Adhesion, invasion, and agglutination mediated by two trimeric autotransporters in the human uropathogen Proteus mirabilis

Abstract

Fimbriae of the human uropathogen Proteus mirabilis are the only characterized surface proteins that contribute to its virulence by mediating adhesion and invasion of the uroepithelia. PMI2122 (AipA) and PMI2575 (TaaP) are annotated in the genome of strain HI4320 as trimeric autotransporters with "adhesin-like" and "agglutinating adhesin-like" properties, respectively. The C-terminal 62 amino acids (aa) in AipA and 76 aa in TaaP are homologous to the translocator domains of YadA from Yersinia enterocolitica and Hia from Haemophilus influenzae. Comparative protein modeling using the Hia three-dimensional structure as a template predicted that each of these domains would contain four antiparallel beta sheets and that they formed homotrimers. Recombinant AipA and TaaP were seen as ∼28 kDa and ∼78 kDa, respectively, in Escherichia coli, and each also formed high-molecular-weight homotrimers, thus supporting this model. E. coli synthesizing AipA or TaaP bound to extracellular matrix proteins with a 10- to 60-fold-higher level of affinity than the control strain. Inactivation of aipA in P. mirabilis strains significantly (P < 0.01) reduced the mutants' ability to adhere to or invade HEK293 cell monolayers, and the functions were restored upon complementation. A 51-aa-long invasin region in the AipA passenger domain was required for this function. E. coli expressing TaaP mediated autoagglutination, and a taaP mutant of P. mirabilis showed significantly (P < 0.05) more reduced aggregation than HI4320. Gly-247 in AipA and Gly-708 in TaaP were indispensable for trimerization and activity. AipA and TaaP individually offered advantages to P. mirabilis in a murine model. This is the first report characterizing trimeric autotransporters in P. mirabilis as afimbrial surface adhesins and autoagglutinins.

FIG. 1.

In silico analyses of AipA and TaaP. (A) Schematic representation of 280-amino-acid-long AipA (PMI2122) and 741-amino-acid-long TaaP (PMI2575) proteins. Predicted domains in both proteins and amino acid numbers are given. The N-terminal 48 residues in AipA and 29 amino acids in TaaP form the putative signal peptide (gray box), which is followed by the passenger domain, or alpha (α) domain (aa 49 to 218 for AipA and 30 to 665 for TaaP), which for each protein contains regions homologous to invasin/Hep_Hag (spotted box) and hemagglutinin motifs (black boxes). Amino acids 219 to 280 in AipA and 666 to 741 in TaaP constitute the putative translocator, or beta (β) domain, with 4 transmembrane beta sheets in each (solid black bars). The theoretical mass of each protein is given in kDa. (B) Multiple-sequence alignment by ClustalW of trimeric autotransporter Hia of H. influenzae (2GR7) with the C-terminal regions of TaaP (PMI2575) and AipA (PMI2122) (AipA). Gray-shaded areas are predicted β-sheets, and black-shaded areas are actual β-sheets in the crystal structure. (C) Ribbon representation of the trimeric model of AipA or TaaP. Helices are shown in red, β-strands in yellow, and turns in blue. For better visibility, two monomers are shown in gray. (D) Multiple-sequence alignment of residues in the beta domains of YadA (Y. enterocolitica O:8; GI:23630568), Hia (H. influenzae; GI:21536216), PMI2122 (AipA), and PMI2575 (P. mirabilis HI4320) (TaaP). The arrow indicates the glycine homologous to the conserved Gly-389 in YadA. (E) Ribbon representation of the trimer models of AipA and TaaP, viewed from the extracellular space through the central helical barrel. Gly-247 and Gly-708 are shown in a space-filling representation (gray ribbons). The Gaussian molecular surface is shown at 7 Å around Gly-247 and Gly-708, indicating the existence of one cavity per monomer inside the proteins.

FIG. 2.

Synthesis and cellular localization of the recombinant proteins AipA and TaaP. (A) AipA Gly-247 and TaaP Gly-708, each homologous to the conserved Gly-389 in YadA, were changed to His to yield AipA* (AipAG247H) and TaaP* (TaaPG708H), respectively. In-frame deletion of the 51-amino-acid-long Hep_Hag/invasin region in the passenger domain of AipA was predicted to yield a shorter protein, AipAΔInv. The theoretical mass of each protein is given in kDa. (B) Whole-cell proteins (6 μg) of E. coli BL21plysS carrying the empty pET21A vector (CTL) or synthesizing either AipA or TaaP or their mutant forms were analyzed by SDS-PAGE and stained with Coomassie brilliant blue. Molecular mass markers (lane M) are shown, and their measurements in kilodaltons are indicated on the left. (C, left panel) Enriched outer membrane protein fractions (8 μg) from E. coli BL21plyS carrying only the pET21A vector (CTL) or the indicated form of TaaP subjected to SDS-PAGE followed by Coomassie blue staining. (Right panel) Immunoblot of enriched outer membrane protein fractions (8 μg) of E. coli carrying the control plasmid (CTL) or the indicated form of AipA stained with monoclonal anti-6×His followed by a goat anti-rabbit IgG-AP conjugate. Arrows indicate bands corresponding to monomeric or multimeric forms of each protein.

FIG. 3.

