Kingella kingae expresses type IV pili that mediate adherence to respiratory epithelial and synovial cells

Kingella kingae is a gram-negative bacterium that colonizes the respiratory tract and is a common cause of septic arthritis and osteomyelitis. Despite the increasing frequency of K. kingae disease, little is known about the mechanism by which this organism adheres to respiratory epithelium and seeds joints and bones. Previous work showed that K. kingae expresses long surface fibers that vary in surface density. In the current study, we found that these fibers are type IV pili and are necessary for efficient adherence to respiratory epithelial and synovial cells and that the number of pili expressed by the bacterium correlates with the level of adherence to synovial cells but not with the level of adherence to respiratory cells. In addition, we established that the major pilin subunit is encoded by a pilA homolog in a conserved region of the chromosome that also contains a second pilin gene and a type IV pilus accessory gene, both of which are dispensable for pilus assembly and pilus-mediated adherence. Upon examination of the K. kingae genome, we identified two genes in physically separate locations on the chromosome that encode homologs of the Neisseria PilC proteins and that have only a low level homology to each other. Examination of mutant strains revealed that both of the K. kingae PilC homologs are essential for a wild-type level of adherence to both respiratory epithelial and synovial cells. Taken together, these results demonstrate that type IV pili and the two PilC homologs play important roles in mediating K. kingae adherence

Evolutionary and functional relationships among the nontypeable Haemophilus influenzae HMW family of adhesins

Nontypeable Haemophilus influenzae (NTHi) is a common cause of localized respiratory tract disease and initiates infection by colonizing the nasopharynx. Approximately 75 to 80% of NTHi clinical isolates produce proteins that belong to the HMW family of adhesins, which are believed to facilitate colonization. The prototype HMW adhesins are designated HMW1 and HMW2 and were identified in NTHi strain 12. HMW1 and HMW2 are 71% identical and 80% similar overall, yet display differing cellular binding specificities. In the present study we set out to define more clearly the relationships between HMW1 and HMW2 and other members of the HMW family of adhesins. PCR analysis of 49 epidemiologically distinct isolates revealed that all strains possessing hmw genes as determined by Southern analysis contain two hmw loci in conserved, unlinked physical locations on the chromosome. Functional analysis of the HMW adhesins produced by three unrelated strains demonstrated that each isolate possesses one protein with HMW1-like adherence properties and another with HMW2-like adherence properties. These findings suggest that the hmw1 and hmw2 loci may have arisen via a gene duplication event in an ancestral strain. In addition, they support the hypothesis that the distinct binding specificities of HMW1 and HMW2 emerged early and have persisted over time, suggesting an ongoing selective advantage

The Haemophilus influenzae HMW1C protein is a glycosyltransferase that transfers hexose residues to asparagine sites in the HMW1 adhesin

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

The C-terminal fragment of the internal 110-kilodalton passenger domain of the Hap protein of nontypeable Haemophilus influenzae is a potential vaccine candidate

Nontypeable Haemophilus influenzae is a major causative agent of bacterial otitis media in children. H. influenzae Hap autotransporter protein is an adhesin composed of an outer membrane Hapβ region and a moiety of an extracellular internal 110-kDa passenger domain called Hap(S). The Hap(S) moiety promotes adherence to human epithelial cells and extracellular matrix proteins, and it also mediates bacterial aggregation and microcolony formation. A recent work (D. L. Fink, A. Z. Buscher, B. A. Green, P. Fernsten, and J. W. St. Geme, Cell. Microbiol. 5:175-186, 2003) demonstrated that Hap(S) adhesive activity resides within the C-terminal 311 amino acids (the cell binding domain) of the protein. In this study, we immunized mice subcutaneously with recombinant proteins corresponding to the C-terminal region of Hap(S) from H. influenzae strains N187, P860295, and TN106 and examined the resulting immune response. Antisera against the recombinant proteins from all three strains not only recognized native Hap(S) purified from strain P860295 but also inhibited H. influenzae Hap-mediated adherence to Chang epithelial cells. Furthermore, when mice immunized intranasally with recombinant protein plus mutant cholera toxin CT-E29H were challenged with strain TN106, they were protected against nasopharyngeal colonization. These observations demonstrate that the C-terminal region of Hap(S) is capable of eliciting cross-reacting antibodies that reduce nasopharyngeal colonization, suggesting utility as a vaccine antigen for the prevention of nontypeable H. influenzae diseases

Structure and function of the Haemophilus influenzae autotransporters

Autotransporters are a large class of proteins that are found in the outer membrane of gram-negative bacteria and are almost universally implicated in virulence. These proteins consist of a C-terminal β-domain that is embedded in the outer membrane and an N-terminal domain that is exposed on the bacterial surface and is endowed with effector function. In this article, we review and compare the structural and functional characteristics of the Haemophilus influenzae IgA1 protease and Hap monomeric autotransporters and the H. influenzae Hia and Hsf trimeric autotransporters. All of these proteins play a role in colonization of the upper respiratory tract and in the pathogenesis of H. influenzae disease

