The Kingella kingae PilC1 MIDAS Motif Is Essential for Type IV Pilus Adhesive Activity and Twitching Motility.

Kingella kingae is an emerging pathogen that has recently been identified as a leading cause of osteoarticular infections in young children. Colonization with K. kingae is common, with approximately 10% of young children carrying this organism in the oropharynx at any given time. Adherence to epithelial cells represents the first step in K. kingae colonization of the oropharynx, a prerequisite for invasive disease. Type IV pili and the pilus-associated PilC1 and PilC2 proteins have been shown to mediate K. kingae adherence to epithelial cells, but the molecular mechanism of this adhesion has remained unknown. Metal ion-dependent adhesion site (MIDAS) motifs are commonly found in integrins, where they function to promote an adhesive interaction with a ligand. In this study, we identified a potential MIDAS motif in K. kingae PilC1 which we hypothesized was directly involved in mediating type IV pilus adhesive interactions. We found that the K. kingae PilC1 MIDAS motif was required for bacterial adherence to epithelial cell monolayers and extracellular matrix proteins and for twitching motility. Our results demonstrate that K. kingae has co-opted a eukaryotic adhesive motif for promoting adherence to host structures and facilitating colonization.

Kingella kingae RtxA toxin interacts with sialylated gangliosides.

The membrane-damaging RTX family cytotoxin RtxA is a key virulence factor of the emerging pediatric pathogen Kingella kingae, but little is known about the mechanism of RtxA binding to host cells. While we have previously shown that RtxA binds cell surface glycoproteins, here we demonstrate that the toxin also binds different types of gangliosides. The recognition of gangliosides by RtxA depended on sialic acid side groups of ganglioside glycans. Moreover, binding of RtxA to epithelial cells was significantly decreased in the presence of free sialylated gangliosides, which inhibited cytotoxic activity of the toxin. These results suggest that RtxA utilizes sialylated gangliosides as ubiquitous cell membrane receptor molecules on host cells to exert its cytotoxic action and support K. kingae infection.

Binding of Kingella kingae RtxA Toxin Depends on Cell Surface Oligosaccharides, but Not on ß2 Integrins.

The Gram-negative coccobacillus Kingella kingae is increasingly recognized as an important invasive pediatric pathogen that causes mostly bacteremia and skeletal system infections. K. kingae secretes an RtxA toxin that belongs to a broad family of the RTX (Repeats in ToXin) cytotoxins produced by bacterial pathogens. Recently, we demonstrated that membrane cholesterol facilitates interaction of RtxA with target cells, but other cell surface structures potentially involved in toxin binding to cells remain unknown. We show that deglycosylation of cell surface structures by glycosidase treatment, or inhibition of protein N- and O-glycosylation by chemical inhibitors substantially reduces RtxA binding to target cells. Consequently, the deglycosylated cells were more resistant to cytotoxic activity of RtxA. Moreover, experiments on cells expressing or lacking cell surface integrins of the ß2 family revealed that, unlike some other cytotoxins of the RTX family, K. kingae RtxA does not bind target cells via the ß2 integrins. Our results, hence, show that RtxA binds cell surface oligosaccharides present on all mammalian cells but not the leukocyte-restricted ß2 integrins. This explains the previously observed interaction of the toxin with a broad range of cell types of various mammalian species and reveals that RtxA belongs to the group of broadly cytolytic RTX hemolysins.

Defining the Mechanical Determinants of Kingella kingae Adherence to Host Cells.

