vhp Is a Fibrinogen-Binding Protein Related to vWbp in Staphylococcus aureus

Abstract

Staphylococcus aureus can target a variety of tissues, causing life-threatening infections. The basis for this diversity stems from the microorganism's ability to spread in the vascular system throughout the body. To survive in blood, S. aureus coats itself with a fibrinogen (Fg)/fibrin shield. The protective shield is assembled by the coordinated actions of a number of Fg-binding bacterial proteins that manipulate the host's blood coagulation system. Several of the Fg binders appear redundant, sharing similar functional motifs. This observation led us to screen for the presence of novel proteins with significant amino acid identities to von Willebrand factor-binding protein (vWbp), a key component in the shield assembly machinery. One identified protein showed significant sequence identity with the C-terminal region of vWbp, and we consequently named it vWbp homologous protein (vhp). The vhp gene lies within a cluster of genes that encode other virulence factors in S. aureus. Although each isolate only contains one copy of the vhp gene, S. aureus has at least three distinct alleles, vhpA, B, and C, that are present in the core genome. All three vhp isoforms bind Fg with high affinity, targeting a site located in the D fragment of Fg. We further identified an ∼79 amino acid-long, conserved segment within the C-terminal region of vWbp that shares high sequence identities (54 to 67%) with the vhps and binds soluble Fg with high affinity. Further analysis of this conserved motif and the intact vhps revealed intriguing differences in the Fg binding behavior, perhaps suggesting that these proteins have similar but discrete functions in the shield assembly. IMPORTANCE The life-threatening diseases caused by multidrug-resistant Staphylococcus aureus strains are a worldwide medical problem due to treatment limitations and the lack of an effective vaccine. The ability of S. aureus to coat itself with a protective fibrinogen (Fg)/fibrin shield allows the organism to survive in blood and to disseminate and cause invasive diseases. This process represents a promising target for novel antistaphylococcal treatment strategies but is incompletely understood. S. aureus expresses a number of Fg-binding proteins. Some of these proteins have apparently redundant functions. Proteins with similar functions often share a structural or functional motif with each other. In this study, we identified a protein homologous to the C-terminal of von Willebrand factor-binding protein (vWbp), a key contributor in the Fg shield assembly that also binds Fg. Further analysis allowed us to identify a common Fg-binding motif.

Keywords: Staphylococcus aureus; fibrinogen-binding proteins; vWbp homolog; vhp.

FIG 1

vhp is homologous to vWbp-C_{(386 to 482)}. (A) Clustal alignment of C-terminal vWbp, covering residues 242 to 482, and full-length vhpA, vhpB, and vhpC. Underlined residues indicate the conserved region. (B) Percent amino acid identity of vWbp_{(314 to 482)} S. aureus Newman (AAK52333.1), vhpA S. aureus N315 (BAB41978.1), vhpB S. aureus USA300_ FPR3757 (ABD21761.1), and vhpC S. aureus TCH60 (ADQ77850.1).

FIG 2

The vhp gene is located in a gene cluster encoding known virulence factors. A schematic representation of the organization of genes in the gene cluster. Blue arrowhead indicates a gene and the direction of translation. ST, sequence type.

FIG 3

The vhp isoforms bind to immobilized and soluble Fg. (A) ELISA binding of soluble Fg to immobilized vhp isoforms (0.5 μg/well). (B) ELISA binding of vhp isoforms to immobilized Fg (0.5 μg/well). Error bars represent standard error of the mean (SEM). The graphs are representative of three independent experiments.

FIG 4

The vhp isoforms bind to the D fragment of Fg. ELISA binding of immobilized Fg fragments (0.5 μg/well) to vhp isoforms vhpA (A), vhpB (B), and vhpC (C); red, full-length Fg; purple, Fg D fragment; black, Fg E fragment. Error bars represent standard error of the mean (SEM). The graphs are representative of three independent experiments.

FIG 5

The C-terminal of vWbp harbors an Fg-binding site at residues 386 to 482. (A) Schematic overview of the vWbp constructs generated in this study. Gray represents the predicted unordered region; SP, signal peptide; D₁D₂, prothrombin-binding domain; Tag, N-terminal His₆-MBP tag. (B) ELISA binding of soluble Fg to immobilized vWbp constructs (0.5 μg/well). (C) ELISA binding of vWbp constructs to immobilized Fg (0.5 μg/well).