Background

Moraxella catarrhalis, formerly known as both Neisseria catarrhalis and Branhamella catarrhalis [1], is a gram-negative bacterium that can frequently be isolated from the nasopharynx of healthy persons [2–4]. For many years, M. catarrhalis was considered to be a harmless commensal [1–4]. About twenty years ago, it was acknowledged that M. catarrhalis was a pathogen of the respiratory tract [5], and since then much evidence has accumulated which indicates that M. catarrhalis causes disease in both adults and children. M. catarrhalis is one of the three most important causes of otitis media in infants and very young children [3, 6]. In adults, this bacterium can cause infectious exacerbations of chronic obstructive pulmonary disease (COPD), and one recent study estimates that, in the United States alone, M. catarrhalis may cause 2 million-4 million infectious exacerbations of COPD annually [7].

The ability of M. catarrhalis to colonize the mucosa of the upper respiratory tract (i.e., nasopharynx) is undoubtedly linked to its expression of different adhesins for various human cells and antigens [8–15]. In addition, this bacterium clearly has the metabolic capability to survive and grow in this environment in the presence of the normal flora. A recent study [16] identified a number of different metabolic pathways encoded by the M. catarrhalis ATCC 43617 genome which could be involved in the colonization process. It is likely that M. catarrhalis forms a biofilm in concert with these other bacteria in the nasopharynx [17], although only a few M. catarrhalis gene products relevant to biofilm formation have been identified to date [13, 18, 19]. Similarly, there is little known about what extracellular gene products are synthesized by M. catarrhalis and released into the extracellular milieu. A study from Campagnari and colleagues [15] found that one or two very large proteins with some similarity to the filamentous hemagglutinin (FhaB) of Bordetella pertussis could be found in M. catarrhalis culture supernatant fluid. Using the nucleotide sequence of the genome of M. catarrhalis ATCC 43617, Murphy and co-workers [20] identified a large number (i.e., 348) of proteins that had signal sequences, among which may be proteins that are released from the M. catarrhalis cell. Another group showed that M. catarrhalis culture supernatant fluid contained several different proteins as detected by SDS-PAGE analysis, but the identity of the individual proteins was not determined [21].

In the present study, we report the first identification of a bacteriocin that is produced by M. catarrhalis. Bacteriocins are proteins or peptides secreted or released by some bacteria that can effect both intraspecies and interspecies killing, and are responsible for some types of bacterial antagonism [for reviews see [22, 23]]. The locus encoding this peptide bacteriocin was identified initially in a M. catarrhalis plasmid and subsequently shown to be present in the chromosome of some M. catarrhalis strains. Four genes encoding the bacteriocin, relevant secretion factors, and a host immunity factor were shown to form a polycistronic operon (mcbABCI). This bacteriocin was shown to be active against M. catarrhalis strains lacking this operon. Recombinant methods were used to confirm the identity of the cognate immunity factor which does not resemble other proteins in the databases. In competitive co-culture assays, a M. catarrhalis strain expressing this bacteriocin became the predominant member of a mixed culture in which the other strain lacked the mcbABCI locus.

Results

M. catarrhalis strain E22 produces a factor that inhibits the growth of M. catarrhalisstrain O35E

Wild-type M. catarrhalis strain E22 was originally described as the host for the plasmid pLQ510 [24]. As reported previously [25], two of the ORFs in this plasmid were predicted to encode products with similarity to proteins involved in secretion of bacteriocins in other bacteria. Upon testing the E22 strain in a bacteriocin production assay using wild-type M. catarrhalis strain O35E as the indicator strain, the growth of the indicator strain was inhibited in the area immediately around the E22 strain (Figure 1C). In control experiments, O35E did not kill either itself (Figure 1A) or E22 (Figure 1B) and E22 did not kill itself (Figure 1D). This result indicated that strain E22 was capable of producing one or more factors that inhibited the growth of strain O35E.

