Proteomic study of non-typable Haemophilus influenzae

Non-typable Haemophilus influenzae (NTHi) are small, gram-negative bacteria and are strictly human pathogens, causing acute otitis media, sinusitis and community-acquired pneumonia. There is no vaccine available for NTHi, as there is for H. influenzae type b. Recent advances in proteomic techniques are finding novel applications in the field of vaccinology. There are several protein separation techniques available today, each with inherent advantages and disadvantages. We employed a combined proteomics approach, including sequential extraction and analytical two-dimensional polyacrylamide electrophoresis (2D PAGE), and two-dimensional semi-preparative electrophoresis (2D PE), in order to study protein expression in the A4 NTHi strain. Although putative vaccine candidates were identified with both techniques, 11 of 15 proteins identified using the 2D PE approach were not identified by 2D PAGE, demonstrating the complementarily of the two methods.     

Carcinoembryonic antigens are targeted by diverse strains of typable and non-typable Haemophilus influenzae

Haemophilus influenzae (Hi), a commensal of the human respiratory mucosa, is an important cause of localized and systemic infections. We show that distinct strains belonging to typable (THi) and non-typable (NTHi) H. influenzae target human carcinoembryonic antigens (the membrane associated CEA family of cell adhesion molecules, are now termed CEACAMs). All strains of H. influenzae biogroup aegyptius (Hi-aeg) and more than 70% of THi and NTHi strains tested specifically recognize CEACAMI-Fc soluble constructs. Furthermore, transfection of Chinese hamster ovary cells with human CEACAM1 cDNA alone was sufficient for promoting Hi interactions with the transfected cells. The majority of the Hi-aeg strains tested interacted with soluble constructs containing only the N-terminal domain. In contrast, several THi and NTHi strains reacted with soluble constructs only when additional extracellular A and B domains of the receptor were present. The use of monoclonal antibodies confirmed that THi and NTHi strains also interact primarily at the N-domain. We used site-directed mutants of CEACAM1 that contained substitutions at surface exposed amino acids and a molecular model of the N-domain to identify the residues involved in interactions with Hi ligands. The studies show that a common region exposed at the CFG face of the molecule is targeted by diverse Hi strains. However, mutation at distinct sites within this area affected the interactions of distinct strains signifying the potential for tissue tropism via this receptor. Analyses of the molecular basis of interaction with human cell lines and purified CEA show that Hi strains, especially those belonging to Hi-aeg, interact with multiple CEACAMs. Because Neisseria meningitidis (Nm) strains are also known to bind at the CFG face of the receptor, we used Nm and Hi strains in co-infection experiments and demonstrate competition between these mucosal pathogens in colonization of target cells via CEACAMs.     

Identification of a novel Haemophilus influenzae protein important for adhesion to epithelial cells

Non-typable Haemophilus influenzae (NTHi) is an important human-specific respiratory pathogen colonizing the mucosa of the upper respiratory tract. The bacterium is a common cause of acute otitis media in children and exacerbations in patients with chronic obstructive pulmonary disease (COPD). An immunoglobulin (Ig) D-lambda myeloma protein was found to detect a 16 kDa surface protein that we designated protein E (PE). The pe gene was cloned using an NTHi genomic DNA library, and a truncated PE-derived protein lacking the endogenous signal peptide (PE22-160) was synthesized and produced in large amounts in Escherichia coli. Interestingly, PE was expressed at the bacterial surface of NTHi as revealed by flow cytometry using the IgD-lambda myeloma protein or PE-specific polyclonal antibodies. A PE-deficient NTHi mutant was produced and lost 50% of its adhesive capacity as compared to the wild-type counterpart when analysed for adhesion to type II lung alveolar epithelial cells. In parallel, E. coli expressing full-length PE1-160 adhered significantly more efficiently to epithelial cells as compared to wild-type E. coli. Recombinant IgD that recognized the chemical dansyl-chloride did not interact with PE indicating that the IgD-lambda myeloma protein most likely was an antibody directed against the H. influenzae surface epitope. In conclusion, we have discovered a novel NTHi outer membrane protein with adhesive properties using an IgD-myeloma protein.     

