Cytotoxic activity of Kingella kingae RtxA toxin depends on post-translational acylation of lysine residues and cholesterol binding.

Kingella kingae is a member of the commensal oropharyngeal flora of young children. Improvements in detection methods have led to the recognition of K. kingae as an emerging pathogen that frequently causes osteoarticular infections in children and a severe form of infective endocarditis in children and adults. Kingella kingae secretes a membrane-damaging RTX (Repeat in ToXin) toxin, RtxA, which is implicated in the development of clinical infections. However, the mechanism by which RtxA recognizes and kills host cells is largely unexplored. To facilitate structure-function studies of RtxA, we have developed a procedure for the overproduction and purification of milligram amounts of biologically active recombinant RtxA. Mass spectrometry analysis revealed the activation of RtxA by post-translational fatty acyl modification on the lysine residues 558 and/or 689 by the fatty-acyltransferase RtxC. Acylated RtxA was toxic to various human cells in a calcium-dependent manner and possessed pore-forming activity in planar lipid bilayers. Using various biochemical and biophysical approaches, we demonstrated that cholesterol facilitates the interaction of RtxA with artificial and cell membranes. The results of analyses using RtxA mutant variants suggested that the interaction between the toxin and cholesterol occurs via two cholesterol recognition/interaction amino acid consensus motifs located in the C-terminal portion of the pore-forming domain of the toxin. Based on our observations, we conclude that the cytotoxic activity of RtxA depends on post-translational acylation of the K558 and/or K689 residues and on the toxin binding to cholesterol in the membrane.

Phasevarion-Regulated Virulence in the Emerging Pediatric Pathogen Kingella kingae.

Kingella kingae is a common etiological agent of pediatric osteoarticular infections. While current research has expanded our understanding of K. kingae pathogenesis, there is a paucity of knowledge about host-pathogen interactions and virulence gene regulation. Many host-adapted bacterial pathogens contain phase variable DNA methyltransferases (mod genes), which can control expression of a regulon of genes (phasevarion) through differential methylation of the genome. Here, we identify a phase variable type III mod gene in K. kingae, suggesting that phasevarions operate in this pathogen. Phylogenetic studies revealed that there are two active modK alleles in K. kingae Proteomic analysis of secreted and surface-associated proteins, quantitative PCR, and a heat shock assay comparing the wild-type modK1 ON (i.e., in frame for expression) strain to a modK1 OFF (i.e., out of frame) strain revealed three virulence-associated genes under ModK1 control. These include the K. kingae toxin rtxA and the heat shock genes groEL and dnaK Cytokine expression analysis showed that the interleukin-8 (IL-8), IL-1ß, and tumor necrosis factor responses of THP-1 macrophages were lower in the modK1 ON strain than in the modK1::kan mutant. This suggests that the ModK1 phasevarion influences the host inflammatory response and provides the first evidence of this phase variable epigenetic mechanism of gene regulation in K. kingae.

First identification of a chromosomally located penicillinase gene in Kingella kingae species isolated in continental Europe.

Kingella kingae is the major pathogen causing osteoarticular infections (OAI) in young children in numerous countries. Plasmid-borne TEM-1 penicillinase production has been sporadically detected in a few countries but not in continental Europe, despite a high prevalence of K. kingae infections. We describe here for the first time a K. kingae ß-lactamase-producing strain in continental Europe and demonstrate the novel chromosomal location of the blaTEM-1 gene in K. kingae species.

Genotyping, local prevalence and international dissemination of ß-lactamase-producing Kingella kingae strains.

ß-lactamase production has been sporadically reported in the emerging Kingella kingae pathogen but the phenomenon has not been studied in-depth. We investigated the prevalence of ß-lactamase production among K. kingae isolates from different geographical origins and genetically characterized ß-lactamase-producing strains. Seven hundred and seventy-eight isolates from Iceland, the USA, France, Israel, Spain and Canada were screened for ß-lactamase production and, if positive, were characterized by PFGE and MLST genotyping, as well as rtxA, por, blaTEM and 16S rRNA sequencing. ß-lactamase was identified in invasive strains from Iceland (n=4/14, 28.6%), the USA (n=3/15, 20.0%) and Israel (n=2/190, 1.1%) and in carriage strains in the USA (n=5/17, 29.4%) and Israel (n=66/429, 15.4%). No French, Spanish or Canadian isolates were ß-lactamase producers. Among ß-lactamase producers, a perfect congruency between the different typing methods was observed. Surprisingly, all US and Icelandic ß-lactamase-producing isolates were almost indistinguishable, belonged to the major international invasive PFGE clone K/MLST ST-6, but differed from the four genetically unrelated Israeli ß-lactamase-producing clones. Representative strains of different genotypes produced the TEM-1 enzyme. K. kingae ß-lactamase producers exhibit a clear clonal distribution and have dissimilar invasive potential. The presence of the enzyme in isolates belonging to the major worldwide invasive clone K/ST-6 highlights the possible spread of ß-lactam resistance, and emphasizes the importance of routine testing of all K. kingae clinical isolates for ß-lactamase production.

Beta-lactamase production by Kingella kingae in Israel is clonal and common in carriage organisms but rare among invasive strains.

The purpose of this study was to investigate the prevalence of ß-lactamase and the genomic clonality of a large collection of Kingella kingae isolates from Israeli patients with a variety of invasive infections and asymptomatic pharyngeal carriers. ß-lactamase production was studied by the nitrocefin method and the minimum inhibitory concentrations (MICs) of penicillin and amoxicillin-clavulanate were determined by the epsilon (Etest) method. The genotypic clonality of isolates was investigated by pulsed-field electrophoresis (PFGE). ß-lactamase was found in 2 of 190 (1.1 %) invasive isolates and in 66 of 429 (15.4 %) randomly chosen carriage organisms (p < 0.001). Overall, 73 distinct PFGE clones were identified (33 among invasive organisms and 56 among carriage isolates). ß-lactamase production was found to be limited to four distinct PFGE clones, which were common among carriage strains but rare among invasive strains, and all organisms in the collection belonging to these four clones expressed ß-lactamase. The penicillin MIC of ß-lactamase-producing isolates ranged between 0.094 and 2 mcg/mL (MIC50: 0.25 mcg/mL; MIC90: 1.5 mcg/mL) and that of amoxicillin-clavulanate between 0.064 and 0.47 mcg/mL (MIC50: 0.125 mcg/mL; MIC90: 0.125 mcg/mL). The penicillin MIC of ß-lactamase non-producing isolates ranged between <0.002 and 0.064 mcg/mL (MIC50: 0.023 mcg/mL; MIC90: 0.047 mcg/mL). Although ß-lactamase production is prevalent among K. kingae organisms carried by healthy carriers, the low invasive potential of most colonizing clones results in infrequent detection of the enzyme in isolates from patients with clinical infections. The exceptional presence of ß-lactamase among invasive organisms correlates with the favorable response of K. kingae infections to the administration of ß-lactamase-susceptible antibiotics.