Chromosomal Expression of the Haemophilus influenzae Hap Autotransporter Allows Fine-Tuned Regulation of Adhesive Potential via Inhibition of Intermolecular Autoproteolysis

The Haemophilus influenzae Hap autotransporter is a nonpilus adhesin that promotes adherence to respiratory epithelial cells and selected extracellular matrix proteins and facilitates bacterial aggregation and microcolony formation. Hap consists of a 45-kDa outer membrane translocator domain called Hapβ and a 110-kDa extracellular passenger domain called HapS. All adhesive activity resides within HapS, which also contains protease activity and directs its own secretion from the bacterial cell surface via intermolecular autoproteolysis. In the present study, we sought to determine the relationship between the magnitude of Hap expression, the efficiency of Hap autoproteolysis, and the level of Hap-mediated adherence and aggregation. We found that a minimum threshold of Hap precursor was required for autoproteolysis and that this threshold approximated expression of Hap from a chromosomal allele, as occurs in H. influenzae clinical isolates. Chromosomal expression of wild-type Hap was sufficient to promote significant adherence to epithelial cells and extracellular matrix proteins, and adherence was enhanced substantially by inhibition of autoproteolysis. In contrast, chromosomal expression of Hap was sufficient to promote bacterial aggregation only when autoproteolysis was inhibited, indicating that the threshold for Hap-mediated aggregation is above the threshold for autoproteolysis. These results highlight the critical role of autoproteolysis and an intermolecular mechanism of cleavage in controlling the diverse adhesive activities of Hap

HMW1C-like proteins in two categories: Those encoded by loci that contain obvious substrate genes and those encoded by isolated genes without adjacent substrate genes.

<p>Numbers below <i>hmw1C</i>-like genes represent translated protein sequence percent identity/similarity when compared to <i>H. influenzae</i> HMW1C. (<b>A</b>) HMW1C-like enzymes encoded in apparent TPS systems. (<b>B</b>) HMW1C-like enzymes encoded in loci without obvious surface protein targets for glycosylation. Abbreviations: <i>Hi</i>, <i>H. influenzae</i> 86-028NP; <i>Bsp</i>, <i>Burkholderia</i> species GCE1003; <i>Ec</i>, Enterotoxigenic <i>E. coli</i> H10407; <i>Yp</i>, <i>Y. pseudotuberculosis</i> YPIII; <i>Ap</i>, <i>Actinobacillus pleuropneumoniae</i> L20; <i>Hd</i>, <i>H. ducreyi</i> HD35000; <i>Kk</i>, <i>K. kingae</i> 269–492; <i>hyp</i>, hypothetical with no conserved domains; <i>hyp</i>¹, predicted lipoprotein; <i>hyp</i>², predicted UDP-glcNAc carboxyvinyltransferase; <i>hyp</i>³, predicted 2 C-methyl-D erythritol-4-phosphate cytidyltransferase; <i>hyp</i>⁴, predicted deoxyguanosinetriphosphate triphosphohydrolase.</p

Glycosylation by HMW1C may play several roles in promoting HMW1 stability, export, folding, and function.

<p>HMW1C has the potential to contribute to several different processes that occur during HMW1 synthesis and transit across the inner and outer membranes. First of all, HMW1C glycosylates the HMW1 adhesin in the cytoplasm and is likely to be involved in the stability of the HMW1 adhesin during or after its synthesis. Glycosylation may contribute to stability of HMW1 in the (<b>A</b>) cytoplasm or (<b>C</b>) periplasm <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003977#ppat.1003977-Grass1" target="_blank">[5]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003977#ppat.1003977-StGeme1" target="_blank">[7]</a>. Alternatively, the HMW1C protein may improve stability of HMW1 by acting as a (<b>B</b>) chaperone prior to secretion of the adhesin. It is unlikely that the activity of HMW1C is required for export of the adhesin across either the inner or outer membrane, as fully processed HMW1 is found in the supernatant in the absence of HMW1C <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003977#ppat.1003977-Grass1" target="_blank">[5]</a>. It is unclear whether glycosylation influences interaction of HMW1 with the (<b>D</b>) HMW1B periplasmic domain prior to transit, (<b>E</b>) the HMW1B pore during transit, or (<b>F</b>) the docking region of HMW1B upon surface tethering <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003977#ppat.1003977-Grass1" target="_blank">[5]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003977#ppat.1003977-Buscher2" target="_blank">[11]</a>. It is also unclear whether glycosylation participates in (<b>G</b>) protein folding upon export. Evidence from the nonglycosylated <i>Bordetella</i> prototypic, two-partner, secreted adhesin FHA indicates that this adhesin remains unfolded in the cytoplasm and folds very rapidly upon export via its TpsB secretion pore <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003977#ppat.1003977-Hodak1" target="_blank">[30]</a>. One hypothesis is that the energy generated by this rapid folding is at least part of what drives export of TpsA proteins across the outer membrane <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003977#ppat.1003977-JacobDubuisson1" target="_blank">[2]</a>. Finally, glycosylation of HMW1 may be required for (<b>H</b>) adherence to host cells or host interaction in a particular niche.</p