The Apoptogenic Toxin AIP56 Is Secreted by the Type II Secretion System of Photobacterium damselae subsp. piscicida

Abstract

AIP56 (apoptosis-inducing protein of 56 kDa) is a key virulence factor of Photobacterium damselae subsp. piscicida (Phdp), the causative agent of a septicaemia affecting warm water marine fish species. Phdp-associated pathology is triggered by AIP56, a short trip AB toxin with a metalloprotease A domain that cleaves the p65 subunit of NF-κB, an evolutionarily conserved transcription factor that regulates the expression of inflammatory and anti-apoptotic genes and plays a central role in host responses to infection. During infection by Phdp, AIP56 is systemically disseminated and induces apoptosis of macrophages and neutrophils, compromising the host phagocytic defence and contributing to the genesis of pathology. Although it is well established that the secretion of AIP56 is crucial for Phdp pathogenicity, the protein secretion systems operating in Phdp and the mechanism responsible for the extracellular release of the toxin remain unknown. Here, we report that Phdp encodes a type II secretion system (T2SS) and show that mutation of the EpsL component of this system impairs AIP56 secretion. This work demonstrates that Phdp has a functional T2SS that mediates secretion of its key virulence factor AIP56.

Keywords: T2SS; exotoxin; polar localisation; secretion.

Figure 1

P. damselae subsp. piscicida encodes a T2SS. Physical map of the P. damselae subsp. piscicida MT1415 12-gene cluster encoding the T2SS.

Figure 2

Deletion of epsL impairs AIP56 secretion. (A) Growth curves of wild type (WT) or isogenic ΔepsL mutant strain grown in TSB-1 at 25 °C. The curves were generated from three replicates for each strain. (B,C) Secreted protein profiles of WT and ΔepsL strain. SDS-PAGE analysis of culture supernatants from WT, ΔepsL (three independent clones) and pEpsL complemented strains. Bacterial strains were grown in TSB-1 at 25 °C to an OD 600 nm of approximately 1 or 0.35 for (B,C), respectively. Supernatants (ECPs) were collected, filtered and subjected to TCA precipitation. TCA precipitates were dissolved in running buffer and equivalents to 0.3 or 1.5 mL cultures (for (B,C), respectively) were subjected to SDS-PAGE. The gel was stained with Coomassie-blue. Numbers at the left indicate the molecular weight of the markers (M) in kDa. Asterisks indicate the AIP56 band. Data are representative of three independent experiments. (D) In epsL deleted strains, AIP56 is synthesised but retained inside the cells. WT and ΔepsL strains were grown in TSB-1 at 25 °C to an OD 600 nm of approximately 1 (exponential phase). ECPs were collected, filtered and subjected to TCA precipitation. AIP56 in the bacterial pellets and ECPs was detected by western blotting. Upper panel: western blotting (AIP56). Lower panel: total protein loading (Ponceau S).

Figure 3

In ΔepsL mutants, AIP56 accumulates in the periplasm. (A) Visualization of AIP56 localization in WT, ΔepsL and pEpsL complemented strains by fluorescence microscopy. Labelling was performed with an anti-AIP56^286–497 rabbit antibody and the anti-rabbit secondary antibody was conjugated to Alexa-488 (green on the coloured panels). The cells were counterstained with DAPI (blue in the coloured panels). (B) Results of the quantification of AIP56 positive cells in the immunofluorescence micrographs of WT, ΔepsL and ΔepsL + pEpsL strains. A minimum of 600 cells per condition were counted in each experiment, using Fiji software. Values are mean ± SD of two independent experiments. One-way ANOVA followed by Tukey post hoc test, **** p < 0.0001. (C) Transmission electron micrographs of ultrathin sections of WT and ΔepsL cells stained with an anti-AIP56^286–497 rabbit antibody followed by a gold-conjugated secondary antibody (15 nm gold). Bar = 200 nm. (D) Western blotting analysis of AIP56 in the WT ECPs and ΔepsL cells under reducing (+DTT) and non-reducing (−DTT) conditions. Samples equivalent to 75 μL initial culture were loaded in each lane. Upper panel: western blotting (AIP56). Lower panel: total protein loading (Ponceau S). Numbers at the left indicate the position and molecular weight of the markers (in kDa).