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

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 orcis-intoxication) or ii) by inhibiting intoxication due to “friendly-fire” translocation from neighboring sister cells (fratricide ortrans-intoxication). Here, we set out to distinguish between these two protection mechanisms in the case of the bactericidalXanthomonas citriType 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 ofX. citrimutants lacking multiple X-Tfe/X-Tfi pairs to show that X-Tfis are not absolutely required to protect againsttrans-intoxication. Our investigation then focused on thein vivofunction of the lysozyme-like effector X-Tfe^XAC2609and its cognate immunity protein X-Tfi^XAC2610. We observe the accumulation of damage in theX. citricell envelope and inhibition of biofilm formation due to the action of X-Tfe^XAC2609in the absence of X-Tfi^XAC2610. We show that X-Tfe^XAC2609toxicity is independent of an active X-T4SS and that X-Tfi^XAC2610protects the cell colony against X-Tfe^XAC2609-inducedcis-intoxication via autolysis.In vitroassays employing X-Tfi^XAC2610mutants were used to test and validate an AlphaFold2-derived model of the X-Tfe^XAC2609-X-Tfi^XAC2610complex which presents topological similarities with the distantly related Tse1/Tsi1 complex fromP. aeruginosaand the the i-type lysozyme fromMeretrix lusoria(MI-iLys) in complex with PliI-Ah fromAeromonas 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 againstcis-intoxication.