Cytotoxic effects of Kingella kingae outer membrane vesicles on human cells

Abstract

Kingella kingae is an emerging pathogen causing osteoarticular infections in pediatric patients. Electron microscopy of K. kingae clinical isolates revealed the heterogeneously-sized membranous structures blebbing from the outer membrane that were classified as outer membrane vesicles (OMVs). OMVs purified from the secreted fraction of a septic arthritis K. kingae isolate were characterized. Among several major proteins, K. kingae OMVs contained virulence factors RtxA toxin and PilC2 pilus adhesin. RtxA was also found secreted as a soluble protein in the extracellular environment indicating that the bacterium may utilize different mechanisms for the toxin delivery. OMVs were shown to be hemolytic and possess some leukotoxic activity while high leukotoxicity was detected in the non-hemolytic OMV-free component of the secreted fraction. OMVs were internalized by human osteoblasts and synovial cells. Upon interaction with OMVs, the cells produced increased levels of human granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleuskin 6 (IL-6) suggesting that these cytokines might be involved in the signaling response of infected joint and bone tissues during natural K. kingae infection. This study is the first report of OMV production by K. kingae and demonstrates that OMVs are a complex virulence factor of the organism causing cytolytic and inflammatory effects on host cells.

RTX Toxin Plays a Key Role in Kingella kingae Virulence in an Infant Rat Model

“…The thymuses of pups receiving KKNB100 appeared to be normal. The pathologies observed in spleen, bone marrow, and thymus are consistent with the cytotoxicity that we have demonstrated previously in vitro for PYKK081 with cultured promyoblasts, T lymphoblasts, T lymphocytes, and megakaryoblasts (35).…”

“…K. kingae strain PYKK081 was isolated in 1991 in Israel from the ankle joint of an 8-month-old boy with septic arthritis. This strain was previously used in our studies (35) and in genome sequencing (36). The bacteria were grown on Columbia agar (CA) (Oxoid Ltd., Hampshire, England) with 5% sheep blood (Hemostat Laboratories, Dixon, CA) or on AAGM agar (37) at 37°C with 10% CO 2 and were stored in AAGM broth with 10% dimethyl sulfoxide (DMSO) at Ϫ80°C.…”

“…(i) RtxA purification. The bacteria were grown on AAGM plates for 25 h, and biomass was collected and subjected to centrifugation at 150,000 ϫ g for 2 h to separate the bacterial pellet and liquid secreted fraction (35). One milliliter of the resultant supernatant was filtered through a 0.22-m filter and was then loaded into a G-100 column (bed volume, 45 ml), equilibrated with 20 mM Tris-HCl-250 mM NaCl-2 mM CaCl 2 (pH 6.8) buffer, and then eluted with the same buffer in 1-ml fractions.…”

“…Skin samples were fixed in 4% neutral buffered formaldehyde and embedded in paraffin. Sections were treated in an automated pressure cooker (Retriever 2100; Electron Microscopy Sciences, Hatfield, PA) in citrate buffer (pH 6.0), rinsed, stained with polyclonal anti-K. kingae outer membrane vesicle (OMV) antibody (35) (1:100 dilution) in Superblock buffer (Pierce, Rockford, IL) for 1 h at room temperature, rinsed with Superblock, and stained with goat antirabbit IgG labeled with Alexa 488 (1:200 dilution) for 2 h at room temperature. Nuclei were stained with 7-aminoactinomycin D (7-AAD) (Invitrogen, Carlsbad, CA) for 30 min.…”

Respiratory Colonization by Kingella kingae, Person-to-Person Transmission, and Pathogenesis of Invasive Infection

“…Sequencing of the locus genes revealed that rtxA, rtxB, and rtxC genes encode proteins that share >70% identity with their homologs in Moraxella bovis, whereas rtxD and tolC genes encode proteins that share homology with their Neisseria meningitidis counterparts [23]. The K. kingae RTX toxin exhibits wide range of cytotoxic activity, especially to macrophage-like and synovial cells, and to a lesser degree to respiratory epithelial cells and appears to be secreted as a soluble protein in the extracellular environment as well as as a component of outer membrane vesicles that are internalized by host's cells [24]. These biological features suggest that the RTX toxin may play an important role in establishing colonization of the pharynx by disrupting the respiratory epithelium and perhaps, in the enhanced effect in the setting of a viral coinfection, causing mucosal erosions, and promoting survival of the bacterium in the bloodstream and invasion of skeletal system tissues [23].…”