The genus Kingella comprises four common species: the commensals K. oralis and K. denitrificans, the emerging paediatric pathogen K. kingae and the novel species K. negevensis. Improved diagnostic methods have led to the recognition of K. kingae as a major cause of septic arthritis in young children. The key virulence factor responsible for the pathogenesis of K. kingae is its cytotoxin RtxA, which is thought to facilitate host invasion. After binding to target cells, RtxA inserts into the host cell membrane and forms ion-conducting membrane pores that disrupt normal cell physiology and ultimately lead to cell death. In this study, we analysed the pro-inflammatory response of oral epithelial cells to a clinical isolate of K. kingae, its isogenic RtxA-deficient mutant and the commensals K. oralis and K. denitrificans, which do not produce RtxA. The results show that infection of cells with K. kingae, but not with the RtxA-deficient mutant and the commensal species, leads to increased expression and secretion of certain pro-inflammatory cytokines and chemokines. Furthermore, the RtxA-producing K. kingae, but not the RtxA-deficient mutant, upregulated the expression of DEFB4A and SLPI genes encoding antimicrobial peptides. These findings demonstrate that the RtxA toxin induces an innate immune response in oral epithelial cells.

Faculty of Science Charles University Prague Czech Republic

Institute of Microbiology of the Czech Academy of Sciences Prague Czech Republic

Zobrazit více v PubMed

Akdis M., Burgler S., Crameri R., Eiwegger T., Fujita H., Gomez E., Klunker S., Meyer N., O'Mahony L., Palomares O., Rhyner C., Ouaked N., Schaffartzik A., Van De Veen W., Zeller S., Zimmermann M., Akdis C.A. Interleukins, from 1 to 37, and interferon-gamma: receptors, functions, and roles in diseases. J. Allergy Clin. Immunol. 2011;127(3):701–721. e1-70. PubMed

Amit U., Porat N., Basmaci R., Bidet P., Bonacorsi S., Dagan R., Yagupsky P. Genotyping of invasive Kingella kingae isolates reveals predominant clones and association with specific clinical syndromes. Clin. Infect. Dis. 2012;55(8):1074–1079. PubMed

Anderson de la Llana R., Dubois-Ferriere V., Maggio A., Cherkaoui A., Manzano S., Renzi G., Hibbs J., Schrenzel J., Ceroni D. Oropharyngeal Kingella kingae carriage in children: characteristics and correlation with osteoarticular infections. Pediatr. Res. 2015;78(5):574–579. PubMed

Antezack A., Boxberger M., Rolland C., Monnet-Corti V., La Scola B. Isolation and characterization of Kingella bonacorsii sp. nov., A novel Kingella species detected in a stable periodontitis subject. Pathogens. 2021;10(2):240. PubMed PMC

Basmaci R., Yagupsky P., Ilharreborde B., Guyot K., Porat N., Chomton M., Thiberge J.M., Mazda K., Bingen E., Bonacorsi S., Bidet P. Multilocus sequence typing and rtxA toxin gene sequencing analysis of Kingella kingae isolates demonstrates genetic diversity and international clones. PLoS. One. 2012;7(5) PubMed PMC

Basmaci R., Deschamps K., Levy C., Mathy V., Corrard F., Thollot F., Bechet S., Sobral E., Bidet P., Cohen R., Bonacorsi S. Prevalence of Kingella kingae oropharyngeal carriage and predominance of type a and type b polysaccharide capsules among French young children. Clin. Microbiol. Infect. 2019;25(1):114–116. PubMed

Bassinet L., Fitting C., Housset B., Cavaillon J.M., Guiso N. Bordetella pertussis adenylate cyclase-hemolysin induces interleukin-6 secretion by human tracheal epithelial cells. Infect. Immun. 2004;72(9):5530–5533. PubMed PMC

Bhakdi S., Muhly M., Korom S., Schmidt G. Effects of Escherichia coli hemolysin on human monocytes. Cytocidal action and stimulation of interleukin 1 release. J. Clin. Invest. 1990;85(6):1746–1753. PubMed PMC

Brown A.M., Rothburn M.M., Roberts C., Nye F.J. Septicaemia with probable endocarditis caused by Kingella denitrificans. J. Infect. 1987;15(3):225–228. PubMed