Molecular determinants of the pathogenesis of disease due to non-typable Haemophilus influenzae

Non-typable Haemophilus influenzae is a common commensal organism in the human upper respiratory tract and an important cause of localized respiratory tract disease. The pathogenesis of disease begins with bacterial colonization of the nasopharynx, a process that involves establishment on the mucosal surface and evasion of local immune mechanisms. Under the proper circumstances, the organism spreads contiguously to the middle ear, the sinuses, or the lungs, and then stimulates a brisk inflammatory response, producing symptomatic infection. In this review, we summarize our present understanding of the molecular determinants of this sequence of events. Continued investigation of the molecular mechanism of non-typable H. influenzae pathogenicity should facilitate development of novel approaches to the treatment and prevention of H. influenzae disease.     

Identification of two iron-repressed periplasmic proteins in Haemophilus influenzae.

Protein expression by Haemophilus influenzae under iron-limiting growth conditions was examined. The five type b strains and four nontypeable strains studied all expressed a new protein of about 40 kDa when deprived of iron during growth. Most strains also expressed a protein of about 31 kDa under the same growth conditions. Both the 40- and 31-kDa proteins were not expressed by cells grown in iron-replete medium. The 40- and 31-kDa proteins were not expressed in iron-deficient medium to which an excess of ferric nitrate had been added, and therefore it was concluded that their expression was iron regulated. These iron-repressed proteins were localized to the periplasmic space. The amino-terminal sequences of both proteins were determined. The N-terminal sequence of the 40-kDa protein had 81% similarity to the N terminus of Fbp, the major iron-binding protein of Neisseria gonorrhoeae and N. meningitidis. The 31-kDa protein sequence showed no homology with any known protein sequence. As no plasmids were found in the strains, it was concluded that these proteins were chromosomally encoded.     

Construction of chimaeric genes for mapping a surface-exposed epitope on the pilus of non-typable Haemophilus influenzae strain M37

A murine monoclonal antibody (mAb), designated 3H12, reacts with a surface-exposed conformational epitope on the pilus of non-typable Haemophilus influenzae strain M37. This antibody does not recognize the related pilus from H. influenzae type b, strain MinnA. Although mAb 3H12 does not recognize strain M37 pilin on Western blots, mAb 3H12 recognizes the recombinant M37 pilin protein expressed by Escherichia coli. In order to map the epitope recognized by mAb 3H12, we constructed a series of chimaeric genes. The chimaeric genes were expressed in E. coli and the chimaeric proteins characterized with respect to their reactivity with mAb 3H12. Residues between 37 and 100 of the M37 pilin protein are essential for the expression of the mAb 3H12 epitope. Residues in the carboxyl half of the M37 protein enhance the reactivity of mAb 3H12 when expressed in the presence of residues 37-100. Construction of chimaeric genes may provide a general methodology for mapping of conformational epitopes expressed by one of a related pair of proteins.     

Identification of a lipopolysaccharide alpha-2,3-sialyltransferase from Haemophilus influenzae

