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Moraxella catarrhalis is a common commensal of the human respiratory tract that has been associated with a number of disease states, including acute otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. During studies to investigate the outer membrane proteins of this bacterium, two novel major proteins, of approximately 19 kDa and 16 kDa (named OMP J1 and OMP J2, respectively), were identified. Further analysis indicated that these two proteins possessed almost identical gene sequences, apart from two insertion/deletion events in predicted external loops present within the putative barrel-like structure of the proteins. The development of a PCR screening strategy found a 100% (96/96) incidence for the genes encoding the OMP J1 and OMP J2 proteins within a set of geographically diverse M. catarrhalis isolates, as well as a significant association of OMP J1/OMP J2 with both the genetic lineage and the complement resistance phenotype (Fisher's exact test; P < 0.01). Experiments using two ΔompJ2 mutants (one complement resistant and the other complement sensitive) indicated that both were less easily cleared from the lungs of mice than were their isogenic wild-type counterparts, with a significant difference in bacterial clearance being observed for the complement-resistant isolate but not for its isogenic ΔompJ2 mutant (unpaired Student's t test; P < 0.001 and P = 0.32). In this publication, we characterize a novel outer membrane protein of Moraxella catarrhalis which exists in two variant forms associated with particular genetic lineages, and both forms are suggested to contribute to bacterial clearance from the lungs.
The gram-negative bacterium Moraxella catarrhalis is a common commensal of the human upper respiratory tract which has been associated with a number of disease states, including acute otitis media in children (9, 14) and both acute and chronic bronchitis in adults (16, 32). Nosocomial outbreaks of this pathogen have also been reported (10, 34), as well as casesof nearly fatal pneumonia (11). The morbidity burden of M.catarrhalis is particularly noticeable in young children suffering from recurrent otitis media episodes (13) and in adults presenting with chronic obstructive pulmonary disease (35).
One particularly important virulence trait of M. catarrhalis is serum resistance (21), with several outer membrane proteins (OMPs) being implicated in the expression of this particular phenotype. Of particular importance is the UspA2 protein, a vitronectin binding protein whose N-terminal half may confer complement resistance on certain isolates (1, 33, 41). Other OMPs associated with virulence include the iron acquisition protein CopB (20), a hemagglutinin (28), and the lipooligosaccharide (44). Interestingly, there is increasing evidence to suggest that particular virulence traits are associated with distinct subpopulations of M. catarrhalis (8, 12, 42).
Several OMPs of M. catarrhalis have been shown to elicit an antibody response in humans and have therefore been suggested as potential vaccine candidates; these include the immunoglobulin D-binding protein (15) and the major heat-modifiable protein Omp CD (31, 43). However, an ideal vaccine candidate has not yet been described.
In this article, a novel outer membrane protein of M. catarrhalis that exists in two major forms (OMP J1 and OMP J2) is described and characterized. The sequence variation of the two forms and their relationship to both genetic lineage and the complement resistance phenotype are discussed. Preliminary investigations into the role of the protein were performed by comparing the clearance of two OMP J2 knockout (ΔompJ2) mutants with that of their isogenic wild-type counterparts in a mouse model of pulmonary infection.
In total, a group of 96 M. catarrhalis isolates were utilized in this study, comprising 35 isolates from The Netherlands (1989-1997), 6 isolates from Ghana (1995), and 55 isolates from the United States (1991-1994; kindly supplied by H. Faden, Department of Pediatrics, Children's Hospital of Buffalo, Buffalo, New York). All of the isolates were cultured from children on Columbia blood agar, apart from six of the Dutch isolates, which were cultured from adults.
One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed using standard techniques, with 100 μg protein loaded per gel lane. Membrane fractions of M.catarrhalis were isolated by ultrasonic treatment and extraction in 1% sarcosyl according to the methodology previously described by Klingman and Murphy (24). Tandem mass spectrometry was performed on trypsin digests of the 19-kDa and 16-kDa proteins (OMP J1 and OMP J2, respectively) to identify their amino acid sequences, which were then compared to an in silico translation (all six reading frames) of the nonannotated M. catarrhalis genome sequence available at GenBank (accession numbers AX067426 to AX067466, comprising 41 contigs ranging in size from 429 bp to 261,300 bp). Identification of the relevant protein and gene sequences allowed PCR screening to be performed and sequencing primers to be designed.