Figure 1
figure 1

Killing of M. catarrhalis O35E by M. catarrhalis E22 carrying pLQ510. Test strains and indicator strains were grown on BHI agar plates as described in Materials and Methods. Panels: A, O35E test strain on O35E indicator; B, O35E test strain on E22 indicator; C, E22 test strain on O35E indicator; D, E22 test strain on E22 indicator. The white arrow in panel C indicates the zone of killing of the indicator strain by the test strain. Panel E, schematic of pLQ510 indicating the four ORFs located in the mcb locus. The nucleotide sequence of pLQ510 is available at GenBank under accession no. AF129811. The positions of the restriction sites used to insert kanamycin resistance cassettes in the mcbB and mcbC genes are indicated.

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Characterization of relevant protein products encoded by pLQ510

In a previous publication [25], ORF1 (now designated as M. c atarrhalis bacteriocin gene A or mcbA) in pLQ510 (Figure 1E) was described as encoding a protein with homology to the colicin V secretion protein of E. coli [26] whereas ORF2 (now designated mcbB) (Figure 1E) encoded a protein that was most similar to the colicin V secretion ATP-binding protein CvaB [26]. Analysis of the similarities between the amino acid sequences of the McbA and McbB proteins and those of proteins in sequence databases was next assessed using BLAST [blastp and PSI-BLAST [27]]. Both McbA and McbB were found to be members of well-populated protein families. McbA belongs to the HlyD family of so-called membrane-fusion proteins (MFPs). These proteins form a periplasm-spanning tube that extends from an ABC-type transporter in the plasma membrane to a TolC-like protein in the outer membrane [28]. An alignment [29] of McbA to E. coli HlyD showed that the two proteins are approximately 19% identical. Likewise, the primary structure of McbB is similar to that of the E. coli protein HlyB protein; their sequence identity is ~27%. HlyB is an ABC-type transporter that is presumably dimeric. It has two main domains: the N-terminal domain spans the plasma membrane, facilitating the export of its cognate substrate, while the C-terminal domain uses the energy of ATP hydrolysis to drive the export of the substrate against a concentration gradient [28]. Although the degree of sequence identity between the M. catarrhalis and E. coli proteins is modest, it is not unreasonable to assume that they may share analogous functions.

Identification of the M. catarrhalisbacteriocin and immunity factor genes

Immediately downstream from mcbB, two overlap** and small putative ORFs were detected. The first of these, designated mcbC (Figure 1E), contained 303-nt in pLQ510 and was predicted to encode a protein containing 101 amino acids (Figure 2A). BLAST analysis showed that this polypeptide had little similarity to other proteins or known bacteriocins. However, examination of the sequence of amino acids 25-39 in this protein revealed that it was similar to the leader sequence of the double-glycine (GG) bacteriocin family including E. coli colicin V (CvaC) and other double-glycine peptides of both gram-negative and gram-positive bacteria [50] using primers AA262 and AA250. The new PCR product was used in a plate transformation system [51] to transform M. catarrhalis strain O12E. Transformants were screened by colony-PCR using primers AA262 and AA251 (5'-AGATTGCTCACTCGTCCAC-3'); this latter primer binds downstream of AA250. One transformant shown to contain the desired deletion in the mcbA gene was designated O12EΔmcbA.