Translocation of heterologous passenger domain. (A) Schematic representation of fusion proteins. DNA encoding the 710-amino-acid-long passenger domain of Proteus toxic agglutinin (Ptaα) (without its helical region) was fused to the region encoding AipA_β or TaaP_β to synthesize fusion proteins Ptaα-AipAβ (773 aa) and Ptaα-TaaPβ (787 aa). Native Pta (1,072 amino acids) with its various domains is shown: Subtilisin, the subtilisin domain; AP, the alkaline phosphatase domain; and Beta, the translocator domain. The gray boxes represent the signal sequences of Pta. (B) The enriched outer membrane protein fraction (6 μg) of E. coli synthesizing native Pta, Ptaα alone, either of the fusion proteins, or the protein carrying only the empty vector (CTL) was analyzed by immunoblotting using polyclonal rabbit anti-Pta serum followed by a goat anti-rabbit IgG-horseradish peroxidase (HRP) conjugate. The Pta in each lane is indicated by arrowheads. The molecular mass of the markers is given in kilodaltons. The ∼50-kDa nonspecific band served as the loading control. (C) E. coli cells synthesizing native Pta, the passenger domain alone (Ptaα), or the fusion forms (Ptaα-AipA or Ptaα-TaaPβ) were individually overlaid on a monolayer of HeLa cells at an MOI of 80:1. Lysis of HeLa cells in each case was determined by measuring the intracellular lactate dehydrogenase released and is presented as a percentage of cell lysis obtained with Triton X-100 (considered 100%). Data presented are means ± SEMs of the results of three independent studies, each performed in triplicate. **, P < 0.01.

FIG. 4.

Binding to ECM proteins. (A) Binding of E. coli carrying AipA (Ec-AipA) or TaaP (Ec-TaaP) and P. mirabilis HI4320 (HI4320) to individual ECM proteins presented as the fold increase over the level of binding by Ec-CTL (y axis). (B) Relative abilities of bovine serum albumin (BSA) or purified wild-type AipA (AipA) or AipA lacking the invasin domain (AipAΔInv) to reduce the binding ability of HI4320 to collagen I. Binding in each case is expressed as the fold increase over that determined for Ec-CTL. Concentrations (nM) of competing proteins are given. Means ± SEMs from experiments conducted in triplicate are shown. **, P < 0.01, as determined using Student's t test.

FIG. 5.

Autotransporter-mediated interaction of E. coli with eukaryotic cells. E. coli cells carrying pET21A as a control (Ec-CTL), Ec-AipA, or Ec-TaaP were overlaid on confluent monolayers of human embryonic kidney cells (HEK293) (MOI, 80 to 100:1) and incubated for 1 h. Giemsa-stained cells are shown at a magnification of ×100. Bar, 100 μm. Arrows point to bacterial cells.

FIG. 6.

Adhesion of bacterial cells to a eukaryotic cell monolayer. (A) Quantitative estimation of host cell adhesion by E. coli synthesizing wild-type or mutant forms of the autotransporter protein. Ec-AipA, E. coli synthesizing AipA; Ec-AipA*, E. coli synthesizing AipA (G247H); Ec-AipAΔInv, E. coli synthesizing AipA lacking the invasion domain; and Ec-TaaP, E. coli synthesizing TaaP. (B) Estimation of host cell adhesion by P. mirabilis HI4320(hpmA), the hpmA aipA double mutant, or the double mutant complemented with wild-type aipA in trans [hpmA aipA (p-aipA)]. Adhesion is given as the fold increase over that determined for Ec-CTL (y axis). The eukaryotic cells used were human embryonic kidney epithelial cells (HEK293), UMUC-3 human bladder epithelial cells, and Vero monkey kidney epithelial cells. Means ± SEMs from experiments conducted in triplicate are shown. *, P < 0.05; **, P < 0.01, calculated using Student's t test.

FIG. 7.

Bacterial invasion of the host cell. An HEK293, UMUC-3, or Vero cell monolayer was individually incubated with E. coli or P. mirabilis cells (80 to 100:1) for 1 h, followed by 2 h of incubation with gentamicin (30 μg/ml). Internalized bacterial cells were enumerated by plating ddH₂O extracts of washed eukaryotic cells. The y axis represents the fold increase in invasion over that for Ec-CTL. Means ± SEMs of the results from experiments conducted in triplicate are shown. *, P < 0.05; **, P < 0.01. Significance was calculated using Student's t test.

FIG. 8.

Autoaggregation of bacterial cells. Autoaggregation of E. coli (A) or P. mirabilis HI4320 (B) was determined by measuring the decrease in optical density of the uppermost layer of suspended cells over time. The OD₆₀₀ at t₀ was considered to be 100%. The percent decrease in the optical density of suspended bacterial cells (y axis) was plotted against time in hours (x axis). Cells were cultured and incubated at pH 7.2 or pH 8.5 (as indicated). Means ± SDs of the results of three independent experiments are given. The inset in panel A shows tubes containing Ec-AipA, Ec-TaaP*, and Ec-TaaP, respectively (from left to right), after 8 h.

FIG. 9.

Cochallenges of CBA/J mice with P. mirabilis HI4320 and the isogenic aipA or taaP mutant. Assessment of virulence of the aipA::kn (A) or taaP::kn (B) strain in a CBA/J mouse model of ascending UTI 7 days after cochallenge with the parent strain. Each symbol represents log₁₀ CFU per ml of urine or g of tissue from an individual mouse. Solid symbols represent counts for the wild-type HI4320, and open symbols represent counts for the mutants. The dotted lines indicate the limit of detection. Solid black bars represent median values. Two-tailed P values were determined by the Wilcoxon matched-pairs signed-rank test. *, P < 0.05; **, P < 0.01.