Phase Variation in HMW1A Controls a Phenotypic Switch in Haemophilus influenzae Associated with Pathoadaptation during Persistent Infection

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

Kingella kingae Virulence Factors and Insights into Pathogenicity

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

Kingella negevensis shares multiple putative virulence factors with Kingella kingae.

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

Kingella kingae Surface Polysaccharides Promote Resistance to Neutrophil Phagocytosis and Killing

Kingella kingae is a Gram-negative commensal in the oropharynx and represents a leading cause of joint and bone infections in young children. The mechanisms by which K. kingae evades host innate immunity during pathogenesis of disease remain poorly understood. In this study, we established that the K. kingae polysaccharide capsule and exopolysaccharide function independently to protect K. kingae against reactive oxygen species (ROS) production, neutrophil phagocytosis, and antimicrobial peptides. These results demonstrate the intricacies of K. kingae innate immune evasion and provide valuable information that may facilitate development of a polysaccharide-based vaccine against K. kingae.Bacterial pathogens have evolved strategies that enable them to evade neutrophil-mediated killing. The Gram-negative coccobacillus Kingella kingae is an emerging pediatric pathogen and is increasingly recognized as a common etiological agent of osteoarticular infections and bacteremia in young children. K. kingae produces a polysaccharide capsule and an exopolysaccharide, both of which are important for protection against complement-mediated lysis and are required for full virulence in an infant rat model of infection. In this study, we examined the role of the K. kingae polysaccharide capsule and exopolysaccharide in protection against neutrophil killing. In experiments with primary human neutrophils, we found that the capsule interfered with the neutrophil oxidative burst response and prevented neutrophil binding of K. kingae but had no effect on neutrophil internalization of K. kingae. In contrast, the exopolysaccharide resisted the bactericidal effects of antimicrobial peptides and efficiently blocked neutrophil phagocytosis of K. kingae. This work demonstrates that the K. kingae polysaccharide capsule and exopolysaccharide promote evasion of neutrophil-mediated killing through distinct yet complementary mechanisms, providing additional support for the K. kingae surface polysaccharides as potential vaccine antigens. In addition, these studies highlight a novel interplay between a bacterial capsule and a bacterial exopolysaccharide and reveal new properties for a bacterial exopolysaccharide, with potential applicability to other bacterial pathogens

Expression of Kingella kingae Type IV Pili Is Regulated by σ54, 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 σ54 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

Characterization of the Kingella kingae polysaccharide capsule and exopolysaccharide.

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

Acquisition, co-option, and duplication of the rtx toxin system and the emergence of virulence in Kingella

Abstract 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

Examination of Type IV Pilus Expression and Pilus-Associated Phenotypes in Kingella kingae Clinical Isolates▿

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

Correction: Transformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in Haemophilus influenzae

The PLOS Pathogens Staff. Notice of Republication.This article was republished on May 13, 2016, to correct the title which was incorrectly published as “Transformed Recombinant Enrichment Profiling Rapidly Identifies HMW1 as an Intracellular Invasion Locus in Haemophilus influenza”. The publisher apologizes for the errors. Please download this article again to view the correct version. The originally published, uncorrected article and the republished, corrected articles are provided here for reference.This work was supported by National Institutes of Health Ruth Kirschstein postdoctoral fellowship F32AI084427 (to JCM); a Canadian Institute of Health Research operating grant (to RJR); Genome British Columbia grant SOF122 (to RJR and JCM); the Faculty of Pharmaceutical Sciences, Canadian Foundation for Innovation (to CN); National Institutes of Heath R01 grant DC002873 (to JWSG); and (to JG) grants from Ministerio Economía y Competitividad-MINECO SAF2012-31166 and SAF2015-66520-R, Dpto. Salud Gobierno Navarra 359/2012 and Ministerio de Educación PRX12/00191.Peer reviewe