For the construction of an in-frame deletion in the mcbB ORF, primers AA247 (5'-TGCCATTGCCAAAGAGAC-3') and AA346 (5'-AATATTCTTTAAAAAATC CAT-3') were used to amplify 830 nt upstream of the mcbB ORF together with the first 21 nt of the mcbB ORF using chromosomal DNA from strain O12E as the template. In addition, primers AA347 (5'-TTTTTAAAGAATATTAGCACTGATT GGGTACTGAACCTTGGTTAA-3') and AA254 (5'-GGGCTTTGGGCGGTA GGTTATTA-3') were used to amplify the last 30 nt of the mcbB ORF and 769 nt of the downstream region. Both PCR fragments were used as templates for an overlap** extension PCR using primers AA247 and AA254; the resultant amplicon was designated 247-254. Wild-type strain O12E was first transformed with a PCR amplicon obtained by using primers AA248 (5'-CTGTTGCCAAAACTGCTC-3') and AA252 (5'-GCACATTGTTCCACCCATTCA-3') with plasmid pLQ510.mcbB::kan as the template; this amplicon contained the mcbB gene and the inserted kan cartridge. One of the resultant kanamycin-resistant transformants (O12E.mcbB::kan) was subsequently transformed with the 247-254 amplicon. Transformants were screened for the loss of kanamycin resistance and one kanamycin-sensitive transformant was selected for further study and designated as O12EΔmcbB.

To construct an in-frame deletion in the mcbC ORF, the same strategy was employed as was used for construction of the O12EΔmcbB mutant. The primer pair AA249 (5'-TTAGACCC AAGTGCTGGAC-3') and AA344 (5'-ACGCATAATATATTCCTTT AT-3') and the primer pair AA345 (5'-GAATATATTATGCGTATTATGGTTG GAGTTACTAAAAAATGGTAA-3') and AA254 were used in the initial PCR reactions with O12E chromosomal DNA, and the final amplicon containing a deletion in the mcbC ORF was used to transform an O12E mutant which had a kanamycin resistance cassette in its mcbC ORF (i.e., O12E.mcbC::kan). One kanamycin-sensitive transformant was selected for further characterization and was designated O12EΔmcbC. PCR and nucleotide sequence analysis were used to confirm that these three deletion mutations (i.e., in O12EΔmcbA, O12EΔmcbB, and O12EΔmcbC) were in-frame.

Reverse transcriptase-PCR

Possible transcriptional linkage among the ORFs in the mcb locus in pLQ510 was assessed by the use of reverse transcriptase-PCR. Total RNA was isolated from mid-logarithmic phase cells of M. catarrhalis E22 by using the RNeasy midi kit (Qiagen). RNA samples were treated with DNase I (Message Clean Kit, GenHunter Corp, Nashville, TN) to remove any DNA contamination. To amplify the region between the mcbA and mcbB ORFs, primers mcb A/B fw (5'-TAGCAGTTGGCATGACC TTG-3') and mcb A/B rv (5'-AGCAAGACAGGCTAGACCACA-3') were used. For the region between mcbB and mcbC, primers mcb B/C fw (5'-AGAGCGCTGATTG GGTACTG-3'), and mcb B/C rv (5'-CAT GCCATTGACTGACCAAC-3'), were used, and for the region between mcbC and mcbI, primers mcb C/I fw (5'-TCCTA ATAGATTGTCATATGGTGGTT-3') and mcb C/I rv (5'-CAAAACG TGCACA ATTAGGG-3') were used. The reverse transcriptase reaction was carried out using MultiScribe reverse transcriptase (Applied Biosystems, Foster City, CA) followed by PCR amplification. In addition, the reaction was also performed using either chromosomal DNA alone as the template or with the RNA template in the absence of reverse transcriptase.

Construction of a plasmid encoding a His-tagged McbC protein

In order to construct a plasmid that expressed the McbC protein with a C-terminal His-tag, the primer pair AA357 (5'-TGGGATCCGGTACTATTTAATGTACTAA GATTTT-3') (BamHI site underlined) and AA359 (5'-GTGGTGGTGGTGGTGGTG CCATTTTTTAGTAACTCCAACCATAAT-3') and the primer pair AA358 (5'-CACCACCACCACCACCACTAAAGACAATAGGTTTAGCATGGATAT-3') (mcbC translational stop codon underlined) and AA354 (5'-GGTTGAGCTCCCA TTTAAGTGATTTTGTTATATCAAT-3') (SacI site underlined) were used to generate two PCR products using O12E chromosomal DNA as the template. The resultant two PCR products were used as templates for an overlap** extension PCR involving primers AA357 and AA354. The final PCR amplicon was then digested with both BamHI and SacI and ligated into pWW115 [52] that had been digested with these same restriction enzymes. The ligation mixture was used to transform O12E.mcbC::kan. A plasmid isolated from a spectinomycin-resistant colony and which expressed the His-tagged McbC protein was designated pAA111. Plasmid pWW115 was used to transform M. catarrhalis O12E.mcbC::kan to provide a negative control.