Demirel I., Persson A., Brauner A., Sarndahl E., Kruse R., Persson K. Activation of the NLRP3 inflammasome pathway by uropathogenic Escherichia coli is virulence factor-dependent and influences colonization of bladder epithelial cells. Front. Cell Infect. Microbiol. 2018;8:81. PubMed PMC

Dewhirst F.E., Chen C.K., Paster B.J., Zambon J.J. Phylogeny of species in the family Neisseriaceae isolated from human dental plaque and description of Kingella oralis sp. nov [corrected] Int. J. Syst. Bacteriol. 1993;43(3):490–499. PubMed

Dewhirst F.E., Chen T., Izard J., Paster B.J., Tanner A.C., Yu W.H., Lakshmanan A., Wade W.G. The human oral microbiome. J. Bacteriol. 2010;192(19):5002–5017. PubMed PMC

Dikshit N., Kale S.D., Khameneh H.J., Balamuralidhar V., Tang C.Y., Kumar P., Lim T.P., Tan T.T., Kwa A.L., Mortellaro A., Sukumaran B. NLRP3 inflammasome pathway has a critical role in the host immunity against clinically relevant Acinetobacter baumannii pulmonary infection. Mucosal. Immunol. 2018;11(1):257–272. PubMed

Doumas S., Kolokotronis A., Stefanopoulos P. Anti-inflammatory and antimicrobial roles of secretory leukocyte protease inhibitor. Infect. Immun. 2005;73(3):1271–1274. PubMed PMC

Dunne A., Ross P.J., Pospisilova E., Masin J., Meaney A., Sutton C.E., Iwakura Y., Tschopp J., Sebo P., Mills K.H. Inflammasome activation by adenylate cyclase toxin directs Th17 responses and protection against Bordetella pertussis. J. Immunol. 2010;185(3):1711–1719. PubMed

El Houmami N., Bakour S., Bzdrenga J., Rathored J., Seligmann H., Robert C., Armstrong N., Schrenzel J., Raoult D., Yagupsky P., Fournier P.E. Isolation and characterization of Kingella negevensis sp. nov., a novel Kingella species detected in a healthy paediatric population. Int. J. Syst. Evol. Microbiol. 2017;67(7):2370–2376. PubMed

Fan J., Heller N.M., Gorospe M., Atasoy U., Stellato C. The role of post-transcriptional regulation in chemokine gene expression in inflammation and allergy. Eur. Respir. J. 2005;26(5):933–947. PubMed

Filipi K., Rahman W.U., Osickova A., Osicka R. Kingella kingae RtxA cytotoxin in the context of other RTX toxins. Microorganisms. 2022;10(3):518. PubMed PMC

Fujita T., Yoshimoto T., Kajiya M., Ouhara K., Matsuda S., Takemura T., Akutagawa K., Takeda K., Mizuno N., Kurihara H. Regulation of defensive function on gingival epithelial cells can prevent periodontal disease. Jpn. Dent. Sci. Rev. 2018;54(2):66–75. PubMed PMC

Gleason T.G., Houlgrave C.W., May A.K., Crabtree T.D., Sawyer R.G., Denham W., Norman J.G., Pruett T.L. Hemolytically active (acylated) alpha-hemolysin elicits interleukin-1beta (IL-1beta) but augments the lethality of Escherichia coli by an IL-1- and tumor necrosis factor-independent mechanism. Infect. Immun. 1998;66(9):4215–4221. PubMed PMC

Hasan S., Kulkarni N.N., Asbjarnarson A., Linhartova I., Osicka R., Sebo P., Gudmundsson G.H. Bordetella pertussis adenylate cyclase toxin disrupts functional integrity of bronchial epithelial layers. Infect. Immun. 2018;86(3) e00445-17. PubMed PMC

Henriksen S., Bovre K. Transfer of Moraxella kingae Henriksen and Bøvre to the genus kingella gen. nov. In the family Neisseriaceae. Int. J. Syst. Evol. Microbiol. 1976;26(4):447–450.

