Riemerella Anatipestifer Gene AS87 08785 Encodes a Functional Component, Gldk, of the Type IX Secretion System
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Veterinary Microbiology 231 (2019) 93–99 Contents lists available at ScienceDirect Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic Riemerella anatipestifer gene AS87_08785 encodes a functional component, GldK, of the type IX secretion system T ⁎ Kanwar Kumar Malhia, Xiaolan Wanga, Zongchao Chena, Chan Dinga, Shengqing Yua,b, a Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Shanghai, China b Jiangsu Agri-Animal Husbandry Vocational College, Veterinary Bio-Pharmaceutical, Jiangsu Key Laboratory for High-Tech Research and Development of Veterinary Biopharmaceuticals, Taizhou, Jiangsu, China ARTICLE INFO ABSTRACT Keywords: Riemerella anatipestifer is an important pathogen of waterfowl, causing septicemic and exudative diseases. In our Riemerella anatipestifer previous study, we demonstrated that the deletion of the AS87_08785 gene significantly reduced the virulence of GldK R. anatipestifer strain Yb2, but the mechanism remained unclear. In this study, R. anatipestifer strains with mu- Gliding motility tated or complemented AS87_08785 genes were constructed and characterized. A sequence analysis indicated Type IX secretion system that the AS87_08785 gene encoded a putative GldK protein, which localized to the membrane fraction in a Bacterial virulence western blotting analysis. The mutant strain Yb2ΔgldK displayed defective gliding motility on agar plates, re- duced protease activity, and a reduced capacity for protein secretion. RNA sequencing and quantitative PCR analyses indicated that the transcription of 13 genes was downregulated in mutant Yb2ΔgldK. Animal experi- ments showed that the bacterial loads in the blood of Yb2ΔgldK-infected ducks were significantly reduced re- lative to those in wild-type strain Yb2 infected ducks. Most of the defective biological properties of the mutant were restored in complementation strain cYb2ΔgldK. Our results demonstrated that R. anatipestifer gene AS87_08785 encoded a component of the type IX secretion system, GldK, which functioned in bacterial gliding motility, protein secretion, and bacterial virulence. 1. Introduction system (T9SS) or Por secretion system, which is associated with gliding motility and the secretion of virulence factors, has recently been iden- Riemerella anatipestifer is a Gram-negative, non-spore-forming, rod- tified in R. anatipestifer (McBride and Zhu, 2013). Motility plays a major shaped, capsulated bacterium with gliding motility, belonging to the role in the pathogenesis in many members of the phylum Bacteroidetes. family Flavobacteriaceae in the phylum Bacteroidetes (Segers et al., T9SS and bacterial gliding motility are also associated with the secre- 1993). It has a worldwide distribution, and causes acute or chronic tion of proteins that are thought to be virulence factors in many pa- septicemia, fibrous pericarditis, airsacculitis, perihepatitis, caseous thogens (Sato et al., 2013; Shrivastava et al., 2012). Genetic analyses salpingitis, and vegetative disorders. Infection with R. anatipestifer have shown that the core of the T9SS gene cluster includes gldK, gldL, predominantly causes disease in domestic ducks, and is responsible for gldM, gldN, sprA, sprE, sprT, sov, porW, porU, and porV (Kharade and major economic losses in the poultry industry (Segers et al., 1993). McBride, 2015; Shrivastava et al., 2013). Proteins secreted by T9SS Twenty-one serotypes have been identified with agglutination tests, but have predicted N-terminal type 1 signal peptides, which allow their no significant cross-protection has been identified (Pathanasophon transit across the cytoplasmic membrane via the general secretion et al., 2002). Several virulence factors, including VapD, CAMP cohe- system. They also have conserved C-terminal domains (CTDs) that ap- molysin, and lipopolysaccharide-synthesis-associated genes, have been pear to direct them to the T9SS for secretion across the outer membrane reported (Chang et al., 1998; Crasta et al., 2002). Recently, 49 virulence (Shrivastava et al., 2012; Slakeski et al., 2011). sprT , which encodes a genes have been identified in R. anatipestifer strain Yb2 using random core protein of T9SS, is involved in proteins secretion in R. anatipestifer transposon mutagenesis (Wang et al., 2015). The virulence of the mu- and contributes to its virulence by exporting key proteins (Guo et al., tant strain RA256, in which the AS87_08785 gene was deleted, was 2017). Bacteria with a functional motility system form spreading co- reduced over 7,000-fold, but the mechanism remains unknown. lonies on agar plates, which allows the easy identification of motility The novel protein secretion system known as the type IX secretion mutants (McBride and Zhu, 2013). In this study, we showed that the ⁎ Corresponding author at: Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Shanghai, China. E-mail address: [email protected] (S. Yu). https://doi.org/10.1016/j.vetmic.2019.03.006 Received 24 November 2018; Received in revised form 6 March 2019; Accepted 