Kingella kingae PilC1 and PilC2 are adhesive multifunctional proteins that promote bacterial adherence, twitching motility, DNA transformation, and pilus biogenesis.

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.

Immunization with HMW1 and HMW2 adhesins protects against colonization by heterologous strains of nontypeable Haemophilus influenzae.

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.

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 Surface Polysaccharides Promote Resistance to Human Serum and Virulence in a Juvenile Rat Model.

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.

Kingella kingae Expresses Four Structurally Distinct Polysaccharide Capsules That Differ in Their Correlation with Invasive Disease.

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-(1→5)-ß-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)-ß-(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)-ß-Ribf-(1→2)-ß-Ribf-(1→2)-ß-Ribf-(1→4)-ß-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)[ß-Galp-(1→4)]-ß-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.

Transformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in Haemophilus influenza.

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.

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.

Hematogenous Osteomyelitis in Infants and Children: Imaging of a Changing Disease.

In children, hematogenous osteomyelitis is an infection that primarily affects the most vascularized regions of the growing skeleton. The disease has increased in frequency, virulence, and degree of soft-tissue involvement. The change in clinical manifestations and management over the past 2 decades should be reflected in the current imaging approach to the disease. Imaging of infection must depict the location of a single focus or of multiple foci of involvement and the presence of drainable collections. This review provides an overview of the imaging implications directed by the changing epidemiology, the newer insights of anatomy and pathophysiology, the imaging characteristics with emphasis on specific locations and disease complications, and the differential diagnosis considerations. In addition, basic imaging guidelines for appropriate extent of area to image based on patient age are provided. © RSNA, 2017.

The HMW1 and HMW2 Adhesins Enhance the Ability of Nontypeable Haemophilus influenzae To Colonize the Upper Respiratory Tract of Rhesus Macaques.

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.

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.

Early-Life Intranasal Colonization with Nontypeable Haemophilus influenzae Exacerbates Juvenile Airway Disease in Mice.

Accumulating evidence suggests a connection between asthma development and colonization with nontypeable Haemophilus influenzae (NTHi). Specifically, nasopharyngeal colonization of human infants with NTHi within 4 weeks of birth is associated with an increased risk of asthma development later in childhood. Monocytes derived from these infants have aberrant inflammatory responses to common upper respiratory bacterial antigens compared to those of cells derived from infants who were not colonized and do not go on to develop asthma symptoms in childhood. In this study, we hypothesized that early-life colonization with NTHi promotes immune system reprogramming and the development of atypical inflammatory responses. To address this hypothesis in a highly controlled model, we tested whether colonization of mice with NTHi on day of life 3 induced or exacerbated juvenile airway disease using an ovalbumin (OVA) allergy model of asthma. We found that animals that were colonized on day of life 3 and subjected to induction of allergy had exacerbated airway disease as juveniles, in which exacerbated airway disease was defined as increased cellular infiltration into the lung, increased amounts of inflammatory cytokines interleukin-5 (IL-5) and IL-13 in lung lavage fluid, decreased regulatory T cell-associated FOXP3 gene expression, and increased mucus production. We also found that colonization with NTHi amplified airway resistance in response to increasing doses of a bronchoconstrictor following OVA immunization and challenge. Together, the murine model provides evidence for early-life immune programming that precedes the development of juvenile airway disease and corroborates observations that have been made in human children.

Structural determinants of the interaction between the TpsA and TpsB proteins in the Haemophilus influenzae HMW1 two-partner secretion system.