PCR screening of M.catarrhalis isolates for ompJ and its genetic variants was performed using standard PCR techniques and the primer pair 19kDres.f and 19kDres.r (details for all primers used are given in Table Table1).1). The expected PCR product sizes were approximately 363 bp (ompJ1) and 333 bp (ompJ2). PCR sequencing of the ompJ genes from 14 M. catarrhalis isolates (representative of the total group of 96 isolates used in this study) was performed using the PCR sequencing primers 19kDseqf, 19kDseqf2, 19kDseqr, and 19kDseqr2. Details of the 14 representative isolates chosen for ompJ sequencing are shown in Table Table2.2. Pulsed-field gel electrophoresis (PFGE) genotypes and complement resistance phenotypes were obtained by reference to the work of Verduin et al. and Hays et al. (18, 42).
The regions immediately flanking the ompJ gene were amplified using primer pair 19kDKO1f.Bam/19kDKO1r.Pst (700-bp upstream fragment) and primer pair 19kDKO2r.Bam/19kDKO2f (500-bp downstream fragment). Both fragments were then digested with PstI and ligated, and the 1,200-bp product (minus an internal 410-bp fragment of ompJ) was reamplified using primers 19kDKO1f.Bam and 19kDKO2r.Bam. The PCR product was digested with BamHI, ligated into plasmid pGEM-7zf(+) (Promega Corporation), and used to transform One Shot TOP10 Escherichia coli cells (Invitrogen). Plasmids containing the insert were selected by PCR analysis of white, ampicillin-resistant E. coli colonies. After extraction, the plasmid was digested using PstI, and an internal kanamycin resistance gene cassette (obtained by PstI digestion of plasmid pUC4K [Amersham Pharmacia Biotech]) was ligated into the ompJ PstI site. The construct was used to transform One Shot TOP10 E. coli cells, with selection on Mueller-Hinton (MH) agar containing 5 μg/ml kanamycin. M. catarrhalis isolates were subsequently naturally transformed with PCR amplification products [primer pair 19kDKO1f.Bam/19kDKO2r.Bam, with pGEM-7zf(+) ΩompJ ΔkanR as the template] and selected on MH agar containing 5 μg/ml kanamycin. The presence of the ΔompJ knockout construct in the chromosomes of kanamycin-resistant M.catarrhalis colonies was confirmed by PCR (primers 19kDaKO.ctrlf, 19kDaKO.ctrlr, KanR1, and KanR2) as well as by the absence of OMP J protein expression (established using one-dimensional SDS-PAGE analysis of outer membrane protein extracts). ΔompJ2 knockout constructs were prepared in M.catarrhalis isolates 3.9 and 3.18.
In order to determine the effect of OMP J on pulmonary clearance in a mouse model, two M. catarrhalis ompJ2-containing gene knockout mutants (3.9ΔompJ2 and 3.18ΔompJ2) were constructed and compared to their isogenic wild-type isolates in a mouse pulmonary clearance study. The mouse pulmonary clearance protocol was based on those published by Forsgren et al. (15) and Unhanand et al. (39). Basically, M. catarrhalis isolates were grown overnight at 37°C on either MH agar (wild-type isogenic isolates) or MH agar incorporating 5 μg/ml kanamycin (ΔompJ2 mutants) and then grown to mid-log phase in MH broth. A 50-μl volume containing 1 × 108 CFU of each isolate was inoculated intranasally into the lungs of anesthetized BALB/c mice, which were sacrificed at 0.5 and 3 h postinfection. Colony counts of surviving M.catarrhalis cells were performed on MH agar after overnight incubation at 37°C. Five mice were sacrificed for each isolate tested (total = 2 × 20 mice). Survival values (percentages) at 3 h postinoculation were calculated by taking the average CFU count of each isolate at the 0.5-h time point as 100%. Statistical analysis was performed by using a two-tailed unpaired student’s t test to compare the difference in lung log10 CFU/ml bacterial survivors 0.5 and 3 h after nasal inoculation. Further analysis involved generating growth curves for both the wild type and the isogenic ΔompJ2 mutants in order to verify that no significant difference in growth rates existed. The animal studies described in this publication were performed in accordance with the ethical and legal requirements of the Erasmus MC, Rotterdam, The Netherlands, and with the approval of the Animal Studies Ethics Committee of the same institution.
