Analysis of the expression of the putatively virulence-associated neisserial protein RmpM (class 4) in commensal Neisseria and Moraxella catarrhalis strains

Abstract

The RmpM protein has been reported to be present only in pathogenic Neisseria species. In the present study we demonstrate that this protein is also present at least in N. lactamica and N. sicca strains. The N. lactamica protein reacts with a RmpM-specific monoclonal antibody (185, H-8), having a molecular mass (∼31 kDa) slightly lower than that of the meningococcal RmpM, and mouse antibodies from sera against outer membrane vesicles from both N. lactamica and N. sicca strains cross-react with the meningococcal RmpM. PCR and hybridization experiments with a complete rmpM probe agree with the immunodetection experiments. Our results strongly suggest that the meningococcal RmpM should not be considered a virulence marker, and the presence of this protein in the commensal species agrees with its role as a structural protein, proposed for the RmpM, which should be considerably conserved in the Neisseria species.

Neisseria meningitidis, Commensal Neisseria, P4, Class 4 protein, RmpM

1 Introduction

Pathogenic and commensal species of the genus Neisseria produce several major outer membrane proteins (OMPs), which play important roles in several functions of these bacteria, especially in the pathogenic species, N. meningitidis and N. gonorrhoeae. The meningococcal OMPs have been classified into five groups, 1–5, on the basis of their molecular mass [1]. Classes 1, 2 and 3 are outer membrane porins; class 5 are Opa proteins, with a role in adhesion and invasion of the host cells. The class 4 protein, also termed RmpM (reduction-modifiable protein M) due to its characteristic electrophoretic behavior after reduction, has been shown to be associated with some other OMPs such as the lactoferrin (LbpA), transferrin (TbpA) and siderophore (FrpB) receptor components, apparently acting as a stabilizer for the receptor complexes [2]. It has also been reported that antibodies to the RmpM are able to block the bactericidal activity of other antibodies against meningococcal proteins [3], although recent work with purified human anti-RmpM and monoclonal antibodies (mAbs) showed no evidence of such a blocking effect [4].

Apart from the proposed role of the RmpM in the stabilization of several meningococcal and gonococcal OMPs, mentioned above, the function of this protein in physiological or pathogenic aspects of Neisseria is still unknown, although it has been proposed that it must be implicated in virulence because the rmpM gene has been reported to be found exclusively in the chromosomal DNA of the pathogenic species [5]. Due to the inefficacy of current meningococcal vaccines, especially for serogroup B, the investigation of virulence-associated proteins has been growing in recent years. The complete DNA sequences of two meningococcal strains have been published recently [6,7], making the comparison of the rmpM sequences possible and confirming their high similarity (99.2% identity) and so, the conservation of the gene in this species. An approach to the identification of putative virulence-associated proteins was done on the basis of the DNA sequence of the serogroup B strain, and some of them have been experimentally characterized [8], demonstrating that they are also present in N. lactamica and N. cinerea strains.

In a previous work, we demonstrated that many meningococcal antigenic outer membrane vesicles (OMVs) are cross-reactive with commensal species, mainly N. lactamica, and vice versa. One of the cross-reactive antigens had a molecular mass of 32 kDa, coincident with that of the RmpM. So, in this work we investigate the identity of the 32-kDa antigen using specific mAbs against the RmpM and a mutant strain not expressing the rmpM gene.

2 Materials and methods

2.1 Strains and culture conditions

Eleven N. meningitidis, 12 commensal Neisseria, and six Moraxella catarrhalis strains were used in this study (Table 1). N. meningitidis strains 44/76, M982, and B16B6 are reference strains widely used in many studies. All other strains were from our laboratory collection. The bacteria were cultured under iron restriction in Mueller–Hinton broth supplemented with 39 μM ethylenediamine-di(o-hydroxy-phenylacetic acid) [9].

