Summary
The Gram-negative cell envelope is a remarkably diverse structure with core components that include an inner membrane, an outer membrane, and a peptidoglycan layer in the periplasmic space between. We show that a conserved DUF1849-family protein, EipB, is secreted to the periplasmic space of Brucella, a monophyletic group of intracellular pathogens. In the periplasm, EipB folds into an unusual fourteen-stranded β-spiral structure that contains a conserved disulfide bond. EipB has structural features that resemble the LolA and LolB lipoprotein delivery system, though the overall topology and fold of EipB is distinct from LolA/LolB. Deletion of eipB results in defects in Brucella cell envelope integrity in vitro and in maintenance of spleen colonization in a mouse model of B. abortus infection. Transposon disruption of ttpA, which encodes a periplasmic tetratricopeptide repeat (TPR) protein, is synthetically lethal with eipB deletion in B. abortus. ttpA is a known virulence determinant in B. melitensis, and our studies of ttpA deletion and overexpression strains provide evidence that ttpA, like eipB, contributes to cell envelope function in Brucella. We conclude that eipB and ttpA function in the Brucella periplasmic space to maintain cell envelope integrity and to facilitate survival in a mammalian host.
Importance Brucella species cause brucellosis, a global zoonosis. A gene encoding a conserved uncharacterized protein, EipB, is present in all sequenced Brucella and several other genera in the class Alphaproteobacteria. To our knowledge, this study presents the first functional and structural characterization of a protein from the DUF1849 family, to which EipB belongs. EipB is secreted to the periplasm where it forms a spiral-like anti-parallel structure. Deletion of Brucella eipB results in defects of the cell envelope and in reduced virulence in an animal model of disease. eipB genetically interacts with ttpA, which also encodes a periplasmic protein. We propose that EipB and TtpA function as part of a system required for cell envelope homeostasis in select Alphaproteobacteria.
