RESUMEN
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 ΔpilC1ΔpilC2 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.
Asunto(s)
Kingella kingae , Adhesinas Bacterianas/genética , Adhesivos , Adhesión Bacteriana , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Niño , Preescolar , ADN , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/genética , Fimbrias Bacterianas/metabolismo , Humanos , Kingella kingae/genéticaRESUMEN
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.
Asunto(s)
Adhesinas Bacterianas/inmunología , Infecciones por Haemophilus/microbiología , Haemophilus influenzae/patogenicidad , Adhesinas Bacterianas/genética , Pruebas de Aglutinación , Animales , Anticuerpos Antibacterianos/sangre , Anticuerpos Antibacterianos/inmunología , Adhesión Bacteriana , Femenino , Infecciones por Haemophilus/inmunología , Infecciones por Haemophilus/prevención & control , Haemophilus influenzae/genética , Haemophilus influenzae/inmunología , Inmunización , Interleucina-17/sangre , Ratones Endogámicos BALB C , Nasofaringe/microbiologíaRESUMEN
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.
Asunto(s)
Kingella kingae , Infecciones por Neisseriaceae , Niño , Humanos , Preescolar , Proteínas Fimbrias/genética , Proteínas Fimbrias/metabolismo , Kingella kingae/genética , Kingella kingae/metabolismo , Adhesión Bacteriana , Fimbrias Bacterianas/metabolismo , Células Epiteliales/microbiología , Metales/metabolismo , Infecciones por Neisseriaceae/microbiologíaRESUMEN
Kingella kingae is a Gram-negative coccobacillus that is increasingly being recognized as an important cause of invasive disease in young children. The pathogenesis of K. kingae disease begins with colonization of the oropharynx, followed by invasion of the bloodstream, survival in the intravascular space, and dissemination to distant sites. Recent studies have revealed that K. kingae produces a number of surface factors that may contribute to the pathogenic process, including a polysaccharide capsule and an exopolysaccharide. In this study, we observed that K. kingae was highly resistant to the bactericidal effects of human serum complement. Using mutant strains deficient in expression of capsule, exopolysaccharide, or both in assays with human serum, we found that elimination of both capsule and exopolysaccharide was required for efficient binding of IgG, IgM, C4b, and C3b to the bacterial surface and for complement-mediated killing. Abrogation of the classical complement pathway using EGTA-treated human serum restored survival to wild-type levels by the mutant lacking both capsule and exopolysaccharide, demonstrating that capsule and exopolysaccharide promote resistance to the classical complement pathway. Consistent with these results, loss of both capsule and exopolysaccharide eliminated invasive disease in juvenile rats with an intact complement system but not in rats lacking complement. Based on these observations, we conclude that the capsule and the exopolysaccharide have important redundant roles in promoting survival of K. kingae in human serum. Each of these surface factors is sufficient by itself to fully prevent serum opsonin deposition and complement-mediated killing of K. kingae, ultimately facilitating intravascular survival and promoting K. kingae invasive disease.
Asunto(s)
Actividad Bactericida de la Sangre/fisiología , Kingella kingae , Infecciones por Neisseriaceae/microbiología , Polisacáridos Bacterianos/farmacología , Animales , Cápsulas Bacterianas/química , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismo , Proteínas del Sistema Complemento , Humanos , Polisacáridos Bacterianos/metabolismo , Ratas , Ratas Sprague-Dawley , VirulenciaRESUMEN
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)-ß-GalpNAc-(1â5)-ß-Kdop-(2â] 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-(1â5)-ß-(8-OAc)Kdop-(2â] (type b). The csc locus contains the cscA, cscB, and cscC genes and is associated with a capsule that is a polymer of [3)-ß-Ribf-(1â2)-ß-Ribf-(1â2)-ß-Ribf-(1â4)-ß-Kdop-(2â] (type c). The csd locus contains the csdA, csdB, and csdC genes and is associated with a capsule that is a polymer of [P-(Oâ3)[ß-Galp-(1â4)]-ß-GlcpNAc-(1â3)-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.