1

Characteristics of the strains used

Strain	Serogroup/serotypea	Origin
N. meningitidis
NmP0	W135: nt; P1.3,6	Oropharynx
NmP5	B: nt; P1.3,6	Oropharynx
NmP7	B: 4; P1.15	Oropharynx
NmP27	AA: 15; P1.7,16	Oropharynx
Nm30	C: 2b; P1.2	Cerebrospinal fluid
Nm14	B: 4; P1.15	Cerebrospinal fluid
Nm16	B: nt	Cerebrospinal fluid
Nm17	C: 2b; P1.2,5	Blood culture
Nm18	B: 4; P1.2,5	Cerebrospinal fluid
M982	B: 9; P1.22,9	Reference strain; cerebrospinal fluid
B16B6	B: 2a; P1.2	Reference strain; cerebrospinal fluid
44/76	B: 15; P1.7,16	Reference strain; blood culture
44/76 RmpM−	B: 15; P1.7,16	RmpM− mutant derived from 44/76
N. lactamica
NlP2	na	Oropharynx
NlP3	na	Oropharynx
NlP4	na	Oropharynx
NlP5	na	Oropharynx
NlP6	na	Oropharynx
NlP8	na	Oropharynx
NlP9	na	Oropharynx
N. sicca
NsP1	na	Oropharynx
NsP3	na	Oropharynx
NsP4	na	Oropharynx
NsP12	na	Oropharynx
N. mucosa
NmuP0	na	Oropharynx
NmuP1	na	Oropharynx
NmuP2	na	Oropharynx
N. perflava
NpfP0	na	Oropharynx
M. catarrhalis
McP0	na	Oropharynx
McP1	na	Oropharynx
McP2	na	Oropharynx
McP3	na	Oropharynx
McP5	na	Oropharynx
McP8	na	Oropharynx

^aAA, autoagglutinable; nt, not typeable; nst, not subtypeable; na, not applicable.

2.2 Extraction of OMVs

OMVs from most strains were obtained as described previously [10]. Briefly, the growth from liquid cultures was centrifuged at 10 000×g for 10 min at 4°C and the pellet resuspended in 0.1 M acetate buffer–0.2 M LiCl, pH 5.8 (5 ml of buffer per gram of cells). The suspension was then incubated for 2 h at 45°C in a shaking water bath and passed three times in a French pressure cell operating at 1.1×10⁸ Pa. Membrane fragments were separated from the cellular debris by centrifugation at 10 000×g for 10 min at 4°C, and the OMVs in the supernatant were collected by ultracentrifugation at 200 000×g for 10 min at 4°C and suspended in distilled water. Protein content in the suspensions was determined with the bicinchoninic acid protein assay reagent [11]. OMVs from the strains N. meningitidis 44/76 and 44/76 RmpM⁻ were obtained after sodium deoxycholate extraction of cells grown in modified Frantz's medium as described previously [12].

2.3 Immune sera

The production and characterization of mAb 185, H-8 specific for the RmpM, produced at the National Institute of Public Health, Oslo, Norway, have been described previously [4]. Mouse immune sera were obtained using OMVs from iron-restricted cultures [13]. For each strain, five 1-month-old BALB/c mice were injected intraperitoneally each with 20 μg of OMVs in 100 μl of complete Freund's adjuvant on day 0 and 20 μg of OMVs in 100 μl of incomplete Freund's adjuvant on day 14. Finally, 20 μg of OMVs in 100 μl of phosphate-buffered saline was given intravenously on day 28. Mice were bled 5 days later through the retro-orbital plexus and the bloods pooled, allowed to clot at room temperature and centrifuged at 2000×g for 10 min. All the sera were heat-inactivated at 56°C for 30 min and stored in 250-μl aliquots at −20°C until use.

