The Orientia tsutsugamushi ScaB Autotransporter Protein Is Required for Adhesion and Invasion of Mammalian Cells

Abstract

Autotransporter proteins are widely present in Gram-negative bacteria. They play a pivotal role in processes related to bacterial pathogenesis, including adhesion, invasion, colonization, biofilm formation, and cellular toxicity. Bioinformatics analysis revealed that Orientia tsutsugamushi, the causative agent of scrub typhus, encodes six different autotransporter genes (scaA-scaF). Although four of these genes (scaA, scaC, scaD, and scaE) are present in diverse strains, scaB and scaF have been detected in only a limited number of strains. Previous studies have demonstrated that ScaA and ScaC are involved in the adherence of host cells. However, the putative function of other O. tsutsugamushi Sca proteins has not been studied yet. In this study, we show that scaB is transcribed and expressed on the surface of O. tsutsugamushi Boryong strain. Using a heterologous Escherichia coli expression system, we demonstrated that ScaB-expressing E. coli can successfully mediate adherence to and invasion into non-phagocytic cells, including epithelial and endothelial cells. In addition, pretreatment with a recombinant ScaB polypeptide inhibits the entry of O. tsutsugamushi into cultured mammalian cells. Finally, we also identified the scaB gene in the Kuroki and TA686 strains and observed high levels of sequence variation in the passenger domains. Here, we propose that the ScaB protein of O. tsutsugamushi can mediate both adhesion to and invasion into host cells in the absence of other O. tsutsugamushi genes and may play important roles in bacterial pathogenesis.

Keywords: adhesion; autotransporter; invasion; scrub typhus; vaccine.

Figure 1

Expression of ScaB by Orientia tsutsugamushi. (A) Reverse transcriptase PCR (RT-PCR) of scaB mRNA from L929 cells infected with O. tsutsugamushi. Primer only without template (lane 1), total RNA (without reverse transcription) isolated from infected cells (lane 2), from genomic DNA (lane 3), from cDNA from uninfected cells (lane 4), and from cDNA from infected cells (lane 5). PCR product is 1950 bp in size. (B) Specificity of anti-ScaB antisera. BSA (lane 1) and recombinant ScaB protein (amino acids 23–1,372; lane 2) were stained with Coomassie blue (left panel) and immunoblotted with the anti-ScaB antisera. Recombinant ScaB protein is 36 kDa in size. (C) Western immunoblot analysis of O. tsutsugamushi whole-cell lysate probed with mouse preimmune sera (left panel) and anti-ScaB serum (right panel). Full-length ScaB protein is 77 kDa in size. (D) Immunofluorescence confocal microscopy using preimmune serum or anti-ScaB serum in the O. tsutsugamushi-infected L929 cells. The left-hand panels show bacteria stained with pooled serum from scrub typhus patients (∝-OT). DIC, differential interference contrast. Scale bars, 10 μm.

Figure 2

Recombinant ScaB protein-binding assay to HeLa cells. (A) Recombinant glutathione S-transferase (GST) or GST-ScaB_23–372 protein was incubated with HeLa cells for 1 h. Cells were visualized by fluorescence microscopy after incubated with anti-GST followed by Alexa488-conjugated goat anti-mouse IgG antibody (green) and ToPro-3 (blue). Scale bars, 10 μm. (B) Flow cytometric analysis of the GST (black) and GST-ScaB (red) protein binding to HeLa cells. The gray histogram represents untreated cells

Figure 3

Expression of ScaB in Escherichia coli is sufficient to mediate adherence to mammalian cells. (A) Immunofluorescence microscopy using an anti-scaB antibody revealed the presence of ScaB on the surface of the recombinant E. coli (lower panel). Preimmune serum did not detect the recombinant protein (upper panel). Scale bars, 5 μm. (B) Expression of O. tsutsugamushi ScaB on the surface of E. coli. Immunoblot analysis of outer membrane fractions of induced E. coli harboring the empty vector (lane 1), ScaB (lane 2), and recombinant ScaB_23–372 protein was performed using an anti-ScaB serum. (C) E. coli transformed with the pET28a vector or with ScaB domain was induced with isopropyl β-D-thiogalactoside (IPTG) and incubated with HeLa cells. Confluent monolayers of HeLa cells were infected for 30 min at 37°C, washed repeatedly with phosphate-buffered saline (PBS), and then stained with an anti-E. coli antibody (red), Phalloidin (gray), and ToPro-3 for nuclear staining (blue). Scale bars, 50 μm. (D) Colony-forming unit (CFU)-based quantification of adherent E. coli transformed with the vector (black bars) or ScaB (red bars) was performed for different host cells (HeLa, A549, and ECV304 cell lines). ^*p < 0.05 and ^****p < 0.0001. The data presented are representative of at least three independent assays for each cell line. Error bars represent the SD of each data set.

Figure 4

Expression of ScaB in E. coli is mediate invasion of mammalian cells. (A) Confluent cell monolayers of HeLa cells were infected for 1 h with E. coli transformed with the vector or ScaB and assessed for invasion by gentamicin protection assay. Cells were stained with an anti-scaB antisera (red), Phalloidin (gray), and ToPro-3 for nuclear (blue). The image is a projection of a 1 μm z-stack collected through x60 objective. Scale bars, 50 μm. (B) CFU-based quantification of bacterial invasion into mammalian host cells. Invasion is presented as the percentage of bacteria recovered after the gentamicin challenge out of the inoculums. The data presented are representative of at least two independent experiments for each individual cell line. Error bars represent the SD of each data set. ^*p < 0.05, ^**p < 0.01, and ^***p < 0.001.

Figure 5

Inhibition of O. tsutsugamushi interactions with host cells by the ScaB polypeptide. (A) Preincubation of HeLa cells with GST-ScaB (lower panels) significantly inhibited O. tsutsugamushi interactions with host cells compared with preincubation with GST (upper panels). After incubation of the polypeptides with the host cells, O. tsutsugamushi was added, and incubation continued for a further 30 min. Cells were visualized by immunofluorescence microscopy. Orientia tsutsugamushi (green) and Nuclei (blue). (B) The numbers of bacteria associated with each of 100 randomly selected host cells. The bars indicate the means ± SDs of triplicate experiments. ^****p < 0.0001.

Figure 6

Comparison of scaB gene from indicated O. tsutsugamushi strains. (A) Amino acids sequence alignments for constructing phylogenetic trees were processed by Clustal W with the maximum likelihood method. (B) The similarity and identity of those nucleotides and amino acids were calculated through Matrix Global Alignment Tool [MatGAT; left: full-length of ScaB, middle: signal peptide(SP)-passenger(P) domain, and right: autotransporter (AT) domain]. (C) Similarity plot comparing ScaB sequence among the indicated O. tsutsugamushi strains and schematic representation above the graph indicates the relative sizes of translated ScaB amino acids sequence from Boryong strain and their sequence motiffs. (D) Secondary/tertiary structure of ScaB protein was predicted by HHPred, PSIPRED, and I-TASSER program. Predicted tertiary of ScaB protein (upper) and predicted secondary structure of passenger domain (Lower). BR, Boryong; KK, Kuroki; SD, Sido; 686, TA686; 716, TA716.