Asunto(s)
Cápsulas Bacterianas/química , Proteínas Bacterianas/química , Kingella kingae/patogenicidad , Infecciones por Neisseriaceae/patología , Polisacáridos Bacterianos/química , Cromatografía en Gel , Cromatografía de Gases y Espectrometría de Masas , Genes Bacterianos , Glicosiltransferasas/genética , Kingella kingae/genética , Virulencia/fisiologíaRESUMEN
Many bacterial species actively take up and recombine homologous DNA into their genomes, called natural competence, a trait that offers a means to identify the genetic basis of naturally occurring phenotypic variation. Here, we describe "transformed recombinant enrichment profiling" (TREP), in which natural transformation is used to generate complex pools of recombinants, phenotypic selection is used to enrich for specific recombinants, and deep sequencing is used to survey for the genetic variation responsible. We applied TREP to investigate the genetic architecture of intracellular invasion by the human pathogen Haemophilus influenzae, a trait implicated in persistence during chronic infection. TREP identified the HMW1 adhesin as a crucial factor. Natural transformation of the hmw1 operon from a clinical isolate (86-028NP) into a laboratory isolate that lacks it (Rd KW20) resulted in ~1,000-fold increased invasion into airway epithelial cells. When a distinct recipient (Hi375, already possessing hmw1 and its paralog hmw2) was transformed by the same donor, allelic replacement of hmw2AHi375 by hmw1A86-028NP resulted in a ~100-fold increased intracellular invasion rate. The specific role of hmw1A86-028NP was confirmed by mutant and western blot analyses. Bacterial self-aggregation and adherence to airway cells were also increased in recombinants, suggesting that the high invasiveness induced by hmw1A86-028NP might be a consequence of these phenotypes. However, immunofluorescence results found that intracellular hmw1A86-028NP bacteria likely invaded as groups, instead of as individual bacterial cells, indicating an emergent invasion-specific consequence of hmw1A-mediated self-aggregation.
Asunto(s)
Adhesinas Bacterianas/genética , Perfilación de la Expresión Génica/métodos , Infecciones por Haemophilus/microbiología , Western Blotting , Células Epiteliales/microbiología , Haemophilus influenzae/genética , Humanos , Espacio Intracelular/microbiología , Microscopía Fluorescente , Reacción en Cadena de la PolimerasaRESUMEN
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.
Asunto(s)
Adhesión Bacteriana/fisiología , Proteínas Bacterianas/metabolismo , Interacciones Huésped-Patógeno/fisiología , Kingella kingae/metabolismo , Células A549 , Adhesinas Bacterianas/metabolismo , Línea Celular Tumoral , Células Epiteliales/microbiología , Humanos , Microscopía Electrónica de Transmisión/métodosRESUMEN
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.
Asunto(s)
Cápsulas Bacterianas/química , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Genoma Bacteriano , Glicosiltransferasas/genética , Kingella kingae/genética , Acetilgalactosamina/química , Acetilgalactosamina/metabolismo , Cápsulas Bacterianas/genética , Cápsulas Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Elementos Transponibles de ADN , Escherichia coli/genética , Escherichia coli/metabolismo , Prueba de Complementación Genética , Glicosiltransferasas/química , Glicosiltransferasas/metabolismo , Kingella kingae/metabolismo , Mutación , Operón , Polisacáridos Bacterianos/biosíntesis , Polisacáridos Bacterianos/química , Dominios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Azúcares Ácidos/química , Azúcares Ácidos/metabolismoRESUMEN
Nontypeable Haemophilus influenzae (NTHi) initiates infection by colonizing the upper respiratory tract and is a common cause of localized respiratory tract disease. Previous work has established that the NTHi HMW1 and HMW2 proteins are potent adhesins that mediate efficient in vitro adherence to cultured human respiratory epithelial cells. In this study, we used a rhesus macaque model to assess the contributions of HMW1 and HMW2 to in vivo colonization. In experiments involving inoculation of individual isogenic derivatives of NTHi strain 12, the parent strain expressing both HMW1 and HMW2 and the mutant strains expressing either HMW1 or HMW2 were able to colonize more frequently than the double mutant strain lacking HMW1 and HMW2. In competition experiments, the parent strain efficiently outcompeted the double mutant lacking HMW1 and HMW2. Colonization with strains expressing HMW2 resulted in development of antibody against HMW2 in a number of the animals, demonstrating that colonization can stimulate an antibody response. In conclusion, we have established that the HMW1 and HMW2 adhesins play a major role in facilitating colonization of the upper respiratory tract of rhesus macaques, in some cases associated with stimulation of an immune response.
