Effects of OxyR regulator on oxidative stress, Apx toxin secretion and virulence of Actinobacillus pleuropneumoniae

Abstract

Introduction: Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, poses a significant threat to global swine populations due to its high prevalence, mortality rates, and substantial economic ramifications. Understanding the pathogen's defense mechanisms against host-produced reactive oxygen species is crucial for its survival, with OxyR, a conserved bacterial transcription factor, being pivotal in oxidative stress response.

Methods: This study investigated the presence and role of OxyR in A. pleuropneumoniae serovar 1-12 reference strains. Transcriptomic analysis was conducted on an oxyR disruption mutant to delineate the biological activities influenced by OxyR. Additionally, specific assays were employed to assess urease activity, catalase expression, ApxI toxin secretion, as well as adhesion and invasion abilities of the oxyR disruption mutant on porcine 3D4/21 and PT cells. A mice challenge experiment was also conducted to evaluate the impact of oxyR inactivation on A. pleuropneumoniae virulence.

Results: OxyR was identified as a conserved regulator present in A. pleuropneumoniae serovar 1-12 reference strains. Transcriptomic analysis revealed the involvement of OxyR in multiple biological activities. The oxyR disruption resulted in decreased urease activity, elevated catalase expression, enhanced ApxI toxin secretion-attributed to OxyR binding to the apxIBD promoter-and reduced adhesion and invasion abilities on porcine cells. Furthermore, inactivation of oxyR reduced the virulence of A. pleuropneumoniae in a mice challenge experiment.

Discussion: The findings highlight the pivotal role of OxyR in influencing the virulence mechanisms of A. pleuropneumoniae. The observed effects on various biological activities underscore OxyR as an essential factor contributing to the pathogenicity of this bacterium.

Keywords: Actinobacillus pleuropneumoniae; Apx toxins; oxidative stress; oxyR gene; virulence.

Figure 1

Prevalence and sequence alignment analysis of OxyR in A. pleuropneumoniae. (A) Analyses the distribution of OxyR in serovar 1-12 of A. pleuropneumoniae by Western blot. (B) Multiple alignments of OxyR transcriptional regulators from A. pleuropneumoniae 4074 (AWG95833.1), Haemophilus (WP_071610708.1), Haemophilus ducreyi (WP_010944668.1) and E. coli (CAA36893.1). Conserved amino acid residues among these four proteins are highlighted by red, the proposed helix-turn-helix DNA-binding domain is marked by green, and the conserved reactive Cys-199 residues between E. coli and 4074 are marked by an asterisk. (C) Phylogenetic analysis of OxyR among different bacterial species. The asterisk (*) is used to mark the location of conserved cysteine residues in the OxyR protein.

Figure 2

Intron-based mutagenesis of oxyR gene in A. pleuropneumoniae strain 4074. (A) The oxyR gene location in A. pleuropneumoniae genome. (B) The mature ribonucleoprotein complex (RNP) recognizes the target DNA by the principle of base complementary pairing. The intron sequence was reverse transcribed into cDNA and inserted into the target site by the reverse splicing process. (C) Identification of the oxyR gene mutant by Western blot using anti-OxyR specific antibody.

Figure 3

Transcriptomic analysis of the oxyR mutant. (A) Volcano plot showing gene expression. Red, green, and gray points represent upregulated, downregulated, and nonsignificant genes, respectively. (B) Pathways of differentially expressed genes analyzed by Gene Ontology.

Figure 4

Effects of oxyR inactivation on the growth, catalase and urease activities of A. pleuropneumoniae. (A) Influence of H₂O₂ and FeCl₃ on growth of WT A. pleuropneumoniae and the ΔoxyR strains. H₂O₂ was added to cultures of the wild-type and mutant strain to the final concentrations indicated. The cultures were kept on a shaker under the same growth conditions. (B) Catalase activity between the WT and △oxyR strains. (C) Urease activity between the WT and △oxyR strains. Data presented are the mean ± S.D. from three independent experiments performed in duplicate. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 5

Secretion and expression levels of Apx toxins in WT and mutant strains. (A) Western blot analysis on secretion of ApxI and ApxII toxins in bacterial culture supernatants of WT and △oxyR strains. (B) Fold change in the expression level of apx genes in △oxyR by comparison with the WT strain.

Figure 6

Identification of the potential OxyR binding motif. (A) Potential OxyR binding motif was identified by MEME. Representative sequences bound by OxyR in the EMSAs are listed below. The conserved sequence is shown in colors. (B) OxyR specifically binds to the oxyR, apxIBD and catalase promoters and the interactions between the protein and DNA were dissociated by unlabeled probe.

Figure 7

Cell adhesion and invasion assays. (A) Adhesion and (B) invasion of △oxyR strain. The strains were incubated with 3D4/21 and PT cells separately at the MOI of 100 for 2 h of incubation at 37°C. After washing out the unbound bacteria, the 3D4/21 and PT cells were lysed separately and the bacterial counts in the lysates were determined. Data are shown as mean ± SD (N = 6). ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Figure 8

Virulence and colonization of the A. pleuropneumoniae △oxyR strains in a BALB/c mouse infection model. (A) Survival curves for A. pleuropneumoniae infected mice. Wild-type and △oxyR treated mice were monitored over a 32 hours period post infection. (B) Bacterial loads in the lungs. Mice were intraperitoneally inoculated with A. pleuropneumoniae strains. Lung samples were isolated to determine bacterial loads at 6 hours post infection. Significant differences are indicated by ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.