Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks almost all the biosynthetic enzymes necessary for the de novo synthesis of that cofactor. Factor V can be provided as either nicotinamide adenosine dinucleotide (NAD), nicotinamide mononucleotide (NMN), or nicotinamide riboside (NR) in vitro, but little is known about the source or the mechanism of uptake of these substrates in vivo. As shown by us earlier, at least two gene products are involved in the uptake of NAD, the outer membrane lipoprotein e (P4), which has phosphatase activity and is encoded by hel, and a periplasmic NAD nucleotidase, encoded by nadN. It has also been observed that the latter gene product is essential for H. influenzae growth on media supplemented with NAD. In this report, we describe the functions and substrates of these two proteins as they act together in an NAD utilization pathway. Data are provided which indicate that NadN harbors not only NAD pyrophosphatase but also NMN 5-nucleotidase activity. The e (P4) protein is also shown to have NMN 5-nucleotidase activity, recognizing NMN as a substrate and releasing NR as its product. Insertion mutants of nadN or deletion and site-directed mutants of hel had attenuated growth and a reduced uptake phenotype when NMN served as substrate. A hel and nadN double mutant was only able to grow in the presence of NR, whereas no uptake of NMN was observed.Haemophilus influenzae, a gram-negative facultative anaerobic bacterium, is responsible for significant morbidity and mortality in young children (9, 35). In order to cultivate H. influenzae, complex medium is required, and if it is not blood based, it must contain two growth factors: nicotinamide adenine dinucleotide (NAD) and hemin (6). Early biochemical investigations established that nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) can substitute for NAD, whereas nicotinamide, niacin, or other nicotine-based intermediates of the Preiss-Handler pathway cannot (10, 20, 31). The NAD dependency of H. influenzae was confirmed by the absence of the genes encoding the enzymes necessary for the de novo biosynthesis of NAD (8). Accumulation of nicotinamide nucleotides derived from NAD or NR has been demonstrated in H. influenzae and Haemophilus parainfluenzae (4, 11). For H. parainfluenzae the K m for transport is about 0.55 M for NAD and 0.14 M for NR, while the V max for NR is about four times that of NAD (4). This implies that NR is the substrate for an as-yet-unidentified inner membrane transporter, a proposal that is supported by the observation that NAD cannot be taken up into the cytosolic compartment as an intact molecule. Limited NAD salvage capacity resides within the H. influenzae cytosol, which can be demonstrated if cell extracts are incubated with NR or NMN, indicating the presence of an NMN adenylyl transferase or an NAD pyrophosphorylase activity (5, 16).

Nontypeable Haemophilus influenzae (NTHI) initiates infection by colonizing the upper respiratory tract mucosa. NTHI disease frequently occurs in the context of respiratory tract inflammation, where organisms encounter damaged epithelium and exposed basement membrane. In this study, we examined interactions between the H. influenzae Hap adhesin and selected extracellular matrix proteins. Hap is an autotransporter protein that undergoes autoproteolytic cleavage, with release of the adhesive passenger domain, Hap s , from the bacterial cell surface. We found that Hap promotes bacterial adherence to purified fibronectin, laminin, and collagen IV and that Hap-mediated adherence is enhanced by inhibition of autoproteolysis. Adherence is inhibited by pretreatment of bacteria with a polyclonal antiserum recognizing Hap s . Purified Hap s binds with high affinity to fibronectin, laminin, and collagen IV but not to collagen II. Binding of Hap s to fibronectin involves interaction with the 45-kDa gelatin-binding domain but not the 30-kDa heparin-binding domain of fibronectin. Taken together, these observations suggest that interactions between Hap and extracellular matrix proteins may play an important role in NTHI colonization of the respiratory tract.Extracellular matrix (ECM) consists of a diverse group of proteins that form the scaffolding responsible for the development, growth, and maintenance of mammalian tissues. In epithelial tissues, certain ECM proteins function together with cells to form barriers intended to prevent penetration of these tissues by microorganisms. Both pathogenic and commensal bacteria have evolved mechanisms designed to subvert epithelial barriers (3,5,14,23,28). These mechanisms include bacterial surface proteins called adhesins that bind to ECM and bacterial proteases that degrade ECM components and permit migration of bacteria to deeper tissue spaces where they may gain easier access to nutrients and safe harbor from the host immune response.Haemophilus influenzae is a gram-negative bacterium that is often found as a commensal inhabitant of the respiratory tract in healthy adults but also represents a common cause of both localized respiratory tract and invasive systemic disease (25). In studies examining interactions between H. influenzae clinical isolates and human respiratory tract tissue, bacteria were often associated with damaged epithelium and exposed ECM (16,17). Furthermore, examination of bronchial biopsies from patients with persistent H. influenzae bronchitis revealed organisms in the subepithelial compartment, suggesting that this pathogen is capable of penetrating the basement membrane (11). Additional analysis of interactions between clinical isolates and purified ECM components demonstrated that many H. influenzae strains were capable of binding to fibronectin, laminin, and various collagens (2, 27). In recent work, Virkola and coworkers found that H. influenzae hemagglutinating pili mediated attachment to both fibronectin and heparin-binding growth-associated molecule, alt...