7 March 2019 0378-1135/ © 2019 Elsevier B.V. All rights reserved. K.K. Malhi, et al. Veterinary Microbiology 231 (2019) 93–99 AS87_08785 gene encoded a putative gliding-motility protein, GldK, described previously (Hu et al., 2012). The plasmid was first introduced which was required for bacterial protein secretion, gliding motility, and into E. coli S17-1 by transformation. It was then transferred into the virulence. mutant strain by conjugation. Transformants were selected on TSA containing 5 μg/mL cefoxitin and 50 μg/mL kanamycin, and identified 2. Materials and methods by PCR amplification with primers AS87_08785-comp-F/AS87_08785- comp-R and RA 16S rRNA-F/RA 16S rRNA-R. The complementation 2.1. Ethics statement strain was designated cYb2ΔgldK. The study protocol was approved by the Institutional Animal Care 2.5. Bacterial growth, adherence, and invasion assays and Use Committee of Shanghai Veterinary Research Institute, the Chinese Academy of Agricultural Sciences (approval no. Shvri-po- A growth curve was constructed as described previously (Hu et al., 2017090876), and was conducted in strict accordance with the re- 2012). Briefly, the wild-type strain Yb2, mutant strain Yb2ΔgldK, and commendations outlined in the Guide for the Care and Use of complementation strain cYb2ΔgldK were grown in TSB at 37 °C for 8 h, Laboratory Animals. One-day-old Cherry Valley ducks were obtained with shaking. Equal amounts of each bacterial culture were then from the Zhuang Hang Duck Farm (Shanghai, China) and housed in transferred into fresh TSB in a ratio of 1:100 (vol/vol) and incubated at cages under a controlled temperature of 28–30 °C under biosafety 37 °C, with shaking at 200 rpm. Bacterial growth was measured as de- conditions, with water and food ad libitum. scribed previously (Wang et al., 2015), by counting the numbers of bacterial colony-forming units (CFU) at 1 h intervals for 18 h. 2.2. Bacterial strains, plasmids, and culture conditions Adhesion and invasion assays were performed in Vero cells (ATCC CCL-81), as described previously (Hu et al., 2012). Briefly, a 24-well The bacterial strains and plasmids are listed in Supplementary Table tissue culture plate was seeded with 105 cells/well in Dulbecco’s S1. Riemerella anatipestifer Yb2 was the virulent wild-type strain used in Modified Eagle’s Medium (DMEM Biowest, France) containing 10% this study. The mutant strain, Yb2ΔgldK, in which the AS87_08785 gene fetal bovine serum. After incubation at 37 °C under 5% CO2 for 24 h, the was deleted, was constructed by the insertion of transposon Tn4351 cell monolayer was rinsed with phosphate-buffered saline (PBS) and (Wang et al., 2015). The complementation strain, cYb2ΔgldK, was infected with bacteria at a multiplicity of infection (MOI) of 100 by constructed using plasmid pCP29, as described previously. The R. centrifugation at 100 × g for 5 min at room temperature. The infected anatipestifer strains were grown at 37 °C in tryptic soy agar (TSA) or cells were incubated at 37 °C under 5% CO2 for 1.5 h, washed three tryptic soy broth (TSB) (Difco Laboratories, Detroit, MI, USA). The Es- times with sterile PBS, and lysed with 0.1% trypsin (100 μL/well). The cherichia coli strains were grown at 37 °C on Luria–Bertani (LB) plates or cell suspensions were diluted 10-fold and plated onto TSA plates to in LB broth. Antibiotics were added at the following concentrations: determine the numbers of viable bacterial cells. For the invasion assay, ampicillin (100 μg/mL), chloramphenicol (30 μg/mL), erythromycin the extracellular bacteria were killed by incubating the monolayer with (0.5 μg/mL), kanamycin (50 μg/mL), and cefoxitin (5 μg/mL). DMEM supplemented with 100 μg/mL gentamycin for an additional 1 h. The bacteria were then incubated and washed three times with PBS. All 2.3. Identification of mutant strain Yb2ΔgldK these assays were performed in triplicate and replicated three times. The transposon Tn4351 was introduced into the R. anatipestifer 2.6. GldK expression, antibody production, and western blotting analysis wild-type strain Yb2 from E. coli BW19851 by conjugation, as described previously (Hu et al., 2012). The primer pairs RA 16S rRNA-F/RA 16S A 1,410-bp fragment of the gldK gene was amplified with primers rRNA-R, specific for R. anatipestifer 16S rRNA, and Erm-F/Erm-R, spe- GldK-segmental-pro-F/GldK-segmental-pro-R (Supplementary Table cific for Tn4351, are described in Supplementary Table S1 and were S1), digested with BamHI and SalI, and ligated into the pET30a vector used to identify the Tn4351 insertion in the R. anatipestifer mutant with to construct the recombinant plasmid pET-gldK. The construction of PCR amplification. pET-gldK was confirmed with DNA sequencing. The expression of the A Southern blotting analysis confirmed one insertion of transposon histidine-tagged recombinant GldK protein (rGldK) was induced in E. Tn4351 in the Yb2 genome, as described previously (Wang et al., 2015). coli strain BL21(DE3) cells by treatment with 1 mM isopropyl-β-D-1- The site of transposon insertion in the mutant strain was determined to thiogalactopy-ranoside (IPTG) for 6 h at 37 °C, with shaking.