Whole genome sequencing of Moraxella bovis strains from North America reveals two genotypes with different genetic determinants.

BACKGROUND: Moraxella bovis and Moraxella bovoculi both associate with infectious bovine keratoconjunctivitis (IBK), an economically significant and painful ocular disease that affects cattle worldwide. There are two genotypes of M. bovoculi (genotypes 1 and 2) that differ in their gene content and potential virulence factors, although neither have been experimentally shown to cause IBK. M. bovis is a causative IBK agent, however, not all strains carry a complete assortment of known virulence factors. The goals of this study were to determine the population structure and depth of M. bovis genomic diversity, and to compare core and accessory genes and predicted outer membrane protein profiles both within and between M. bovis and M. bovoculi. RESULTS: Phylogenetic trees and bioinformatic analyses of 36 M. bovis chromosomes sequenced in this study and additional available chromosomes of M. bovis and both genotype 1 and 2 M. bovoculi, showed there are two genotypes (1 and 2) of M. bovis. The two M. bovis genotypes share a core of 2015 genes, with 121 and 186 genes specific to genotype 1 and 2, respectively. The two genotypes differ by their chromosome size and prophage content, encoded protein variants of the virulence factor hemolysin, and by their affiliation with different plasmids. Eight plasmid types were identified in this study, with types 1 and 6 observed in 88 and 56% of genotype 2 strains, respectively, and absent from genotype 1 strains. Only type 1 plasmids contained one or two gene copies encoding filamentous haemagglutinin-like proteins potentially involved with adhesion. A core of 1403 genes was shared between the genotype 1 and 2 strains of both M. bovis and M. bovoculi, which encoded a total of nine predicted outer membrane proteins. CONCLUSIONS: There are two genotypes of M. bovis that differ in both chromosome content and plasmid profiles and thus may not equally associate with IBK. Immunological reagents specifically targeting select genotypes of M. bovis, or all genotypes of M. bovis and M. bovoculi together could be designed from the outer membrane proteins identified in this study.

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identification of Moraxella bovoculi and Moraxella bovis isolates from cattle.

Infectious bovine keratoconjunctivitis (IBK) is an economically significant disease caused by Moraxella bovis. Moraxella bovoculi, although not reported to cause IBK, has been isolated from the eyes of cattle diagnosed with IBK. Identification of M. bovis and M. bovoculi can be performed using biochemical or DNA-based approaches, both of which may be time consuming and inconsistent between laboratories. We conducted a comparative evaluation of M. bovoculi and M. bovis identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with a database provided by Bruker Daltonics (termed the BDAL database), the BDAL database supplemented with spectra generated in our study (termed the UNLVDC database), and with PCR-restriction-fragment length polymorphism (PCR-RFLP) typing. M. bovoculi ( n = 250) and M. bovis ( n = 18) isolates from cattle with or without IBK were used. MALDI-TOF MS using the UNLVDC database correctly identified 250 of 250 (100%) of M. bovoculi and 17 of 18 (94%) of M. bovis isolates. With the BDAL database, MALDI-TOF MS correctly identified 249 of 250 (99%) of M. bovoculi and 7 of 18 (39%) of M. bovis isolates. In comparison, the PCR-RFLP test correctly identified 210 of 250 (84%) of M. bovoculi and 12 of 18 (66%) of M. bovis isolates. Thus, MALDI-TOF MS with the UNLVDC database was the most effective identification methodology for M. bovis and M. bovoculi isolates from cattle.

Identification and characterisation of a biosynthetic locus for Moraxella bovis lipo-oligosaccharide.

Moraxella bovis is a Gram-negative gammaproteobacterium and is one of the causative agents of infectious bovine keratoconjunctivitis. The structure of lipooligosaccharide (LOS) from strain Epp63 was recently elucidated. In the present study a genetic locus of seven encoding genes with high similarity to glycosyltransferases has been identified. Mutation of these putative glycosyltransferase genes resulted in M. bovis mutant bacteria that expressed truncated LOS structures. The structures of the oligosaccharide (OS) expressed by the mutant strains were elucidated and demonstrated the role of the glycosyltransferase enzymes in the LOS biosynthesis of M. bovis. The glycosyltransferase genes designated lgt1, lgt3, and lgt6 are highly similar to the genes in the related bacterium M. catarrhalis. In addition, there are syntenic similarities with the corresponding LOS biosynthesis locus in M. catarrhalis and other members of Moraxellaceae.

