[PubMed] [Google Scholar] 41. the expression of individual OMP was used to determine the identities of OMP giving rise to IgA bands. Human saliva was shown consistently to exhibit IgA-binding activity for oligomeric UspA2 ( 250 kDa), hemagglutinin (200 kDa), monomeric UspA1 (120 kDa), transferrin-binding protein B (TbpB), monomeric UspA2, CopB, and presumably OMP CD. TbpB, oligomeric UspA2, and CopB formed a cluster of bands at about 80 kDa. These data indicate that the human salivary IgA response is directed consistently against a small number of major OMP, some of which are presently considered vaccine candidates. The functional properties of these mucosal antibodies remain to be elucidated. is a major mucosal pathogen of the human respiratory tract TTA-Q6(isomer) (24). This gram-negative organism is the third most common cause of otitis media in children (15, 25) and is associated with acute exacerbations of chronic obstructive pulmonary disease in adults (30). The interaction between and the human host is incompletely understood. Colonization and infection with induce a systemic humoral immune response (4, 21, 29, 42, 43). Serum immunoglobulin G (IgG) concentrations increase with age and are functionally active through the induction of complement-mediated bacterial killing (5, 6). Target antigens of serum antibodies include various outer membrane proteins (OMP) and lipooligosaccharide (4, 6, 7, 10, 13, 31). In contrast, little is known about the mucosal immune response against surface determinants of (4). However, the occurrence and specificity of mucosal antibodies against surface determinants of in healthy adults have not been investigated comprehensively. The aim of the present study was to characterize sIgA antibodies against OMP of in saliva IL22R samples from healthy adults. The identities of specific OMP targeted by IgA antibodies were determined by constructing isogenic knockout mutants for various candidate OMP. MATERIALS AND METHODS Bacterial strains and TTA-Q6(isomer) culture conditions. O35E and its isogenic knockout mutants used in this study are described in Table ?Table1.1. All strains were routinely cultured at 37C and 200 rpm in brain heart infusion (BHI) broth or on BHI agar plates in an atmosphere of 5% CO2. Media were supplemented with kanamycin (20 mg/liter), chloramphenicol (0.5 mg/liter), or erythromycin (1 mg/liter) for culturing of isogenic mutants (Table ?(Table1).1). DH5, the host strain for the plasmid constructs in this study, was grown in Luria-Bertani broth or on Luria-Bertani agar plates supplemented with ampicillin (100 mg/liter), kanamycin (100 mg/liter), or erythromycin (500 mg/liter). All antibiotics were purchased from Sigma Chemical Co., St. Louis, Mo. TABLE 1. Strains used in this study mutant; kanamycin resistant1, 2O35E.2Isogenic mutant; kanamycin resistant1O35E.12Isogenic double mutant; kanamycin and chloramphenicol resistant1O35E.mutant; kanamycin resistant32O35E.mutant; kanamycin resistant32O35E.mutant; kanamycin resistant32O35E.12-hag triple mutant; resistant to kanamycin, chloramphenicol, and erythromycinThis studyDH5 (DH5 was transformed as described previously (17). Restriction enzymes were purchased from New England Biolabs, Inc., Beverly, Mass. Electrocompetent was prepared and DNA was electroporated into these bacteria as described previously (20). DNA sequencing was performed by using an ABI PRISM 310 genetic analyzer (PE Biosystems, Rotkreuz, Switzerland) with a Big Dye Terminator cycle sequencing ready reaction kit (PE Biosystems). Sequences were analyzed and aligned by using the Lasergene software package (DNASTAR Inc., Madison, Wis.). Construction of the isogenic triple knockout mutant O35E.12-O35E was amplified with forward primer hagF3 (5-CAGGGCAAGTTGGCAGTGTATG-3) and reverse primer hagB3 (5-TGGAGACAAAGTCAACCGCTTC-3). The PCR product was ligated into plasmid pGEM-T-Easy (Promega). An erythromycin resistance cassette amplified by PCR from plasmid pJDC9 (9) was ligated into the was used for electroporation of competent O35E.12, the double knockout mutant (Table ?(Table1).1). Transformants were selected on BHI agar plates containing 1 mg of erythromycin/liter. Insertional inactivation of was confirmed by PCR analysis, sequencing, Southern blot analysis, and a phenotypic autoagglutination test (37). Saliva sampling. The study was approved by the local ethics committee, and informed consent was TTA-Q6(isomer) given by the study participants. Unstimulated saliva samples were collected from 14 healthy adult volunteers by using a Salivette collection system (Sarstedt, Nmbrecht, Germany). Saliva-soaked cotton tampons were centrifuged for 10 min at 2,200 O35E. OMP of strain O35E were resolved by SDS-PAGE and transferred to PVDF membranes. OMP strips were incubated with saliva samples from 14 healthy adult volunteers. All saliva samples contained sIgA directed against OMP of O35E. OMP were separated by SDS-7.5% PAGE, transferred to PVDF membranes, and incubated with saliva samples at a 1:20 dilution or with monoclonal antibody 17C7 hybridoma culture supernatant (recognizes both UspA1 and UspA2) at a 1:4 dilution. Numbers.