In vivo gene expression in Mannheimia haemolytica A1 during a time-course trial in the bovine host.

The objective of this study is to examine the expression of Mannheimia haemolytica genes over time during the early stage of infection. In addition, gene expression at different sites of infection in the bovine host was examined. A time-course experiment was designed to collect pharyngeal swabs and lung washings from the same animals over two time points. Six calves were experimentally challenged with M. haemolytica A1; pharyngeal swabs were collected from all animals 5h post infection. Three calves were euthanized at 6h; pharyngeal swabs were collected from the remaining 3 calves at 12h and the calves were euthanized. Lung washings were recovered from all animals at necropsy. Total RNA was prepared from the pharyngeal swabs and lung washings and primers for eight well characterized virulence-associated genes were used in qRT-PCR to examine mRNA levels. The expression of key virulence genes such as lktA, gcp and tbpB was higher in vivo compared to in vitro with the highest changes observed from 6 to 12h. The expression of lktA and gapA increased while expression of fbpA, gs60, nmaA and tbpB was found to decreased over time in the 6h period. Gene expression profiles in the lungs versus the pharynx also differed, with most genes (fbpA, tbpB, nmaA, gs60, lktA and narP) showing higher expressing in lung washings. This is the first study to follow gene expression by M. haemolytica in the same animal over time during an infection.

Serum IgG response in calves to the putative pneumonic virulence factor Gs60 of Mannheimia haemolytica A1.

Bovine pneumonic pasteurellosis vaccines incorporate various antigens of Mannheimia haemolytica, including the acknowledged virulence factor leukotoxin (Lkt), and Gs60, a surface lipoprotein. To examine the role of antibodies to Gs60 in protection, an enzyme-linked immunosorbent assay (ELISA) was developed for retrospective analysis of serum samples from previous trials in which vaccines containing native or recombinant Gs60 were administered parenterally. The analysis revealed a positive correlation between the titer of antibodies to Gs60 and protection against experimental challenge in both vaccinates and naturally exposed controls. There was a strong correlation between production of IgG antibodies to Gs60 and Lkt neutralizing antibodies. Analysis of the relationship between the serum antibody titers and resistance to experimental challenge using linear statistical models revealed a significant association between prechallenge titers of serum antibodies to Lkt and protection. Further analysis suggested that antibodies against Gs60 were beneficial when Lkt neutralizing antibody titers were low.

Les vaccins pour la pneumonie bovine à Pasteurella contiennent divers antigènes de Mannheimia haemolytica incluant la leucotoxine (Lkt), facteur de virulence reconnu, ainsi que Gs60, une lipoprotéine de surface. Afin d'examiner le rôle des anticorps contre Gs60 dans la protection, une épreuve immunoenzymatique (ELISA) a été développée pour analyse rétrospective d'échantillons de sérum provenant d'études antérieures au cours desquelles des vaccins contenant la Gs60 native ou recombinante étaient administrés par voie parentérale. L'analyse a révélé une corrélation positive entre le titre d'anticorps contre Gs60 et la protection contre une infection expérimentale autant chez des animaux vaccinés que des témoins exposés naturellement. Il y avait une forte corrélation entre la production d'anticorps de type IgG contre Gs60 et des anticorps neutralisants Lkt. Une analyse de la relation entre les titres d'anticorps sériques et la résistance à une infection expérimentale utilisant des modèles statistiques linéaires a révélé une association significative entre les titres d'anticorps sériques pré-infection avec Lkt et la protection. Des analyses supplémentaires ont suggéré que les anticorps contre Gs60 étaient bénéfiques lorsque les titres d'anticorps neutralisants anti-Lkt étaient bas.(Traduit par Docteur Serge Messier).

A snap-shot of Mannheimia hemolytica A1 gene expression during infection in the bovine host.

It is expected that Mannheimia hemolytica A1 expresses a particular collection of genes during infection in the host. The bacterial gene products are produced in the in vivo environment to facilitate growth and survival. Here, we examined gene expression by M. hemolytica A1 in the bovine host after 6 days of infection. Total RNA from M. hemolytica A1 recovered from pneumonic lungs of two animals was used to produce cDNA to screen a custom M. hemolytica A1 microarray. The expression profile was compared to a RNA sample from an in vitro grown culture. The data showed that 44 genes were differentially expressed by more than eightfold when compared with the in vitro sample. Seventeen genes were found to have higher expression in vivo and 27 genes had lower expression. Several virulence-associated genes including those encoding leukotoxin, a capsule biosynthetic enzyme and the serotype-specific antigen, Ssa, had reduced expression, suggesting that their products may not be important during the later stages of infection. Most of the genes up-regulated in vivo encoded hypothetical or conserved hypothetical proteins. Three Mu-like bacteriophage-related genes were up-regulated in the in vivo sample, suggesting that the prophage may be transcriptionally active. The results provide a glimpse of gene expression by the bacterium in the host after pulmonary infection has been established.