Phylogenetic analysis and genetic diversity of 3' region of rtxA gene from geographically diverse strains of Moraxella bovis, Moraxella bovoculi and Moraxella ovis.

The cytotoxin A (MbxA) is one of the main virulence factors of Moraxella bovis involved in the pathogenesis of infectious bovine keratoconjunctivitis (IBK). Moraxella ovis and Moraxella bovoculi, suspected to be associated with infectious keratitis in sheep and cattle respectively, also have a gene that encodes the cytotoxin A (movA and mbvA, respectively). The aim of this study was to determine the molecular sequence of the 3' region of the cytotoxin gene of Moraxella spp. strains isolated from clinical cases to establish phylogenetic and evolutionary comparisons. PCR amplification, nucleotide sequencing (nt) and amino acid (aa) sequence prediction were performed, followed by the sequences comparison, identity level calculation and selective pressure analysis. The phylogenetic reconstruction based on nt and aa sequences clearly differentiate M. bovis (n=15), M. bovoculi (n=11) and M. ovis (n=7) and their respective reference strains. An alignment of 843nt revealed high similarity within bacterial species (MbxA=99.9% nt and aa; MbvA=99.3% nt and 98.8% aa; MovA=99.5% nt and 99.3% aa). The similarity of partial sequences (nt 1807-2649) of MbxA in relation to MbvA and MovA ranged from 76.3 to 78.5%; similarity between MbvA and MovA ranged from 95.7 to 97.5%. A negative selection on mbvA and movA sequences was revealed by the molecular evolution analysis. The phylogenetic analysis of movA and mbvA allowed different strains of Moraxella spp. to be grouped according to the period of isolation. Sequence analysis of cytotoxin may provide insights into genetic and evolutionary relationships and into the genetic/molecular basis of Moraxella spp.

RNA editing by T7 RNA polymerase bypasses InDel mutations causing unexpected phenotypic changes.

DNA-dependent T7 RNA polymerase (T7 RNAP) is the most powerful tool for both gene expression and in vitro transcription. By using a Next Generation Sequencing (NGS) approach we have analyzed the polymorphism of a T7 RNAP-generated mRNA pool of the mboIIM2 gene. We find that the enzyme displays a relatively high level of template-dependent transcriptional infidelity. The nucleotide misincorporations and multiple insertions in A/T-rich tracts of homopolymers in mRNA (0.20 and 0.089%, respectively) cause epigenetic effects with significant impact on gene expression that is disproportionally high to their frequency of appearance. The sequence-dependent rescue of single and even double InDel frameshifting mutants and wild-type phenotype recovery is observed as a result. As a consequence, a heterogeneous pool of functional and non-functional proteins of almost the same molecular mass is produced where the proteins are indistinguishable from each other upon ordinary analysis. We suggest that transcriptional infidelity as a general feature of the most effective RNAPs may serve to repair and/or modify a protein function, thus increasing the repertoire of phenotypic variants, which in turn has a high evolutionary potential.

Molecular characterization of plasmid pMbo4.6 of Moraxella bovis ATCC 10900.