Kingella kingae is an important pathogen in young children and initiates infection by colonizing the posterior pharynx. Adherence to pharyngeal epithelial cells is an important first step in the process of colonization. In the present study, we sought to elucidate the interplay of type IV pili (T4P), a trimeric autotransporter adhesin called Knh, and the polysaccharide capsule in K. kingae adherence to host cells. Using adherence assays performed under shear stress, we observed that a strain expressing only Knh was capable of higher levels of adherence than a strain expressing only T4P. Using atomic force microscopy and transmission electron microscopy (TEM), we established that the capsule had a mean depth of 700 nm and that Knh was approximately 110 nm long. Using cationic ferritin capsule staining and thin-section transmission electron microscopy, we found that when bacteria expressing retractile T4P were in close contact with host cells, the capsule was absent at the point of contact between the bacterium and the host cell membrane. In a T4P retraction-deficient mutant, the capsule depth remained intact and adherence levels were markedly reduced. These results support the following model: T4P make initial contact with the host cell and mediate low-strength adherence. T4P retract, pulling the organism closer to the host cell and displacing the capsule, allowing Knh to be exposed and mediate high-strength, tight adherence to the host cell surface. This report provides the first description of the mechanical displacement of capsule enabling intimate bacterial adherence to host cells.IMPORTANCE Adherence to host cells is an important first step in bacterial colonization and pathogenicity. Kingella kingae has three surface factors that are involved in adherence: type IV pili (T4P), a trimeric autotransporter adhesin called Knh, and a polysaccharide capsule. Our results suggest that T4P mediate initial contact and low-strength adherence to host cells. T4P retraction draws the bacterium closer to the host cell and causes the displacement of capsule. This displacement exposes Knh and allows Knh to mediate high-strength adherence to the host cell. This work provides new insight into the interplay of T4P, a nonpilus adhesin, and a capsule and their effects on bacterial adherence to host cells.

Genetic and Molecular Basis of Kingella kingae Encapsulation.

Kingella kingae is a common cause of invasive disease in young children and was recently found to produce a polysaccharide capsule containing N-acetylgalactosamine (GalNAc) and ß-3-deoxy-d-manno-octulosonic acid (ßKdo). Given the role of capsules as important virulence factors and effective vaccine antigens, we set out to determine the genetic determinants of K. kingae encapsulation. Using a transposon library and a screen for nonencapsulated mutants, we identified the previously identified ctrABCD (ABC transporter) operon, a lipA (kpsC)-like gene, a lipB (kpsS)-like gene, and a putative glycosyltransferase gene designated csaA (capsule synthesis type a gene A). These genes were found to be present at unlinked locations scattered throughout the genome, an atypical genetic arrangement for Gram-negative bacteria that elaborate a capsule dependent on an ABC-type transporter for surface localization. The csaA gene product contains a predicted glycosyltransferase domain with structural homology to GalNAc transferases and a predicted capsule synthesis domain with structural homology to Kdo transferases, raising the possibility that this enzyme is responsible for alternately linking GalNAc to ßKdo and ßKdo to GalNAc. Consistent with this conclusion, mutation of the DXD motif in the GalNAc transferase domain and of the HP motif in the Kdo transferase domain resulted in a loss of encapsulation. Examination of intracellular and surface-associated capsule in deletion mutants and complemented strains further implicated the lipA (kpsC)-like gene, the lipB (kpsS)-like gene, and the csaA gene in K. kingae capsule production. These data define the genetic requirements for encapsulation in K. kingae and demonstrate an atypical organization of capsule synthesis, assembly, and export genes.

Pore forming activity of the potent RTX-toxin produced by pediatric pathogen Kingella kingae: Characterization and comparison to other RTX-family members.

Pediatric septic arthritis in patients under age of four is frequently caused by the oral Gram-negative bacterium Kingella kingae. This organism may be responsible for a severe form of infective endocarditis in otherwise healthy children and adults. A major virulence factor of K. kingae is RtxA, a toxin that belongs to the RTX (Repeats-in-ToXin) group of secreted pore forming toxins. To understand the RtxA effects on host cell membranes, the toxin activity was studied using planar lipid bilayers. K. kingae strain PYKK081 and its isogenic RtxA-deficient strain, KKNB100, were tested for their ability to form pores in artificial membranes of asolectin/n-decane. RtxA, purified from PYKK081, was able to rapidly form pores with an apparent diameter of 1.9nm as measured by the partition of nonelectrolytes in the pores. The RtxA channels are cation-selective and showed strong voltage-dependent gating. In contrast to supernatants of PYKK081, those of KKNB100 did not show any pore forming activity. We concluded that RtxA toxin is the only secreted protein from K. kingae forming large channels in host cell membranes where it induces cation flux leading to programmed cell death. Furthermore, our findings suggested that the planar lipid bilayer technique can effectively be used to test possible inhibitors of RTX toxin activity and to investigate the mechanism of the toxin binding to the membrane.

Genome Analysis of Kingella kingae Strain KWG1 Reveals How a ß-Lactamase Gene Inserted in the Chromosome of This Species.