We have identified a gene for the addition of N-acetylneuraminic acid (Neu5Ac) in an alpha-2,3-linkage to a lactosyl acceptor moiety of the lipopolysaccharide (LPS) of the human pathogen Haemophilus influenzae. The gene is one that was identified previously as a phase-variable gene known as lic3A. Extracts of H. influenzae, as well as recombinant Escherichia coli strains producing Lic3A, demonstrate sialyltransferase activity in assays using synthetic fluorescent acceptors with a terminal galactosyl, lactosyl or N-acetyl-lactosaminyl moiety. In the RM118 strain of H. influenzae, Lic3A activity is modulated by the action of another phase-variable glycosyltransferase, LgtC, which competes for the same lactosyl acceptor moiety. Structural analysis of LPS from a RM118:lgtC mutant and the non-typeable strain 486 using mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy confirmed that the major sialylated species has a sialyl-alpha-(2-3)-lactosyl extension off the distal heptose. This sialylated glycoform was absent in strains containing a lic3A gene disruption. Low amounts of sialylated higher molecular mass glycoforms were present in RM118:lgtC lic3A, indicating the presence of a second sialyltransferase. Lic3A mutants of H. influenzae strains show reduced resistance to the killing effects of normal human serum. Lic3A, encoding an alpha-2,3-sialyltransferase activity, is the first reported phase-variable sialyltransferase gene.     

Ampicillin resistance and penicillin-binding proteins of Haemophilus influenzae

Penicillin-binding protein (PBP) alterations have been associated with non-beta-lactamase-mediated ampicillin resistance in Haemophilus influenzae. We evaluated the PBP profiles of several ampicillin-susceptible and -resistant clinical isolates of H. influenzae to determine how consistently the described alterations occurred, and to document the reproducibility of the PBP profiles for this species. The MIC of ampicillin ranged from 0.06 to 0.13 microgram ml-1 for the susceptible isolates at an inoculum of 100,000 c.f.u. when tested by broth dilution, and was 0.5 microgram ml-1 for all four isolates when tested by agar dilution. The MIC for the resistant isolates ranged from 4 to 8 micrograms ml-1 when tested by broth dilution, and from 1.5 to 16 micrograms ml-1 when tested by agar dilution. At least eight distinct PBPs with molecular masses ranging from 27 to 90 kDa were detected both in cell membrane preparations and whole cell (in vivo) binding assays done on cells in the exponential growth phase. PBP variability was evident both in the ampicillin-susceptible and -resistant isolates; however, much greater variability existed within the four resistant strains. The differences in PBP patterns included (1) electrophoretic mobility, (2) binding capacity for the antibiotic and (3) the presence of additional PBPs in two of the resistant isolates. However, decreased binding capacity was consistently demonstrated in PBP 5 (56 kDa) of all of the resistant isolates. Saturation curves with both penicillin and ampicillin indicated that PBP 5 had decreased affinity for the antibiotics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Urogenital, maternal and neonatal isolates of Haemophilus influenzae: identification of unusually virulent serologically non-typable clone families and evidence for a new Haemophilus species

A collection of 117 strains of Haemophilus influenzae, including 112 non-typable isolates recovered predominantly in the USA and France from genital, obstetric and neonatal sources, was characterized by the electrophoretic mobilities of 10 metabolic enzymes. Eighty-six distinctive multilocus chromosomal genotypes (electrophoretic types, ETs) were distinguished on the basis of allele profiles at the enzyme loci. Isolates of five allied biotype IV ETs were highly divergent from all other strains and hybridization of chromosomal DNA revealed that they undoubtedly represent a previously unrecognized species of Haemophilus. Isolates representing these ETs were recovered predominantly from obstetric infections and serious neonatal diseases and apparently possess specific tropism for the genital tract. Strains of these five ETs were present in samples from both the USA and France, but only in the USA did they cause bacteraemia and meningitis, an occurrence which probably reflects differences in patient management between the two countries. Although strains assigned to H. influenzae (sensu stricto) were strongly polymorphic in multilocus enzyme genotype, 69% of isolates recovered from patients with meningitis and/or septicaemia were assigned to only two clone families, a result suggesting that some serologically nontypable strains of H. influenzae originating from the genital tract are unusually virulent.     

