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Kingella kingae is a Gram-negative bacterium that has emerged as a leading cause of invasive disease in children between 6 months and 4 years of age. K. kingae initiates infection by colonizing the oropharynx, then breaches the oropharyngeal epithelium, enters the bloodstream, and disseminates to distant sites to cause disease, including osteomyelitis, septic arthritis, and endocarditis. To survive in the bloodstream and disseminate to sites of invasive disease, K. kingae produces a polysaccharide capsule and an exopolysaccharide that inhibit opsonin deposition and mediate resistance to complement-mediated serum killing. However, elimination of these extracellular polysaccharides only partially reduces K. kingae survival in human serum, suggesting that additional factors contribute to serum resistance. In this study, we found that K. kingae binds human factor H (FH), a negative regulator of the alternative complement pathway. In experiments using rat serum as a source of complement, we observed that K. kingae was able to utilize human FH to resist killing. Introduction of exogenous human FH into the juvenile rat infection model of K. kingae disease enhanced virulence in vivo, demonstrating the importance of FH binding in the pathogenesis of disease. Far-western blot analysis identified a 37-kDa outer membrane protein designated KK02920 that was responsible for FH binding and enhanced virulence in vivo in the presence of human FH. Loss of KK02920 virtually abrogated serum resistance, indicating that KK02920 is the major determinant of K. kingae serum resistance. Additional analysis revealed the presence of KK02920 across a collection of serum-resistant invasive and carrier K. kingae isolates, all of which can utilize human FH to resist complement-mediated killing. This work demonstrates the importance of a complement-regulator binding protein as a major mechanism of serum resistance in an encapsulated organism.

The bacterial genus Kingella includes two pathogenic species, namely Kingella kingae and Kingella negevensis , as well as strictly commensal species. Both K. kingae and K. negevensis secrete a toxin called RtxA that is absent in the commensal species. Here we present a phylogenomic study of the genus Kingella , including new genomic sequences for 88 clinical isolates, genotyping of another 131 global isolates, and analysis of 52 available genomes. The phylogenetic evidence supports that the toxin-encoding operon rtxCA was acquired by a common ancestor of the pathogenic Kingella species, and that a preexisting type-I secretion system was co-opted for toxin export. Subsequent genomic reorganization distributed the toxin machinery across two loci, with 30-35% of K. kingae strains containing two copies of the rtxA toxin gene. The rtxA duplication is largely clonal and is associated with invasive disease. Assays with isogenic strains show that a single copy of rtxA is associated with reduced cytotoxicity in vitro. Thus, our study identifies key steps in the evolutionary transition from commensal to pathogen, including horizontal gene transfer, co-option of an existing secretion system, and gene duplication. The bacterial genus Kingella includes pathogenic species that secrete a toxin called RtxA, which is absent in commensal species. Here, Morreale et al. identify key steps in the evolutionary transition from commensal to pathogen, including horizontal gene transfer of the toxin-encoding genes, co-option of an existing secretion system, and gene duplication.

Kingella negevensis is a newly described gram-negative bacterium in the Neisseriaceae family and is closely related to Kingella kingae, an important cause of pediatric osteoarticular infections and other invasive diseases. Like K. kingae, K. negevensis can be isolated from the oropharynx of young children, although at a much lower rate. Due to the potential for misidentification as K. kingae, the burden of disease due to K. negevensis is currently unknown. Similarly, there is little known about virulence factors present in K. negevensis and how they compare to virulence factors in K. kingae. Using a variety of approaches, we show that K. negevensis produces many of the same putative virulence factors that are present in K. kingae, including a polysaccharide capsule, a secreted exopolysaccharide, a Knh-like trimeric autotransporter, and type IV pili, suggesting that K. negevensis may have significant pathogenic potential.