The multivalent pneumococcal conjugate vaccine is effective against both systemic disease and otitis media caused by serotypes contained in the vaccine. However, serotypes not covered by the current conjugate vaccine may still cause pneumococcal disease. To address these serotypes and the remaining otitis media due to Streptococcus pneumoniae, we have been evaluating antigenically conserved proteins from S. pneumoniae as vaccine candidates. A previous report identified a 20-kDa protein with putative human complement C3-proteolytic activity. By utilizing the publicly released pneumococcal genomic sequences, we found the gene encoding the 20-kDa protein to be part of a putative open reading frame of approximately 2,400 bp. We recombinantly expressed a 79-kDa fragment (rPhpA-79) that contains a repeated HxxHxH motif and evaluated it for vaccine potential. The antibodies elicited by the purified rPhpA-79 protein were cross-reactive to proteins from multiple strains of S. pneumoniae and were against surface-exposed epitopes. Immunization with rPhpA-79 protein adjuvanted with monophosphoryl lipid A (for subcutaneous immunization) or a mutant cholera toxin, CT-E29H (for intranasal immunization), protected CBA/N mice against death and bacteremia, as well as reduced nasopharyngeal colonization, following intranasal challenge with a heterologous pneumococcal strain. In contrast, immunization with the 20-kDa portion of the PhpA protein did not protect mice. These results suggest that rPhpA-79 is a potential candidate for use as a vaccine against pneumococcal systemic disease and otitis media.

Nontypeable Haemophilus influenzae is a common cause of respiratory tract disease and initiates infection by colonizing the nasopharynx. The H. influenzae Hap adhesin is an autotransporter protein that was discovered because it promotes intimate interaction with human epithelial cells. Hap contains an extracellular domain called Hap(s) that has adhesive and protease activity and an outer membrane domain called Hap(beta) that serves to present Hap(s) on the surface of the cell. Hap(s) purified from nontypeable H. influenzae strain P860295 was used to immunize BALB/c mice intranasally. Immunization stimulated significant mucosal and serum anti-Hap(s) antibody titers, which were augmented by the addition of mutant cholera toxin (CT-E29H) as an adjuvant. Immunization was associated with a marked reduction in the density of nasopharyngeal colonization when mice were challenged with a heterologous strain of nontypeable H. influenzae. These results suggest that intranasal immunization with Hap formulated with CT-E29H may be a valuable vaccine strategy for the prevention of nontypeable H. influenzae disease.

The multivalent pneumococcal conjugate vaccine is effective against both systemic disease and otitis media caused by serotypes contained in the vaccine. However, serotypes not covered by the present conjugate vaccine may still cause pneumococcal disease. To address these serotypes, and the remaining otitis media due to Streptococcus pneumoniae, efforts have been devoted to identifying protective protein antigens. Immunity to conserved surface proteins important for adhesion, nutrient acquisition, or other functions could result in a reduction of colonization and a lower disease potential. We have been searching for conserved surface-exposed proteins from S. pneumoniae that may be involved in pathogenesis to test as vaccine candidates. Here, an ϳ20-kDa protein that has significant homology to a nonheme iron-containing ferritin protein from Listeria innocua and other bactoferritins was identified as pneumococcal protective protein A (PppA). We expressed and purified recombinant PppA (rPppA) and evaluated its potential as a vaccine candidate. The antibodies elicited by purified rPppA were cross-reactive with PppA from multiple strains of S. pneumoniae and were directed against surface-exposed epitopes. Intranasal immunization of BALB/c mice with PppA protein and either a synthetic monophosphoryl lipid A analog, RC529AF, or a cholera toxin mutant, CT-E29H, used as an adjuvant reduced nasopharyngeal colonization in mice following intranasal challenge with a heterologous pneumococcal strain. PppA-specific systemic and local immunoglobulin G (IgG) and IgA antibody responses were induced. The antisera reacted with whole cells of a heterologous S. pneumoniae type 3 strain. These observations indicate that PppA may be a promising candidate for inclusion in a vaccine against pneumococcal otitis media.Infections with Streptococcus pneumoniae are a major cause of human diseases, such as otitis media, bacteremia, meningitis, and fatal pneumonia, worldwide (9). The rapid emergence of multidrug-resistant pneumococcal strains throughout the world has led to an increased emphasis on prevention of pneumococcal infections by vaccination (18). The presently available 23-valent pneumococcal capsular polysaccharide vaccine is not effective in children less than 2 years of age or immunocompromised patients, two of the major populations at risk for pneumococcal infection (14). A seven-valent pneumococcal polysaccharide-protein conjugate vaccine, recently licensed in the United States, was shown to be highly effective in infants and children against systemic pneumococcal disease caused by the vaccine serotypes and against cross-reactive capsular serotypes (36). However, parenteral immunization with the sevenvalent vaccine was only 60% effective against serotype-specific otitis media (17), demonstrating the need for additional immunization strategies (e.g., intranasal [i.n.] immunization), additional noncapsular antigens, or both. Therefore, there is an immediate need for a cost-effective vaccine to cover most or all of the disease-causin...