Expression of urease by Haemophilus influenzae during human respiratory tract infection and role in survival in an acid environment

<p>Abstract</p> <p>Background</p> <p>Nontypeable <it>Haemophilus influenzae </it>is a common cause of otitis media in children and lower respiratory tract infection in adults with chronic obstructive pulmonary disease (COPD). Prior studies have shown that <it>H. influenzae </it>expresses abundant urease during growth in the middle ear of the chinchilla and in pooled human sputum, suggesting that expression of urease is important for colonization and infection in the hostile environments of the middle ear and in the airways in adults. Virtually nothing else is known about the urease of <it>H. influenzae</it>, which was characterized in the present study.</p> <p>Results</p> <p>Analysis by reverse transcriptase PCR revealed that the <it>ure </it>gene cluster is expressed as a single transcript. Knockout mutants of a urease structural gene (<it>ureC</it>) and of the entire <it>ure </it>operon demonstrated no detectable urease activity indicating that this operon is the only one encoding an active urease. The <it>ure </it>operon is present in all strains tested, including clinical isolates from otitis media and COPD. Urease activity decreased as nitrogen availability increased. To test the hypothesis that urease is expressed during human infection, purified recombinant urease C was used in ELISA with pre acquisition and post infection serum from adults with COPD who experienced infections caused by <it>H. influenzae</it>. A total of 28% of patients developed new antibodies following infection indicating that <it>H. influenzae </it>expresses urease during airway infection. Bacterial viability assays performed at varying pH indicate that urease mediates survival of <it>H. influenzae </it>in an acid environment.</p> <p>Conclusions</p> <p>The <it>H. influenzae </it>genome contains a single urease operon that mediates urease expression and that is present in all clinical isolates tested. Nitrogen availability is a determinant of urease expression. <it>H. influenzae </it>expresses urease during human respiratory tract infection and urease is a target of the human antibody response. Expression of urease enhances viability in an acid environment. Taken together, these observations suggest that urease is important for survival and replication of <it>H. influenzae </it>in the human respiratory tract.</p

Proteomic expression profiling of Haemophilus influenzae grown in pooled human sputum from adults with chronic obstructive pulmonary disease reveal antioxidant and stress responses

<p>Abstract</p> <p>Background</p> <p>Nontypeable <it>Haemophilus influenzae </it>colonizes and infects the airways of adults with chronic obstructive pulmonary disease, the fourth most common cause of death worldwide.Thus, <it>H. influenzae</it>, an exclusively human pathogen, has adapted to survive in the hostile environment of the human airways.To characterize proteins expressed by <it>H. influenzae </it>in the airways, a prototype strain was grown in pooled human sputum to simulate conditions in the human respiratory tract.The proteins from whole bacterial cell lysates were solubilized with a strong buffer and then quantitatively cleaned with an optimized precipitation/on-pellet enzymatic digestion procedure.Proteomic profiling was accomplished by Nano-flow liquid chromatography/mass spectroscopy with low void volume and high separation efficiency with a shallow, long gradient.</p> <p>Results</p> <p>A total of 1402 proteins were identified with high confidence, including 170 proteins that were encoded by genes that are annotated as conserved hypothetical proteins.Thirty-one proteins were present in greater abundance in sputum-grown conditions at a ratio of > 1.5 compared to chemically defined media.These included 8 anti-oxidant and 5 stress-related proteins, suggesting that expression of antioxidant activity and stress responses is important for survival in the airways.Four proteins involved in uptake of divalent anions and 9 proteins that function in uptake of various molecules were present in greater abundance in sputum-grown conditions.</p> <p>Conclusions</p> <p>Proteomic expression profiling of <it>H. influenzae </it>grown in pooled human sputum revealed increased expression of antioxidant, stress-response proteins and cofactor and nutrient uptake systems compared to media grown cells.These observations suggest that <it>H. influenzae </it>adapts to the oxidative and nutritionally limited conditions of the airways in adults with chronic obstructive pulmonary disease by increasing expression of molecules necessary for survival in these conditions.</p

Antigenic Specificity of the Mucosal Antibody Response to Moraxella catarrhalis in Chronic Obstructive Pulmonary Disease

Moraxella catarrhalis is an important human mucosal pathogen causing otitis media in children and lower respiratory tract infection in adults with chronic obstructive pulmonary disease (COPD). Little is known about the mucosal antibody response to M. catarrhalis in adults with COPD. In this study, 10 pairs of well-characterized sputum supernatant samples from adults with COPD who had acquired and subsequently cleared M. catarrhalis from their respiratory tracts were studied in detail in an effort to begin to elucidate potentially protective immune responses. Flow cytometry analysis was used to study the distribution of immunoglobulin isotypes in paired preacquisition and postclearance sputum samples. The results showed that immunoglobulin A (IgA) is the predominant M. catarrhalis-specific immunoglobulin isotype and that the sputum IgA contains a secretory component, indicating that it is locally produced at the mucosal site. Most patients made new sputum IgA responses to the adhesins UspA1 and Hag, along with the surface protein UspA2. A smaller proportion of patients made new sputum IgA responses to the iron-regulated proteins TbpB and CopB and to lipooligosaccharide. These results have important implications in understanding the mucosal immune response to M. catarrhalis in the setting of COPD and in elucidating the elements of a protective immune response