The gram-negative bacterium Kingella kingae is a leading cause of osteoarticular infections in young children and initiates infection by colonizing the oropharynx. Adherence to respiratory epithelial cells represents an initial step in the process of K . kingae colonization and is mediated in part by type IV pili. In previous work, we observed that elimination of the K . kingae PilC1 and PilC2 pilus-associated proteins resulted in non-piliated organisms that were non-adherent, suggesting that PilC1 and PilC2 have a role in pilus biogenesis. To further define the functions of PilC1 and PilC2, in this study we eliminated the PilT retraction ATPase in the Δ pilC1 Δ pilC2 mutant, thereby blocking pilus retraction and restoring piliation. The resulting strain was non-adherent in assays with cultured epithelial cells, supporting the possibility that PilC1 and PilC2 have adhesive activity. Consistent with this conclusion, purified PilC1 and PilC2 were capable of saturable binding to epithelial cells. Additional analysis revealed that PilC1 but not PilC2 also mediated adherence to selected extracellular matrix proteins, underscoring the differential binding specificity of these adhesins. Examination of deletion constructs and purified PilC1 and PilC2 fragments localized adhesive activity to the N-terminal region of both PilC1 and PilC2. The deletion constructs also localized the twitching motility property to the N-terminal region of these proteins. In contrast, the deletion constructs established that the pilus biogenesis function of PilC1 and PilC2 resides in the C-terminal region of these proteins. Taken together, these results provide definitive evidence that PilC1 and PilC2 are adhesins and localize adhesive activity and twitching motility to the N-terminal domain and biogenesis to the C-terminal domain.

Kingella kingae is an emerging pediatric pathogen and a leading cause of osteoarticular infections in children 6 months to 4 years of age. To establish infection, K. kingae relies on T4P, dynamic surface structures that mediate host cell adherence, motility, and DNA uptake. T4P are expressed by a wide range of bacterial pathogens beyond K. kingae , including Pseudomonas aeruginosa , Neisseria gonorrhoeae , Neisseria meningitidis , and Legionella pneumophila , among others. The type IV pilus is composed of pilin subunits, including a major pilin that displays significant antigenic diversity and low-abundance minor pilins that are highly conserved. This study demonstrates the importance of eight minor pilins in K. kingae virulence properties. Given the conservation of minor pilins across diverse bacterial species, targeting minor pilin complexes may provide a foundation for a new class of broad-spectrum antivirulence therapies that prevent bacterial colonization and disease.

The Haemophilus influenzae HMW1 adhesin is a high-molecular weight protein that is secreted by the bacterial two-partner secretion pathway and mediates adherence to respiratory epithelium, an essential early step in the pathogenesis of H. influenzae disease. In recent work, we discovered that HMW1 is a glycoprotein and undergoes N-linked glycosylation at multiple asparagine residues with simple hexose units rather than N-acetylated hexose units, revealing an unusual N-glycosidic linkage and suggesting a new glycosyltransferase activity. Glycosylation protects HMW1 against premature degradation during the process of secretion and facilitates HMW1 tethering to the bacterial surface, a prerequisite for HMW1-mediated adherence. In the current study, we establish that the enzyme responsible for glycosylation of HMW1 is a protein called HMW1C, which is encoded by the hmw1 gene cluster and shares homology with a group of bacterial proteins that are generally associated with two-partner secretion systems. In addition, we demonstrate that HMW1C is capable of transferring glucose and galactose to HMW1 and is also able to generate hexose-hexose bonds. Our results define a new family of bacterial glycosyltransferases.