Hernandez-Ruperez M.B., Suarez-Arrabal M.D.C., Villa-Garcia A., Zarzoso-Fernandez S., Navarro-Gomez M., Santos-Sebastian M.D.M., Garcia-Martin A., Marin M., Gonzalez-Martinez F., Narbona-Carceles J., Cervera-Bravo P., Gonzalez-Lopez J.L., Hernandez-Sampelayo T., Saavedra-Lozano J. Kingella kingae as the main cause of septic arthritis: importance of molecular diagnosis. Pediatr. Infect. Dis. J. 2018;37(12):1211–1216. PubMed

Hollis D.G., Wiggins G.L., Weaver R.E. An unclassified gram-negative rod isolated from the pharynx on Thayer-Martin medium (selective agar) Appl. Microbiol. 1972;24(5):772–777. PubMed PMC

Chang D.W., Nudell Y.A., Lau J., Zakharian E., Balashova N.V. RTX toxin plays a key role in Kingella kingae virulence in an infant rat model. Infect. Immun. 2014;82(6):2318–2328. PubMed PMC

Chen C. Distribution of a newly described species, Kingella oralis, in the human oral cavity. Oral Microbiol. Immunol. 1996;11(6):425–427. PubMed

Cherkaoui A., Ceroni D., Emonet S., Lefevre Y., Schrenzel J. Molecular diagnosis of Kingella kingae osteoarticular infections by specific real-time PCR assay. J. Med. Microbiol. 2009;58(Pt 1):65–68. PubMed

Kaplan J.B., Lo C., Xie G., Johnson S.L., Chain P.S., Donnelly R., Kachlany S.C., Balashova N.V. Genome sequence of Kingella kingae septic arthritis isolate PYKK081. J. Bacteriol. 2012;194(11):3017. PubMed PMC

Kehl-Fie, T.E., St Geme J.W. Identification and characterization of an RTX toxin in the emerging pathogen Kingella kingae. J. Bacteriol. 2007;189(2):430–436. PubMed PMC

Khaliq H., Osickova A., Lichvarova M., Sulc M., Navarrete K.M., Espinosa-Vinals C., Masin J., Osicka R. Structural and functional significance of two conserved lysine residues in acylated sites of Kingella kingae RtxA cytotoxin. Biochimie. 2024;S0300-9084(24):00320–00321. PubMed

Khan J.A., Sharp S., Mann K.R., Brewer J. Kingella denitrificans prosthetic endocarditis. Am. J. Med. Sci. 1986;291(3):187–189. PubMed

Khattak M., Chakkalakumbil S.V., Stevenson R.A., Bryson D.J., Reidy M.J., Talbot C.L., George H. Kingella kingae septic arthritis: maintaining a high level of suspicion in children with atypical presentation. Bone Joint J. 2021;103(3):584–588. PubMed

Klein Klouwenberg, P., Bont L. Neonatal and infantile immune responses to encapsulated bacteria and conjugate vaccines. Clin. Dev. Immunol. 2008 2008. PubMed PMC

Lawson P.A., Malnick H., Collins M.D., Shah J.J., Chattaway M.A., Bendall R., Hartley J.W. Description of Kingella potus sp. nov., an organism isolated from a wound caused by an animal bite. J. Clin. Microbiol. 2005;43(7):3526–3529. PubMed PMC

Lebedev, K.A., Poniakina I.D. [Immunophysiology of epithelial cells and pattern-recognition receptors] Fiziol. Cheloveka. 2006;32(2):114–126. PubMed

Lehours P., Freydiere A.M., Richer O., Burucoa C., Boisset S., Lanotte P., Prere M.F., Ferroni A., Lafuente C., Vandenesch F., Megraud F., Menard A. The rtxA toxin gene of Kingella kingae: a pertinent target for molecular diagnosis of osteoarticular infections. J. Clin. Microbiol. 2011;49(4):1245–1250. PubMed PMC

Linhartova I., Bumba L., Masin J., Basler M., Osicka R., Kamanova J., Prochazkova K., Adkins I., Hejnova-Holubova J., Sadilkova L., Morova J., Sebo P. RTX proteins: a highly diverse family secreted by a common mechanism. FEMS Microbiol. Rev. 2010;34(6):1076–1112. PubMed PMC

Livak, K.J., Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods. 2001;25(4):402–408. PubMed

