SecSPARC

The Type II secretion systems (T2SS) promote secretion of toxins, hydrolytic enzymes, adhesins or cytochromes in Gram-negative bacteria. Together with the closely-related type IV pili (T4P), they are key virulence factors in many plant, animal and human pathogens. T2SSs and T4P share highly similar architecture and mechanism. While T4P are long surface fibers that extend and retract to promote bacterial adherence, colonization and motility, in T2SS, short periplasmic fibers called endopili drive secretion of folded proteins. Understanding the molecular mechanisms of their assembly and dynamics is important from the basic knowledge standpoint, but may also lead to biotechnological applications and rational design of new antimicrobial strategies. The T2SS endopili are composed of 4 minor and one major abundant pilin subunit. They polymerize at their base in the inner membrane of Gram-negative (diderm) bacteria. Pilus biogenesis is primed by self-assembly of three minor pilins embedded in the inner membrane; this is followed by assembly motor-dependent assembly of the fourth minor and multiple major pilins which build a helical polymer. This process is coordinated and powered by the assembly platform and ATPase motor. However, our knowledge on the architecture of this complex and the sequential steps during pilus assembly is limited. In addition, it remains unclear how endopilus assembly promotes protein secretion in T2SS. This project focuses on the central complex involved in endopilus assembly, designated here as the Active site of the T2SS complex. The Active site includes the assembly platform and motor complex, the nascent pilus and the pilin monomers, as substrates of the assembly reaction. We will apply an integrative approach to investigate the structure and dynamic interactions of Active site components. We will isolate Active site protein complexes in nanodisc which preserve the native membrane environment for study by cryoEM. Advanced modeling and NMR will be used to complete the structural information and define a detailed architecture of Active site during endopilus assembly initiation and elongation. Structure-based mutagenesis and functional studies will define assembly intermediates and their functional roles. Endopilus dynamics will be studied by live fluorescence microscopy. To investigate the T2SS substrate specificity, we will compare two T2SS models from Klebsiella, a human, and Dickeya, a plant pathogen. The strength of this proposal lies in several major preliminary findings, and in integration of in silico, in vitro and in vivo approaches, nurturing each other.