<p>Nontypeable Haemophilus influenzae (NTHi) is a common commensal in the human nasopharynx that can cause localized respiratory tract diseases such as otitis media, bronchitis, and pneumonia. NTHi adheres to respiratory epithelial cells, a critical step in the process of colonization enabled by bacterial surface adhesive structures called adhesins. One group of NTHi adhesins are autotransporters, proteins that have an N-terminal signal sequence, a C-terminal &#946;-barrel domain, and an internal passenger domain with effector function. The goal of this work was to increase our understanding of two NTHi autotransporters, Hap and Hia.</p><p>Hap is a monomeric autotransporter that mediates adherence to epithelial cells and extracellular matrix (ECM) proteins. Hap also self-associates with protein on neighboring bacteria, resulting in bacterial aggregation and microcolony formation. The Hap passenger domain contains the regions responsible for adhesive activity. To define the molecular mechanism of Hap adhesive activity, we crystallized the Hap passenger domain. Characterization of the crystal structure revealed an N-terminal globular domain and a more ordered, prism-like C-terminal domain. Interestingly, Hap crystallized as a multimer, suggesting that Hap-Hap interactions occurred in the passenger domain. Progressive deletions of the &#946;-loops that comprise the C-terminal region disrupted Hap-Hap interactions and led to a defect in bacterial settling. To further support that the C-terminal domain was responsible for Hap-Hap interactions,</p><p>7</p><p>we purified the wild type and truncated passenger domains and conjugated the proteins to latex beads. By light microscopy we visualized bead aggregation when the wild type passenger domain was conjugated to the beads, but not when the truncated passenger domain was conjugated. These results show that the C-terminal portion of the Hap passenger domain is responsible for Hap-Hap interactions leading to multimerization. Hap multimerization could be important in microcolony formation that leads to biofilm formation in vivo.</p><p>The ECM binding domain in located in the final 511 amino acids of the Hap passenger domain. To pin-point the region of the ECM protein fibronectin that is recognized by Hap, we spotted small fragments of fibronectin onto nitrocellulose membranes and incubated the membrane with purified Hap passenger domain. Far Western analysis using Hap antibody revealed that the smallest fibronectin region necessary for binding was comprised of the first two type III repeats, FNIII(1-2). To define the regions of Hap responsible for interaction with fibronectin, we mutated motifs in the Hap passenger domain that are important for fibronectin binding in other bacterial proteins. Based on assessment by ELISA, many of the mutations located between amino acids 525-725 caused reduced bacterial binding to fibronectin. However, no mutation totally ablated binding, suggesting that a larger Hap region is involved in fibronectin binding.</p><p>8</p><p>In an additional study, we identified a relationship between Hap levels in the outer membrane and the expression of lipopolysaccharide (LPS) biosynthesis enzymes. Through Western and qPCR analysis, we found that mutation of the rfaF, pgmB, lgtC, kfiC, orfE, rfbP, lsgB and lsgD genes involved in the synthesis of LPS oligosaccharide core in H. influenzae strain Rd/HapS243A resulted in loss of Hap in the bacterial outer membrane and a decrease in hap transcript. In contrast, the same mutations had no effect on outer membrane localization of H. influenzae P5 and IgA1 protease or levels of the p5 or iga1 transcripts, suggesting a Hap-specific effect. Elimination of the HtrA periplasmic protease resulted in a return of Hap to the outer membrane and restoration of wild type levels of hap transcript. We speculate that the lack of certain LPS biosynthesis enzymes causes Hap to mislocalize and accumulate in the periplasm, where it is degraded by HtrA. This degradation then leads to a decrease in hap transcript. lgtC is one of several phase variable LPS biosynthesis genes. Using an antibody against the epitope formed in part by the lgtC gene product, we identified lgtC phase-off bacteria by Western analysis of colony blots. Consistent with our previous observations, in lgtC phase off bacteria Hap was absent from the outer membrane and hap transcript was reduced. By analyzing a lgtC/lic2A double mutant, we found that Hap localization in the outer membrane and hap transcript levels were not related to LPS size but instead to the functions of the LPS synthesis enzymes themselves. This relationship could be beneficial to bacteria in vivo as a way to regulate Hap expression.</p><p>9</p><p>Early models suggested that autotransporters do not require accessory factors for folding and OM insertion. However, mounting recent evidence has suggested that the Bam complex is required for OM localization of most &#946;-barrel proteins, including autotransporters. We studied the role of the Bam complex in OM localization of the trimeric autotransporter Hia. We expressed Hia in E. coli strains with mutations in the Bam complex and found that BamA and BamD were needed for Hia localization, while BamB, BamC, and BamE were not necessary. In further studies, we mutated the C-terminus of Hia and found that the final and third-to-last amino acids were the most important for outer membrane localization.</p><p>In summary, this work provides insights into the regulation and adhesive activity of Hap and the outer membrane localization of Hia. We have learned important details about these factors that shed light on aspects of H. influenzae disease and could lead to new antimicrobial therapies.</p> / Dissertation