May A.K., Sawyer R.G., Gleason T., Whitworth A., Pruett T.L. In vivo cytokine response to Escherichia coli alpha-hemolysin determined with genetically engineered hemolytic and nonhemolytic E. coli variants. Infect. Immun. 1996;64(6):2167–2171. PubMed PMC

Momose F., Araida T., Negishi A., Ichijo H., Shioda S., Sasaki S. Variant sublines with different metastatic potentials selected in nude mice from human oral squamous cell carcinomas. J. Oral Pathol. Med. 1989;18(7):391–395. PubMed

Morreale D.P., Porsch E.A., Kern B.K., St Geme J.W., Planet P.J. Acquisition, co-option, and duplication of the rtx toxin system and the emergence of virulence in Kingella. Nat. Commun. 2023;14(1):4281. PubMed PMC

Murik O., Zeevi D.A., Mann T., Kashat L., Assous M.V., Megged O., Yagupsky P. Whole-genome sequencing reveals differences among Kingella kingae strains from carriers and patients with invasive infections. Microbiol. Spectr. 2023;11(3) PubMed PMC

Murthy A.M.V., Phan M.D., Peters K.M., Nhu N.T.K., Welch R.A., Ulett G.C., Schembri M.A., Sweet M.J. Regulation of hemolysin in uropathogenic Escherichia coli fine-tunes killing of human macrophages. Virulence. 2018;9(1):967–980. PubMed PMC

Osickova A., Balashova N., Masin J., Sulc M., Roderova J., Wald T., Brown A.C., Koufos E., Chang E.H., Giannakakis A., Lally E.T., Osicka R. Cytotoxic activity of Kingella kingae RtxA toxin depends on post-translational acylation of lysine residues and cholesterol binding. Emerg. Microbes. Infect. 2018;7(1):178. PubMed PMC

Porta H., Cancino-Rodezno A., Soberon M., Bravo A. Role of MAPK p38 in the cellular responses to pore-forming toxins. Peptides. 2011;32(3):601–606. PubMed PMC

Proctor, D.M., Relman D.A. The landscape ecology and microbiota of the human nose, mouth, and throat. Cell Host. Microbe. 2017;21(4):421–432. PubMed PMC

Rahman W.U., Fiser R., Osicka R. Kingella kingae RtxA toxin interacts with sialylated gangliosides. Microb. Pathog. 2023;181 PubMed

Rahman W.U., Osickova A., Klimova N., Lora J., Balashova N., Osicka R. Binding of Kingella kingae RtxA toxin depends on cell surface oligosaccharides, but not on beta(2) integrins. Int. J. Mol. Sci. 2020;21(23):9092. PubMed PMC

Raman D., Sobolik-Delmaire T., Richmond A. Chemokines in health and disease. Exp. Cell Res. 2011;317(5):575–589. PubMed PMC

Reekmans A., Noppen M., Naessens A., Vincken W. A rare manifestation of Kingella kingae infection. Eur. J. Intern. Med. 2000;11(6):343–344. PubMed

Rolle U., Schille R., Hormann D., Friedrich T., Handrick W. Soft tissue infection caused by Kingella kingae in a child. J. Pediatr. Surg. 2001;36(6):946–947. PubMed

Ruzickova E., Lichvarova M., Osickova A., Filipi K., Jurnecka D., Khaliq H., Espinosa-Vinals C., Pompach P., Masin J., Osicka R. Two pairs of back-to-back alpha-helices of Kingella kingae RtxA toxin are crucial for the formation of a membrane pore. Int. J. Biol. Macromol. 2024;283(Pt 1) PubMed

Samara E., Lutz N., Zambelli P.Y. Kingella kingae spinal infections in children. Children. (Basel) 2022;9(5):705. PubMed PMC

Schroeder, H.E., Listgarten M.A. The gingival tissues: the architecture of periodontal protection. Periodontol. 2000. 1997;13:91–120. PubMed

Senel S. An overview of physical, microbiological and immune barriers of oral mucosa. Int. J. Mol. Sci. 2021;22(15):7821. PubMed PMC

Shim B.S., Choi Y., Cheon I.S., Song M.K. Sublingual delivery of vaccines for the induction of mucosal immunity. Immune Netw. 2013;13(3):81–85. PubMed PMC

Schroder, J.M., Harder J. Human beta-defensin-2. Int. J. Biochem. Cell Biol. 1999;31(6):645–651. PubMed

Snell, J.J.S., Lapage S.P. Transfer of some saccharolytic Moraxella species to Kingella Henriksen and Bøvre 1976, with descriptions of Kingella indologenes sp. nov. and Kingella denitrificans sp. nov. Int. J. Syst. Evol. Microbiol. 1976;26(4):451–458.

