwardsiellosis.

Keywords: Edwardsiella piscicida; EvpQ; effector; mobile genetic element; regulation; type VI secretion system.

FIGURE 1

EvpQ is secreted by Edwardsiella piscicida in a manner different from T3SS effectors. Five percent of bacterial pellets (TBPs) and 10% of culture supernatants (ECPs) from similar amounts of WT evpQ:2HA and ΔesaB evpQ:2HA strains grown in Dulbecco’s modified Eagle’s medium (DMEM) were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene fluoride (PVDF) membranes for probing with anti-HA (EvpQ), anti-EseG, anti-EseJ, anti-DnaK, and anti-EvpC.

FIGURE 2

EvpQ is secreted at a T6SS-dependent manner and is translocated into eukayotic host. (A) EvpQ is secreted at a T6SS-dependent manner. TBPs and ECPs from each strain grown in DMEM were harvested at stationary phase, and equal amount of TBPs and ECPs were loaded for separating by SDS-PAGE and transferred onto PVDF membranes for immunoblotting. (B) Extracellular and intracellular profiles of E. piscicida strains. ECPs from similar amounts of E. piscicida strains grown in DMEM were separated using SDS-PAGE and stained with Coomassie blue. EvpI, EvpP, and EvpC are T6SS proteins secreted. (C) EvpQ does not depend on T3SS for its translocation. The EPC cells were infected with E. piscicida strains transformed with pACYC-evpQ::cyaA. At 2 h post-infection, the EPC monolayers were processed to examine the intracellular cAMP level as described in section “Materials and Methods.” Means ± SD from one representative experiment was shown. ^{* **}P < 0.001; NS, not significant. (D) EvpQ is translocated into host cells. EPC cells were infected, respectively, with WT evpQ::2HA strain and ΔevpQ strain before fractionation by mechanical disruption. Cell lysates of EPC cells and bacteria were analyzed by immunoblotting using anti-HA, anti-EseE, and anti-actin antibodies. Lanes 1–2, E. piscicida cell lysates of WT evpQ::2HA strain and ΔevpQ strain, respectively; lane 3, protein marker; lanes 4–5, cell lysates of EPC cells infected, respectively, with WT evpQ::2HA strain and ΔevpQ strain.

FIGURE 3

The ΔevpQ strain is less competitive than wild-type E. piscicida. Six naïve blue gourami fish were injected intramuscularly with a mixture of equal numbers of wild-type and ΔevpQ::km strains and killed 48 h post-injection. CIs from spleen and head kidney were given for individual fish, and means ± SD were shown by the horizontal lines. Student’s t test was used to calculate the P-value with the hypothetical mean of 1.0. ***P < 0.001; **P < 0.01.

FIGURE 4

EvpQ is encoded by a genomic island far outsides T6SS gene cluster. Gene organization of T6SS gene cluster and the GI that encodes EvpQ. Data were from reference (Zheng and Leung, 2007) and our laboratory. Open reading frames with identifiable orthologs in the horizontal transferred fragment are labeled. Yellow arrows, T6SS effector genes; azure arrows, T6SS apparatus genes; green arrows, transposase genes; orange arrows, tRNA genes; white arrows, hypothetical genes.

FIGURE 5

EvpQ is under the control of EsrC, RpoS, Fur, and temperature. (A) Comparison of the transcriptional level of evpQ in WT, ΔesrC, ΔrpoS, and Δfur strains. The DNA fragment (bp -594 to -1) upstream of evpQ was inserted into the promoterless gfp shuttle vector pFPV25 to create pFPV-evpQ_{–594 to –1}. This plasmid was then introduced into E. piscicida strains. At 8, 18, and 24 hps in DMEM, the fluorescence intensity of those E. piscicida strains were evaluated by using a microplate reader. Means ± SD from one representative experiment was shown. ***P < 0.001; **P < 0.01; NS, not significant. (B) Examination on the steady-state protein level of EvpQ in different E. piscicida strains at 8-, 18-, and 24-h post-subculture in DMEM. This experiment was repeated separately for at least three times, and one representative blot was shown. (C) The expression and secretion of EvpQ from E. piscicida wild type cultured in DMEM at different temperatures. ECPs and TCPs from similar amounts of the WT evpQ:2HA cultured at 25, 35, or 37°C were probed with anti-HA, anti-EvpC, anti-EseG, and anti-DanK antibodies. (D) The transcription levels of evpQ from E. piscicida wild type cultured in DMEM at different temperatures. The mRNA levels of evpQ from the wild-type strain cultured at 25, 35, or 37°C were examined by qRT-PCR. 16S rRNA was used as the reference gene. Transcription levels of evpQ relative to that of 16S rRNA are presented (i.e., relative fold changes in gene expression). Data are presented as means ± SD. **P < 0.01.

FIGURE 6

Schematic diagram of regulatory network of EsrC, RpoS, and Fur on EvpQ and T6SS gene cluster in E. piscicida PPD130/91, based on reference papers and our results from the current study. PhoQ senses the change of ambient temperature (23∼35°C) and transmits the signal to PhoP, and the phosphorylated PhoP binds directly to the PhoP box within the promoter region of esrB to activate its transcription (Chakraborty et al., 2010). The activated EsrB protein upregulates the transcription of T6SS through EsrC (Zheng et al., 2005; Chakraborty et al., 2010). High concentration of iron activates the Fur protein, and activated Fur binds directly to the Fur box in the promoter of T6SS effector gene evpP. The binding of Fur inhibits the binding of EsrC to the same region (Chakraborty et al., 2011). The EvpQ encoded by the genomic island is also negatively regulated by Fur and positively regulated by EsrC, while it remains to be resolved whether EsrC and Fur directly bind the promoter of evpQ. The sigma factor RpoS, antagonizing the expression of esrB (Yin et al., 2018), negatively controls the expression of EvpQ.