Asunto(s)
Adhesinas Bacterianas/fisiología , Adhesión Bacteriana/fisiología , Infecciones por Haemophilus/microbiología , Haemophilus influenzae/patogenicidad , Infecciones del Sistema Respiratorio/microbiología , Animales , Anticuerpos Antibacterianos , Adhesión Bacteriana/inmunología , Línea Celular , Modelos Animales de Enfermedad , Infecciones por Haemophilus/inmunología , Haemophilus influenzae/inmunología , Macaca mulatta , Faringe/microbiología , Infecciones del Sistema Respiratorio/inmunología , Tráquea/microbiologíaRESUMEN
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.
Asunto(s)
Adhesión Bacteriana/fisiología , Calcio/metabolismo , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/fisiología , Kingella kingae/metabolismo , Sitios de Unión , Proteínas Fimbrias/genética , Fimbrias Bacterianas/clasificación , Regulación Bacteriana de la Expresión Génica/fisiología , Kingella kingae/genética , Movimiento , Fenotipo , Plásmidos , Unión ProteicaRESUMEN
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.
Asunto(s)
Toxinas Bacterianas , Kingella kingae , Filogenia , Virulencia/genética , Toxinas Bacterianas/genética , Kingella/genética , Kingella kingae/genéticaRESUMEN
BACKGROUND: Improvements in blood culture techniques and molecular-based diagnostics have led to increased recognition of Kingella kingae as an invasive human pathogen causing bacteremia, septic arthritis, osteomyelitis and endocarditis in young children. Serious disease and potentially life-threatening complications of infection due to K. kingae necessitate timely identification and appropriate antimicrobial therapy. Ceftaroline is a fifth-generation broad spectrum cephalosporin that possesses activity against Gram-negative and Gram-positive pathogens similar to third-generation cephalosporins, but also includes methicillin-resistant Staphylococcus aureus . This study reports the in vitro activity of ceftaroline and comparator agents against an international collection of K. kingae isolates. METHODS: A collection of 308 K. kingae isolates was obtained primarily from children with bacteremia, endocarditis, osteoarticular infections or from asymptomatic pediatric carriers. Isolates were tested for antibiotic susceptibility using Clinical and Laboratory Standard Institute broth microdilution methodology and screened for ß-lactamase production using a nitrocefin chromogenic test. RESULTS: Ceftaroline inhibited all K. kingae isolates at ≤0.06 mg/L (MIC 50/90 , 0.015/0.03 mg/L). Ceftaroline MICs were similar to results with ceftriaxone (MIC 50/90 , 0.015/0.015 mg/L), meropenem (MIC 50/90 , 0.015/0.015 mg/L) and ampicillin-sulbactam (MIC 50/90 , 0.06/0.06 mg/L). Ceftaroline MICs were slightly lower than MICs for cefuroxime and amoxicillin/clavulanate (MIC 50/90 , 0.06/0.12 mg/L). MICs were high for clindamycin (MIC 50/90 , 2/4 mg/L) and oxacillin (MIC 50/90 , 4/8 mg/L). Sixteen isolates (5.2%) yielded a positive nitrocefin test indicating production of ß-lactamase; ceftaroline demonstrated equivalent MICs against ß-lactamase - positive and ß-lactamase - negative strains (MIC 50/90 , 0.015/0.3 mg/L). CONCLUSIONS: The potent activity of ceftaroline against this large international collection of K. kingae isolates supports further clinical evaluation in children.
Asunto(s)
Bacteriemia , Endocarditis , Kingella kingae , Staphylococcus aureus Resistente a Meticilina , Humanos , Niño , Preescolar , Antibacterianos/farmacología , Cefalosporinas/farmacología , beta-Lactamasas , Pruebas de Sensibilidad Microbiana , CeftarolinaRESUMEN
The emergence of Kingella kingae as an important etiology of pediatric osteoarticular infections over the past three decades has led to significant research efforts focused on understanding the pathogenicity of this fastidious Gram-negative bacterium. This work has uncovered multiple virulence factors that likely play key roles in the ability of the organism to colonize the upper respiratory tract, breach the epithelial barrier, and disseminate to distal sites of infection. Herein the current body of knowledge about K. kingae virulence factors is reviewed in the context of K. kingae disease pathogenesis. The work summarized here has identified multiple targets for therapeutic intervention as well as potential vaccine antigens.
RESUMEN
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.