2.4 Detection of the reduction-modifiable protein RmpM (class 4)

The presence of RmpM in the OMVs of the strains was detected after SDS–PAGE and electrotransfer. Aliquots of OMV suspensions containing 20 μg of protein were diluted 1:1 with double-strength sample buffer, heated at 95°C for 10 min and applied to 9–18% gradient acrylamide gels using the discontinuous buffer system of Laemmli [14]. After separation at 200 V constant voltage, the OMV proteins were transferred to PVDF membranes (100 V constant voltage). The membranes were blocked for 1 h at room temperature with 5% BLOTTO (Bio-Rad Laboratories S.A., Madrid, Spain) in Tris-buffered saline (TBS; 20 mM Tris–HCl, pH 7.5, with 500 mM NaCl) and washed three times with TBS-Tween (0.05% Tween 20 in TBS). The membranes were then incubated with the immune sera diluted in antibody buffer (1% BLOTTO in TBS-Tween), washed again with TBS-Tween, and finally incubated with horseradish peroxidase (HRP)-conjugated rabbit anti-mouse immunoglobulins in antibody buffer. HRP was developed with 4-chloro-1-naphthol [15]. Calculation of apparent molecular masses was done using the Bio-Rad Gel-Doc 1000 system (Bio-Rad Laboratories S.A., Madrid, Spain).

OMVs from the strains N. meningitidis 44/76 and 44/76 RmpM⁻ were used as RmpM-positive and -negative controls in all experiments.

2.5 Detection of the rmpM gene

The rmpM gene was detected by standard polymerase chain reaction (PCR) experiments and by dot hybridization using the digoxigenin-labeled rmpM whole gene, isolated from the strain N. meningitidis M982, as the probe. Primers for PCR (pairs R1/L1 and R2/L2; Fig. 1) were built up from published serogroup B N. meningitidis rmpM sequence data [7] obtained from public libraries (The Institute for Genomic Research; locus NMB0382; http://www.tigr.org). Standard reactions for PCR were carried out using the following conditions: one cycle (94°C/5 min, 58°C/2 min and 72°C/1.5 min), 30 cycles (94°C/45 s, 58°C/45 s and 72°C/1.5 min), and a final elongation cycle (72°C/5 min).

1

rmpM cassette (N. meningitidis OMP class 4). R1/L1 and R2/L2 are the primer pairs used for PCR and for purification of the rmpM gene. The N. gonorrhoeae PIII probe used by Wolff and Stern [5] is indicated (bp 286–304).

Chromosomal DNA preparations for dot hybridization assays were obtained from the Neisseria strains grown in agar plates as described previously [16] and hybridization reactions were performed on positively charged nylon membranes. The rmpM probe was obtained from strain N. meningitidis M982 in a two-stage PCR procedure. In the first stage, primers R1 and L1 were used to synthesize a specific fragment that contains the complete rmpM gene and, in the second stage, primers R2 and L2 were used to reamplify a fragment of about 729 bp long corresponding to the rmpM open reading frame (ORF) region (Fig. 1). The rmpM ORF fragment was separated in low-melting-point agarose, purified using the Wizard DNA Clean-Up System (Promega Corp., Madison, WI, USA), and labelled with the DIG DNA Labelling Kit (Boehringer Mannheim, Barcelona, Spain).

3 Results

3.1 Detection of the RmpM with specific mAb in commensal species

Probing OMV proteins after SDS–PAGE and electrotransfer with the specific anti-RmpM 185, H-8 mAb demonstrates that all the N. meningitidis and N. lactamica strains tested synthesize the RmpM or a RmpM-like protein sharing the mAb-specific epitope (Fig. 2A), whereas none of the other commensal Neisseria (Fig. 2B) nor the M. catarrhalis strains (not shown) contained a protein expressing the epitope for mAb 185, H-8. The apparent molecular mass of the RmpM in the N. lactamica strains is lower (∼31 kDa) than that of the meningococcal RmpM (∼34 kDa).

2

Reactivity of the anti-RmpM mAb 185, H-8 with proteins from OMVs obtained from different strains. A: OMVs from N. meningitidis (a–f: strains NmP5, M986, B16B6, NmP0, NmP27, and Nm30), and N. lactamica (g–k: strains NlP2, NlP3, NlP4, NlP8, and NlP9). B: OMVs from N. meningitidis (a–e: strains M982, Nm14, Nm16, NmHC17, and Nm18), N. sicca (f–h: strains NsP1, NsP3, and NsP4), N. mucosa (i, j: strains NmuP1 and NmuP2), and N. perflava (k: strain NpfP0). Molecular mass markers (M) and OMVs from N. meningitidis 44/76 (C; RmpM⁺ control) are also included.