We report the characterization of a small cryptic plasmid unlike any previously described from Moraxella bovis ATCC 10900, a Gram-negative bacterium belonging to the family Moraxellaceae. The complete nucleotide sequence of the plasmid pMbo4.6 was determined. The plasmid was analyzed and found to be 4658 in size with a G+C content of 38.6 mol %. Computer analysis of the sequence data revealed four major open reading frames encoding putative proteins of 10.1 (ORF1), 64.2 (ORF2), 45.7 (ORF3), and 12.1 kDa (ORF4). ORF1 and ORF2 encode proteins that show a high level of amino acid sequence similarity (44 %) with some mobilization proteins. ORF3 encodes a protein showing a relatively high amino acid sequence similarity (about 40 %) with several plasmid replication initiator proteins. Upstream of ORF3, a 320-bp intergenic region, constituting the putative origin of replication that contained an AT-rich region followed by four direct repeats, was identified. This set of repeated sequences resembles iteron structures and plays an important role in the control of plasmid replication by providing a target site for the initiation of transcription and replication factors (IHF and RepA). Several palindromic sequences, inverted repeats, and hairpin-loop structures, which might confer regulatory effects on the replication of the plasmid, were also noted. ORF4 encodes an uncharacterized protein, conserved in bacteria, belonging to the DUF497 family. Sequence analysis and structural features indicate that pMbo4.6 replicates by a theta mechanism.

Randomized controlled field trial to assess efficacy of a Moraxella bovis pilin-cytotoxin-Moraxella bovoculi cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis.

OBJECTIVE: To evaluate efficacy of a recombinant Moraxella bovis pilin-cytotoxin-Moraxella bovoculi cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK). ANIMALS: 107 beef steers. PROCEDURES: 2 groups of calves were inoculated SC with an immunostimulating complex (ISCOM) matrix adjuvant (control group; n = 54) or a recombinant M bovis pilin-cytotoxin-M bovoculi cytotoxin subunit antigen with the ISCOM matrix adjuvant (vaccine group; 53); calves received booster injections 21 days later. Calves were examined once weekly for 16 weeks. Investigators and herd managers were not aware of the inoculum administered to each calf throughout the trial. Primary outcome of interest was the cumulative proportion of calves that developed IBK. Serum samples were obtained before inoculation (day 0) and on days 42 and 112. Serum hemolysin-neutralizing titers against native M bovis and M bovoculi cytotoxin were determined. RESULTS: No difference was detected between groups for the cumulative proportion of calves that developed IBK at weeks 8 and 16 after inoculation. Non-IBK-affected calves in the vaccine group had a significantly higher fold change in serum hemolysin-neutralizing titer against native M bovoculi cytotoxin from day 0 to 42 compared to control calves. CONCLUSIONS AND CLINICAL RELEVANCE: The M bovis pilin-cytotoxin-M bovoculi cytotoxin subunit vaccine with the ISCOM matrix adjuvant was not effective at preventing naturally occurring IBK. It is likely that the incorporation of additional protective antigens in a recombinant Moraxella spp subunit vaccine will be required to yield a product that can be used for effective immunization of cattle against IBK.

The role of lactoferrin binding protein B in mediating protection against human lactoferricin.

Bacteria that inhabit the mucosal surfaces of the respiratory and genitourinary tracts of mammals encounter an iron-deficient environment because of iron sequestration by the host iron-binding proteins transferrin and lactoferrin. Lactoferrin is also present in high concentrations at sites of inflammation where the cationic, antimicrobial peptide lactoferricin is produced by proteolysis of lactoferrin. Several Gram-negative pathogens express a lactoferrin receptor that enables the bacteria to use lactoferrin as an iron source. The receptor is composed of an integral membrane protein, lactoferrin binding protein A (LbpA), and a membrane-bound lipoprotein, lactoferrin binding protein B (LbpB). LbpA is essential for growth with lactoferrin as the sole iron source, whereas the role of LbpB in iron acquisition is not yet known. In this study, we demonstrate that LbpB from 2 different species is capable of providing protection against the killing activity of a human lactoferrin-derived peptide. We investigated the prevalence of lactoferrin receptors in bacteria and examined their sequence diversity. We propose that the protection against the cationic antimicrobial human lactoferrin-derived peptide is associated with clusters of negatively charged amino acids in the C-terminal lobe of LbpB that is a common feature of this protein.

Molecular and phenotypic analysis of Moraxella spp. associated with infectious bovine keratoconjunctivitis in Uruguay.

