Nina Baltes

Actinobacillus pleuropneumoniae Iron Transport and Urease Activity: Effects on Bacterial Virulence and Host Immune Response

ABSTRACT Actinobacillus pleuropneumoniae, a porcine respiratory tract pathogen, has been shown to express transferrin-binding proteins and urease during infection. Both activities have been associated with virulence; however, their functional role for infection has not yet been elucidated. We used two isogenic A. pleuropneumoniaesingle mutants (ΔexbB and ΔureC) and a newly constructed A. pleuropneumoniae double (ΔureC ΔexbB) mutant in aerosol infection experiments. Neither the A. pleuropneumoniaeΔexbB mutant nor the double ΔureCΔexbB mutant was able to colonize sufficiently long to initiate a detectable humoral immune response. These results imply that the ability to utilize transferrin-bound iron is required for multiplication and persistence of A. pleuropneumoniae in the porcine respiratory tract. TheA. pleuropneumoniae ΔureC mutant and the parent strain both caused infections that were indistinguishable from one another in the acute phase of disease; however, 3 weeks postinfection the A. pleuropneumoniae ΔureCmutant, in contrast to the parent strain, could not be isolated from healthy lung tissue. In addition, the local immune response—as assessed by fluorescence-activated cell sorter and enzyme-linked immunosorbent spot analyses—revealed a significantly higher number ofA. pleuropneumoniae-specific B cells in the bronchoalveolar lavage fluid (BALF) of pigs infected with the A. pleuropneumoniae ΔureC mutant than in the BALF of those infected with the parent strain. These results imply thatA. pleuropneumoniae urease activity may cause sufficient impairment of the local immune response to slightly improve the persistence of the urease-positive A. pleuropneumoniaeparent strain.

Enzymes involved in anaerobic respiration appear to play a role in Actinobacillus pleuropneumoniae virulence.

ABSTRACT Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia, is able to survive on respiratory epithelia, in tonsils, and in the anaerobic environment of encapsulated sequesters. It was previously demonstrated that a deletion of the anaerobic dimethyl sulfoxide reductase gene (dmsA) results in attenuation in acute disease (N. Baltes, S. Kyaw, I. Hennig-Pauka, and G. F. Gerlach, Infect. Immun. 71:6784-6792, 2003). In the present study, using two-dimensional polyacrylamide gel electrophoresis and quadrupole time-of-flight mass spectrometry, we identified an aspartate ammonia-lyase (AspA) which is upregulated upon induction with bronchoalveolar lavage fluid (BALF). This enzyme is involved in the production of fumarate, an alternative electron acceptor under anaerobic conditions. The coding gene (aspA) was cloned and shown to be present in all A. pleuropneumoniae serotype reference strains. The transcriptional start point was identified downstream of a putative FNR binding motif, and BALF-dependent activation of aspA was confirmed by construction of an isogenic A. pleuropneumoniae mutant carrying a chromosomal aspA::luxAB transcriptional fusion. Two aspA deletion mutants, A. pleuropneumoniae ΔaspA and A. pleuropneumoniae ΔaspAΔdmsA, were constructed, both showing reduced growth under anaerobic conditions in vitro. Pigs challenged with either of the two mutants in an aerosol infection model showed a lower lung lesion score than that of the A. pleuropneumoniae wild-type (wt) controls. Pigs challenged with A. pleuropneumoniae ΔaspAΔdmsA had a significantly lower clinical score, and this mutant was rarely reisolated from unaltered lung tissue; in contrast, A. pleuropneumoniae ΔaspA and the A. pleuropneumoniae wt were consistently reisolated in high numbers. These results suggest that enzymes involved in anaerobic respiration are necessary for the pathogens ability to persist on respiratory tract epithelium and play an important role in A. pleuropneumoniae pathogenesis.

Identification of Dimethyl Sulfoxide Reductase in Actinobacillus pleuropneumoniae and Its Role in Infection

ABSTRACT Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is capable of persisting in oxygen-deprived surroundings, namely, tonsils and sequestered necrotic lung tissue. Utilization of alternative terminal electron acceptors in the absence of oxygen is a common strategy in bacteria under anaerobic growth conditions. In an experiment aimed at identification of genes expressed in vivo, the putative catalytic subunit DmsA of anaerobic dimethyl sulfoxide reductase was identified in an A. pleuropneumoniae serotype 7 strain. The 90-kDa protein exhibits 85% identity to the putative DmsA protein of Haemophilus influenzae, and its expression was found to be upregulated under anaerobic conditions. Analysis of the unfinished A. pleuropneumoniae genome sequence revealed putative open reading frames (ORFs) encoding DmsB and DmsC proteins situated downstream of the dmsA ORF. In order to investigate the role of the A. pleuropneumoniae DmsA protein in virulence, an isogenic deletion mutant, A. pleuropneumoniae ΔdmsA, was constructed and examined in an aerosol infection model. A. pleuropneumoniae ΔdmsA was attenuated in acute disease, which suggests that genes involved in oxidative metabolism under anaerobic conditions might contribute significantly to A. pleuropneumoniae virulence.

