Joseph W. St. Geme

Identification of a second family of high-molecular-weight adhesion proteins expressed by non-typable Haemophilus influenzae.

We previously reported that two surface‐exposed high‐molecular‐weight proteins, HMW1 and HMW2, expressed by a prototypic strain of non‐typable Haemophilus influenzae (NTHI), mediate attachment to human epithelial cells. These proteins are members of a family of highly immunogenic proteins common to 70–75% of NTHI strains. NTHI strains that lack HMW1/ HMW2‐like proteins remain capable of efficient attachment to cultured human epithelial cells, suggesting the existence of additional adhesion molecules. We reasoned that characterization of high‐molecular‐weight immunogenic proteins from an HMW1/HMW2‐deficient strain might identify additional adhesion proteins. A genomic library was prepared in λEMBL3 with chromosomal DNA from non‐typable Haemophilus strain 11, a strain that lacks HMW1/HMW2‐like proteins. The library was screened immunologically with convalescent serum from a child naturally infected with strain 11, and phage clones expressing high‐molecular‐weight recombinant proteins were identified by Western blot analysis. One clone was identified that expressed a protein with an apparent molecular mass greater than 200 kDa. Transformation of non‐adherent Escherichia coli strain DH5α with plasmids containing the genetic locus encoding this protein gave rise to E. colitransformants that adhered avidly to Chang conjunctival cells. Subcloning and mutagenesis studies localized the DNA conferring the adherence phenotype to a 4.8 kbp fragment, and nucleotide sequence analysis further localized the gene encoding the adhesion protein to a 3.3 kbp open reading frame predicted to encode a protein of 114kDa. The gene was designated hia for Haemophilus influenzae adhesin. Southern analysis revealed an hia homologue in 13 of 15 HMW1/HMW2‐deficient non‐typable H. influenzae strains. In contrast, the hia gene was not present in any of 23 non‐typable H. influenzae strains which expressed HMW1/HMW2‐like proteins. Identification of this second family of high‐molecular‐weight adhesion proteins suggests the possibility of developing vaccines based upon a combination of HMW1/HMW2‐like proteins and Hia‐like proteins which would be protective against disease caused by most or all non‐typable H. influenzae

A Haemophilus influenzae IgA protease‐like protein promotes intimate interaction with human epithelial cells

Haemophilus influenzae represents a common cause of human disease and an important source of morbidity and mortality. Disease caused by this organism begins with colonization of the upper respiratory tract. Several studies indicate that H. influenzae is capable of binding to and entering cultured human cells, properties which are potentially of relevance to the process of colonization. In the present study, we isolated an H. influenzae gene designated hap, which is associated with the capacity for In vitro attachment and entry. Analysis of the derived amino acid sequence of hap demonstrated significant homology with the serine‐type lgA1 proteases expressed by H. influenzae and Neisseria gonorrhoeae. It is notable that the hap product shares the catalytic domain of the lgA1 proteases and appears to be processed and secreted in an analogous manner. We speculate that the hap gene product is an important determinant of colonization, perhaps enabling the organism to evade the local immune response and thereby persist within the respiratory tract.

Structure of the outer membrane translocator domain of the Haemophilus influenzae Hia trimeric autotransporter

Autotransporter proteins are defined by the ability to drive their own secretion across the bacterial outer membrane. The Hia autotransporter of Haemophilus influenzae belongs to the trimeric autotransporter subfamily and mediates bacterial adhesion to the respiratory epithelium. In this report, we present the crystal structure of the C‐terminal end of Hia, corresponding to the entire Hia translocator domain and part of the passenger domain (residues 992–1098). This domain forms a β‐barrel with 12 transmembrane β‐strands, including four strands from each subunit. The β‐barrel has a central channel of 1.8 nm in diameter that is traversed by three N‐terminal α‐helices, one from each subunit. Mutagenesis studies demonstrate that the transmembrane portion of the three α‐helices and the loop region between the α‐helices and the neighboring β‐strands are essential for stability of the trimeric structure of the translocator domain, and that trimerization of the translocator domain is a prerequisite for translocator activity. Overall, this study provides important insights into the mechanism of translocation in trimeric autotransporters.