Serum bactericidal survival testing of M. catarrhalis isolates 3.9, 3.18, and their isogenic ΔompJ2 mutants was based on a protocol described by Attia et al. (1). Briefly, bacterial cultures were grown to mid-log phase (approximately 5 × 108 CFU/ml) in MH broth and diluted 1/1,000 in Veronal-buffered saline containing 0.1% (wt/vol) gelatin. Twenty microliters of this bacterial suspension was added to 160 μl Veronal-buffered saline-gelatin, and 20 μl of human pooled sera (HPS) (or 20 μl heat-inactivated pooled human sera that were previously incubated at 56°C for 30 min) was added. Ten-microliter aliquots of each reaction mix were plated onto MH agar after 0 and 30 min of incubation at 37°C. Four independent experiments were performed per bacterial isolate, using HPS obtained from eight healthy adult volunteers. Survival values (percentages) were calculated by comparing the means of results after 0 min and 30 min of exposure to HPS from four independent experiments. Statistical differences between wild-type and ΔompJ2 isolates were calculated using log10 CFU/ml survival values after 30 min of HPS exposure and two-tailed unpaired Student's t test (after first ensuring that there was no significant difference in CFU/ml results after 0 min of exposure to HPS).
One-dimensional SDS-PAGE analysis of outer membrane protein extracts from 48 of the 96 M. catarrhalis isolates used in this study revealed the presence of two putative proteins (of approximately 19 kDa and 16 kDa) and an association between protein and a complement-resistant or -sensitive phenotype (Fig. (Fig.1).1). Subsequent tandem mass spectroscopy analysis indicated that the two proteins consisted mainly of identical polypeptide sequences, indicating that both proteins were in fact two variants of the same protein. These sequences were found in only one translated open reading frame (ORF) within the whole M. catarrhalis proteome, allowing the protein and its genomic location to be identified. Using these data, the surrounding ORFs of ompJ were found to include genes encoding proteins involved in the recognition and processing of DNA lesions (uvrC) and glycolate metabolism (pgp), a glutamyl-tRNA synthetase (gluRS), and a protein involved in the suppression of a DnaK-like heat shock protein (data not shown).
Sequence analysis of the ompJ genes from 14 geographically distinct M.catarrhalis isolates indicated that two distinct phylogenetic lineages existed (GenBank accession numbers DQ008974 to DQ008986 and DQ105644), with 90% identity (over 579 bp) between the two most divergent sequences (Fig. (Fig.2).2). This clustering occurred independent of the geographical origin of the isolates. Translation of the 14 gene sequences showed three insertion/deletion events which were common to each cluster, occurring at amino acid positions 69 to 71, 82 to 97, 138 to 144, and 151 to 153 of the isolate F3.57 sequence. Sequence comparisons of OMP J1 with known protein sequences indicated that the protein was similar to a hypothetical protein found in a closely related Psychrobacter sp. (GenBank accession number gi|52853456|ref|ZP_00145674.2|) but contained little sequence similarity with other known proteins. Secondary structure analysis revealed the putative positions of α-helix and β-sheet regions within the OMP J sequence, providing indications that OMP J may form a beta-barrel-type structure with considerable structural similarity to a superfamily of proteins which include the Omp21 protein from Comamonas (Deftia) acidovorans (3, 4), the Neisseria opacity-associated protein (Opa) (17), its homologue Neisseria surface protein A (NspA) (40), and Enterobacter cloacae outer membrane protein X (OmpX) (37, 38). The positions of two of the major insertion/deletion events occurred in a putative external loop (loop 2) of the predicted barrel-like structure.