Identification of putative two-component regulatory systems in the bovine pathogen Mannheimia haemolytica A1, and preliminary characterization of the NarQ/P system.

The complete genome sequence of the bovine pathogen Mannheimia haemolytica A1 was analyzed by blast searches for the presence of two-component regulatory system proteins. Five complete sets of putative two-component systems were identified, and the NarQ/P system was further investigated. in silico analysis of the NarQ and NarP proteins showed features that are typical of the sensor and response regulator proteins. A narP knock-out mutant was constructed. The narP mutant has lost its ability to respond to NaNO(3) in the media and fail to alter the expression of several proteins. One of the proteins that showed increased production in the parent strain in response to NaNO(3) was analyzed by matrix-assisted laser desorption ionization time-of-flight MS. Unexpectedly, the protein was identified to be FbpA, a periplasmic component of the iron transporter system. Sequence analysis of the promoter region of fbpA identified motifs typical for NarP-regulated genes. The expression of the leukotoxin gene was also altered in the narP mutant as shown by Western immunoblot analysis and reverse transcription-PCR.

Characterization of Staphylococcus aureus isolates with a partial or complete absence of staphylococcal cassette chromosome elements.

Detection of methicillin-resistant Staphylococcus aureus (MRSA) by single-locus PCR assays that target the extremity of the staphylococcal cassette chromosome-mec (SCCmec) and part of the adjacent S. aureus-specific open reading frame gene (orfX) is a significant diagnostic advancement, since it provides real-time detection directly from screening specimens. However, isolates harboring mecA deletions within SCCmec may result in false-positive identification of MRSA in these assays. We characterized 24 methicillin-susceptible S. aureus (MSSA) isolates that tested positive in one such assay to investigate this phenomenon. Seven isolates resembled USA100 and carried SCCmec II elements with mecA deletions that spanned 20 to 46 kbp. The mecA excisions in USA100-resembling isolates appeared to be linked with IS431 transposable elements present in SCCmec II. For 17 isolates that resembled USA400 and/or MSSA476, the identity and possible excision of SCC elements could not be confirmed. The downstream common sequence (dcs) shared by SCCmec I, II, and IV elements was detected in these isolates. Sequence analysis of the chromosomal regions flanking the missing SCC element revealed an intact SCC integration site, a duplicate dcs, and the enterotoxin gene cluster downstream of orfX. An annealing sequence for one of the SCCmec-specific primers (mecii574) in the single-locus PCR assay was identified in the duplicate dcs. In the absence of SCC, a 176-bp amplicon can be generated from this mecii574 annealing sequence to yield a false-positive result. In conclusion, partial SCCmec II excisions via IS431 elements in strains that resembled USA100 and the presence of a duplicate mecii574 annealing sequence in strains that resembled USA400/MSSA476 were identified as causes for false-positive results in a single-locus PCR assay that targets the SCCmec/orfX junction.

Analysis of a collagen-binding trimeric autotransporter adhesin from Mannheimia haemolytica A1.

A locus that codes for a high-molecular-weight adhesin was previously isolated from Mannheimia haemolytica A1. In this study, we showed that this locus, named ahs, codes for two proteins (AhsA and AhsB) that exhibit characteristics of a trimeric autotransporter adhesin. Sequence analysis of AhsA showed the presence of 21 collagen-binding motifs in the protein. Collagen-binding assays showed that M. haemolytica A1 binds to collagen in a dose-dependent manner. This binding activity is trypsin sensitive and can be inhibited by anti-AhsA antibody. AhsB is the cognate transporter for AhsA. The C-terminal of AhsB showed highly conserved amino acids typical of trimeric autotransporters. Experimental data showed that the C-terminal 120 amino acids of AhsB could indeed form trimeric molecules. Western immunoblots showed the presence of anti-AhsA antibodies in the sera of calves that had been challenged with M. haemolytica A1, suggesting that AhsA is expressed and immunogenic in cattle.

Expression of a modified Mannheimia haemolytica GS60 outer membrane lipoprotein in transgenic alfalfa for the development of an edible vaccine against bovine pneumonic pasteurellosis.

