Identification of FtpA, a Dps-Like Protein Involved in Anti-Oxidative Stress and Virulence in Actinobacillus pleuropneumoniae

Abstract

Bacteria have evolved a variety of enzymes to eliminate endogenous or host-derived oxidative stress factors. The Dps protein, first identified in Escherichia coli, contains a ferroxidase center, and protects bacteria from reactive oxygen species damage. Little is known of the role of Dps-like proteins in bacterial pathogenesis. Actinobacillus pleuropneumoniae causes pleuropneumonia, a respiratory disease of swine. The A. pleuropneumoniae ftpA gene is upregulated during shifts to anaerobiosis, in biofilms and, as found in this study, in the presence of H₂O₂. An A. pleuropneumoniae ftpA deletion mutant (ΔftpA) had increased H₂O₂ sensitivity, decreased intracellular viability in macrophages, and decreased virulence in a mouse infection model. Expression of ftpA in an E. coli dps mutant restored wild-type H₂O₂ resistance. FtpA possesses a conserved ferritin domain containing a ferroxidase site. Recombinant rFtpA bound and oxidized Fe²⁺ reversibly. Under aerobic conditions, the viability of an ΔftpA mutant was reduced compared with the wild-type strain after extended culture, upon transition from anaerobic to aerobic conditions, and upon supplementation with Fenton reaction substrates. Under anaerobic conditions, the addition of H₂O₂ resulted in a more severe growth defect of ΔftpA than it did under aerobic conditions. Therefore, by oxidizing and mineralizing Fe²⁺, FtpA alleviates the oxidative damage mediated by intracellular Fenton reactions. Furthermore, by mutational analysis, two residues were confirmed to be critical for Fe²⁺ binding and oxidization, as well as for A. pleuropneumoniae H₂O₂ resistance. Taken together, the results of this study demonstrate that A. pleuropneumoniae FtpA is a Dps-like protein, playing critical roles in oxidative stress resistance and virulence. IMPORTANCE As a ferroxidase, Dps of Escherichia coli can protect bacteria from reactive oxygen species damage, but its role in bacterial pathogenesis has received little attention. In this study, FtpA of the swine respiratory pathogen A. pleuropneumoniae was identified as a new Dps-like protein. It facilitated A. pleuropneumoniae resistance to H₂O₂, survival in macrophages, and infection in vivo. FtpA could bind and oxidize Fe²⁺ through two important residues in its ferroxidase site and protected the bacteria from oxidative damage mediated by the intracellular Fenton reaction. These findings provide new insights into the role of the FtpA-based antioxidant system in the pathogenesis of A. pleuropneumoniae, and the conserved Fe²⁺ binding ligands in Dps/FtpA provide novel drug target candidates for disease prevention.

Keywords: Actinobacillus pleuropneumoniae; Dps; Fenton reaction; FtpA; ferroxidase; oxidative stress; virulence.

FIG 1

FtpA has a role in A. pleuropneumoniae virulence and resistance to H₂O₂. (A) DNA sequence alignment of ftpA of A. pleuropneumoniae and dps of E. coli. The common ferritin domain (black dotted box), FOC sites (red box), and tryptophan free radical (green box) are shown. (B) The survival rates of mice infected with WT or ΔftpA. Four-week-old mice (n = 6) were infected nasally with 1 × 10⁸ CFU (log phase). Survival rates were continuously recorded until 48 h postinfection. (C) Bacterial burden in the lungs of mice infected with 2 × 10⁶ CFU of WT or ΔftpA at 6 and 12 h postinfection. Data are shown as means ± SD (n = 6). (D) The H₂O₂ sensitivities of WT, ΔftpA, and CΔftpA. Bacteria at 10,000 CFU were incubated with or without 500 μM 2,2′-bipyridine (Bpy) for 20 min, then treated with 250 μM H₂O₂ for 10 min. A control group without H₂O₂ treatment was also used (data not shown). (E) H₂O₂ sensitivities of PCN033, Δdps, CΔdps, and CΔdps^ftpA. Strains at 10,000 CFU were treated with 250 μM H₂O₂ for 10 min. A control group without H₂O₂ treatment was also used (data not shown). (F) Intracellular survival of WT, ΔftpA, and CΔftpA in macrophages. Bacteria were incubated with RAW 264.7 cells with an MOI of 10:1 for 4 h. Supernatants of the cells were discarded and the remainder were treated with antibiotics and incubated with fresh medium for an additional 0.5 h. Viable counts of bacteria inside macrophages were determined after lysis of the cells. Survival percentages of the strains are displayed as means ± SD (n = 3). (G) Influence of H₂O₂ on the expression level of the ftpA gene. The cDNA used as the templates were extracted from WT cultures (log phase) which had been incubated with 100 μM H₂O₂ for 0, 10, and 20 min. qRT-PCR was performed to determine the transcription level of ftpA using the 2^−ΔΔCt method normalized to 16s rRNA genes. Data are shown as means ± SD (n = 4). *, P < 0.05; **, P < 0.01, ***; P < 0.001, ns, not significant, as shown by Student's t test. Significant difference in the mortality of mice was analyzed by log rank (Mantel-Cox) test.

