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Type VI protein secretion systems (T6SSs) are specialized transport apparatus which can target both eukaryotic and prokaryotic cells and play key roles in host⁻pathogen⁻microbiota interactions. Therefore, T6SSs have attracted much attention as a research topic during the past ten years. In this review, we particularly summarized the T6SS antibacterial function, which involves an interesting offensive and defensive mechanism of the effector⁻immunity (E⁻I) pairs. The three main categories of effectors that target the cell wall, membranes, and nucleic acids during bacterial interaction, along with their corresponding immunity proteins are presented. We also discuss structural analyses of several effectors and E⁻I pairs, which explain the offensive and defensive mechanisms underpinning T6SS function during bacterial competition for niche-space, as well as the bioinformatics, proteomics, and protein⁻protein interaction (PPI) methods used to identify and characterize T6SS mediated E⁻I pairs. Additionally, we described PPI methods for verifying E⁻I pairs.

Effector–Immunity Pairs Provide the T6SS Nanomachine its Offensive and Defensive Capabilities