Purification and detection of the His-tagged McbC protein

M. catarrhalis O12E.mcbC::kan(pWW115) and M. catarrhalis O12E.mcbC::kan(pAA111) were grown independently in 1 L BHI overnight at 37°C with shaking. The cultures were subjected to centrifugation to pellet the bacterial cells and the supernatant fluid was filter-sterilized. Two columns each containing 1.5 mL of NiNTA agarose beads (Qiagen, Valencia, CA) were washed with washing buffer (50 mM NaH2PO4, 200 mM NaCl, 5 mM imidazole [pH 7.9]). The culture supernatant fluids were passed through the columns twice after which the columns were washed with washing buffer again. The His-tagged protein was eluted using elution buffer (50 mM NaH2PO4, 200 mM NaCl, 200 mM imidazole [pH 7.9]). Selected fractions were pooled and dialyzed against PBS. SDS-digestion buffer was added to a final concentration of 1× to each sample. For Western blot analysis, proteins were resolved by SDS-PAGE using 15% (wt/vol) polyacrylamide separating gels and transferred to polyvinylidene difluoride membranes. The anti-His tag antibody HIS.H8 (Millipore, Temecula, CA) was used at a dilution of 1:2,000 in PBS-Tween containing 3% (wt/vol) dried milk and incubated with the membrane for 2 h at room temperature. Horseradish peroxidase-conjugated goat anti-mouse antibody (Jackson Immunoresearch, West Grove, PA) was used as the secondary antibody. The antigen-antibody complexes were detected by using Western Lightning Chemiluminescence Reagent Plus (New England Nuclear, Boston, MA).

Construction of a plasmid containing the mcbIgene

Primers AA353 (5'-ATGGATCCGAAAACTCATTGGGGAGATAGAGGGAT-3') (BamHI site underlined) and AA378 (5'-TTGTGAGCTCGCTCGGATTTGCTATTATTGA-3') (SacI site underlined) were used to PCR-amplify a 288-bp fragment containing the mcbI gene from M. catarrhalis O12E chromosomal DNA. The resultant PCR product was digested with both BamHI and SacI and ligated into pWW115 which had been digested with the same two restriction enzymes. This ligation mixture was used to transform M. catarrhalis strain O35E and transformants were selected for spectinomycin resistance. The plasmid from one of these transformants was designated pAA113.

Competitive index-based broth growth experiments

A streptomycin-resistant mutant of the wild-type strain O12E (O12E-Smr) [53] and the spectinomycin-resistant recombinant strains O35E(pWW115) and O35E(pAA113) were grown separately in MH broth to a density of approximately 108 CFU/ml. Equal volumes of O12E-Smrand the individual recombinant O35E strains were mixed in a 1:1 ratio. Serial dilutions of this mixture were plated on BHI agar plates containing the appropriate antibiotic to determine the relative percentages of each strain in the input mixture. Either a 1 ml or a 0.5 ml portion of the mixture was used to inoculate either 250 ml or 125 ml of MH broth, respectively, which was then allowed to grow overnight at 37°C with aeration. The cells were harvested after 18 h of growth, serially diluted, and plated on agar-based media containing the appropriate antibiotic to determine the relative percentage of each strain in the output mixture. A second set of competition experiments involving O12E-Smr and the spectinomycin-resistant mutant O35EΔmapA [34] was performed similarly. Each co-culture experiment was done three times independently; the data are the mean of the three experiments.