The GS60 antigen is one of the protective antigens of Mannheimia haemolytica A1. GS60 contains conserved domains belonging to the LppC family of bacterial outer membrane lipoproteins. A high antibody titer to GS60 has been shown to be significantly correlated with resistance to pneumonic pasteurellosis. Calves vaccinated with a commercial vaccine (Presponse) and demonstrating protection against M. haemolytica A1 produced antibodies directed against GS60. Alfalfa was chosen as the platform for an edible vaccine. Agrobacterium tumefaciens was used to mediate the transformation of alfalfa with sequences encoding a slightly shortened derivative of the GS60 antigen (GS60(54)). Stable transgenic alfalfa lines were recovered and production of GS60(54) was examined by Western immunoblot analysis. The antigen is stable in dried transgenic plant material stored at ambient temperature for more than a year. The plant-produced GS60(54) protein was shown to be immunogenic when injected into rabbits. Feeding of the dried transgenic alfalfa expressing the GS60(54) to rabbits is capable of inducing seroconversion, suggesting that GS60(54) could be an effective oral antigen for stimulating mucosal immune responses.

The outer membrane protein OmpA of Mannheimia haemolytica A1 is involved in the binding of fibronectin.

An enzyme-linked immunosorbent assay using bovine fibronectin as the substrate was used to demonstrate that Mannheimia haemolytica A1 binds to fibronectin. This binding to fibronectin was specific as no binding was observed with bovine fibrinogen. The binding to fibronectin was not observed if the M. haemolytica A1 cells were pretreated with trypsin or proteinase K, suggesting that it involved a protein molecule on the cell surface. Interestingly, the fibronectin-binding activity was found to be higher in an acapsular mutant compared with its parent strain. The fibronectin-binding protein was shown to be present in the outer membrane fraction of M. haemolytica A1. A 45 kDa outer membrane protein that binds to fibronectin was identified by Far-Western immunoblot analysis. This protein was purified and subjected to MS matrix-assisted laser desorption ionization time-of-flight analysis. The results identified it to be outer membrane OmpA based on comparison with the M. haemolytica A1 genomic sequence.

Analysis of in vivo expressed genes in Mannheimia haemolytica A1.

The expression of Mannheimia haemolytica A1 genes during in vivo growth was examined by reverse transcriptase-polymerase chain reaction (RT-PCR) using total RNA extracted directly from M. haemolytica A1 recovered from pneumonic lungs of cattle. Primers specific for three groups of genes were used. Group 1 includes virulence-related genes: lktC, tbpB, ahs, nmaA, gs60 and gcp. Group 2 includes genes that code for putative two-component regulatory systems: narP, narQ, ttrR, ttrS, phoB and phoR. Group 3 includes genes involved in regular cellular functions such as plp4, thiL and rrf. The RT-PCR data were examined in conjunction with the percent pneumonic lesion in each lung scored during necropsy. The analysis showed that lungs with a higher percent pneumonic score exhibit expression of more M. haemolytica A1 genes. For group 1 genes, lktC was expressed in the majority of samples, whereas the other genes were only expressed in some samples. This was not unexpected as the leukotoxin is a major virulence factor of the bacterium. The genes encoding the response regulators for the putative two-component regulatory systems were found to be expressed in more samples than the genes encoding the sensor proteins. The regulator proteins may be required in higher levels to regulate expression of target genes.

Cloning of a gene fragment encoding bovine complement component C3d with expression and characterization of derived fusion proteins.

The gene fragment coding for bovine C3d gene (boC3d) was cloned and expressed as a component of fusion proteins destined for use in vaccine studies in cattle, and for in vitro experiments. This fragment of complement protein C3 (C3d) has been shown to enhance B cell responses when complexed with antigen. Three potential vaccine constructs were engineered to contain one, two or three boC3d units linked to a fragment of the leukotoxin of Mannheimia haemolytica A1, an economically important pathogen of cattle that causes a fibrinous pneumonia in calves. A recombinant biotinylated boC3d protein (for use in in vitro studies) was generated by endogenous biotinylation in Escherichia coli by means of the BirA holoenzyme synthetase. All recombinant proteins incorporated polyhistidine tags and were purified by nickel-agarose chromatography, then analyzed by SDS-PAGE and Western immunoblot. The identity of boC3d was confirmed by mass spectrometry, since monoclonal antibodies to boC3d were not available. To date, published research into the adjuvant activities of C3d has been limited to experiments in mice and rabbits, using antigens unrelated to diseases occurring naturally in these species. The boC3d fusion proteins expressed in this study will provide the basis for immunization trials in cattle and studies of receptor binding and cell activation of bovine lymphocytes.

Construction and analysis of a Mannheimia haemolytica A1 luxS mutant.