Structural analysis of lipopolysaccharide oligosaccharide epitopes expressed by non-typeable Haemophilus influenzae strain 176

The structure of the lipopolysaccharide (LPS) from non-typeable Haemophilus influenzae strain 176 has been investigated. Electrospray ionization-mass spectrometry (ESIMS) on O-deacylated LPS (LPS-OH) and core oligosaccharide (OS) samples obtained after mild-acid hydrolysis of LPS provided information on the composition and relative abundance of the glycoforms. ESIMS tandem-mass spectrometry on LPS-OH confirmed the presence of minor sialylated and disialylated glycoforms. Oligosaccharide samples were studied in detail using high-field NMR techniques. It was found that the LPS contains the common inner-core element of H. influenzae, L-alpha-D-Hepp-(1-->2)-[PEtn-->6]-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp-(1-->4)]-L-alpha-D-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdop-(2-->6)-Lipid A having glycosyl substitution at the O-3 position of the terminal heptose as recently observed for non-typeable H. influenzae strain 486 [M?nsson, M.; Bauer, S. H. J.; Hood, D. W.; Richards, J. C.; Moxon, E. R.; Schweda, E. K. H., Eur. J. Biochem. 2001, 268, 2148--2159]. The following LPS structures were identified as the major glycoforms, the most significant being indicated with an asterisk (*) (glycoforms are partly substituted with Gly at the terminal Hep):     

Identification of a novel family of cell-surface proteins expressed in human vascular endothelium

Vascular endothelial cells (EC) play a key role in a variety of pathophysiologic processes, such as angiogenesis, inflammation, cancer metastasis, and vascular diseases. As part of a strategy to identify all genes expressed in human EC, a full-length cDNA encoding a potential secreted protein harboring 10 epidermal growth factor (EGF)-like domains and one CUB domain at the carboxyl terminus (termed, SCUBE1 for Signal peptide-CUB-EGF-like domain containing protein 1) was identified. SCUBE1 shares homology with several protein families, including members of the fibrillin and Notch families, and the anticoagulant proteins, thrombomodulin and protein C. SCUBE1 mRNA is found in several highly vascularized tissues such as liver, kidney, lung, spleen, and brain and is selectively expressed in EC by in situ hybridization. SCUBE1 is a secreted glycoprotein that can form oligomers and manifests a stable association with the cell surface. A second gene encoding a homologue (designated SCUBE2) was also identified and is expressed in EC as well as other cell types. SCUBE2 is also a cell-surface protein and can form a heteromeric complex with SCUBE1. Both SCUBE1 and SCUBE2 are rapidly down-regulated in EC after interleukin-1beta and tumor necrosis factor-alpha treatment in vitro and after lipopolysaccharide injection in vivo. Thus, SCUBE1 and SCUBE2 define an emerging family of human secreted proteins that are expressed in vascular endothelium and may play important roles in development, inflammation, and thrombosis.     

Distribution of bacterial proteins in biofilms formed by non-typeable Haemophilus influenzae.

The ability to preserve the fragile ultrastructural organization of bacterial biofilms using cryo-preparation methods for electron microscopy has enabled us to probe sections through non-typeable Haemophilus influenzae (NTHi) biofilms and determine the localization of NTHi-specific lipooligosaccharide (LOS) and proteins within these structures. Some of the proteins we examined are currently being considered as candidates for vaccine development, so it is important that their distribution and accessibility within the biofilms formed by NTHi be determined. We have localized LOS to the extracellular matrix (ECM) of the biofilm and the P6 outer membrane protein to the membrane of what appear to be viable bacteria within the biofilm. The Hap and HWM1/HMW2 adhesive proteins were associated with bacteria within the biofilm and were present in the biofilm ECM. The IgA1 protease is a secreted protein that was also associated with NTHi in the biofilm and was in the ECM, but was more concentrated in the top region of the biofilm, suggesting a role in protecting biofilm bacteria from antibody attack.     

Structural analysis of the lipopolysaccharide oligosaccharide epitopes expressed by a capsule-deficient strain of Haemophilus influenzae Rd.

