Defining the Ail Ligand-Binding Surface: Hydrophobic Residues in Two Extracellular Loops Mediate Cell and Extracellular Matrix Binding To Facilitate Yop Delivery

Abstract

Yersinia pestis, the causative agent of plague, binds host cells to deliver cytotoxic Yop proteins into the cytoplasm that prevent phagocytosis and generation of proinflammatory cytokines. Ail is an eight-stranded β-barrel outer membrane protein with four extracellular loops that mediates cell binding and resistance to human serum. Following the deletion of each of the four extracellular loops that potentially interact with host cells, the Ail-Δloop 2 and Ail-Δloop 3 mutant proteins had no cell-binding activity while Ail-Δloop 4 maintained cell binding (the Ail-Δloop 1 protein was unstable). Using the codon mutagenesis scheme SWIM (selection without isolation of mutants), we identified individual residues in loops 1, 2, and 3 that contribute to host cell binding. While several residues contributed to the binding of host cells and purified fibronectin and laminin, as well as Yop delivery, three mutations, F80A (loop 2), S128A (loop 3), and F130A (loop 3), produced particularly severe defects in cell binding. Combining these mutations led to an even greater reduction in cell binding and severely impaired Yop delivery with only a slight defect in serum resistance. These findings demonstrate that Y. pestis Ail uses multiple extracellular loops to interact with substrates important for adhesion via polyvalent hydrophobic interactions.

Ail provides multiple mechanisms of serum resistance to Yersinia pestis

“…While the Δ ail strain was ~100,000‐fold defective for survival in NHS, chromosomally expressed wild‐type Ail, Ail‐F80A or Ail‐F130A conferred 100% serum resistance similar to previous findings (Tsang et al ., ). Furthermore, Ail‐F80A/F130A had only a modest (sixfold) survival defect in NHS compared to the wild‐type Ail as previously reported (Tsang et al ., ). Serum inactivated for CP and LP (NHS‐AP) had no statistically significant difference in killing of Δ ail or Ail‐F80A/F130A compared to NHS, as determined by the two‐way ANOVA analysis (Fig.…”

“…Since a Y. pestis strain expressing Ail‐F80A/F130A exhibited only sixfold less serum resistance than a strain expressing wild‐type Ail, while remaining ~10,000‐fold more serum resistant than Δ ail (Fig. , [Tsang et al ., ]) , we determined whether residues F80 and F130 contribute to Y. pestis Ail‐mediated recruitment of complement regulatory proteins as a mechanism of serum resistance. Co‐sedimentation assays were performed with strains expressing Ail‐F80A, Ail‐F130A and Ail‐F80A/F130A to assess vitronectin and factor H binding.…”

“…High‐level septicemia allows for transmission to a new flea vector during a blood meal (Lorange et al ., ). Ail is necessary and sufficient to confer resistance to serum to Y. pestis , providing 100,000‐fold greater evasion of complement‐mediated killing than a strain lacking ail (Kolodziejek et al ., ; Bartra et al ., ; Tsang et al ., ). In this study, we show that a Δ ail mutant is efficiently killed in 80% NHS during in vitro serum resistance assays and this killing is attributed to the alternative pathway of complement (Fig.…”

“…Defining the amino acids of Ail involved in preventing complement‐mediated lysis further elucidates the role of Ail during host infection. We have found residues in Ail (F80 and F130) that not only mediate cell binding, binding to ECM and Yop delivery (Tsang et al ., ), but also facilitate the binding/recruitment of complement regulatory proteins (Vn, factor H and C4BP) to the bacterial surface. We found that Ail residues (E43 and G122), when mutated in combination decrease serum resistance, while regulatory protein binding remains relatively unchanged, implicating an alternative mechanism of serum resistance in addition to/instead of complement regulatory protein recruitment.…”

“…In addition to serum resistance, previous studies have demonstrated the importance of Y. pestis Ail in host cell binding, extracellular matrix (ECM) binding and Yop (cytotoxin) delivery, (Felek and Krukonis, 2009;Felek et al, 2010;Tsang et al, 2010;Yamashita et al, 2011). Tsang et al recently described the contribution of surface exposed hydrophobic residues, F80, S128 and F130 to these functions (Tsang et al, 2017). Cumulative mutation of these residues (Ail-F80A/F130A and Ail-F80A/S128A/ F130A) resulted in substantial defects in cell adhesion, ECM binding, Yop delivery and auto-aggregation, while Ail-F94 played a particularly critical role in fibronectin binding (Tsang et al, 2017).…”