UNLABELLED: The two-partner secretion (TPS) pathway in Gram-negative bacteria consists of a TpsA exoprotein and a cognate TpsB outer membrane pore-forming translocator protein. Previous work has demonstrated that the TpsA protein contains an N-terminal TPS domain that plays an important role in targeting the TpsB protein and is required for secretion. The nontypeable Haemophilus influenzae HMW1 and HMW2 adhesins are homologous proteins that are prototype TpsA proteins and are secreted by the HMW1B and HMW2B TpsB proteins. In the present study, we sought to define the structural determinants of HMW1 interaction with HMW1B during the transport process and while anchored to the bacterial surface. Modeling of HMW1B revealed an N-terminal periplasmic region that contains two polypeptide transport-associated (POTRA) domains and a C-terminal membrane-localized region that forms a pore. Biochemical studies demonstrated that HMW1 engages HMW1B via interaction between the HMW1 TPS domain and the HMW1B periplasmic region, specifically, the predicted POTRA1 and POTRA2 domains. Subsequently, HMW1 is shuttled to the HMW1B pore, facilitated by the N-terminal region, the middle region, and the NPNG motif in the HMW1 TPS domain. Additional analysis revealed that the interaction between HMW1 and HMW1B is highly specific and is dependent upon the POTRA domains and the pore-forming domain of HMW1B. Further studies established that tethering of HMW1 to the surface-exposed region of HMW1B is dependent upon the external loops of HMW1B formed by residues 267 to 283 and residues 324 to 330. These observations may have broad relevance to proteins secreted by the TPS pathway. IMPORTANCE: Secretion of HMW1 involves a recognition event between the extended form of the HMW1 propiece and the HMW1B POTRA domains. Our results identify specific interactions between the HMW1 propiece and the periplasmic HMW1B POTRA domains. The results also suggest that the process of HMW1 translocation involves at least two discrete steps, including initial interaction between the HMW1 propiece and the HMW1B POTRA domains and then a separate translocation event. We have also discovered that the HMW1B pore itself appears to influence the translocation process. These observations extend our knowledge of the two-partner secretion system and may be broadly relevant to other proteins secreted by the TPS pathway.

Crystal structure of the Haemophilus influenzae Hap adhesin reveals an intercellular oligomerization mechanism for bacterial aggregation.

Bacterial biofilms are complex microbial communities that are common in nature and are being recognized increasingly as an important determinant of bacterial virulence. However, the structural determinants of bacterial aggregation and eventual biofilm formation have been poorly defined. In Gram-negative bacteria, a major subgroup of extracellular proteins called self-associating autotransporters (SAATs) can mediate cell-cell adhesion and facilitate biofilm formation. In this study, we used the Haemophilus influenzae Hap autotransporter as a prototype SAAT to understand how bacteria associate with each other. The crystal structure of the H. influenzae Hap(S) passenger domain (harbouring the SAAT domain) was determined to 2.2 Å by X-ray crystallography, revealing an unprecedented intercellular oligomerization mechanism for cell-cell interaction. The C-terminal SAAT domain folds into a triangular-prism-like structure that can mediate Hap-Hap dimerization and higher degrees of multimerization through its F1-F2 edge and F2 face. The intercellular multimerization can give rise to massive buried surfaces that are required for overcoming the repulsive force between cells, leading to bacterial cell-cell interaction and formation of complex microcolonies.

The Haemophilus cryptic genospecies Cha adhesin has at least two variants that differ in host cell binding, bacterial aggregation, and biofilm formation properties.

The Haemophilus cryptic genospecies (HCG) causes genital tract infections in pregnant and postpartum women and respiratory infections in neonates. The major surface adhesin in HCG is called Cha, which mediates bacterial adherence to cultured human epithelial cells. In this study, we report that there are two antigenically distinct variants of Cha, dubbed Cha1 and Cha2. These variants are encoded by the same genetic locus in diverse strains and have nearly identical N-terminal export and C-terminal surface anchoring domains but significantly different internal adhesive domains. Based on the comparison of derivatives of a laboratory strain of Haemophilus influenzae expressing either surface-associated Cha1 or surface-associated Cha2, Cha1 mediates a higher level of adherence to cultured human epithelial cells and Cha2 mediates a higher level of adherence to abiotic surfaces. We hypothesize that variation in the Cha1 and Cha2 internal region results in changes in binding specificity or binding affinity and may be associated with adaptation to different host environments during colonization and disease.