Biofilm formation by the human pathogen Neisseria meningitidis was analyzed. Biofilm-forming meningococcal strains were identified and quantitated by crystal violet staining. Laser scanning confocal microscopy of the meningococcal biofilm revealed variable layers up to 90 m in thickness. A total of 39 meningococcal isolates were studied; 23 were nasopharyngeal-carriage isolates, and 16 were invasive-disease isolates. Thirty percent of carriage isolates and 12.5% of invasive-disease isolates formed biofilms proficiently on a polystyrene surface. Generally, the strains that formed biofilms showed high-level cell surface hydrophobicity, characteristic of strains lacking a capsule. The inhibitory role of capsule in biofilm formation was further confirmed by comparing the biofilm-forming capabilities of a serogroup B wild-type strain of a disease-associated isolate to those of its capsule-deficient mutant (ctrA). Some strains of meningococci form biofilms, and this process is likely important in menigococcal colonization.Bacterial biofilms are sessile bacterial communities that adhere to each other and solid surfaces and are enclosed in an exopolysaccharide matrix (6). Biofilms are the predominant communities of many bacterial species in numerous ecosystems. Formation of biofilms involves participation of the extracellular-matrix and cellular-surface molecules, including membrane proteins. Biofilm formation also requires considerable bacterial energy and resources. The formation of biofilms begins with the attachment of the planktonic cells to a suitable surface, followed by replication and spreading. Eventually, the biofilms mature to differentiated forms. Exopolysaccharides play a key role in the establishment of biofilm architecture (6).In clinical settings, bacteria in biofilms are less susceptible to antimicrobial agents and host immune responses, thereby becoming persistent colonizers or sources of chronic infections (8). Bacteria are released from biofilms as individual planktonic cells or as a result of the sloughing of the biofilms. While many biofilms form on abiotic surfaces such as medical devices, some also develop on living tissues, as in the case of endocarditis or cystic fibrosis (8).Studies of biofilm formation by the Neisseria species are very limited, and most of those species examined have been oral commensals (4,20,24,26,38). Biofilm formation by Neisseria meningitidis, an etiologic agent of epidemic sepsis and bacterial meningitis, has not been documented. Meningococci are isolated from 5 to 10% of the normal population, and the colonization of the human nasopharyngeal mucosal surface by meningococci is the first step of the host-parasite interaction.Successful meningococcal colonization requires initial attachment facilitated by pili and subsequent interaction of other secondary-surface molecules with the host mucosal surface (12,31,36,43).In this study, the formation of the biofilms by N. meningitidis was assessed. In addition, the roles of the bacterial-surface molecules (pilus, capsule, and ...
Nontypeable Haemophilus influenzae (NTHI) causes recurrent respiratory tract infections in patients with chronic bronchitis. To elucidate the human immune response to NTHI, sera from 2 patients with exacerbations of chronic bronchitis due to NTHI were characterized. Both patients developed new bactericidal antibodies following infection. Immunoblot assays with homologous strains revealed antibodies to many antigens, with minimal difference between pre- and postexacerbation sera. By contrast, whole cell radioimmunoprecipitation, which detects antibodies exclusively to epitopes exposed on the bacterial surface, revealed that both patients made new antibodies to a limited number of antigens following infection, including P2, the major outer membrane protein of NTHI. Adsorption experiments showed that strain-specific, surface-exposed epitopes on the P2 molecule are targets for bactericidal antibodies. These results indicate that new bactericidal antibodies following infection by NTHI recognize antigenically heterogeneous surface-exposed epitopes on P2 and other surface proteins of NTHI.
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 protein is the major outer membrane protein of nontypeable Haemophilus influenzae and is a potential target of a protective immune response. Nine monoclonal antibodies (MAbs) to P2 were developed by immunizing mice with nontypeable H. influenzae whole organisms. Each MAb reacted exclusively with the homologous strain in a whole-cell immunodot assay demonstrating exquisite strain specificity. All nine MAbs recognized abundantly expressed surface-exposed epitopes on the intact bacterium by immunofluorescence and immunoelectron microscopy. Each MAb was bactericidal to the homologous strain in an in vitro complementmediated killing assay. Immunoblot assay of cyanogen bromide cleavage products of purified P2 indicated that MAb 5F2 recognized the 10-kDa fragment, and the other eight MAbs recognized the 32-kDa fragment. Competitive ELISAs confirmed that 5F2 recognized an epitope that is different from the other eight MAbs. To further localize epitopes, MAbs 5F2 and 6G3 were studied in protein footprinting by using reversed-phase high-performance liquid chromatography. Three potential epitope-containing peptides which were reactive in an enzyme-linked immunosorbent assay with both 5F2 and 6G3 were isolated. These peptides were identified by N-terminal amino acid sequence and localized to loops 5 and 8 of the proposed model for P2. Fusion proteins consisting of glutathione S-transferase fused with variable-length peptides from loops 5 and 8 were expressed in the pGEX-2T vector. Immunoblot assay of fusion peptides of loops 5 and 8 confirmed that SF2 recognized an epitope within residues 338 to 354 of loop 8; 6G3 and the remaining MAbs recognized an epitope within residues 213 to 229 of loop 5. These studies indicate that nontypeable H. influenzae contains bactericidal epitopes which have been mapped to two different surface-exposed loops of the P2 molecule. These potentially protective epitopes are strain specific and abundantly expressed on the surface of the intact bacterium.
Nontypeable Haemophilus influenzae (NTHI) frequently causes recurrent infections of the respiratory tract in humans. Previous indirect evidence suggested that a strain-specific immune response occurs following infection and that this immune response is directed at an immunodominant epitope on the bacterial surface. To test this hypothesis, mice and rabbits were immunized with whole cells of a strain of NTHI and the antiserum was characterized to identify the antigens to which antibodies were directed. All animals made a prominent antibody response to the loop 5 region of the P2 molecule, which is the major outer membrane protein. Rabbit serum showed complement-dependent bactericidal activity. Adsorption of the immune serum with the loop 5 fusion peptide removed bactericidal activity and also abolished reactivity to P2 detected by an immunoblot assay, an enzyme-linked immunosorbent assay, and a radioimmunoprecipitation assay. These data indicate that immunization with whole cells of NTHI results in a prominent antibody response which is directed at epitopes on the loop 5 region of the P2 molecule. Thus, a strain-specific immune response to NTHI occurs as a result of the expression of an immunodominant epitope on the P2 molecule.
Studies of meningococcal pathogenesis have been severely restricted due to the absence of an adequate animal model. Given the significance of iron in meningococcal pathogenesis, we developed a model of Neisseria meningitidis colonization in outbred adult mice that included daily administration of iron dextran. While receiving iron, the animals were inoculated intranasally with the initial doses of bacterial suspension. Meningococci were recovered from the animals by nasopharyngeal washes. Approximately half of the animals inoculated with 10 7 CFU remained colonized 13 days after the initial bacterial inoculation. The model was further evaluated with genetically defined isogenic serogroup B mutant strains, and the colonization capabilities of the mutants were compared to that of the wild-type parent. A mutant that produces truncated lipooligosaccharide (KDO 2 -lipid A) and a mutant defective in capsule transport were dramatically impaired in colonization. A mutant defective in pilus transport (pilQ) showed moderately impaired colonization. The immunological aspect of the model was also evaluated by challenging mice after immunization with homologous whole-cell meningococci. The immunized mice were protected from colonization of the homologous strain. In this model, long-term meningococcal colonization was maintained, allowing us to study the effects of specific genetic mutation on colonization. In addition, this model allows investigation of the role of active immune response against meningococci.
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