BrucellaEipB is a periplasmic β-spiral protein required for envelope stress resistance and infection

Abstract

28The Gram-negative cell envelope is a remarkable structure with core components that include an 29 inner membrane, an outer membrane, and a peptidoglycan layer in the periplasmic space between. 30Multiple molecular systems function to maintain integrity of this essential barrier between the 31 interior of the cell and its surrounding environment. We show that a conserved DUF1849-family 32 protein, EipB, is secreted to the periplasmic space of Brucella, a monophyletic group of intracellular 33 pathogens. In the periplasm, EipB folds into an unusual fourteen-stranded b-spiral structure that 65 envelope stressors and determines B. abortus virulence in a mouse model of infection (8). In this 66 study, we report a functional and structural analysis of envelope integrity protein B (EipB), a 67 member of the uncharacterized gene family DUF1849. 68 69 3 DUF1849 (Pfam: PF08904, (9)) is widespread among the Rhizobiales, Rhodospirillales and 70 Rhodobacterales (Figure 1). To our knowledge, no functional data have been reported for this gene 71 family other than results from a recent multi-species Tn-seq study that showed stress sensitivity in 72 Sinorhizobium meliloti DUF1849 (locus SMc02102) mutant strains (10). Here we show that the 73 Brucella DUF1849 protein, EipB (locus tag bab1_1186; RefSeq locus BAB_RS21600), is a 280-74 residue periplasmic protein that folds into a 14-stranded, open β-barrel structure containing a 75 conserved disulfide bond. We term this novel barrel structure a β-spiral and show that it resembles 76 the lipoprotein chaperone LolB, though its overall fold is distinct. Replication and survival of a B. 77abortus strain in which we deleted eipB was attenuated in a mouse infection model, and deletion 78 of eipB in both B. abortus and Brucella ovis enhanced sensitivity to compounds that affect the 79 integrity of the cell envelope. We have further shown that B. abortus eipB deletion is synthetically 80 lethal with transposon disruption of gene locus bab1_0430, which encodes a periplasmic 81 tetratricopeptide-repeat (TPR) containing-protein that we have named TtpA. The Brucella 82 melitensis ortholog of TtpA (locus tag BMEI1531) has been previously described as a molecular 83 determinant of mouse spleen colonization (11), while a Rhizobium leguminosarum TtpA homolog 84 (locus tag RL0936) is required for proper cell envelope function (12). We propose that TtpA and 85 EipB coordinately function in the Brucella periplasm to ensure cell envelope integrity and to enable 86 cell survival in the mammalian host niche. 88 Results 89B. abortus eipB is required for maintenance of mouse spleen colonization 90As part of a screen to evaluate the role of conserved Alphaproteobacterial genes of unknown 91 function in B. abortus infection biology, we infected THP-1 macrophage-like cells with wild-type B. 92abortus, an eipB deletion strain (∆eipB), and a genetically complemented ∆eipB strain. Infected 93 macrophages were lysed and colony forming units (CFU) were enumerated on tryptic soy agar 94 plates (TSA) at 1, 24 and...