Mannheimia haemolytica A1 is the causative agent of bovine pneumonic pasteurellosis, a major cause of sickness, death, and economic loss to the feedlot cattle industry. M. haemolytica A1 produces autoinducer-2 (AI-2) like molecules that are capable of inducing quorum sensing system 2 of Vibrio harveyi. This interspecies quorum sensing system has been shown to regulate the expression of virulence genes in several pathogenic bacteria. The protein central to the production of AI-2 is LuxS. To determine if quorum sensing is involved in the regulation of virulence genes in M. haemolytica A1, a luxS mutant was constructed by replacing luxS with a cat cassette. This mutant was verified by PCR analysis, Southern hybridization, as well as its inability to induce bioluminescence in the V. harveyi reporter strain. RT-PCR analysis showed there was no difference in leukotoxin (lktC) mRNA levels, however there were increased mRNA levels of putative virulence associated genes, transferrin binding protein B (tbpB), adhesin (ahs) and capsule biosynthesis (nmaA). Electron microscopy showed that the level of encapsulation in the mutant is higher than the parent. Additionally, the mutant was slightly more adherent to bovine tracheal cells than the parent. In vitro competition assays showed the mutant out-competed the parent under iron-restricted conditions. However, in a calf challenge, the parent was the dominant isolate recovered.

Efficacy of recombinant sialoglycoprotease in protection of cattle against pneumonic challenge with Mannheimia (Pasteurella) haemolytica A1.

Secreted recombinant sialoglycoprotease fusion protein (Gcp-F) of Mannheimia (Pasteurella) haemolytica A1 was examined for its ability to protect cattle from experimental challenge with M. haemolytica A1. Five M. haemolytica vaccines were compared including Gcp-F, logarithmic phase culture supernate (Presponse) and Presponse enriched with Gcp-F, recombinant leukotoxin (rLkt) or both. All calves receiving Gcp-F had significant serum antibody responses to this antigen, measured by ELISA, prior to challenge. Those vaccinated with Gcp-F alone had significantly lower percent pneumonic tissue than unvaccinated controls and a trend (P=0.085, one-tailed test) to lower clinical scores. Calves receiving Presponse with Gcp-F and rLkt had lower percent pneumonic tissue than those receiving Presponse alone, and calves receiving Presponse enriched with Gcp-F and/or rLkt had lower mean clinical scores, but the differences were not significant. This trial demonstrates the protective capacity of sialoglycoprotease. While, remarkably, recombinant Gcp-F provided some protection alone the results support its practical potential as a component of a multiple antigen vaccine.

Novel protease produced by a Pasteurella trehalosi serotype 10 isolate from a pneumonic bighorn sheep: characteristics and potential relevance to protection.

A strain of Pasteurella trehalosi serotype 10, E(CO)-100, isolated from a bighorn sheep that had succumbed to pneumonic pasteurellosis during an epizootic, was compared to well-characterized strains of P. trehalosi serotype 10 and Mannheimia haemolytica serotype 1. The gene for leukotoxin A (lktA) from E(CO)-100 was sequenced and found to be identical on an amino acid basis to a published sequence for lktA from P. trehalosi serotype 10. However, the toxic activity in culture supernatant measured over time for E(CO)-100 was quite different from reference strains. Typically, the ability of the supernatant to lyse target cells increases over time corresponding to the logarithmic growth of the organism, peaks at mid to late phase, then declines gradually. Supernatant from E(CO)-100 exhibited a sharp decline in toxicity after mid-logarithmic growth to undetectable levels. Investigation of this anomaly using a commercial kit with a porcine gelatin/bovine albumin substrate matrix revealed high protease activity in the supernatant of this strain compared to another P. trehalosi serotype 10 and to a M. haemolytica serotype 1. Protease activity was also visualized using gelatin based zymogram gels. This protease was not substrate specific as it was shown to degrade leukotoxin. Activity was neutralized by bighorn sera in a titratable manner. There was an association between the ability to neutralize protease and low pneumonic lung scores in bighorn sheep experimentally challenged with E(CO)-100 (r=0.5, P=0.1). This previously unidentified protease may be an important protective antigen in vaccines designed to prevent pneumonic pasteurellosis resulting from P. trehalosi in bighorn sheep.

Mannheimia haemolytica serotype 1 and Pasteurella trehalosi serotype 10 culture supernatants contain fibrinogen-binding proteins.

Fibrinogen-binding proteins were found in the culture supernatants of Mannheimia haemolytica serotype 1 (ATCC 43270) and Pasteurella trehalosi serotype 10 (ECO-100). Sheep fibrinogen was biotinylated and shown to bind to proteins in the culture supernatants by modified western blot. Fibrinogen-binding proteins in the culture supernatant may be important virulence factors leading to the characteristic fibrinous pneumonia caused by these organisms and may be critical antigenic targets for immune prophylaxis.

