The Metabolic Adaptation in Response to Nitrate Is Critical for Actinobacillus pleuropneumoniae Growth and Pathogenicity under the Regulation of NarQ/P

Abstract

Nitrate metabolism is an adaptation mechanism used by many bacteria for survival in anaerobic environments. As a by-product of inflammation, nitrate is used by the intestinal bacterial pathogens to enable gut infection. However, the responses of bacterial respiratory pathogens to nitrate are less well understood. Actinobacillus pleuropneumoniae is an important bacterial respiratory pathogen of swine. Previous studies have suggested that adaptation of A. pleuropneumoniae to anaerobiosis is important for infection. In this work, A. pleuropneumoniae growth and pathogenesis in response to the nitrate were investigated. Nitrate significantly promoted A. pleuropneumoniae growth under anaerobic conditions in vitro and lethality in mice. By using narQ and narP deletion mutants and single-residue-mutated complementary strains of ΔnarQ, the two-component system NarQ/P was confirmed to be critical for nitrate-induced growth, with Arg50 in NarQ as an essential functional residue. Transcriptome analysis showed that nitrate upregulated multiple energy-generating pathways, including nitrate metabolism, mannose and pentose metabolism, and glycerolipid metabolism via the regulation of NarQ/P. Furthermore, narQ, narP, and its target gene encoding the nitrate reductase Nap contributed to the pathogenicity of A. pleuropneumoniae. The Nap inhibitor tungstate significantly reduced the survival of A. pleuropneumoniae in vivo, suggesting that Nap is a potential drug target. These results give new insights into how the respiratory pathogen A. pleuropneumoniae utilizes the alternative electron acceptor nitrate to overcome the hypoxia microenvironment, which can occur in the inflammatory or necrotic infected tissues.

Keywords: Actinobacillus pleuropneumoniae; NarP; NarQ; growth; nitrate; pathogenicity; regulation.

FIG 1

Analysis of nitrate sensing and utilization pathways in A. pleuropneumoniae genome and the effects of nitrate on growth and pathogenicity. (A) Nitrate sensing and metabolism pathways in E. coli and A. pleuropneumoniae based on genome analysis. Each rectangle is colored according to the protein present in E. coli (green) and A. pleuropneumoniae (purple). Arrows indicate the direction of the pathways, and solid dots indicate products of the process. (B) Growth curves of A. pleuropneumoniae cultured under aerobic conditions with shaking (O₂) and anaerobic conditions in a static anaerobic chamber (−O₂) with or without nitrate in NAD-supplemented TSB. The OD₆₀₀ of bacterial cultures was determined every 2 h. (C) Survival rate of mice intranasally infected with A. pleuropneumoniae with different doses of nitrate (7 mice per group); (D) viable counts of different serovars of A. pleuropneumoniae at mid-log phase (4 h) cultured under anaerobic conditions in NAD-supplemented TSB in a static anaerobic chamber with additional nitrate. For growth curves and viable counts of bacteria, data are shown as means ± SD from three independent replicates. The two-tailed t test was used for statistical analysis. For comparisons of mortalities of mice, a log rank (Mantel-Cox) test was performed for statistical analysis (**, P < 0.01; ***, P < 0.001; ns, not significant).

FIG 2

Schematic representation of A. pleuropneumoniae metabolic pathways differentially regulated by nitrate. +p, transfer of phosphate groups. Red letters indicate upregulated genes, and blue letters indicate downregulated genes.

FIG 3

Roles of NarP, NarQ and its nitrate sensing residue in the growth enhancement of A. pleuropneumoniae in response to nitrate. (A and B) Growth curves of WT, ΔnarP, ΔnarQ, and complementary strains supplemented with or without nitrate; (C) sequence alignment of NarQ and NarX from different species. The aligned partial sensor domain sequences of NarQ are shown. The P-box is indicated by a red rectangle. The numbers ahead of sequences are amino acid positions of A. pleuropneumoniae NarQ. Highly conserved sites are highlighted in blue. The Arg residues reported to bind with nitrate are shown in red. Genome names are shown as italicized abbreviations (Ec for E. coli, St for Salmonella Typhimurium, Hi for Haemophilus influenzae, Ap for A. pleuropneumoniae). (D) Growth curves of the WT, ΔnarQ, complementary, and mutated complementary strains with single-residue mutations at Arg50 supplemented with or without nitrate. R50K indicates mutation of Arg50 into Lys, while R50S indicates mutation of Arg50 into Ser. The bacterial strains were cultured anaerobically in NAD-supplemented TSB in a static anaerobic chamber. Data are shown as means ± SD from three independent replicates.

