The Type II Secretory System Mediates Phage Infection in Vibrio cholerae

Abstract

Attachment and specific binding to the receptor on the host cell surface is the first step in the process of bacteriophage infection. The lytic phage VP2 is used in phage subtyping of the Vibrio cholerae biotype El Tor of the O1 serogroup; however, its infection mechanism is poorly understood. In this study, we aimed to identify its receptor on V. cholerae. The outer membrane protein EpsD in the type II secretory system (T2SS) was found to be related to VP2-specific adsorption to V. cholerae, and the T2SS inner membrane protein EpsM had a role in successful VP2 infection, although it was not related to adsorption of VP2. The tail fiber protein gp20 of VP2 directly interacts with EpsD. Therefore, we found that in V. cholerae, in addition to the roles of the T2SS as the transport apparatus of cholera toxin secretion and filamentous phage release, the T2SS is also used as the receptor for phage infection and probably as the channel for phage DNA injection. Our study expands the understanding of the roles of the T2SS in bacteria.

Keywords: EpsD; Vibrio cholerae; bacteriophage; receptor; type II secretory system.

Figure 1

Transposon insertion and VP2 infection of the mutants. (A) Transposon insertion site of VP2-resistant mutants in the gene cluster of the T2SS in N16961. The eps genes are colored according to the classification of functions and positions of the proteins in T2SS in V. cholerae (Korotkov et al., 2012), including inner-membrane plateform proteins (light blue), outer-membrane secretin (yellow), pseudopilin (light green), others (light brown) and unknown (grey). The black arrow represents the site where the transposon was inserted into the epsM. (B) Detection of VP2 infection in V. cholerae mutants by double-layer plaque assay and EOP assay. The wild-type V. cholerae El Tor strain N16961 was used as the control for plaque formation. N-ΔepsM showed VP2 resistance (no plaque formation). The strain ΔepsM-C, carrying the EpsM expression plasmid cloned into the strain N-ΔepsM, was sensitive to VP2. N-ΔepsD showed VP2 resistance (no plaque formation). The strain ΔepsD-C, carrying the EpsD expression plasmid cloned into the strain N-ΔepsD, was sensitive to VP2. The values of EOP were shown in the top half of the figure.

Figure 2

VP2 adsorption by the wild-type strain N16961 and its mutants. The VP2 phage (10⁶ CFU/mL) was mixed with fresh N16961 culture, N-ΔepsM, N-ΔepsD, N-ΔepsD+C (10⁸ CFU/mL) respectively for 3 min, 5min,10 min at 37°C, and then, each sample was centrifuged at 6000 rpm for 8 min. LB culture medium containing only VP2 phage was used as a negative control, and the phage titer in the control supernatant was set to 100%. The experiment was repeated three times. The mean of three independent assays was shown and error bars represent the standard deviation. **P =0.0026, ***P = 0.0001, ****P <0.0001, ns, no significance (Student’s t test).

Figure 3

Binding capacity of the vp2 tail filament protein with wild-type N16961 and N-ΔepsD. (A): Wild type+ His6-tagged gp20+anti-His6 tag antibody; (B): N-ΔepsD + His6-tagged gp20+anti-His6 tag antibody; (C): wild type +anti-His6 tag antibody; (D): N-ΔepsD +anti-His6 tag antibody; (E): wild type; (F): N-ΔepsD. Ten micrograms of His6-tagged gp20 protein and 10 μg of Alexa Fluor 488-conjugated anti-His-tag monoclonal antibody (product # MA1-21315-A488) were mixed and incubated for 30 min in the dark at room temperature and then transferred to the wild-type N16961 and N-ΔepsD strains, with OD600 = 1. After induction at 37°C, induced cultures were washed twice with 1 mL of filter-sterilized PBS. Antibodies and no antibodies were added to these two strains as controls. Samples were analyzed using BD flow cytometry. The data shown on the left represent the geometric MFI. The data shown on the right represent the fluorescence intensity distribution of the bacteria analyzed in the experiment shown on the left. The mean of three independent assays is shown and error bars represent the standard deviation. ****P <0.0001 (Student’s t test).

Figure 4

Binding capacity of the VP2 tail filament protein with wild-type N16961 and N-ΔepsD, and analysis of the interactions of EpsD with gp20. (A) Analysis the gp20-EpsD interaction by BACTH. PC, positive control (leucine zipper of GCN4); NC, negative control (vector plasmid only). The resulting recombinant plasmid pair pT25-gp20/pT18C-EpsD was co-transformed into BTH101 cells, and the β-galactosidase activity was measured. ****P <0.0001. ns, no significance. (Student’s t test). (B) Analysis of the interactions of EpsD with gp20 in vitro. Western blot analysis of His pull-down experiments was performed with GST-gp20 immobilized on Ni²⁺ resin, and the gp20 protein was incubated with GST for pull-down analysis as a negative control 0.3 mg of His6-tagged gp20 protein and 0.1 mg of GST-tagged EpsD protein were mixed, then the mixtures were rotated at room temperature for 2 h, bound to Ni2+ resin and incubated for 1 h at 4°C. Lane 1: His-gp20; 2: GST-EpsD; 3: Pull-down of His-gp20 and GST-EpsD; 4 Pull-down of His-gp20 + GST.