Construction and characterization of an acapsular mutant of Mannheimia haemolytica A1.

The nmaA and nmaB genes, which code for UDP-GlcNAc-2-epimerase and UDP-ManNAc-dehydrogenase, respectively, are involved in capsular polysaccharide biosynthesis in Mannheimia haemolytica A1. A chloramphenicol resistance (Cm(r)) cassette cloned behind an M. haemolytica A1 promoter, plpcat, was created and used to interrupt nmaA and nmaB. A 1.3-kbp DNA fragment that encompasses part of nmaA and nmaB was replaced by the 1.0-kbp plpcat, resulting in a knockout mutant which is Cm(r) and unable to synthesize N-acetylmannosamine (ManNAc) and N-acetylmannosaminuronic acid (ManNAcA). The DNA replacement was confirmed by Southern hybridization and PCR analyses of the nmaA and nmaB loci. Electron microscopy examination of the mutant showed the absence of capsular materials compared to the parent strain. The loss of NmaA and NmaB activity was confirmed by analysis of carbohydrate moieties using capillary electrophoresis. Serum sensitivity assays indicated that the acapsular mutant is as resistant as the encapsulated parent to complement-mediated killing by colostrum-deprived calf serum but is more sensitive to killing by immune bovine serum. Analysis of lipopolysaccharide prepared from the acapsular mutant and encapsulated parent confirmed that these strains have long O-polysaccharide chains, possibly conferring resistance to serum-mediated killing.

Studies on the production of quorum-sensing signal molecules in Mannheimia haemolytica A1 and other Pasteurellaceae species.

The bioluminescence assay system using Vibrio harveyi reporter strains were used to examine quorum-sensing autoinducer (AI) activity from Mannheimia haemolytica A1 cell-free culture supernatant. We showed that M. haemolytica A1 cell-free culture supernatant contains molecules that can stimulate the quorum-sensing system that regulates the expression of the luciferase operon in V. harveyi. Specifically, M. haemolytica A1 can stimulate only the quorum system 2 but not system 1, suggesting that the culture supernatant only contains molecules similar to AI-2 of V. harveyi. The bioluminescence assay was also used to show that culture supernatants from related Pasteurellaceae organisms, Pasteurella multocida, Pasteurella trehalosi, Actinobacillus suis and Actinobacillus pleuropneumoniae, also contain AI-2-like molecules. This is consistent with the presence of a luxS homolog in the genomes of P. multocida and A. pleuropneumoniae. A luxS homolog was cloned by PCR from M. haemolytica A1 using sequencing data from the ongoing genome sequencing project. The cloned luxS(M.h.) was able to complement AI-2 production in the Escherichia coli DH5alpha luxS mutant. This is the first report of a quorum-sensing activity in M. haemolytica A1 and suggests that this bacterium utilizes this mechanism to regulate expression of genes under specific conditions.

A putative iron-regulated TonB-dependent receptor of Mannheimia (Pasteurella) haemolytica A1: possible mechanism for phase variation.

A recombinant plasmid that codes for a novel iron receptor protein (Irp) of Mannheimia (Pasteurella) haemolytica A1 was isolated by the partial complementation of an Escherichia coli fur mutant. The deduced amino acid sequence of Irp exhibited characteristics typical of TonB-dependent receptors. These include: a TonB-box at the N-terminal; a 50 amino acid region homologous to the "plug" domain of the E. coli FhuA and FepA receptors; and a C-terminal TonB-dependent signature which likely functions as an outer membrane anchoring domain. Previously uncharacterized Irp homologues were detected by BLAST analysis of available databases and incomplete microbial genomes. When the irp homologues from Neisseria gonorrhoeae and N. meningitidis were cloned by PCR and expressed in E. coli, novel proteins of the predicted size (84kDa) were detected in cell lysates, demonstrating that these are functional genes. The M. haemolytica A1 irp gene undergoes phase variation at a nucleotide region which contain the sequence AAAAAAATTAAAA (7A-2T-4A) flanked by a short inverted repeat. Site-specific mutagenesis of the 7A-2T-4A sequence as well as replacement of the inverted repeats resulted in a stable construct that expressed the Irp protein without phase variation. The expression of irp in M. haemolytica A1 was regulated by iron concentrations and most likely a Fur homologue, consistent with the proposed function of Irp in iron metabolism. The irp genes may represent contingency loci that play a role in iron acquisition during infection.