RESUMEN
Kingella kingae is a leading cause of bone and joint infections and other invasive diseases in young children. A key K. kingae virulence determinant is a secreted exopolysaccharide that mediates resistance to serum complement and neutrophils and is required for full pathogenicity. The K. kingae exopolysaccharide is a galactofuranose homopolymer called galactan and is encoded by the pamABC genes in the pamABCDE locus. In this study, we sought to define the mechanism by which galactan is tethered on the bacterial surface, a prerequisite for mediating evasion of host immune mechanisms. We found that the pamD and pamE genes encode glycosyltransferases and are required for synthesis of an atypical lipopolysaccharide (LPS) O-antigen. The LPS O-antigen in turn is required for anchoring of galactan, a novel mechanism for association of an exopolysaccharide with the bacterial surface. IMPORTANCE Kingella kingae is an emerging pediatric pathogen and produces invasive disease by colonizing the oropharynx, invading the bloodstream, and disseminating to distant sites. This organism produces a uniquely multifunctional exopolysaccharide called galactan that is critical for virulence and promotes intravascular survival by mediating resistance to serum and neutrophils. In this study, we established that at least some galactan is anchored to the bacterial surface via a novel structural interaction with an atypical lipopolysaccharide O-antigen. Additionally, we demonstrated that the atypical O-antigen is synthesized by the products of the pamD and pamE genes, located downstream of the gene cluster responsible for galactan biosynthesis. This work addresses how the K. kingae exopolysaccharide can mediate innate immune resistance and advances understanding of bacterial exopolysaccharides and lipopolysaccharides.
Asunto(s)
Kingella kingae , Infecciones por Neisseriaceae , Humanos , Niño , Preescolar , Kingella kingae/química , Lipopolisacáridos , Antígenos O/genética , Galactanos , Glicosiltransferasas/genética , Infecciones por Neisseriaceae/microbiologíaRESUMEN
Genetic variants arising from within-patient evolution shed light on bacterial adaptation during chronic infection. Contingency loci generate high levels of genetic variation in bacterial genomes, enabling adaptation to the stringent selective pressures exerted by the host. A significant gap in our understanding of phase-variable contingency loci is the extent of their contribution to natural infections. The human-adapted pathogen nontypeable Haemophilus influenzae (NTHi) causes persistent infections, which contribute to underlying disease progression. The phase-variable high-molecular-weight (HMW) adhesins located on the NTHi surface mediate adherence to respiratory epithelial cells and, depending on the allelic variant, can also confer high epithelial invasiveness or hyperinvasion. In this study, we characterize the dynamics of HMW-mediated hyperinvasion in living cells and identify a specific HMW binding domain shared by hyperinvasive NTHi isolates of distinct pathological origins. Moreover, we observed that HMW expression decreased over time by using a longitudinal set of persistent NTHi strains collected from chronic obstructive pulmonary disease (COPD) patients, resulting from increased numbers of simple-sequence repeats (SSRs) downstream of the functional P2hmw1A promoter, which is the one primarily driving HMW expression. Notably, the increased SSR numbers at the hmw1 promoter region also control a phenotypic switch toward lower bacterial intracellular invasion and higher biofilm formation, likely conferring adaptive advantages during chronic airway infection by NTHi. Overall, we reveal novel molecular mechanisms of NTHi pathoadaptation based on within-patient lifestyle switching controlled by phase variation. IMPORTANCE Human-adapted bacterial pathogens have evolved specific mechanisms to colonize their host niche. Phase variation is a contingency strategy to allow adaptation to changing conditions, as phase-variable bacterial loci rapidly and reversibly switch their expression. Several NTHi adhesins are phase variable. These adhesins are required for colonization but also immunogenic, in such a way that bacteria with lower adhesin levels are better equipped to survive an immune response, making their contribution to natural infections unclear. We show here that the major NTHi adhesin HMW1A displays allelic variation, which can drive a phase-variable epithelial hyperinvasion phenotype. Over time, hmw1A phase variation lowers adhesin expression, which controls an NTHi lifestyle switch from high epithelial invasiveness to lower invasion and higher biofilm formation. This reversible loss of function aligns with the previously stated notion that epithelial infection is essential for NTHi infection establishment, but once established, persistence favors gene inactivation, in this case facilitating biofilm growth.