Sokol, C.L., Luster A.D. The chemokine system in innate immunity. Cold. Spring. Harb. Perspect. Biol. 2015;7(5) PubMed PMC

Srikhanta Y.N., Fung K.Y., Pollock G.L., Bennett-Wood V., Howden B.P., Hartland E.L. Phasevarion-regulated virulence in the emerging pediatric pathogen Kingella kingae. Infect. Immun. 2017;85(12) e00319-17. PubMed PMC

Swann, R.A., Holmes B. Infective endocarditis caused by Kingella denitrificans. J. Clin. Pathol. 1984;37(12):1384–1387. PubMed PMC

Syridou G., Giannopoulou P., Charalampaki N., Papaparaskevas J., Korovessi P., Papagianni S., Tsakris A., Trikka-Grafakou E. Invasive infection from Kingella kingae: not only arthritis. IDCases. 2020;20 PubMed PMC

Tang B., Bojesen A.M. Immune suppression induced by gallibacterium anatis GtxA during interaction with chicken macrophage-like HD11 cells. Toxins. (Basel) 2020;12(9):536. PubMed PMC

Thornberry N.A., Bull H.G., Calaycay J.R., Chapman K.T., Howard A.D., Kostura M.J., Miller D.K., Molineaux S.M., Weidner J.R., Aunins J., et al. A novel heterodimeric cysteine protease is required for interleukin-1 beta processing in monocytes. Nature. 1992;356(6372):768–774. PubMed

Uhlen P., Laestadius A., Jahnukainen T., Soderblom T., Backhed F., Celsi G., Brismar H., Normark S., Aperia A., Richter-Dahlfors A. Alpha-haemolysin of uropathogenic E. coli induces Ca2+ oscillations in renal epithelial cells. Nature. 2000;405(6787):694–697. PubMed

Verma V., Kumar P., Gupta S., Yadav S., Dhanda R.S., Thorlacius H., Yadav M. alpha-hemolysin of uropathogenic E. coli regulates NLRP3 inflammasome activation and mitochondrial dysfunction in THP-1 macrophages. Sci. Rep. 2020;10(1) PubMed PMC

Walterspiel J.N. Kingella kingae meningitis with bilateral infarcts of the basal ganglia. Infection. 1983;11(6):307–309. PubMed

Wang C., Li Q., Lv J., Sun X., Cao Y., Yu K., Miao C., Zhang Z.S., Yao Z., Wang Q. Alpha-hemolysin of uropathogenic Escherichia coli induces GM-CSF-mediated acute kidney injury. Mucosal. Immunol. 2020;13(1):22–33. PubMed PMC

Williams N., Cooper C., Cundy P. Kingella kingae septic arthritis in children: recognising an elusive pathogen. J. Child Orthop. 2014;8(1):91–95. PubMed PMC

Xiao M., Liu R., Du J., Liu R., Zhai L., Wang H., Yao S., Xu Y.C. Kingella pumchi sp. nov., an organism isolated from human vertebral puncture tissue. Antonie Van Leeuwenhoek. 2023;116(2):143–151. PubMed

Yagupsky P. Kingella kingae: carriage, transmission, and disease. Clin. Microbiol. Rev. 2015;28(1):54–79. PubMed PMC

Yagupsky P., Porsch E., St Geme J.W. Kingella kingae: an emerging pathogen in young children. Pediatrics. 2011;127(3):557–565. PubMed

Yagupsky P., Dagan R., Howard C.B., Einhorn M., Kassis I., Simu A. Clinical features and epidemiology of invasive Kingella kingae infections in southern Israel. Pediatrics. 1993;92(6):800–804. PubMed