IgA1 (immunoglobulin A1), a predominant immunoglobulin, is at the first defense line against microbial pathogens infection and invasion, to neutralize pathogenic antigens. Some bacterial pathogens, such as Neisseria meningitidis and Haemophilus influenzae, however, secrete site-specific IgA1 proteases to counteract with the human defense system. The protease is capable of cleaving at the hinge region of immunoglobulin A1 to destroy the structure and function of human IgA1, impairing the role of the immunoglobulin from the host defense. The protease has therefore been implicated as a putative virulence factor that contributes to bacterial colonization, but bacterial isolates from patients with invasive diseases contain both positive and negative IgA1 proteases. To clarify the role of IgA1 protease in bacterial infection, this project is designed to reveal the molecular mechanism of the protease in bacterial infection and colonization. To do this, iga genes encoding non-typable H. influenzae type 1, type 3 and Neisseria meningitidis type 3 IgA1 proteases were isolated, sequenced and then expressed in IgA1 protease-negative E. coli BL21 (DE3). The recombinant proteases have been purified to homogeneity using ion exchange chromatography. Comparison of the deduced amino acid sequences from non-typable H. influenzae IgA1 proteases with other published H. influenzae IgA1 protease revealed a high degree of homology. Sequence analysis indicates that both type 1 and type 3 non-typable H. influenzae IgA1 proteases lack £\-protein in comparison with the iga from N. meningitidis. The role of IgA1 protease in relation to deposition and invasion has also been evaluated in human lung carcinoma cell (A549) model. The results suggest that the IgA1 protease plays a role in the adherence of H. influenzae on epithelial cell surface though the best effectiveness varies upon different pathogenic bacterial strains at different concentrations.

Immunoglobulin A¡]IgA¡^, the principal antibody class in secretions that bathe mucosal surfaces, acts as an important first line of defense. However, some pathogenic bacteria such as Haemophilus influenzae can produce IgA1 proteases to impair IgA1, especially in human mucosal immune system. IgA1 proteases are characterized by a polypeptide precursor containing four domains, the signal peptide, protease, linking region and the £]-domain. The function of the protease and the £]-domain (£] core) had been studied extensively, but the linking region is less defined, let alone its function. To complete the project, the DNA fragment for linking region was amplified by PCR from iga gene (Gene Bank DQ683353) spanning from NT3130 to NT4686, and then transferred to pGEX-2T for expression. Recombinant linking region-protein was purified using glutathione-Sepharose column. The proliferation assay showed that purified recombinant protein did not enhance cell growth significantly at the concentration of 1 £gg/ml compared to either the negative control or GST control; but when the concentration of the recombinant protein was increased to 5 £gg/ml or 10 £gg/ml, the cell proliferation was significantly stimulated. These results suggest that recombinant linking region-protein contains special element that stimulates the jurkat cell.

Thesis (Ph. D.)--University of Texas at Austin, 2003. / Vita. Includes bibliographical references. Available also from UMI Company.

Thesis (Ph. D.)--University of Missouri--Columbia, 2000. / Typescript. Vita. Includes bibliographical references (leaves 161-166). Also available on the Internet.

Not available / text

Following an outbreak of multi-resistant Haemophilus influenzae type b (Hib)infections in a tuberculosis hospital, this study was undertaken to determine carriage of Hib in 2 paediatric wards; to characterise all isolates of Hib, determine their antimicrobial susceptibility profile and the antibody response of the children to a conjugate vaccine. Prior to and one month after immunisation, oro- and nasopharyngeal swab specimens as well as venous blood were collected from each child. Isolates were tested for /3-lactamase and chloramphenicol acetyltransferase (CAT)production, their MIC's determined by the agar dilution method and characterisation of Hib isolates was performed by biotyping and analysis of outer membrane protein (OMP) profiles. An ELISA was also developed to determine serum antibody levels to polyribosyl-ribitol-phosphate (PRP), the capsular polysaccharide of Hib. The study population comprised a total of 135 children who had been hospitalised for treatment for tuberculosis. The patients were aged 4 months to 14 years with a median of 37,5 months. During the study period, none of the children developed invasive Hib disease. The overall carriage rate of Hib increased from 38% (51/135) before immunisation to 62% (84/135) after immunisation (P 0,15 /ig/ml. After immunisation, 34%(45) of patients increased their antibody levels to > 1,0 /xg/ml. There was no statistical difference between the mean antibody concentrations of patients who were colonised by Hib and those who were not (p = 0,58). The vaccine did not reduce carriage of Hib in this study population of children being treated for tuberculosis and the immune response to the vaccine was not optimal. Production of /3-lactamase and the prevalence of rifampicin resistance has implications for treatment and chemoprophylaxis in this population. OMP analysis showed a diversity of types. Multi-resistant strains causing invasive disease had the same OMP type as some multiresistant strains which colonised the children. / Thesis (M.Med.Sc.)-University of Natal, Durban, 1996.

Microbiological and molecular epidemiological studies were conducted on 36 multi-resistant Haemophilus influenzae strains, isolated from paediatric patients, over a 26 month period (April 1986 to May 1988). The majority of strains (80,5%) had been isolated from blood and cerebrospinal fluid. More than 80% of isolates tested belonged to biotype II and 90% were of serotype B. Minimal inhibitory concentrations against 6 antibiotics (ampicillin, chloramphenicol, tetracycline, rifampicin, streptomycin and cefotaxime) confirmed the presence of multi-resistant strains. Resistance to rifampicin was confirmed in 6 (16,7%) strains. All strains were susceptible to cefotaxime. Ten transconjugants analysed with respect to their plasmid content were shown to harbour an identical 41 MDa plasmid. Restriction endonuclease digests of these plasmids with Eco R1 and Sst1 revealed almost identical restriction patterns. Outer membrane protein profiles of 19 strains revealed the predominance of one particular subtype. By combining the microbiological and molecular epidemiological findings, it is concluded that one strain of H. influenzae type b is responsible for the nosocomial acquisition of infections amongst paediatric patients. The implifications of these findings are discussed. / Thesis (M.Med.)-University of Natal, Durban, 1988.

Proefschrift Universiteit van Amsterdam. / Met lit. opg. - Met samenvatting in het Nederlands.

Thesis (M.P.H.)--University of Adelaide, Dept. of Public Health, 1999? / Includes bibliographical references (leaves 123-135).