Infectious bovine keratoconjunctivitis (IBK) is a common ocular disease of cattle, which is generally thought to be caused by Moraxella bovis. However, a recently characterized Moraxella, M. bovoculi, has been isolated from animals with IBK. The aim of this study was to identify and characterize strains of Moraxella spp. obtained from IBK cases in different geographic locations within Uruguay. Ribosomal gene sequencing indicated that there were two groups of isolates that showed homology with either M. bovis or M. bovoculi. Phylogenetic analysis confirmed the presence of two species as the isolates grouped in different branches of the dendrogram. Conventional biochemical characterization did not distinguish between the species; only 9/25 isolates which had genetic homology with M. bovoculi showed any differences in biochemistry.

Prevention of naturally occurring infectious bovine keratoconjunctivitis with a recombinant Moraxella bovis pilin-Moraxella bovis cytotoxin-ISCOM matrix adjuvanted vaccine.

To evaluate the efficacy of a recombinant Moraxella bovis pilin-M. bovis cytotoxin subunit vaccine to prevent naturally occurring infectious bovine keratoconjunctivitis (IBK; pinkeye), a randomized, blinded, controlled field trial was conducted during summer 2005 in a northern California herd of beef cattle. One hundred and one steers were vaccinated with ISCOM matrix (adjuvant control), recombinant M. bovis cytotoxin carboxy terminus+ISCOM matrix (MbxA), or recombinant M. bovis pilin-cytotoxin carboxy terminus+ISCOM matrix (pilin-MbxA); calves received secondary vaccinations 21 days later. Calves were examined once weekly for 18 weeks for the development of corneal ulcers associated with IBK. Overall, the pilin-MbxA vaccinated group had the lowest overall cumulative proportion of ulcerated calves. Calves that received MbxA, whether alone or with pilin had significantly higher M. bovis cytotoxin serum neutralizing titers as compared to control calves. Results of ocular cultures suggested that vaccination with an M. bovis antigen affected organism type isolated from an ulcer: M. bovis was cultured more often from the eyes of control calves than from the eyes of calves vaccinated with MbxA and pilin-MbxA. In addition, vaccination of calves with MbxA and pilin-MbxA resulted in a higher prevalence of Moraxella bovoculi sp. nov. in ocular cultures. While no significant difference was observed between a cytotoxin versus pilin+cytotoxin vaccine against IBK, the reduced cumulative proportion of IBK in the pilin-cytotoxin vaccinated calves suggests it may provide an advantage over a cytotoxin vaccine alone. Efficacy of an M. bovis vaccine may be reduced in herds where IBK is associated with M. bovoculi sp. nov.

Relatedness of cytotoxins from geographically diverse isolates of Moraxella bovis.

To determine whether amino acid sequence variation exists in the Moraxella bovis (M. bovis) cytotoxin (MbxA) from geographically diverse M. bovis isolated in the United States, mbxA was amplified and sequenced. The MbxA deduced amino acid sequence from M. bovis originally isolated in California, Washington, North Carolina, and Georgia, as well as reference strains of M. bovis isolated at the National Animal Disease Laboratory, Ames, IA, USA, all encoded a nearly identical 927 amino acid protein. MbxA from two of the four California isolates (SFS 9a and SFS 100a) differed from all other isolates at two sites at which the polar amino acids glutamine (position 666) and asparagine (position 823) were replaced by ionized amino acids glutamic acid and aspartic acid, respectively. Rabbit antiserum to the expressed carboxy terminus (amino acids 590-927) of MbxA from M. bovis (Tifton I) neutralized the hemolytic activity of SFS 9a and SFS 100a. The M. bovis cytotoxin appears to be conserved amongst geographically diverse isolates of M. bovis from the USA. Antiserum against the carboxy terminus of MbxA common to the majority of isolates neutralized the hemolytic activity of two strains with a divergent MbxA deduced amino acid sequence. Vaccines against IBK that incorporate MbxA as antigen may offer protection against geographically diverse strains of M. bovis.

Filamentous-haemagglutinin-like protein genes encoded on a plasmid of Moraxella bovis.