Secretion of virulence determinants by the general secretory pathway in Gram-negative pathogens: an evolving story

Secretion of proteins by the general secretory pathway (GSP) is a two-step process requiring the Sec translocase in the inner membrane and a separate substrate-specific secretion apparatus for translocation across the outer membrane. Gram-negative bacteria with pathogenic potential use the GSP to deliver virulence factors into the extracellular environment for interaction with the host. Well-studied examples of virulence determinants using the GSP for secretion include extracellular toxins, pili, curli, autotransporters, and crystaline S-layers. This article reviews our current understanding of the GSP and discusses examples of terminal branches of the GSP which are utilized by factors implicated in bacterial virulence.

The Haemophilus influenzae HMW1 adhesin is glycosylated in a process that requires HMW1C and phosphoglucomutase, an enzyme involved in lipooligosaccharide biosynthesis

Non‐typeable Haemophilus influenzae is a common respiratory pathogen and an important cause of morbidity in humans. The non‐typeable H. influenzae HMW1 and HMW2 adhesins are related proteins that mediate attachment to human epithelial cells, an essential step in the pathogenesis of disease. Secretion of these adhesins requires accessory proteins called HMW1B/HMW2B and HMW1C/HMW2C. In the present study, we investigated the specific function of HMW1C. Examination of mutant constructs demonstrated that HMW1C influences both the size and the secretion of HMW1. Co‐immunoprecipitation and yeast two‐hybrid assays revealed that HMW1C interacts with HMW1 and forms a complex in the cytoplasm. Additional experiments and homology analysis established that HMW1C is required for glycosylation of HMW1 and may have glycotransferase activity. The glycan structure contains galactose, glucose and mannose and appears to be generated in part by phosphoglucomutase, an enzyme important for lipooligosaccharide biosynthesis. In the absence of glycosylation, HMW1 is partially degraded and is efficiently released from the surface of the organism, resulting in reduced adherence. Based on these results, we conclude that glycosylation is a prerequisite for HMW1 stability. In addition, glycosylation appears to be essential for optimal HMW1 tethering to the bacterial surface, which in turn is required for HMW1‐mediated adherence, thus revealing a novel mechanism by which glycosylation influences cell–cell interactions.

The Haemophilus influenzae Hap Serine Protease Promotes Adherence and Microcolony Formation, Potentiated by a Soluble Host Protein

Haemophilus influenzae initiates infection by colonizing the upper respiratory mucosa. The process of colonization involves adherence to epithelium and evasion of host immunity. In this study, we examined the H. influenzae Hap adhesin, which has serine protease activity and undergoes autoproteolytic cleavage and extracellular release in broth. We found that the uncleaved cell-associated form of Hap mediates adherence to cultured epithelial cells and promotes bacterial aggregation and microcolony formation. Adherence and aggregation are augmented by secretory leukocyte protease inhibitor, a natural component of respiratory secretions that inhibits Hap autoproteolysis. These observations suggest a novel paradigm in host-pathogen relations, in which a soluble host protein whose primary function is to protect host epithelium potentiates properties that facilitate bacterial colonization.

Kingella kingae: an emerging pathogen in young children.

Kingella kingae is being recognized increasingly as a common etiology of pediatric osteoarticular infections, bacteremia, and endocarditis, which reflects improved culture methods and use of nucleic acid–amplification techniques in clinical microbiology laboratories. K kingae colonizes the posterior pharynx of young children and is transmitted from child to child through close personal contact. Day care attendance increases the risk for colonization and transmission, and clusters of K kingae infections among day care center attendees have been reported. Key virulence factors in K kingae include type IV pili and a potent RTX toxin. In previously healthy children, >95% of K kingae infections are diagnosed between the ages of 6 and 48 months. Among children with underlying medical conditions, K kingae disease may occur at older ages as well. The clinical presentation of K kingae disease is often subtle and may be associated with normal levels of acute-phase reactants, which underscores the importance of a high index of suspicion. K kingae is usually susceptible to ß-lactam antibiotics, and infections typically respond well to medical treatment, with the exception of cases of endocarditis.

