Pathogenic determinants of Kingella kingae disease

Abstract

Kingella kingae is an emerging pediatric pathogen and is increasingly recognized as a leading etiology of septic arthritis, osteomyelitis, and bacteremia and an occasional cause of endocarditis in young children. The pathogenesis of K. kingae disease begins with colonization of the upper respiratory tract followed by breach of the respiratory epithelial barrier and hematogenous spread to distant sites of infection, primarily the joints, bones, and endocardium. As recognition of K. kingae as a pathogen has increased, interest in defining the molecular determinants of K. kingae pathogenicity has grown. This effort has identified numerous bacterial surface factors that likely play key roles in the pathogenic process of K. kingae disease, including type IV pili and the Knh trimeric autotransporter (adherence to the host), a potent RTX-family toxin (epithelial barrier breach), and multiple surface polysaccharides (complement and neutrophil resistance). Herein, we review the current state of knowledge of each of these factors, providing insights into potential approaches to the prevention and/or treatment of K. kingae disease.

Keywords: adhesin; capsule; exopolysaccharide; genomics; kingella kingae; lipopolysaccharide; toxin; type IV pili.

Figure 1

Piliation phenotypes in K. kingae mutants. Wild type (WT) K. kingae bacteria (blue) produce high levels of filamentous surface fibers called type IV pili. Deletion of the pilA1 gene (ΔpilA1) encoding the PilA1 major pilin subunit results in abrogation of surface pili. The pilus fiber is assembled by an assembly ATPase PilF, and deletion of the pilF gene (ΔpilF) prevents surface piliation. T4P retraction is controlled by the PilT retraction ATPase, and deletion of the pilT gene (ΔpilT) results in higher levels of surface piliation compared to WT. Expression of the pilA1 gene is controlled by the PilR/S two-component system and the σ factor σ54. Deletion of pilR (ΔpilR) and the gene encoding σ54 (ΔrpoN) abrogates surface pili. However, deletion of pilS (ΔpilS) results in very low levels of surface piliation compared to WT. Elimination of both the PilC1 and PilC2 pilus-associated adhesins (ΔpilC1pilC2) significantly decreases levels of surface pili compared to WT. However, surface piliation is restored to WT levels following additional deletion of the pilT gene (ΔpilC1pilC2pilT), suggesting that PilC1 and PilC2 promote surface piliation through counteracting retraction of the filament.

Figure 2

Activation and secretion of RtxA. The rtxA gene encodes a pro-toxin in K. kingae. Following translation, pro-RtxA remains unfolded and associates with RtxC in the cytosol, possibly near the cell membrane. (i) RtxC catalyzes the addition of an acyl-moiety onto each of two, conserved lysine residues. (ii) As with other RTX toxins, RtxA is thought to remain unfolded until it is exported from the bacterial cell via a type I secretion system comprised of RtxD and RtxB in the inner membrane and TolC in the outer membrane. Export is facilitated by a C-terminal secretion signal, and folding is likely mediated through the interaction of extracellular calcium with the RtxA calcium-binding domain. (iii) Outside of the cell, RtxA is found in a free state in spent media as well as in association with outer membrane vesicles (OMVs). It is unclear if the OMV-associated toxin is within the OMV or associated with the membrane. Both free RtxA and OMV-associated RtxA can cause host cell lysis.

Figure 3

A proposed model depicting the role of each of the known capsule biosynthesis gene products in K. kingae strain KK01 producing capsule type a. (i) The lipB gene encodes a β-Kdo transferase that adds a single β-Kdo residue onto a lipid moiety (phosphatidylglycerol) at the cytoplasmic side of the bacterial membrane. (ii) The lipA gene encodes a β-Kdo transferase that adds multiple β-Kdo residues onto the initial β-Kdo residue and creates a short β-Kdo chain. (iii) The csaA gene encodes a bifunctional enzyme with a GalNAc transferase at the N-terminal region (blue portion) and a Kdo transferase in the middle and C-terminal region (green portion) of the protein. CsaA builds the capsular polysaccharide chain composed of GalNAc and Kdo repeating units onto the chain of β-Kdo residues, with the N-terminal region (blue) adding GalNAc and the middle and C-terminal region (green) adding Kdo. (iv) The capsular polysaccharide is then shuttled to the outer membrane through the ABC-type capsule transporter encoded by ctrABCD.

Figure 4

The relationship between the galactan exopolysaccharide and LPS in K. kingae. (i) The pamABCDE locus is essential for presentation of the galactan exopolysaccharide on the bacterial surface and contains genes encoding four predicted glycosyltransferases (pamA, pamC, pamD, and pamE) and one predicted UDP-galactopyranose mutase (pamB). (ii) The pamABC genes are necessary for synthesis of the galactan homopolymer with the structure of →5)-β-Galf-(1→, which is linked to the atypical O-antigen. The pamDE genes are required for synthesis of the atypical O-antigen. In wild type K. kingae strain KK01, the LPS migrates as two distinct modal clusters: a high molecular weight (HMW) band that contains LPS decorated with both the atypical O-antigen and galactan and a low molecular weight ladder cluster that contains LPS decorated with only the atypical O-antigen.