3.2 Demonstration of antibodies reacting with the meningococcal RmpM in sera raised against OMVs from commensal species

Results obtained probing OMVs from the RmpM⁺ and RmpM⁻ variants of the strain N. meningitidis 44/76 with mouse sera raised against OMVs from two N. lactamica and two N. sicca strains show that, in all cases, the commensal RmpM – or the above mentioned RmpM-like protein – induces in vivo antibodies that cross-react with the meningococcal RmpM (Fig. 3), as proven by the antigenic protein revealed at 34 kDa in the 44/76 (RmpM⁺) OMVs, which is absent in those of the 44/76 RmpM⁻ mutant. Fig. 3 also shows that other cross-reactive antigenic proteins are revealed in both the RmpM⁺ and RmpM⁻ OMVs with the different sera, and, in all cases, a 31-kDa antigen is visible only in the OMVs from the RmpM⁻ mutant. This same behavior is shown when using sera obtained using OMVs from four N. meningitidis strains (not shown). The same assay with mouse sera obtained against OMVs from two M. catarrhalis strains did not react with the RmpM or the 31-kDa antigen mentioned above (not shown).

3

Reactivity of mouse polyclonal antisera against OMVs from two N. lactamica and two N. sicca strains with proteins present in OMVs from the N. meningitidis strains 44/76 (+, RmpM positive control) and 44/76 RmpM⁻ (−, RmpM negative control). Molecular mass markers (M) are also included.

3.3 Demonstration of the presence of the rmpM gene in commensal species

PCR experiments allowed a good detection of the rmpM gene in all N. meningitidis, four of the seven N. lactamica and one of the four N. sicca strains tested (Fig. 4B). The size of the rmpM gene of the N. lactamica strains is similar to that of the meningococcal strains (∼700 bp; reported rmpM ORF size is 728 bp), and that of the N. sicca strain is clearly lower (∼500 bp).

4

Detection of the rmpM gene in commensal Neisseria species. A: Dot hybridization of PCR amplified DNA from N. meningitidis (a–c: strains B16B6, NmP27, and Nm30), N. lactamica (d–i: strains NlP2, NlP4, NlP5, NlP6, NlP8, and NlP9), N. sicca (j–n: strains NsP1, NsP3, NsP4, and NsP12), N. mucosa (o, p: strains NmuP1 and NmuP2), and N. perflava (q: strain Npf0). (+), positive control. B: PCR products examined under UV light after electrophoresis in an agarose gel. Lanes show products from N. meningitidis (a: strain M982), N. lactamica (b–e: strains NlP5, NlP6, NlP3, and NlP9), and N. sicca (f–i: strains NsP12, NsP1, NsP3, and NsP4). L: 100-bp ladder.

Dot-blot hybridization experiments carried out under low stringent conditions show that both chromosomal DNA isolated from the different N. meningitidis, N. lactamica, N. sicca, and N. mucosa strains (not shown) and PCR products amplified with primers R2/L2 react with the DIG-labelled rmpM probe obtained from the strain N. meningitidis M982 (Fig. 4A). PCR products obtained from N. perflava showed a faint reaction with that probe, and those of the M. catarrhalis strains were negative in this assay.

4 Discussion

The search for meningococcal surface proteins appropriate for use as virulence markers and/or useful for the development of more efficient vaccines against meningococcal meningitis has been the center of the efforts of many researchers in recent years. Due to the relatively low efficacy of actual polysaccharide vaccines, research has focused on OMPs and lipooligosaccharides, but the major outer membrane antigens, mainly porins, are not good candidates due to their antigenic variability in most cases, although vaccines containing several porins or strains expressing some of the most relevant isotypes are being considered. Other outer membrane components investigated for these purposes are those that form the membrane receptors for lactoferrin (LbpA and LbpB), transferrin (TbpA and TbpB), and some other molecules such as the H8 protein and lipooligosaccharides, although to date, none of them has given satisfactory results [17].