We describe the genome of a penicillinase-producing Kingella kingae strain (KWG1), the first to be isolated in continental Europe, whose bla(TEM-1) gene was, for the first time in this species, found to be chromosomally inserted. The bla(TEM) gene is located in an integrative and conjugative element (ICE) inserted in Met-tRNA and comprising genes that encode resistance to sulfonamides, streptomycin, and tetracycline. This ICE is homologous to resistance-conferring plasmids of K. kingae and other Gram-negative bacteria.

Calcium binding properties of the Kingella kingae PilC1 and PilC2 proteins have differential effects on type IV pilus-mediated adherence and twitching motility.

Kingella kingae is an emerging bacterial pathogen that is being recognized increasingly as an important etiology of septic arthritis, osteomyelitis, and bacteremia, especially in young children. The pathogenesis of K. kingae disease begins with bacterial adherence to respiratory epithelium, which is dependent on type IV pili and is influenced by two PilC-like proteins called PilC1 and PilC2. Production of either PilC1 or PilC2 is necessary for K. kingae piliation and bacterial adherence. In this study, we set out to further investigate the role of PilC1 and PilC2 in type IV pilus-associated phenotypes. We found that PilC1 contains a functional 9-amino-acid calcium-binding (Ca-binding) site with homology to the Pseudomonas aeruginosa PilY1 Ca-binding site and that PilC2 contains a functional 12-amino-acid Ca-binding site with homology to the human calmodulin Ca-binding site. Using targeted mutagenesis to disrupt the Ca-binding sites, we demonstrated that the PilC1 and PilC2 Ca-binding sites are dispensable for piliation. Interestingly, we showed that the PilC1 site is necessary for twitching motility and adherence to Chang epithelial cells, while the PilC2 site has only a minor influence on twitching motility and no influence on adherence. These findings establish key differences in PilC1 and PilC2 function in K. kingae and provide insights into the biology of the PilC-like family of proteins.

Broad-spectrum biofilm inhibition by Kingella kingae exopolysaccharide.

Cell-free extracts prepared from Kingella kingae colony biofilms were found to inhibit biofilm formation by Aggregatibacter actinomycetemcomitans, Klebsiella pneumoniae, Staphylococcus aureus, Staphylococcus epidermidis, Candida albicans, and K. kingae. The extracts evidently inhibited biofilm formation by modifying the physicochemical properties of the cell surface, the biofilm matrix, and the substrate. Chemical and biochemical analyses indicated that the biofilm inhibition activity in the K. kingae extract was due to polysaccharide. Structural analyses showed that the extract contained two major polysaccharides. One was a linear polysaccharide with the structure →6)-α-d-GlcNAcp-(1→5)-ß-d-OclAp-(2→, which was identical to a capsular polysaccharide produced by Actinobacillus pleuropneumoniae serotype 5. The second was a novel linear polysaccharide, designated PAM galactan, with the structure →3)-ß-d-Galf-(1→6)-ß-d-Galf-(1→. Purified PAM galactan exhibited broad-spectrum biofilm inhibition activity. A cluster of three K. kingae genes encoding UDP-galactopyranose mutase (ugm) and two putative galactofuranosyl transferases was sufficient for the synthesis of PAM galactan in Escherichia coli. PAM galactan is one of a growing number of bacterial polysaccharides that exhibit antibiofilm activity. The biological roles and potential technological applications of these molecules remain unknown.

Expression of Kingella kingae type IV pili is regulated by sigma54, PilS, and PilR.

Kingella kingae is a member of the Neisseriaceae and is being recognized increasingly as an important cause of serious disease in children. Recent work has demonstrated that K. kingae expresses type IV pili that mediate adherence to respiratory epithelial and synovial cells and are selected against during invasive disease. In the current study, we examined the genome of K. kingae strain 269-492 and identified homologs of the rpoN and the pilS and pilR genes that are essential for pilus expression in Pseudomonas aeruginosa but not in the pathogenic Neisseria species. The disruption of either rpoN or pilR in K. kingae resulted in a marked reduction in the level of transcript for the major pilus subunit (pilA1) and eliminated piliation. In contrast, the disruption of pilS resulted in only partial reduction in the level of pilA1 transcript and a partial decrease in piliation. Furthermore, the disruption of pilS in colony variants with high-density piliation resulted in variants with low-density piliation. Mutations in the promoter region of pilA1 and gel shift analysis demonstrated that both sigma(54) and PilR act directly at the pilA1 promoter, with PilR binding to two repetitive elements. These data suggest that the regulation of K. kingae type IV pilus expression is complex and multilayered, influenced by both the genetic state and environmental cues.