The complete nucleotide sequence of a plasmid, pMBO-1, from Moraxella bovis strain Epp63 was determined. We identified 30 open reading frames (ORFs) encoded by the 44,215bp molecule. Two large ORFs, flpA and flpB, encoding proteins with similarity to Bordetella pertussis filamentous haemagglutinin (FHA), were identified on the same plasmid. The gene for a specific accessory protein (Fap), which may play a role in the secretion of Flp protein, was also identified. Reverse transcriptase PCR analysis of total RNA isolated from M. bovis Epp63 indicated that the flpA, flpB, and fap genes are all transcribed. Southern blot analysis indicated that the flp and fap genes are present in other clinical isolates of geographically diverse M. bovis.

The Moraxella bovis RTX toxin locus mbx defines a pathogenicity island.

To characterize flanking regions of the mbx operon in Moraxella bovis, DNA surrounding mbxCABDtolC was sequenced in haemolytic and nonhaemolytic strains of M. bovis. In two haemolytic strains of M. bovis, the mbx operon, including the adjacent M. bovis tolC orthologue, was flanked by approximately 700 bp imperfect repeats. Nonhaemolytic strains of M. bovis had only one or no such repeats, as well as ORFs identical to those flanking the repeats from haemolytic M. bovis. Two nonhaemolytic strains also contained ORFs with deduced amino acid sequence similarity to bacterial araJ genes. The G+C content of the mbxCABDtolC gene region was lower than the flanking regions. The genetic organization and G+C content of mbxCABDtolC genes, and flanking repeats in haemolytic M. bovis, as well as the presence or absence of flanking repeats in nonhaemolytic M. bovis, suggests that this RTX operon is located on a mobile genetic element, and supports the designation of this region as a pathogenicity island, which is believed to be the first such element demonstrated in M. bovis.

Molecular diversity of Moraxella bovis isolated from Brazil, Argentina and Uruguay over a period of three decades.

The molecular profile of 30 Moraxella bovis strains, recovered from outbreaks of infectious bovine keratoconjunctivitis in Argentina, Brazil and Uruguay between 1974 and 2001, was determined through randomly applied polymorphic DNA (RAPD) analysis. Molecular profiles of nine strains recovered after 1990 varied from those recovered before 1990. The profiles of 13 strains (48%) differed from those of three vaccinal strains extensively used since 1984 in Argentina and Uruguay. Eight Argentinean strains, one from Brazil and two from Uruguay had identical RAPD profiles. Strains belonging to different serogroups had identical RAPD profiles, demonstrating that this technique was not able to discriminate among strains with low cross-reactivity indices. RAPD may be helpful in the primary characterization of M. bovis strains, but it does not replace serological characterization.

Crystal structure of MboIIA methyltransferase.

DNA methyltransferases (MTases) are sequence-specific enzymes which transfer a methyl group from S-adenosyl-L-methionine (AdoMet) to the amino group of either cytosine or adenine within a recognized DNA sequence. Methylation of a base in a specific DNA sequence protects DNA from nucleolytic cleavage by restriction enzymes recognizing the same DNA sequence. We have determined at 1.74 A resolution the crystal structure of a beta-class DNA MTase MboIIA (M.MboIIA) from the bacterium Moraxella bovis, the smallest DNA MTase determined to date. M.MboIIA methylates the 3' adenine of the pentanucleotide sequence 5'-GAAGA-3'. The protein crystallizes with two molecules in the asymmetric unit which we propose to resemble the dimer when M.MboIIA is not bound to DNA. The overall structure of the enzyme closely resembles that of M.RsrI. However, the cofactor-binding pocket in M.MboIIA forms a closed structure which is in contrast to the open-form structures of other known MTases.

Molecular cloning and characterization of a 79-kDa iron-repressible outer-membrane protein of Moraxella bovis.

Moraxella bovis expresses an iron-repressible 79-kDa outer-membrane protein, IrpA. DNA and N-terminal amino acid sequence analysis indicate that IrpA is closely related to FrpB of Neisseria meningitidis, FetA of Neisseria gonorrhoeae and CopB of Moraxella catarrhalis. The results of manganese mutagenesis and a gel-shift assay suggested that the transcription of irpA is negatively regulated by the ferric uptake regulator. The insertion of an antibiotic resistance cassette into the irpA gene affected the strain's ability to utilize bovine transferrin and lactoferrin. IrpA was detected in geographically diverse clinical isolates, and the antigenicity of IrpA was conserved in all the isolates tested. Therefore, IrpA may have potential as a candidate vaccine.