The Haemophilus influenzae Hia Adhesin Is an Autotransporter Protein That Remains Uncleaved at the C Terminus and Fully Cell Associated

Nontypeable Haemophilus influenzae is a gram-negative commensal organism that is commonly associated with localized respiratory tract disease. The pathogenesis of disease begins with colonization of the nasopharynx, a process that likely depends on bacterial adherence to respiratory epithelial cells. Hia is the major adhesin expressed by a subset of nontypeable H. influenzae strains and promotes efficient adherence to a variety of human epithelial cell lines. Based on previous work, Hia is transported to the surface of Escherichia coli transformants and is capable of mediating E. coli adherence without the assistance of other H. influenzae proteins. In the present study, we examined the mechanism of Hia secretion. PhoA fusions, deletional mutagenesis, and N-terminal amino acid sequencing established that the signal for Hia export from the cytoplasm resides in the first 49 amino acids, including a 24-amino-acid stretch with striking similarity to the N terminus of a number of proteins belonging to the autotransporter family. Immunoelectron microscopy demonstrated that the Hia internal region defined by amino acids 221 to 779 is exposed on the bacterial surface. Secondary-structure analysis predicted that the C terminus of Hia forms a beta-barrel with a central hydrophilic channel, and site-specific mutagenesis and fusion protein analysis demonstrated that the C terminus targets Hia to the outer membrane and functions as an outer membrane translocator, analogous to observations with autotransporter proteins. In contrast to typical autotransporter proteins, Hia undergoes no cleavage between the internal and C-terminal domains and remains fully cell associated. Together, these results suggest that Hia is the prototype of an important subfamily of autotransporter proteins.

Structural determinants of processing and secretion of the Haemophilus influenzae Hap protein

Haemophilus influenzae elaborates a surface protein called Hap, which is associated with the capacity for intimate interaction with cultured epithelial cells. Expression of hap results in the production of three protein species: outer membrane proteins of approximately 155 kDa and 45 kDa and an extracellular protein of approximately 110 kDa. The 155 kDa protein corresponds to full‐length mature Hap (without the signal sequence), and the 110 kDa extracellular protein represents the N‐terminal portion of mature Hap (designated Haps). In the present study, we examined the mechanism of processing and secretion of Hap. Site‐directed mutagenesis suggested that Hap is a serine protease that undergoes autoproteolytic cleavage to generate the 110 kDa extracellular protein and the 45 kDa outer membrane protein. Biochemical analysis confirmed this conclusion and established that cleavage occurs on the bacterial cell surface. Determination of N‐terminal amino acid sequence and mutagenesis studies revealed that the 45 kDa protein corresponds to the C‐terminal portion of Hap, starting at N1037. Analysis of the secondary structure of this protein (designated Hapβ) predicted formation of a β‐barrel with an N‐terminal transmembrane α‐helix followed by 14 transmembrane β‐strands. Additional analysis revealed that the final β‐strand contains an amino acid motif common to other β‐barrel outer membrane proteins. Upon deletion of this entire C‐terminal consensus motif, Hap could no longer be detected in the outer membrane, and secretion of Haps was abolished. Deletion or complete alteration of the final three amino acid residues had a similar but less dramatic effect, suggesting that this terminal tripeptide is particularly important for outer membrane localization and/or stability of the protein. In contrast, isolated point mutations that disrupted the amphipathic nature of the consensus motif or eliminated the C‐terminal tryptophan had no effect on outer membrane localization of Hap or secretion of Haps. These results provide insight into a growing family of Gram‐negative bacterial exoproteins that are secreted by an IgA1 protease‐like mechanism; in addition, they contribute to a better understanding of the structural determinants of targeting of β‐barrel proteins to the bacterial outer membrane.

Molecular and cellular determinants of non-typeable Haemophilus influenzae adherence and invasion.

Non‐typeable Haemophilus influenzae is a common cause of human disease and initiates infection by colonizing the upper respiratory tract. Based on information from histopathologic specimens and in vitro studies with human cells and tissues in culture, non‐typeable H. influenzae is capable of efficient adherence and appreciable invasion, properties that facilitate the process of colonization. A number of adhesive factors exist, each recognizing a distinct host cell structure and influencing cellular binding specificity. In addition, at least three invasion pathways exist, including one resembling macropinocytosis, a second mediated via the PAF receptor and a third involving β‐glucan receptors. Organisms are also capable of disrupting cell–cell junctions and passing between cells to the subepithelial space.