FIG 2

FtpA possesses the ability to reversibly capture and oxidize ferrous iron. (A) Growth curves of WT, ΔftpA, and CΔftpA under iron-restricted conditions. Growth curves of the bacteria were determined in medium containing 2,2′-bipyridine (solid symbols) or both 2,2′-bipyridine and ferric iron (hollow symbols) at an initial OD₆₀₀ of 0.01. (B) Interaction between rFtpA and Fe²⁺. Molecular interaction was tested in a system containing rFtpA (6 μg) and either Fe²⁺ or Fe³⁺ (0, 1, and 2 mM) (Fig. S4) in PBS. Interaction parameters are presented in the box. (C) Identification of iron capture capacity of rFtpA. After incubation of 2.5 mM FeCl₂ with 1 μM rFtpA at various times ranging from 0 to 90 min, the protein was removed and phenanthroline was added to detect residual Fe²⁺ in the solution of the reaction to measure the amount of Fe³⁺ sequestered per rFtpA dodecamer. (D) Release of iron bound by rFtpA. The rFtpA was preincubated with excess Fe²⁺, then the protein was separated and sodium ascorbate was added to reduce iron sequestered by rFtpA. Phenanthroline was applied to detect the amount of Fe²⁺ released from the rFtpA dodecamer. BSA was used as a negative control. Fe²⁺ added to Tris-HCl instead of protein was used as a blank control.

FIG 3

FtpA alleviates oxidative damage mediated by the intracellular Fenton reaction. WT, ΔftpA, and CΔftpA were continuously cultured aerobically for 13 h (A), or cultured anaerobically for 4 h and then transferred to an aerobic environment for an additional 5 h (B). Strains were cultured aerobically or aerobically for 2 h and then either 2.5 mM Fe²⁺ (C), 100 μM H₂O₂ (D), 100 μM H₂O₂ plus 200 μM deferoxamine (E), or deferoxamine alone (F) was added until stationary phase. Viable bacterial numbers were counted at selected time points. All data are shown as means ± SD (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001, as indicated by Student’s t test.

FIG 4

Predicted structure of FtpA with iron binding ligands. (A) Homologous modeling of FtpA. Using S. coelicolor DpsA15 as the template, the monomer and dodecamer structures are shown. (B) Detailed illustration of binding forms of FtpA. The two monomers form a dimer in a central symmetric structure, with two FOC sites located at each interface. (C) Molecular docking of FtpA with Fe²⁺. The amino acid residues in the Fe²⁺-binding pocket were predicated to be H65, H77, D81, D92, and E96.

FIG 5

Confirmation of critical ligands involved in ferrous iron binding and anti-oxidative stress activity of FtpA. (A and B) Detection of H₂O₂ sensitivity. Complemented strains with point mutations in ftpA (CΔftpA^H65A, CΔftpA^H77A, CΔftpA^D92A, or CΔftpA^E96A) (A) or dps (CΔftpA^dps), dprA (CΔftpA^dprA), or tftpA (a truncated ftpA gene lacking the upstream sequence before the ferritin domain, CΔftpA^tftpA) (B) (500 CFU) were treated with 250 μM H₂O₂ for 10 min. As a control, the WT, ΔftpA, and CΔftpA strains were treated in the same way. (C and D) Intracellular survival of strains. All of the above strains were incubated with RAW 264.7 cells for 4.5 h and viable counts were determined. (E) Comparison of iron oxidative ability of rFtpA and those carrying point mutations. Phenanthroline was used to assay Fe²⁺ concentration after incubation with 1 μM rFtpA or rFtpA^mut at 37°C for 90 min. (F) Comparison of reversibility of iron oxidative abilities of rFtpA and rFtpA^mut. Sodium ascorbate was added to the preincubated mixture containing 1 μM rFtpA or rFtpA^mut with Fe²⁺ to reduce Fe³⁺ in the rFtpA or rFtpA^mut at 37°C for 60 min. BSA was used as a negative control and Fe²⁺ added to PBS alone was used as a blank control. All data are shown as means ± SD (n = 3). The symbols # and * represent the significance of the difference compared with ΔftpA or CΔftpA, respectively. # or *, P < 0.05; ## or **, P < 0.01; ### or ***, P < 0.001, as indicated by Student’s t test.

FIG 6

FtpA has a role in resisting oxidative stress by restricting Fe²⁺ and alleviating oxidative damage mediated by the intracellular Fenton reaction. When exposed to ROS released from phagocytes or endogenous ROS from autoxidation of flavoproteins, H₂O₂ accumulates to inactivate iron-sulfur proteins. The released Fe²⁺ reacts with H₂O₂ in the Fenton reaction to generate toxic HO·. As an anti-oxidative stress protein, FtpA interferes with the Fenton reaction by binding with and oxidizing Fe²⁺, and therefore protects the bacteria from damage by HO·. This process may be important to A. pleuropneumoniae survival in an anaerobic environment in necrotic tissue and/or biofilms.