Structural Insight into How Bacteria Prevent Interference between Multiple Divergent Type IV Secretion Systems
Authors: Gillespie Joseph J. , Phan Isabelle Q. H. , Scheib Holger , Subramanian Sandhya , Edwards Thomas E. , Lehman Stephanie S. , Piitulainen Hanna , Sayeedur Rahman M. , Rennoll-Bankert Kristen E. , Staker Bart L. , Taira Suvi , Stacy Robin , Myler Peter J. , Azad Abdu F. , Pulliainen Arto T. 
Published: December 2015

Prokaryotes use type IV secretion systems (T4SSs) to translocate substrates (e.g., nucleoprotein, DNA, and protein) and/or elaborate surface structures (i.e., pili or adhesins). Bacterial genomes may encode multiple T4SSs, e.g., there are three functionally divergent T4SSs in some
Bartonella
species (
vir
,
vbh
, and
trw
). In a unique case, most rickettsial species encode a T4SS (
rvh
) enriched with gene duplication. Within single genomes, the evolutionary and functional implications of cross-system interchangeability of analogous T4SS protein components remains poorly understood. To lend insight into cross-system interchangeability, we analyzed the VirB8 family of T4SS channel proteins. Crystal structures of three VirB8 and two TrwG
Bartonella
proteins revealed highly conserved C-terminal periplasmic domain folds and dimerization interfaces, despite tremendous sequence divergence. This implies remarkable structural constraints for VirB8 components in the assembly of a functional T4SS. VirB8/TrwG heterodimers, determined via bacterial two-hybrid assays and molecular modeling, indicate that differential expression of
trw
and
vir
systems is the likely barrier to VirB8-TrwG interchangeability. We also determined the crystal structure of
Rickettsia typhi
RvhB8-II and modeled its coexpressed divergent paralog RvhB8-I. Remarkably, while RvhB8-I dimerizes and is structurally similar to other VirB8 proteins, the RvhB8-II dimer interface deviates substantially from other VirB8 structures, potentially preventing RvhB8-I/RvhB8-II heterodimerization. For the
rvh
T4SS, the evolution of divergent VirB8 paralogs implies a functional diversification that is unknown in other T4SSs. Collectively, our data identify two different constraints (spatiotemporal for
Bartonella
trw
and
vir
T4SSs and structural for
rvh
T4SSs) that mediate the functionality of multiple divergent T4SSs within a single bacterium.

IMPORTANCE
Assembly of multiprotein complexes at the right time and at the right cellular location is a fundamentally important task for any organism. In this respect, bacteria that express multiple analogous type IV secretion systems (T4SSs), each composed of around 12 different components, face an overwhelming complexity. Our work here presents the first structural investigation on factors regulating the maintenance of multiple T4SSs within a single bacterium. The structural data imply that the T4SS-expressing bacteria rely on two strategies to prevent cross-system interchangeability: (i) tight temporal regulation of expression or (ii) rapid diversification of the T4SS components. T4SSs are ideal drug targets provided that no analogous counterparts are known from eukaryotes. Drugs targeting the barriers to cross-system interchangeability (i.e., regulators) could dysregulate the structural and functional independence of discrete systems, potentially creating interference that prevents their efficient coordination throughout bacterial infection.