Actinobacillus pleuropneumoniae serotype 7 siderophore receptor FhuA is not required for virulence

A ferrichrome receptor, FhuA, was identified in Actinobacillus pleuropneumoniae serotype 7. An isogenic mutant with a deletion in the ferrichrome uptake receptor gene (fhuA) was constructed and examined in an aerosol infection model. The disease caused by the mutant was indistinguishable from disease induced by A. pleuropneumoniae serotype 7 wild-type; an isogenic mutant lacking expression of the exbB gene that is required for the uptake of transferrin-bound iron retained the ability to utilize ferrichrome, thereby indicating that an energy-coupling mechanism involved in ferrichrome transport remains to be identified.

Deletion of the Anaerobic Regulator HlyX Causes Reduced Colonization and Persistence of Actinobacillus pleuropneumoniae in the Porcine Respiratory Tract

ABSTRACT Actinobacillus pleuropneumoniae, the etiological agent of porcine pleuropneumonia, is able to persist on respiratory epithelia, in tonsils, and in the anaerobic environment of encapsulated lung sequesters. We have demonstrated previously that putative HlyX-regulated genes, coding for dimethyl sulfoxide (DMSO) reductase and aspartate ammonia lyase, are upregulated during infection and that deletions in these genes result in attenuation of the organism. The study presented here investigates the role of HlyX, the fumarate nitrate reductase regulator (FNR) homologue of A. pleuropneumoniae. By constructing an isogenic A. pleuropneumoniae hlyX mutant, the HlyX protein is shown to be responsible for upregulated expression of both DMSO reductase and aspartate ammonia lyase (AspA) under anaerobic conditions. In a challenge experiment the A. pleuropneumoniae hlyX mutant is shown to be highly attenuated, unable to persist in healthy lung epithelium and tonsils, and impaired in survival inside sequestered lung tissue. Further, using an A. pleuropneumoniae strain carrying the luxAB genes as transcriptional fusion to aspA on the chromosome, the airway antioxidant glutathione was identified as one factor potentially responsible for inducing HlyX-dependent gene expression of A. pleuropneumoniae in epithelial lining fluid.

Identification of Genes Transcribed by Actinobacillus pleuropneumoniae in Necrotic Porcine Lung Tissue by Using Selective Capture of Transcribed Sequences

ABSTRACT Genes expressed by Actinobacillus pleuropneumoniae in necrotic porcine lung tissue were identified by selective capture of transcribed sequences analysis. In total, 46 genes were identified, 20 of which have been previously reported to be associated with in vivo expression or virulence in A. pleuropneumoniae or in other organisms.

Actinobacillus pleuropneumoniae Iron Transport: a Set of exbBD Genes Is Transcriptionally Linked to the tbpB Gene and Required for Utilization of Transferrin-Bound Iron

ABSTRACT Upon iron restriction, Actinobacillus pleuropneumoniaehas been shown to express the transferrin-binding proteins TbpB and TbpA, both of which have been implied to be important virulence factors. In order to identify additional iron-regulated proteins, we cloned and analyzed the region upstream of the transferrin-binding protein genes in an A. pleuropneumoniae serotype 7 strain. We located immediately upstream of the tbpB gene two open reading frames which were 43% homologous to the neisserial ExbBD protein genes. By raising specific antibodies, we showed that ExbB is expressed under iron-limiting growth conditions only, and RT-PCR analysis revealed that the exbBD genes and thetbpB gene are transcribed on a single polycistronic mRNA. By constructing an isogenic and nonpolar exbBD mutant, we showed that the exbBD genes are required by A. pleuropneumoniae for utilization of transferrin-bound iron. Using PCR and Western blotting, we showed that the genetic organization found in A. pleuropneumoniae serotype 7 is similar in all 12A. pleuropneumoniae serotype reference strains.

Identification of novel potential virulence associated factors in Haemophilus parasuis

Haemophilus (H.) parasuis is best known as the cause of Glässers disease, a potentially fatal polyserositis in pigs. The mechanisms underlying virulence differences on the molecular level are largely unknown to date. We have compared the serotype 5 (causes polyserositis) and 11 (described as avirulent) reference strains by modified representational difference analysis, and identified five potentially virulence associated factors present in the invasive serotype 5 strain, but not in the avirulent serotype 11 strain. Among these, a putative hemolysin operon, hhdBA, was identified, which is also present in half of the serotype reference strains described as virulent, but not in those reference strains that were reported to cause no disease in animal infection experiments. The presence of all identified genes was investigated in serotype reference strains as well as in 26 field isolates from clinically ill pigs. Determining the presence of these genes may be useful in H. parasuis diagnostics to judge a strains potential to cause disease.

Construction of an Actinobacillus pleuropneumoniae Serotype 2 Prototype Live Negative-Marker Vaccine

ABSTRACT Deletions were introduced into the ureC and apxIIA genes of an Actinobacillus pleuropneumoniae serotype 2 strain by homologous recombination and counterselection. The double-mutant contains no foreign DNA, is highly attenuated, protects pigs from homologous challenge upon a single aerosol application, and facilitates the serological discrimination of immunized and infected herds.