Asunto(s)
Adhesinas Bacterianas/genética , Adhesinas Bacterianas/metabolismo , Variación Genética , Genoma Bacteriano , Haemophilus influenzae/genética , Haemophilus influenzae/metabolismo , Adaptación Fisiológica/genética , Adhesinas Bacterianas/clasificación , Adhesión Bacteriana/genética , Adhesión Bacteriana/fisiología , Biopelículas , Infecciones por Haemophilus/microbiología , Haemophilus influenzae/patogenicidad , Humanos , Regiones Promotoras GenéticasRESUMEN
Kingella kingae is a gram-negative bacterium that is being recognized increasingly as a cause of septic arthritis and osteomyelitis in young children. Previous work established that K. kingae expresses type IV pili that mediate adherence to respiratory epithelial and synovial cells. PilA1 is the major pilus subunit in K. kingae type IV pili and is essential for pilus assembly. To develop a better understanding of the role of K. kingae type IV pili during colonization and invasive disease, we examined a collection of clinical isolates for pilus expression and in vitro adherence. In addition, in a subset of isolates we performed nucleotide sequencing to assess the level of conservation of PilA1. The majority of respiratory and nonendocarditis blood isolates were piliated, while the majority of joint fluid, bone, and endocarditis blood isolates were nonpiliated. The piliated isolates formed either spreading/corroding or nonspreading/noncorroding colonies and were uniformly adherent, while the nonpiliated isolates formed domed colonies and were nonadherent. PilA1 sequence varied significantly from strain to strain, resulting in substantial variability in antibody reactivity. These results suggest that type IV pili may confer a selective advantage on K. kingae early in infection and a selective disadvantage on K. kingae at later stages in the pathogenic process. We speculate that PilA1 is immunogenic during natural infection and undergoes antigenic variation to escape the immune response.
Asunto(s)
Adhesión Bacteriana , Fimbrias Bacterianas/fisiología , Regulación Bacteriana de la Expresión Génica , Kingella kingae/genética , Kingella kingae/patogenicidad , Infecciones por Neisseriaceae/microbiología , Anticuerpos Antibacterianos/inmunología , Artritis Infecciosa/microbiología , Proteínas Bacterianas/genética , Portador Sano/microbiología , ADN Bacteriano/química , ADN Bacteriano/genética , Proteínas Fimbrias/genética , Fimbrias Bacterianas/inmunología , Humanos , Kingella kingae/inmunología , Kingella kingae/aislamiento & purificación , Datos de Secuencia Molecular , Osteomielitis/microbiología , Fenotipo , Polimorfismo Genético , Infecciones del Sistema Respiratorio/microbiología , Análisis de Secuencia de ADNRESUMEN
The ability to combine a selective capture strategy with on chip MALDI-TOF analysis allows for rapid, sensitive analysis of a variety of different analytes. In this overview a series of applications of capture enhanced laser desorption ionization time of flight (CELDI-TOF) mass spectrometry are described. The key feature of the assay is an off-chip capture step that utilizes high affinity bacterial binding proteins to capture a selected ligand. This allows large volumes of sample to be used and provides for a concentration step prior to transfer to a gold chip for traditional mass spectral analysis. The approach can also be adapted to utilize specific antibody as the basis of the capture step. The direct and indirect CELDI-TOF assays are rapid, reproducible and can be a valuable proteomic tool for analysis of low abundance molecules present in complex mixtures like blood plasma.
RESUMEN
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.
Asunto(s)
Kingella kingae/patogenicidad , Kingella/patogenicidad , Infecciones por Neisseriaceae/microbiología , Factores de Virulencia/análisis , Proteínas Bacterianas/análisis , Fimbrias Bacterianas/química , Fimbrias Bacterianas/microbiología , Humanos , Kingella/química , Kingella kingae/química , Polisacáridos Bacterianos/análisis , VirulenciaRESUMEN
Kingella kingae is a gram-negative coccobacillus that is a fastidious commensal organism in the oropharynx and is being recognized increasingly as a common cause of osteoarticular infections and other invasive diseases in young children. The pathogenesis of K. kingae disease begins with bacterial adherence to respiratory epithelium, followed by translocation across the epithelial barrier, survival in the bloodstream, and dissemination to distant sites, including bones, joints, and the endocardium, among others. Characterization of the determinants of K. kingae pathogenicity has revealed a novel model of adherence that involves the interplay of type IV pili, a non-pilus adhesin, and a polysaccharide capsule and a novel model of resistance to serum killing and neutrophil killing that involves complementary functions of a polysaccharide capsule and an exopolysaccharide. These models likely apply to other bacterial pathogens as well.