Contribution of trimeric autotransporter C-terminal domains of oligomeric coiled-coil adhesin (Oca) family members YadA, UspA1, EibA, and Hia to translocation of the YadA passenger domain and virulence of Yersinia enterocolitica

Abstract

The Oca family is a novel class of autotransporter-adhesins with highest structural similarity in their C-terminal transmembrane region, which supposedly builds a beta-barrel pore in the outer membrane (OM). The prototype of the Oca family is YadA, an adhesin of Yersinia enterocolitica and Yersinia pseudotuberculosis. YadA forms a homotrimeric lollipop-like structure on the bacterial surface. The C-terminal regions of three YadA monomers form a barrel in the OM and translocate the trimeric N-terminal passenger domain, consisting of stalk, neck, and head region to the exterior. To elucidate the structural and functional role of the C-terminal translocator domain (TLD) and to assess its promiscuous capability with respect to transport of related passenger domains, we constructed chimeric YadA proteins, which consist of the N-terminal YadA passenger domain and C-terminal TLDs of Oca family members UspA1 (Moraxella catarrhalis), EibA (Escherichia coli), and Hia (Haemophilus influenzae). These constructs were expressed in Y. enterocolitica and compared for OM localization, surface exposure, oligomerization, adhesion properties, serum resistance, and mouse virulence. We demonstrate that all chimeric YadA proteins translocated the YadA passenger domain across the OM. Y. enterocolitica strains producing YadA chimeras or wild-type YadA showed comparable binding to collagen and epithelial cells. However, strains producing YadA chimeras were attenuated in serum resistance and mouse virulence. These results demonstrate for the first time that TLDs of Oca proteins of different origin are efficient translocators of the YadA passenger domain and that the cognate TLD of YadA is essential for bacterial survival in human serum and mouse virulence.

FIG. 1.

(A) Sequence alignment of the TLD monomers of YadA, EibA, Hia, and UspA1. The TLD monomer consists of two main parts: the linker domain and the beta-barrel domain. The linker domain can be separated into a proximal α1 domain, which traverses the beta-barrel domain, and a distal hairpin loop, which connects the α1 domain to the beta-barrel domain. The beta-barrel domain consists of four transmembrane antiparallel beta-sheets (β1 to β4). In this study, the YadA TLD (aa 334 to 422) was replaced with either the EibA (aa 306 to 392), Hia (aa 1009 to 1098), or UspA1 (aa 742 to 832) TLD region. Arrows mark the replaced amino acids for construction of YadA-UspA1-2 (aa 362 to 422 of YadA replaced by aa 770 to 832 of UspA1) (from arrow a to arrow c) and YadA-UspA1-3 (aa 369 to 422 of YadA replaced by aa 777 to 832 of UspA1) (from arrow b to arrow c). (B) Crystal structure model of the TLD monomer and trimer, e.g., three YadA monomers build the trimeric YadA protein (YadA oligomer). Ribbon diagrams were generated with DeepView PDB. (C) Construction scheme of the yadA chimeras. PCR fragments I, II, and III were ligated into a ClaI-SphI cut pUC-A-1 vector backbone (see Materials and Methods). SS, signaling sequence. The asterisks refer to the corresponding stop codons.

FIG. 2.

Comparison of surface expression of wild-type YadA, YadA(D332L, H333E), YadA-EibA, YadA-Hia, and YadA-UspA1 on whole, unfixed Yersinia WA(pYV-O8) cells by an ELISA with MAb 8D1. The asterisk indicates the highly significantly (P < 0.01) diminished surface expression of YadA-UspA1 compared to wild-type YadA. YadA neg., YadA-negative strain WA(pYVO8-A-0); YadA, strain carrying wild-type YadA WA(pYVO8-A-1).

FIG. 3.

Expression, OM localization, and stability of high-molecular-weight complexes of controls YadA negative, YadA positive, and YadA(D332L, H333E) and of YadA-hybrid proteins YadA-UspA1, YadA-EibA, and YadA-Hia (A) and of three different YadA-UspA1 hybrid proteins: YadA-UspA1, YadA-UspA1-2, and YadA-UspA1-3 (B). OM fractions were prepared from Yersinia WA strains harboring the gene for the corresponding YadA-hybrid protein on their pYV virulence plasmids. Strains were grown at 37°C for 6 h before OM preparation, and 8 μg of each sample was solubilized in sample buffer either for 60 min at 37°C or for 10 min at 100°C, separated by SDS-PAGE, transferred to nitrocellulose sheets, and probed with MAb 8D1. The positions of molecular size markers are shown on the left in kilodaltons.

FIG. 4.

Protease sensitivity assay of Y. enterocolitica WA-314 strains carrying YadA and YadA hybrid constructs. Strains were grown at 37°C for 6 h and 10⁷ bacteria were submitted to a 60 min tryptic digest with 2.5 μg of trypsin/ml, subsequently solubilized in sample buffer for 60 min at 37°C (A) or 100°C (B), separated by SDS-PAGE, and probed with MAb 8D1. YadA neg., YadA-negative strain WA(pYVO8-A-0); YadA pos., strain carrying wild-type YadA WA(pYVO8-A-1); +, trypsin added; −, no trypsin added. The positions of molecular size markers are shown on the left in kilodaltons.

FIG. 5.

(A) Type I collagen adherence of Y. enterocolitica strains producing YadA or the YadA hybrid proteins as indicated. Collagen coating concentrations: ▪, 20 μg/ml; □, 2 μg/ml. Yersinia adherence was determined by a YadA-specific immunoassay. (B and C) HEp-2 cell adherence of Y. enterocolitica strains producing YadA or the YadA hybrid proteins, determined by CFU per well (values are the averages of triplicate samples, with the ranges indicated, and reflect similar results from several experiments) (B) or by microscopic counting of cell-associated yersiniae (average number of yersiniae per cell obtained from 30 randomly selected cells, with the ranges indicated) (C) (see Materials and Methods). Highly significant differences in these experiments are indicated by an asterisk for the difference between the YadA negative strain and the YadA wild-type strain, which are also representative for the differences between the YadA-negative strain and each of the YadA hybrid-producing strains (P < 0.01).

FIG. 6.

Virulence of Y. enterocolitica serotype O:8 strains WA(pYVO8-A-0) (YadA-negative virulence plasmid carrying strain), WA(pYVO8-A-1) (YadA wild type), WA(pYVO8-YadA-UspA1), WA(pYVO8-YadA-EibA), and WA(pYVO8-YadA-Hia) in orally (A), intravenously (B), and intraperitoneally (C) infected groups of five BALB/c mice infected with 1 × 10⁹ CFU (A) or 5 × 10⁴ CFU (B and C) of bacteria. After 5 days (A) or 2 and 4 days (B and C), the CFU of bacteria in the small intestine (SI), Peyer's patches (PP), spleen (S), and liver (L) (A) or in the spleen (S) and liver (L) (B and C) were determined. The data are means ± the standard deviations. wt, wild type.