Among the major antigenic meningococcal OMPs, the reduction-modifiable protein (RmpM, also termed class 4 OMP or P4) has been especially studied because it has been described that it is present only in the pathogenic species N. meningitidis and N. gonorrhoeae. Although its role in pathogenicity has been not identified, the association of the RmpM with virulence has been suggested [5]. It has also been reported that antibodies against the RmpM can block the bactericidal activity of antibodies against other proteins [3], although in a recent work it was found that human antibodies against RmpM induced in vivo during natural infections do not have such blocking activity [4]. Other recently described properties of the RmpM are its association with several important OMPs such as porins (PorA and PorB), components of the lactoferrin (LbpA) and transferrin receptors (TbpA), and the siderophore receptor protein (FrpB), and the presence in the RmpM of a peptidoglycan binding domain, thus suggesting that it can act to stabilize OMPs or functional complexes [2]. Our results in this work show that the RmpM is present in all N. lactamica and N. sicca strains tested. Also, the RmpM or a related protein could be present in N. mucosa strains.

In our experiments, the anti-RmpM mAb 185, H-8 clearly reacts with a protein present in all the N. lactamica strains, although this protein has a molecular mass slightly lower (31 kDa) than that of the meningococcal RmpM (34 kDa). On the other hand, our results using the mouse polyclonal antisera obtained by immunization with OMVs from N. lactamica and N. sicca strains demonstrate the presence of the RmpM (or a protein with common epitopes) in both species by an alternative approach. All the sera reacted with the meningococcal RmpM as evidenced by the 33-kDa antigenic band shown in the 44/76 OMVs and the corresponding absence of this band in the 44/76 RmpM⁻ OMVs.

Our PCR and dot-blot hybridization results confirm the above results showing the presence of the rmpM gene in all N. lactamica and N. sicca strains. Although the gene could not be shown by direct analysis of the PCR amplification in agarose gels in all these strains, all PCR products reacted well by hybridization with the meningococcal rmpM DIG-labelled probe, which is congruent with the higher sensitivity of this technique. The N. mucosa PCR products also seem to react with the rmpM probe, although in this case the intensity is low and a more in depth analysis is necessary in order to get definitive conclusions for this species.

The disagreement with the results obtained by Wolff and Stern [5], who reported the absence of the rmpM gene in commensal Neisseria, could be explained if the DNA probe that was used in their work (an 18-nucleotide region inside the rmpM gene) was specific for a sequence not present, or altered, in the gene of the commensal species. This could also account for the difference in the molecular mass of this protein found in the N. lactamica RmpM and in the size of the rmpM gene in N. sicca strains. In the last, the gene must also lack the sequence encoding the epitope for which the 185, H-8 mAb is specific.

In a previously published work [18], we found a highly cross-reactive 32-kDa protein that is present in all N. meningitidis and N. lactamica strains. We now think that this protein is the RmpM, and that it is also present at least in N. sicca strains, which is in agreement with the proposed role for this protein as a structural component of the outer membrane that binds to other functional membrane proteins (PorA, PorB, LbpA, TbpA, FrpB) and to the peptidoglycan layer, stabilizing the complexes that these proteins form in the outer membrane. The 31-kDa antigenic protein detected in the OMVs from the RmpM mutant is probably a fusion of the ermC′ marker, introduced to substitute the RmpM in the transformation plasmid [19], with at least the initial (it is synthesized, so it must have the promoter region), and perhaps also the final fragments of the meningococcal RmpM, which could encode reactive epitopes.

In conclusion, we have shown that the RmpM is present in some commensal Neisseria species, which is in disagreement with the proposed role of this protein in the virulence of the pathogenic species.

Acknowledgements

This work was supported by Grants PB98-0610, from the Dirección General de Enseñanza Superior e Investigación Científica (DGESIC), and PXIDT99PXI20308B, from the Xunta de Galicia, Spain.