Kingella kingae is an encapsulated gram-negative organism that is a common cause of osteoarticular infections in young children. In earlier work, we identified a glycosyltransferase gene called csaA that is necessary for synthesis of the [3)-β-GalpNAc-(1→5)-β-Kdop-(2→] polysaccharide capsule (type a) in K. kingae strain 269-492. In the current study, we analyzed a large collection of invasive and carrier isolates from Israel and found that csaA was present in only 47% of the isolates. Further examination of this collection using primers based on the sequence that flanks csaA revealed three additional gene clusters (designated the csb, csc, and csd loci), all encoding predicted glycosyltransferases. The csb locus contains the csbA, csbB, and csbC genes and is associated with a capsule that is a polymer of [6)-α-GlcpNAc-(1→5)-β-(8-OAc)Kdop-(2→] (type b). The csc locus contains the cscA, cscB, and cscC genes and is associated with a capsule that is a polymer of [3)-β-Ribf-(1→2)-β-Ribf-(1→2)-β-Ribf-(1→4)-β-Kdop-(2→] (type c). The csd locus contains the csdA, csdB, and csdC genes and is associated with a capsule that is a polymer of [P-(O→3)[β-Galp-(1→4)]-β-GlcpNAc-(1→3)-α-GlcpNAc-1-] (type d). Introduction of the csa, csb, csc, and csd loci into strain KK01Δcsa, a strain 269-492 derivative that lacks the native csaA gene, was sufficient to produce the type a capsule, type b capsule, type c capsule, and type d capsule, respectively, indicating that these loci are solely responsible for determining capsule type in K. kingae. Further analysis demonstrated that 96% of the invasive isolates express either the type a or type b capsule and that a disproportionate percentage of carrier isolates express the type c or type d capsule. These results establish that there are at least four structurally distinct K. kingae capsule types and suggest that capsule type plays an important role in promoting K. kingae invasive disease.

Recent evidence indicates that Kingella kingae produces a polysaccharide capsule. In an effort to determine the composition and structure of this polysaccharide capsule, in the current study we purified capsular material from the surface of K. kingae strain 269-492 variant KK01 using acidic conditions to release the capsule and a series of steps to remove DNA, RNA, and protein. Analysis of the resulting material by gas chromatography and mass spectrometry revealed N-acetyl galactosamine (GalNAc), 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo), and galactose (Gal). Further analysis by NMR demonstrated two distinct polysaccharides, one consisting of GalNAc and Kdo with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and the other containing galactose alone with the structure →5)-β-Galf-(1→. Disruption of the ctrA gene required for surface localization of the K. kingae polysaccharide capsule resulted in elimination of GalNAc and Kdo but had no effect on the presence of Gal in bacterial surface extracts. In contrast, deletion of the pamABCDE locus involved in production of a reported galactan exopolysaccharide eliminated Gal but had no effect on the presence of GalNAc and Kdo in surface extracts. Disruption of ctrA and deletion of pamABCDE resulted in a loss of all carbohydrates in surface extracts. These results establish that K. kingae strain KK01 produces a polysaccharide capsule with the structure →3)-β-GalpNAc-(1→5)-β-Kdop-(2→ and a separate exopolysaccharide with the structure →5)-β-Galf-(1→. The polysaccharide capsule and the exopolysaccharide require distinct genetic loci for surface localization.

Nontypeable Haemophilus influenzae (NTHi) is a common cause of localized respiratory tract disease and results in significant morbidity. The pathogenesis of NTHi disease begins with nasopharyngeal colonization, and therefore, the prevention of colonization represents a strategy to prevent disease. The NTHi HMW1 and HMW2 proteins are a family of conserved adhesins that are present in 75 to 80% of strains and have been demonstrated to play a critical role in colonization of the upper respiratory tract in rhesus macaques. In this study, we examined the vaccine potential of HMW1 and HMW2 using a mouse model of nasopharyngeal colonization. Immunization with HMW1 and HMW2 by either the subcutaneous or the intranasal route resulted in a strain-specific antibody response associated with agglutination of bacteria and restriction of bacterial adherence. Despite the specificity of the antibody response, immunization resulted in protection against colonization by both the parent NTHi strain and heterologous strains expressing distinct HMW1 and HMW2 proteins. Pretreatment with antibody against IL-17A eliminated protection against heterologous strains, indicating that heterologous protection is IL-17A dependent. This work demonstrates the vaccine potential of the HMW1 and HMW2 proteins and highlights the importance of IL-17A in protection against diverse NTHi strains