The Gram-negative bacterium Acinetobacter baumannii causes a range of severe nosocomial-acquired infections and many strains are multi-drug resistant. A. baumannii possesses many survival mechanisms allowing it to thrive in competitive polymicrobial environments. These include a Type VI Secretion System (T6SS) that injects effector proteins into other bacteria to give the bacteria a competitive advantage. The T6SS of some species can deliver ‘specialised effectors,’ effectors that are fused directly to T6SS components, such as PAAR proteins. PAAR proteins contain PAAR domains that are predicted to form the piercing tip of the T6SS and are essential for T6SS function. Although no specialised effectors have been identified in A. baumannii, many strains encode multiple PAAR proteins. Analysis of PAAR proteins across the A. baumannii species identified 12 families of PAAR proteins with distinct C-terminal extensions; it is unknown whether these extensions impact PAAR function.
A. baumannii AB307-0294 encodes two PAAR proteins (PAAR1, PAAR2), one of which has a C-terminal extension of 193 amino acids. Mutation of one or both of the PAAR-encoding genes in this strain showed that expression of either PAAR protein was sufficient for T6SS function. We employed a heterologous expression approach to determine whether PAAR proteins from different A. baumannii strains could complement the A. baumannii ∆paar1∆paar2 mutant. All heterologous PAAR proteins tested were able to restore T6SS function. Furthermore, to show that PAAR fusions could be used to deliver artificially cloned protein fragments, we fused Histidine and Streptavidin tags onto PAAR domains and showed that both His- and Strep-tagged PAAR specialised effectors could restore T6SS activity. This data provides evidence that fusion of protein fragments to PAAR proteins in A. baumannii is compatible with a functional T6SS. Successful delivery by this mechanism greatly extends the scope of what can be delivered by the T6SS. As such, proteins fused to PAAR, including user designer proteins could be engineered and delivered by the T6SS directly into target cells for therapeutic uses.