The protective function of an immunity protein against thecis-toxic effects of aXanthomonasType IV Secretion System Effector

Abstract Many bacterial species use specialized secretion systems to translocate proteinaceous toxic effectors into target bacterial cells. In most cases, effectors are encoded in bicistronic operons with their cognate immunity proteins. The current model is that immunity proteins could, in principle, provide protection in two different ways: i) by avoiding self-intoxication (suicide or cis -intoxication) or ii) by inhibiting intoxication due to “friendly-fire” translocation from neighboring sister cells (fratricide or trans -intoxication). Here, we set out to distinguish between these two protection mechanisms in the case of the bactericidal Xanthomonas citri Type IV Secretion System (X-T4SS), where killing is due to the action of a cocktail of secreted effectors (X-Tfes) that are inhibited by their cognate immunity proteins (X-Tfis). We use a set of X. citri mutants lacking multiple X-Tfe/X-Tfi pairs to show that X-Tfis are not absolutely required to protect against trans -intoxication. Our investigation then focused on the in vivo function of the lysozyme-like effector X-Tfe XAC2609 and its cognate immunity protein X-Tfi XAC2610 . We observe the accumulation of damage in the X. citri cell envelope and inhibition of biofilm formation due to the action of X-Tfe XAC2609 in the absence of X-Tfi XAC2610 . We show that X-Tfe XAC2609 toxicity is independent of an active X-T4SS and that X-Tfi XAC2610 protects the cell colony against X-Tfe XAC2609 -induced cis -intoxication via autolysis. In vitro assays employing X-Tfi XAC2610 mutants were used to test and validate an AlphaFold2-derived model of the X-Tfe XAC2609 -X-Tfi XAC2610 complex which presents topological similarities with the distantly related Tse1/Tsi1 complex from P. aeruginosa and the the i-type lysozyme from Meretrix lusoria (MI-iLys) in complex with PliI-Ah from Aeromonas hydrophila . While immunity proteins in other systems have been shown to protect against attacks by sister cells ( trans -intoxication), this is the first description of an antibacterial secretion system in which the immunity proteins are dedicated to protecting cells against cis -intoxication.