Cloning and characterization of the fur gene from Moraxella bovis.

A homologue of the ferric uptake regulator gene (fur) was isolated from Moraxella bovis by degenerate polymerase chain reaction and cloning. Fur protein of M. bovis exhibited 72.1% amino acid identity with Acinetobacter calcoaceticus Fur. Western blot analysis showed a decrease of Fur expression in response to sufficient-iron conditions compared with deficient-iron conditions. An electrophoretic mobility-shift assay indicated that Fur protein binds to DNA fragments containing a putative Fur-box derived from the upstream region of the M. bovis fur gene. Fur of M. bovis may regulate the expression of iron transport systems in response to iron limitation in the environment.

An RTX operon in hemolytic Moraxella bovis is absent from nonhemolytic strains.

Pathogenic isolates of Moraxella bovis express a calcium-dependent transmembrane pore forming cytotoxin that is an RTX toxin encoded by mbxA. The DNA flanking mbxA was cloned and sequenced to determine if M. bovis contained a classical RTX operon. Open reading frames (ORFs) with deduced amino acid sequence homology to putative activation (RTX C) and transport (RTX B and D) proteins were identified and have been designated MbxC, MbxB, and MbxD, respectively. Thus, hemolytic M. bovis contains a typical RTX operon comprised of four genes arranged (5'-3') mbxCABD. In addition, the deduced amino acid sequences of DNA flanking mbxCABD revealed ORFs with amino acid sequence similarity to transposases (5'). At the 3' end of the mbx gene cluster, an ORF with homology to bacterial tolC genes was identified. Thus, as with the cya RTX operon of Bordetella pertussis, M. bovis appears to have a secretion accessory protein linked to RTX genes. Analysis of genomic DNA isolated from 5 nonhemolytic M. bovis strains by PCR and Southern blotting revealed the absence of mbxCABD. These strains did, however, amplify with primers specific for the 5' region flanking mbxC. M. bovis harbors a classical RTX operon that is absent in nonhemolytic strains.

Bacterial lactoferrin receptors: insights from characterizing the Moraxella bovis receptors.

Moraxella bovis is the causative agent of infectious conjunctivitis in cattle. Moraxella bovis isolates were shown to specifically bind bovine lactoferrin (bLf) and bovine transferrin (bTf) and to use these proteins as a source of iron to support the growth of iron-limited cells. Affinity isolation experiments with immobilized bTf yielded two proteins readily resolved by SDS-PAGE analysis, whereas only a single band of approximately 100 kDa was detected when immobilized bLf was used as the affinity ligand. Using a novel cloning strategy, regions containing the genes encoding the lactoferrin (Lf) and transferrin (Tf) receptor proteins were isolated and sequenced, demonstrating that they both consisted of two genes, with the tbpB or lbpB gene preceding the tbpA or lbpA gene. The cloned lbp genes were used to generate isogenic mutants deficient in lactoferrin binding protein A and (or) B, and the resulting strains were tested in growth and binding assays. The isogenic mutants were deficient in their use of bLf for growth and had substantially diminished bLf binding capability. The predicted amino acid sequence from the segment encoding Lf binding protein B revealed an internal amino acid homology suggesting it is a bi-lobed protein, with a C-lobe enriched in acidic amino acids, but without the evident clustering observed in Lf-binding proteins from other species.

Molecular characterization of a secreted enzyme with phospholipase B activity from Moraxella bovis.

A candidate for a vaccine against infectious bovine keratoconjunctivitis (IBK) has been cloned and characterized from Moraxella bovis. The plb gene encodes a protein of 616 amino acids (molecular mass of ~65.8 kDa) that expresses phospholipase B activity. Amino acid sequence analysis revealed that PLB is a new member of the GDSL (Gly-Asp-Ser-Leu) family of lipolytic enzymes.