FIG 4

Clustering analysis revealing divergent data sets of the differential expression patterns of WT and mutant strains (Q, ΔnarQ; P, ΔnarP) with or without nitrate treatment. (A and B) Principal-component analysis (A) and Venn (B) diagram of differentially expressed genes of WT and mutant strains treated with nitrate under anaerobic conditions. Different symbols represent three repetitions (rep) within a group. (C) Heat map of differentially expressed genes from all comparison groups and bidirectional cluster analysis of the samples. The horizontal line represents genes; each column is a sample. Red indicates genes with high expression levels, and green indicates genes with low expression levels. (D) Clusters of differentially expressed genes according to their expression patterns. Each gray line indicates the expression of each gene in a cluster, and each blue line indicates the average expression of all of the genes in a cluster.

FIG 5

Verification of differentially expressed genes by qRT-PCR and EMSA. (A) Pearson coefficient correlation analysis. x axis, log₂ value from qRT-PCR; y axis, log₂ value from RNA-Seq. (B to D) qRT-PCR showed the transcript levels of 14 candidate genes from different clusters in the WT, ΔnarQ, and ΔnarP strains after nitrate treatment compared to the corresponding untreated groups. The A. pleuropneumoniae 16S rRNA gene was used as an endogenous control. The threshold cycle (2^−ΔΔCT) was calculated to determine the relative transcript levels of each gene. Experiments were performed independently in triplicate. Data are shown as means ± SD from four independent replicates. The two-tailed t test was used for statistical analysis (**, P < 0.01; ***, P < 0.001). (E) Assessment of the binding between NarP and the promoter regions of the candidate genes by EMSA. Schematic structures depicting the organization of the candidate genes in each operon are shown above the corresponding EMSA image. Arrows indicate the transcription direction. Colored arrows in orange are differentially expressed genes validated by qRT-PCR. Red asterisks indicate the presence of NarP binding motif. Gray circles indicate the absence of the NarP binding motif.

FIG 6

Function of nitrate reductase Nap and the roles of narP, narQ, and nap in the pathogenicity of A. pleuropneumoniae. (A) Growth curves of the WT, Δnap, and CΔnap strains with or without nitrate supplementation; (B) nitrate-reducing activity of the WT, Δnap, ΔnarQ, and ΔnarP mutant strains, their complementary strains, and the single-residue-mutated complementary strains of the ΔnarQ mutant. The concentrations of NO₂⁻ in the supernatants of the bacterial strains were detected after 2 h of incubation of bacteria with 5 mM NaNO₃. (C) Survival percentage curves of the mice infected by different A. pleuropneumoniae strains. Mice were intranasally infected with WT and mutant strains (20 μL) at 4 × 10⁶ CFU (8 mice per group). The time of death of each mouse was recorded. (D) Bacterial loads in the mice infected by different A. pleuropneumoniae strains. Mice were intranasally infected with WT and mutant strains (20 μL) at 2 × 10⁶ CFU (6 mice per group). The bacterial loads in the lungs of the mice were detected at 12, 24, and 48 h postinfection. (E) Antagonistic effect of molybdoenzyme inhibitor tungstate (W) on nitrate-induced growth of A. pleuropneumoniae; (F) inhibition of nitrate reductase activity of Nap by tungstate; (G) reduction in bacterial loads in mice infected with A. pleuropneumoniae by tungstate (6 mice per group). The bacterial strains were cultured anaerobically in NAD-supplemented TSB in a static anaerobic chamber. Data are shown as means ± SD from three independent replicates. The dotted line indicates the limit of detection (40 CFU/g). A two-tailed Mann-Whitney test was used for statistical analysis in animal experiments. In other experiments, the two-tailed t test was used for statistical analysis (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ns, not significant).

FIG 7

Regulatory model of A. pleuropneumoniae overgrowth and pathogenesis induced by nitrate.