PCR screening primers for ompJ were designed using conserved sequences found in both the ompJ1 and ompJ2 genes and yielded positive PCR products from 100% (96/96) of the M. catarrhalis isolates tested (Fig. (Fig.3).3). Of these, 97% (72/74) of complement-resistant isolates generated shorter PCR products, of approximately 333 bp (ompJ2-like), while 74% (14/22) of complement-sensitive isolates generated larger PCR products, of approximately 363 bp (ompJ1-like). This distribution of ompJ genes between complement resistance phenotypes was found to be highly significant (Fisher's exact test; P < 0.001). Importantly, there also appeared to be a significant correlation between the two major forms of ompJ genes and the genetic lineage (as determined by PFGE). In particular, by cross-referencing the results obtained with 41 isolates previously genotyped by Verduin et al. (42), it was observed that 22/28 isolates from a short-branched lineage harbored the ompJ2 gene and that 9/13 isolates from a longer-branched lineage harbored the larger ompJ1 gene. Further cross-referencing of ompJ PCR data to data for 55 American isolates previously genotyped by Hays et al. (18) indicated that 50/50 isolates from a short-branched lineage harbored the ompJ2-like gene and that 4/5 isolates from a longer-branched lineage harbored the larger ompJ1-like gene (data not shown). These results were highly significant (Fisher's exact test; P = 0.005 and P < 0.0001, respectively). PCR screening results did not reveal the presence of multiple ompJ PCR products within individual isolates. Furthermore, sequence searching of the only (unannotated) publicly available M. catarrhalis whole genome sequence (GenBank accession numbers AX067426 to AX067466) revealed the presence of only a single copy of the ompJ gene (ompJ2) within this isolate.
Attempts to create ΔompJ2 gene knockouts in M. catarrhalis were successful for the complement-resistant isolate 3.9 and the complement-sensitive isolate 3.18 (Fig. (Fig.4),4), with further studies indicating that knocking out these ompJ2 genes did not affect the expression of other outer membrane proteins (Fig. (Fig.55).
The average M. catarrhalis survival in a pulmonary mouse model 3 h after inoculation was measured to be 23%, 78%,89%, and 92% for isolates 3.9, 3.9ΔompJ2, 3.18, and 3.18ΔompJ2, respectively (Fig. (Fig.6).6). Statistical analysis using two-tailed unpaired Student's t test indicated a statistically significant decrease in log10 CFU/ml bacterial survivors after 3 h for isolate 3.9 (P < 0.001), but not for its isogenic ΔompJ2 mutant (P = 0.32), isolate 3.18 (P = 0.25), or the isogenic 3.18ΔompJ2 mutant (P = 0.49). Moreover, both ΔompJ2 mutants survived in greater numbers than their respective wild-type parents. Growth curve comparisons showed no difference in exponential growth rate between the ΔompJ2 mutants and their respective isogenic isolates, although it was noted that the final concentration of ΔompJ2 mutant cells was somewhat lower in the plateau phase of the growth cycle (Fig. (Fig.77).
Serum bactericidal survival results for M. catarrhalis isolates 3.9, 3.18, and their respective isogenic ΔompJ2 mutants are shown in Fig. Fig.8.8. No significant difference was observed between wild-type isolate 3.9 and its ΔompJ2 mutant in either HPS or heat-inactivated HPS (P = 0.44 and 0.16, respectively), although survival values were reduced for the 3.9ΔompJ2 knockout isolate in both HPS and inactivated HPS. The complement-sensitive isolate 3.18 actually showed an increase in survival in both HPS and heat-inactivated HPS. However, the significance of any conclusions that could be drawn was limited by the detection limit of the methodology used (zero colonies were recorded after incubation in HPS).