Structural elucidation of the lipopolysaccharide (LPS) of Haemophilus influenzae, strain Rd, a capsule-deficient type d strain, has been achieved by using high-field NMR techniques and electrospray ionization-mass spectrometry (ESI-MS) on delipidated LPS and core oligosaccharide samples. It was found that this organism expresses heterogeneous populations of LPS of which the oligosaccharide (OS) epitopes are subject to phase variation. ESI-MS of O-deacylated LPS revealed a series of related structures differing in the number of hexose residues linked to a conserved inner-core element, L-alpha-D-Hepp-(1-->2)-L-alpha-D-Hepp-(1-->3)-[beta-D-Glcp- (1-->4)-]- L-alpha-D-Hepp-(1-->5)-alpha-Kdo, and the degree of phosphorylation. The structures of the major LPS glycoforms containing three (two Glc and one Gal), four (two Glc and two Gal) and five (two Glc, two Gal and one GalNAc) hexoses were substituted by both phosphocholine (PCho) and phosphoethanolamine (PEtn) and were determined in detail. In the major glycoform, Hex3, a lactose unit, beta-D-Galp-(1-->4)-beta-D-Glcp, is attached at the O-2 position of the terminal heptose of the inner-core element. The Hex4 glycoform contains the PK epitope, alpha-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-beta-D-Glcp while in the Hex5 glycoform, this OS is elongated by the addition of a terminal beta-D-GalpNAc residue, giving the P antigen, beta-D-GalpNAc-(1-->3)-alpha-D-Galp-(1-->4)-beta-D-Galp-(1-->4)-D-Glc p. The fully extended LPS glycoform (Hex5) has the following structure. [see text] The structural data provide the first definitive evidence demonstrating the expression of a globotetraose OS epitope, the P antigen, in LPS of H. influenzae. It is noteworthy that the molecular environment in which PCho units are found differs from that observed in an Rd- derived mutant strain (RM.118-28) [Risberg, A., Schweda, E. K. H. & Jansson, P-E. (1997) Eur. J. Biochem. 243, 701-707].     

Maturation and secretion of the non-typable Haemophilus influenzae HMW1 adhesin: roles of the N-terminal and C-terminal domains

Non-typable Haemophilus influenzae is a common cause of human disease and initiates infection by colonizing the upper respiratory tract. The non-typable H. influenzae HMW1 and HMW2 adhesins mediate attachment to human epithelial cells, an essential step in the process of colonization. HMW1 and HMW2 have an unusual N-terminus and undergo cleavage of a 441-amino-acid N-terminal fragment during the course of their maturation. Following translocation across the outer membrane, they remain loosely associated with the bacterial surface, except for a small amount that is released extracellularly. In the present study, we localized the signal sequence to the first 68 amino acids, which are characterized by a highly charged region from amino acids 1-48, followed by a more typical signal peptide with a predicted leader peptidase cleavage site after the amino acid at position 68. Additional experiments established that the SecA ATPase and the SecE translocase are essential for normal export and demonstrated that maturation involves cleavage first between residues 68 and 69, via leader peptidase, and next between residues 441 and 442. Site-directed mutagenesis revealed that HMW1 processing, secretion and extracellular release are dependent on amino acids in the region between residues 150 and 166 and suggested that this region interacts with the HMW1B outer membrane translocator. Deletion of the C-terminal end of HMW1 resulted in augmented extracellular release and elimination of HMW1-mediated adherence, arguing that the C-terminus may serve to tether the adhesin to the bacterial surface. These observations suggest that the HMW proteins are secreted by a variant form of the general secretory pathway and provide insight into the mechanisms of secretion of a growing family of Gram-negative bacterial exoproteins.     

Distribution of a family of Haemophilus influenzae genes containing CCAA nucleotide repeating units

A family of genes containing lengths of CCAA nucleotide repeating units directly following the sequence encoding the leader peptide has been identified in Haemophilus influenzae. The length of the CCAA repeats ranges from 6 to 43 and all of the identified genes encode proteins or predicted proteins with a significant homology to bacterial iron- or heme-related outer membrane proteins. We have previously shown that two of these gene products, HgpA and HgpB, bind hemoglobin and the hemoglobin-haptoglobin complex. Studies were performed to define the species distribution of the five identified genes and the CCAA repeats. We show that both the CCAA motif and the structural genes for hemoglobin and hemoglobin-haptoglobin binding are widely distributed among H. influenzae strains.     