Identification of Surface Antigens of Moraxella catarrhalis as Targets of Human Serum Antibody Responses in Chronic Obstructive Pulmonary Disease

Moraxella catarrhalis is an important respiratory tract pathogen, causing otitis media in children and lower respiratory tract infections in adults with chronic obstructive pulmonary disease (COPD). Adults with COPD make antibody responses to M. catarrhalis following infection, but little is known about the identity of the antigens to which these antibodies are directed. In this study, 12 serum samples obtained from adults with COPD who had cleared M. catarrhalis from the respiratory tract following infection and who had developed new serum immunoglobulin G (IgG) to their infecting strain were subjected to a series of assays to identify the antigens to which potentially protective antibodies were directed. Sera were adsorbed with intact bacterial cells, and antibodies were eluted from the surfaces of the bacteria. Analysis by flow cytometry established that adsorption and elution effectively detected antibodies specifically directed to surface-exposed epitopes. Immunoblot assays of adsorbed and eluted serum fractions were performed with purified outer membranes and purified lipooligosaccharide of homologous infecting strains and with a series of mutants deficient in expression of individual outer membrane proteins (OMPs). While heterogeneity in antibody responses among individuals was observed, five major OMPs, UspA1, UspA2, Hag, TbpB, and OMP CD, were identified as targets of antibodies to surface epitopes in the majority of adults with COPD who cleared the organism. These results have important implications in understanding human immune responses to M. catarrhalis and in elucidating the elements of a protective immune response

ATP-Binding Cassette (ABC) Transporters of the Human Respiratory Tract Pathogen, Moraxella catarrhalis: Role in Virulence.

Moraxella catarrhalis is a human respiratory tract pathogen that causes otitis media (middle ear infections) in children and respiratory tract infections in adults with chronic obstructive pulmonary disease. In view of the huge global burden of disease caused by M. catarrhalis, the development of vaccines to prevent these infections and better approaches to treatment have become priorities. In previous work, we used a genome mining approach that identified three substrate binding proteins (SBPs) of ATP-binding cassette (ABC) transporters as promising candidate vaccine antigens. In the present study, we performed a comprehensive assessment of 19 SBPs of 15 ABC transporter systems in the M. catarrhalis genome by engineering knockout mutants and studying their role in assays that assess mechanisms of infection. The capacity of M. catarrhalis to survive and grow in the nutrient-limited and hostile environment of the human respiratory tract, including intracellular growth, account in part for its virulence. The results show that ABC transporters that mediate uptake of peptides, amino acids, cations and anions play important roles in pathogenesis by enabling M. catarrhalis to 1) grow in nutrient-limited conditions, 2) invade and survive in human respiratory epithelial cells and 3) persist in the lungs in a murine pulmonary clearance model. The knockout mutants of SBPs and ABC transporters showed different patterns of activity in the assay systems, supporting the conclusion that different SBPs and ABC transporters function at different stages in the pathogenesis of infection. These results indicate that ABC transporters are nutritional virulence factors, functioning to enable the survival of M catarrhalis in the diverse microenvironments of the respiratory tract. Based on the role of ABC transporters as virulence factors of M. catarrhalis, these molecules represent potential drug targets to eradicate the organism from the human respiratory tract

Moraxella catarrhalis in Chronic Obstructive Pulmonary Disease: Burden of Disease and Immune Response

Rationale: Moraxella catarrhalis is frequently present in the sputum of adults with chronic obstructive pulmonary disease (COPD). Little is known about the role of M. catarrhalis in this common disease. Objective: To elucidate the burden of disease, the dynamics of carriage, and immune responses to M. catarrhalis in COPD. Methods: Prospective cohort study of 104 adults with COPD in an outpatient clinic at the Buffalo Veterans Affairs Medical Center. Measurements: Clinical information, sputum cultures, molecular typing of isolates, and immunoassays to measure antibodies to M. catarrhalis. Main Results: Over 81 months, 104 patients made 3,009 clinic visits, 560 during exacerbations. Molecular typing identified 120 episodes of acquisition and clearance of M. catarrhalis in 50 patients; 57 (47.5%) of the acquisitions were associated with clinical exacerbations. No instances of simultaneous acquisition of a new strain of another pathogen were observed. The duration of carriage of M. catarrhalis was shorter with exacerbations compared with asymptomatic colonization (median, 31.0 vs. 40.4 days; p = 0.01). Reacquisition of the same strain was rare. The intensity of the serum IgG response was greater after exacerbations than asymptomatic colonization (p = 0.009). Asymptomatic colonization was associated with a greater frequency of a sputum IgA response than exacerbation (p = 0.009). Conclusions: M. catarrhalis likely causes approximately 10% of exacerbations of COPD, accounting for approximately 2 to 4 million episodes annually. The organism is cleared efficiently after a short duration of carriage. Patients develop strain-specific protection after clearance of M. catarrhalis from the respiratory tract