Early studies of the outer membrane proteins of M. catarrhalis identified and characterized eight major proteins within this species, ranging from 98 kDa to 21 kDa and named OMP A to OMP H (29). Several of these OMPs have now been characterized and include proteins involved in iron acquisition, e.g., CopB (7), LbpB/A (6), and TbpB (36), fatty acid uptake (5), and adhesion (22, 23, 26). A role for some of these proteins in M. catarrhalis virulence and pathogenicity has been suggested, including the UspA2 (1, 41) and OMP E (30) proteins, which appear to facilitate serum resistance. Other experiments have shown that a CopB-binding monoclonal antibody (MAb10F3) could enhance the clearance of M. catarrhalis in a mouse pulmonary disease model, binding to 70% of M. catarrhalis isolates tested (19). Furthermore, the finding that adults develop new serum immunoglobulin G and mucosal immunoglobulin A to bacterial surface epitopes after exacerbations of chronic obstructive pulmonary disease shows the importance of the humoral immune response to M. catarrhalis-mediated infection (2).
In this study, one-dimensional SDS-PAGE analysis of outer membrane protein extracts from several isolates of M. catarrhalis revealed the presence of a small and novel major outer membrane protein (OMP J) which was found to exist in two major forms, with molecular masses of approximately 19 kDa and 16 kDa (OMP J1 and OMP J2, respectively). Sequence analysis and database searching indicated limited homology between the OMP J protein and ompJ gene and other known protein and gene sequences, with the possible exception being a hypothetical protein found in a closely related Psychrobacter sp. However, secondary structure prediction for OMP J indicated that the protein might possess a barrel-like tertiary structure, which taken in context with the presence of a signal sequence, suggests that OMP J may be an integral membrane protein. Indeed, the sequence/structure results suggest that OMP J belongs to a superfamily of proteins that include the OPA (opacity) family of proteins of Neisseria spp., which mediate bacterial adherence to epithelial cells by interacting with (for example) the receptors for the human carcinoembryonic antigen cell adhesion molecule on human polymorphonuclear phagocytes. Other members of this superfamily include Neisseria surface protein A (NspA), a highly conserved protein of unknown function which is a promising vaccine candidate against both Neisseria meningitidis and Neisseria gonorrhoeae (27, 40). Structurally, the major difference between the two forms of OMP J seems to reside in the deletion of 12 amino acids forming part of a putative loop 2 region, but the consequences of this deletion with respect to the function and antigenic properties of the two proteins have yet to be determined.
PCR screening of isolates suggested that only a single copy of the ompJ gene is present in M. catarrhalis species and that it may be found in 100% of isolates, indicating a significant role for OMP J in the M. catarrhalis life cycle. No clear indication of the likely function of OMP J was obtained by inspecting neighboring ORFs, which appeared to comprise a mix of putative housekeeping genes involved in various metabolic and DNA repair activities. Note, however, that the direction of transcription of the ompJ gene lies in the opposite orientation to that of the neighboring ORFs.
A statistically significant association between the two major forms of OMP J, the genetic lineage, and the complement resistance phenotype was observed in diverse geographical isolates. However, serum resistance experiments using two ΔompJ2 mutants did not indicate a significant role for OMP J2 in facilitating complement resistance. It seems likely that the association of OMP J1 and OMP J2 with the complement phenotype is simply a consequence of their association with different genetic lineages previously associated with the differential expression of virulence traits (8). In fact, most evidence implicates the UspA2 outer membrane protein as the major contributor to the complement resistance phenotype within this species (1, 41).
Previous investigations have shown that alterations in the expression of outer membrane proteins and lipooligosaccharide in M. catarrhalis may significantly impact the in vivo clearance of isogenic mutants in a mouse model of pulmonary infection (25). Studies investigating the role of ompJ2 in the clearance of M. catarrhalis from the lungs of mice showed that the absence of OMP J2 resulted in a reduction in bacterial clearance from the lungs, suggesting that OMP J2 may actually be a target for the immune system.
In this publication, we identified and characterized a novel outer membrane protein (OMP J) of M. catarrhalis which appears to be present in two major lineage-specific forms. Furthermore, the ompJ gene appears to be universally present within the species and may play a role in immune system-mediated bacterial clearance from the lungs.
We thank H. Faden (Department of Pediatrics, Children's Hospital, Buffalo, New York) for kindly supplying the American M. catarrhalis isolates used in this study, as well as A. Ott (Department of Medical Microbiology, Erasmus MC).
This work was funded by the Sophia Children's Hospital Foundation, Erasmus MC, Rotterdam, The Netherlands (grant number 397).