The P2 porin protein is the most abundant protein in the outer membrane of nontypeable Haemophilus influenzae (NTHI). Analysis of sequences of P2 from different strains reveals the presence of both heterogeneous and conserved surface-exposed loops of the P2 molecule among strains. The present study was undertaken to test the hypothesis that antibodies to a conserved surface-exposed loop are bactericidal for multiple strains of NTHI and could thus form the basis of vaccines to prevent infection due to NTHI. Polyclonal antiserum to a peptide corresponding to loop 6 was raised and was immunopurified over a loop 6 peptide column. Analysis of the antibodies to whole organisms and peptides corresponding to each of the eight loops of P2 by immunoassays revealed that the antibodies were highly specific for loop 6 of P2. The immunopurified antibodies bound to P2 of 14 of 15 strains in immunoblot assays. These antibodies to loop 6 demonstrated complement-mediated bactericidal killing of 8 of 15 strains. These results support the concept of using conserved regions of the P2 protein as a vaccine antigen.Nontypeable Haemophilus influenzae (NTHI) is a small, gram-negative bacillus which causes otitis media in children and lower respiratory infections in adults with chronic obstructive pulmonary disease (COPD). In both otitis media and COPD, patients routinely suffer recurrent episodes of disease (15,21). Factors such as health care costs, pain and suffering, and lost work time underscore the need for a vaccine against NTHI (10,14,22).The ability of NTHI to cause recurrent infections is in part attributable to antigenic variability in several surface-exposed loops of major outer membrane protein P2 (2, 5, 26). The P2 protein is a homotrimeric porin which constitutes approximately one-half of the total outer membrane protein of the organism. The loop 5 region is highly heterogeneous among strains and contains almost all of the epitopes to which an antibody response is mounted when animals are immunized with the whole organism (30). Adults with COPD make new antibodies to strain-specific epitopes on P2 following infection by NTHI (31). Thus, immunity against NTHI is most often strain specific, leaving the patient vulnerable to reinfection by other strains.One approach to vaccine development for NTHI has been to study antigenically conserved outer membrane proteins as potential vaccine antigens. In view of the abundant expression of P2 on the bacterial surface, identification of a conserved region on the P2 molecule to which immune responses could be directed would be a significant step towards developing a vaccine against NTHI.In this study, antibodies to a conserved loop of the P2 molecule of NTHI (loop 6) were raised and studied for their ability to recognize the P2 molecules of heterologous strains. Since bactericidal antibody is associated with protection from otitis media due to NTHI (8, 25), antibodies to loop 6 were also assessed for their ability to direct killing of heterologous strains. MATERIALS AND METHODSBacterial s...

The P2 porin is the most abundant protein in the outer membrane of nontypeable Haemophilus influenzae. Analysis of P2 sequences from a limited number of strains reveals the presence of both heterogeneous and conserved surface-exposed loops of the P2 molecule among strains. We have previously shown that antibodies raised against the loop 6 sequence of P2 from strain 5657 are bactericidal against multiple isolates. In this study, we determined the nucleotide sequence of the loop 6 region of the P2 molecule from 108 strains of nontypeable H. influenzae in order to assess more rigorously the degree of conservation of loop 6. Based on this analysis, we identified a conserved sequence, different from that of strain 5657, that occurs in approximately one-third of the strains sequenced. To assess the potential of this peptide as a vaccine antigen, antibodies raised to a multiple antigenic peptide corresponding to this sequence were characterized with respect to specificity for the P2 molecule and reactivity with heterologous strains in immunoblot assay, flow cytometry and bactericidal assays. Antibodies were reactive to the P2 molecule of 16 of 20 strains tested by immunoblot assay. Antibodies recognized nine of the 20 strains in a flow cytometry assay, and 13 of 20 demonstrated complement-mediated killing in bactericidal assays. These results support the concept of using conserved regions of the P2 protein as a vaccine antigen.

Acinetobacter baumannii is a Gram-negative opportunistic nosocomial pathogen that causes pneumonia and soft tissue and systemic infections. Screening of a transposon insertion library of A. baumannii ATCC 19606 T resulted in the identification of the 2010 derivative, which, although capable of growing well in iron-rich media, failed to prosper under iron chelation. Genetic, molecular, and functional assays showed that 2010's iron utilization-deficient phenotype is due to an insertion within the 3= end of secA, which results in the production of a C-terminally truncated derivative of SecA. SecA plays a critical role in protein translocation through the SecYEG membrane channel. Accordingly, the secA mutation resulted in undetectable amounts of the ferric acinetobactin outer membrane receptor protein BauA while not affecting the production of other acinetobactin membrane protein transport components, such as BauB and BauE, or the secretion of acinetobactin by 2010 cells cultured in the presence of subinhibitory concentrations of the synthetic iron chelator 2,2=-dipyridyl. Outer membrane proteins involved in nutrient transport, adherence, and biofilm formation were also reduced in 2010. The SecA truncation also increased production of 30 different proteins, including proteins involved in adaptation/tolerance responses. Although some of these protein changes could negatively affect the pathobiology of the 2010 derivative, its virulence defect is mainly due to its inability to acquire iron via the acinetobactin-mediated system. These results together indicate that although the C terminus of the A. baumannii ATCC 19606 T SecA is not essential for viability, it plays a critical role in the production and translocation of different proteins and virulence.

Bacillus cereus is a common cause of gastrointestinal diseases. The majority of individuals with B cereus-related food poisoning recover without any specific treatment. It can, however, rarely cause invasive disease in immunocompromised patients.