Interbacterial competition and anti‐predatory behaviour of environmental Vibrio cholerae strains

Summary Vibrio cholerae isolates responsible for cholera pandemics represent only a small portion of the diverse strains belonging to this species. Indeed, most V. cholerae are encountered in aquatic environments. To better understand the emergence of pandemic lineages, it is crucial to discern what differentiates pandemic strains from their environmental relatives. Here, we studied the interaction of environmental V. cholerae with eukaryotic predators or competing bacteria and tested the contributions of the haemolysin and the type VI secretion system (T6SS) to those interactions. Both of these molecular weapons are constitutively active in environmental isolates but subject to tight regulation in the pandemic clade. We showed that several environmental isolates resist amoebal grazing and that this anti‐grazing defense relies on the strains' T6SS and its actincross‐linking domain (ACD)‐containing tip protein. Strains lacking the ACD were unable to defend themselves against grazing amoebae but maintained high levels of T6SS‐dependent interbacterial killing. We explored the latter phenotype through whole‐genome sequencing of 14 isolates, which unveiled a wide array of novel T6SS effector and (orphan) immunity proteins. By combining these in silico predictions with experimental validations, we showed that highly similar but non‐identical immunity proteins were insufficient to provide cross‐immunity among those wild strains.

Aux cluster 3 is absent.
Encoded protein with 1164 aa; evolved VgrG1 with an actin crosslinking domain (ACD).
Aux cluster 3 is absent.
Encoded protein with 1164 aa; evolved VgrG1 with an actin crosslinking domain (ACD).
Aux cluster 3 is absent.

C SL6Y
Encoded protein with 977 aa; evolved VgrG3 with a predicted peptidoglycan hydrolase and phage-like lysozyme domains. Etype.
Encoded protein with 1164 aa; evolved VgrG1 with an actin crosslinking domain (ACD).
Aux cluster 3 is absent.

SP6G*
Encoded protein with 1052 aa; evolved VgrG3 with a predicted peptidoglycan hydrolase domain; H-type.
Encoded protein with 1164 aa; evolved VgrG1 with an actin crosslinking domain (ACD).
Aux cluster 3 is absent.

L6G*
Encoded protein with 989 aa; evolved VgrG3 with a predicted peptidoglycan hydrolase and lambda phage-like lysozyme domains; K-type.
Encoded protein with 1167 aa; evolved VgrG1 with an actin crosslinking domain (ACD).
Aux cluster 3 is absent. Aux cluster 3 is absent.
Encoded protein with 1164 aa; evolved VgrG1 with an actin crosslinking domain (ACD).
Aux cluster 3 is absent.

A W10G
Encoded protein with 1018 aa; evolved VgrG3 with predicted peptidoglycan hydrolase domain; A-type.
PacBio sequencing artifact in the effector gene (HPY12_07370). Region was Sanger sequenced and manually corrected before translation to allow typing. Encoded protein with 710 aa; uncharacterized alpha/beta hydrolase domain (DUF2235); C-type.
PacBio sequencing artifact in the effector gene (HPY12_14010). Region was Sanger sequenced and manually corrected before translation to allow typing. Encoded protein with 1086 aa; VasX; pore forming activity 2 ; A-type.
Aux cluster 3 is absent.

B SA3G
Encoded protein with 1018 aa; evolved VgrG3 with predicted peptidoglycan hydrolase domain; A-type.
Aux cluster 3 is absent.
Aux cluster 3 is absent.
Aux cluster 3 is absent.
Aux cluster 3 is absent.
Aux cluster 3 is absent.

Non-pandemic ATCC25872
Encoded protein with 1018 aa; evolved VgrG3 with predicted peptidoglycan hydrolase domain; A-type.
Encoded protein with 1164 aa; evolved VgrG1 with an actin crosslinking domain (ACD).