DNA-binding by Haemophilus influenzae and Escherichia coli YbaB, members of a widely-distributed bacterial protein family

Background  

Genes orthologous to the ybaB loci of Escherichia coli and Haemophilus influenzae are widely distributed among eubacteria. Several years ago, the three-dimensional structures of the YbaB orthologs of both E. coli and H. influenzae were determined, revealing a novel "tweezer"-like structure. However, a function for YbaB had remained elusive, with an early study of the H. influenzae ortholog failing to detect DNA-binding activity. Our group recently determined that the Borrelia burgdorferi YbaB ortholog, EbfC, is a DNA-binding protein. To reconcile those results, we assessed the abilities of both the H. influenzae and E. coli YbaB proteins to bind DNA to which B. burgdorferi EbfC can bind.     

Characteristics of major outer membrane proteins of Haemophilus influenzae.

Several properties of Haemophilus influenzae outer membrane proteins were analyzed to define related proteins in various isolates. H. influenzae type b 760705 had six major outer membrane proteins with the following characteristics. Protein a (Mr, 47,000) demonstrated heat modifiability in sodium dodecyl sulfate; its apparent molecular weight was 34,000 at temperatures below 60 degrees C. This protein was extracted from cell envelopes by using Triton X-100-10 mM MgCl2; in cell envelope preparations, the protein was degraded by trypsin. Proteins b (Mr, 41,000) and c (Mr, 40,000) were insensitive to trypsin degradation, were not heat modifiable in sodium dodecyl sulfate, and were peptidoglycan associated in 0.5% Triton X-100-0.2% sodium dodecyl sulfate. The amount of protein b was reduced in ultrasonically obtained cell envelopes. Protein d (Mr, 37,000) was heat modifiable in sodium dodecyl sulfate with an Mr of 28,000 at temperatures below 100 degrees C and was degraded by trypsin, leaving a membrane-bound fragment of Mr, 27,000. Both the intact and degraded proteins were immunologically cross-reactive with the heat-modifiable OmpA protein of Escherichia coli K-12. Protein d was absent in LiCl-EDTA extracts of cells. Protein e (Mr, 30,000), invariably present in all H. influenzae strains tested, was insensitive to trypsin and absent in LiCl-EDTA extracts of cells. Protein k (Mr, 58,000) was extracted from cell envelopes with 2% Triton X-100-10 mM MgCl2 and, in cell envelopes, appeared to be sensitive to trypsin degradation. Proteins with similar properties to those of proteins a to k were found in 10 other H. influenzae b strains, reference strains with serotype a, c, d, e, and f capsules, and 18 of 20 nonencapsulated strains. Their relative molecular weights, however, varied.     

Biological and biophysical properties of Haemophilus influenzae transforming DNA encapsided by viral proteins

Summary The expression of the transforming ability of Haemophilus influenzae DNA was investigated after its encapsidation by the coat protein of two different plant viruses, Brome Mosaic Virus (BMV) and Alfalfa Mosaic Virus (AMV). The influence of the encapsidation on the various steps of the transformation process was studied, as well as the protection of the DNA molecule inside of the DNA-protein complex particles against nucleolytic attack.The kinetics of uptake and penetration of free and encapsided DNA and their respective competitive abilities were compared in order to explain the differences which appeared between the rates of transformation by free and encapsided high molecular weight DNAs.Finally, some conclusions are drawn concerning the uncoating process of these nucleoprotein complex particles and the strength of the DNA-protein and protein-protein interactions existing in these particles.Abbreviations M.W. molecular weight - T.A. transforming ability