Formylglycine-Generating Enzyme-Like Proteins Constitute a Novel Family of Widespread Type VI Secretion System Immunity Proteins

Abstract

Competition is a critical aspect of bacterial life, as it enables niche establishment and facilitates the acquisition of essential nutrients. Warfare between Gram-negative bacteria is largely mediated by the type VI secretion system (T6SS), a dynamic nanoweapon that delivers toxic effector proteins from an attacking cell to adjacent bacteria in a contact-dependent manner. Effector-encoding bacteria prevent self-intoxication and kin cell killing by the expression of immunity proteins, which prevent effector toxicity by specifically binding their cognate effector and either occluding its active site or preventing structural rearrangements necessary for effector activation. In this study, we investigate Tsi3, a previously uncharacterized T6SS immunity protein present in multiple strains of the human pathogen Acinetobacter baumannii . We show that Tsi3 is the cognate immunity protein of the antibacterial effector of unknown function Tse3. Our bioinformatic analyses indicate that Tsi3 homologs are widespread among Gram-negative bacteria, often encoded within T6SS effector-immunity modules. Surprisingly, we found that Tsi3 homologs are predicted to possess a characteristic formylglycine-generating enzyme (FGE) domain, which is present in various enzymatic proteins. Our data shows that Tsi3-mediated immunity is dependent on Tse3-Tsi3 protein-protein interactions and that Tsi3 homologs from various bacteria do not provide immunity against non-kin Tse3. Thus, we conclude that Tsi3 homologs are unlikely to be functional enzymes. Collectively, our work identifies FGE domain-containing proteins as important mediators of immunity against T6SS attacks and indicates that the FGE domain can be co-opted as a scaffold in multiple proteins to carry out diverse functions. Importance Despite the wealth of knowledge on the diversity of biochemical activities carried out by T6SS effectors, comparably little is known about the various strategies bacteria employ to prevent susceptibility to T6SS-dependent bacterial killing. Our work establishes a novel family of T6SS immunity proteins with a characteristic FGE domain. This domain is present in enzymatic proteins with various catalytic activities. Our characterization of Tsi3 expands the known functions carried out by FGE-like proteins to include defense during T6SS-mediated bacterial warfare. Moreover, it highlights the evolution of FGE domain-containing proteins to carry out diverse biological functions.

The Type VI secretion systemof Stenotrophomonas rhizophilaCFBP13503 limits the transmission ofXanthomonas campestrispv. campestris8004 from radish seeds to seedlings

“…We assume that these genes could encode potential toxins and associated immunity proteins and potential chaperones. The 18kb inter region between tssM and tssA and other structural genes contains a poly-immunity locus with a predicted formylglycine-generating enzyme family immunity protein encoding gene (Lopez et al 2021) and 8 duplications of the putative immunity gene HKJBHOBG_02263 containing a DUF6708 domain.…”

The type VI secretion system of Stenotrophomonas rhizophilaCFBP13503 limits the transmission of Xanthomonas campestris pv. campestris 8004 from radish seeds to seedlings

“…We assume that these genes could encode potential toxins and associated immunity proteins and potential chaperones. The 18 kb region between tssM and tssA and other structural genes contains a poly-immunity locus with a predicted formylglycine-generating enzyme family immunity protein encoding gene (Lopez et al, 2021) and eight duplications of the putative immunity gene HKJBHOBG_02263 containing a DUF6708 domain. (Figure 3).…”

The structure of a tautomerase superfamily member linked to the type VI secretion system of Acinetobacter baumannii

“…This might therefore represent an extension or adaptation of the system noted in Vibrio cholerae whereby quorum sensing contributes a role to control the expression of a T6SS with a link to virulence (Zheng et al, 2010). Intriguingly, enzyme-like proteins have been identified as providing T6SS-dependent immunity proteins (Lopez et al, 2021) and the possibility exists that AbTFM might represent an effector-interacting protein. Finally, we cannot rule out the possibility that AbTFM indeed fulfils a role in a host or target cell.…”