WrpA Is an Atypical Flavodoxin Family Protein under Regulatory Control of the Brucella abortus General Stress Response System

Abstract

The general stress response (GSR) system of the intracellular pathogen Brucella abortus controls the transcription of approximately 100 genes in response to a range of stress cues. The core genetic regulatory components of the GSR are required for B. abortus survival under nonoptimal growth conditions in vitro and for maintenance of chronic infection in an in vivo mouse model. The functions of the majority of the genes in the GSR transcriptional regulon remain undefined. bab1_1070 is among the most highly regulated genes in this regulon: its transcription is activated 20- to 30-fold by the GSR system under oxidative conditions in vitro. We have solved crystal structures of Bab1_1070 and demonstrate that it forms a homotetrameric complex that resembles those of WrbA-type NADH:quinone oxidoreductases, which are members of the flavodoxin protein family. However, B. abortus WrbA-related protein (WrpA) does not bind flavin cofactors with a high affinity and does not function as an NADH:quinone oxidoreductase in vitro. Soaking crystals with flavin mononucleotide (FMN) revealed a likely low-affinity binding site adjacent to the canonical WrbA flavin binding site. Deletion of wrpA (ΔwrpA) does not compromise cell survival under acute oxidative stress in vitro or attenuate infection in cell-based or mouse models. However, a ΔwrpA strain does elicit increased splenomegaly in a mouse model, suggesting that WrpA modulates B. abortus interaction with its mammalian host. Despite high structural homology with canonical WrbA proteins, we propose that B. abortus WrpA represents a functionally distinct member of the diverse flavodoxin family.

Importance: Brucella abortus is an etiological agent of brucellosis, which is among the most common zoonotic diseases worldwide. The general stress response (GSR) regulatory system of B. abortus controls the transcription of approximately 100 genes and is required for maintenance of chronic infection in a murine model; the majority of GSR-regulated genes remain uncharacterized. We present in vitro and in vivo functional and structural analyses of WrpA, whose expression is strongly induced by GSR under oxidative conditions. Though WrpA is structurally related to NADH:quinone oxidoreductases, it does not bind redox cofactors in solution, nor does it exhibit oxidoreductase activity in vitro. However, WrpA does affect spleen inflammation in a murine infection model. Our data provide evidence that WrpA forms a new functional class of WrbA/flavodoxin family proteins.

FIG 1

Regulated expression of wrpA (bab1_1070). (A) Differential expression of wrpA under oxidative stress in B. abortus ΔrpoE1 and ΔlovhK backgrounds relative to the wild type (WT) as measured by transcriptome sequencing (RNA-seq) (49). (B) Cartoon representation of the wrpA promoter region, with the consensus σ^E1 binding site at the −35 and −10 regions. (C) Oxidative stress (H₂O₂) resistance of the B. abortus 2308 wild-type (WT) strain, the ΔwrpA strain, and the ΔrpoE1 strain. CFU ratios between the treated and untreated strains (mock) are presented on a log scale. Experiments were performed in triplicate; error bars represent standard deviations.

FIG 2

Bacterial phenotype microarray analysis. (A) Comparison of cell growth between wild-type B. abortus 2308 (WT) and the B. abortus ΔrpoE1 strain across a range of distinct metabolic and stress conditions. The plot shows tetrazolium reduction (measured at 630 nm) after 72 h in the WT strain versus the ΔrpoE1 strain. Dotted lines show the 99% confidence prediction band; red dots correspond to conditions where the ΔrpoE1 strain showed a difference in growth relative to that of the WT, outside the 99% confidence prediction band, after 72 h. The full list of conditions in which the ΔrpoE1 strain differed from the WT (with data collected at multiple points on the growth curve) is presented in Table S4 in the supplemental material. (B) Comparison of cell growth between wild-type B. abortus 2308 (WT) and the B. abortus ΔwrpA strain across the same set of conditions after 72 h. We detected no significant, repeatable differences between strains after 72 h or at any other time points measured. Each full screen of 1,920 conditions was performed in duplicate for each strain, and results were averaged.

FIG 3

In vitro and in vivo infection assays. (A) Infection of differentiated THP-1 cells with wild-type B. abortus 2308 and ΔwrpA strains. The infected cells were inactivated macrophages (IM), activated macrophages (AM), or immature dendritic cells (IDC). The numbers of B. abortus CFU recovered from different THP-1 cell variants at 1, 12, 24, and 72 h postinfection were plotted. Each infection was performed in triplicate; error bars represent standard errors of the means (SEM). (B) Mice (n = 5 per strain per time point) were injected intraperitoneally with 5 × 10⁴ brucellae (wild-type, ΔwrpA, and ΔwrpA::wrpA strains). Spleens were harvested, weighed (top), and plated for CFU enumeration (bottom) at 1, 4, 8, and 12 weeks postinfection. Error bars represent SEM. Data were analyzed by one-way analysis of variance (ANOVA) followed by Dunnett's posttest (for spleen weight at 4 weeks, P is <0.05).

FIG 4

Biochemical characterization of Brucella abortus WrpA. (A) UV-visible spectra of purified E. coli WrbA (WrbA_Ec) and B. abortus WrpA. (B) Size exclusion chromatography of purified WrpA incubated with or without FMN. The corresponding standard curve used for molecular size calculation is presented in Fig. S1 in the supplemental material. (C) Representative NADH:quinone oxidoreductase assay. Purified WrbA_Ec and WrpA with the FAD or FMN cofactor were subjected to an enzymatic activity assay in which oxidation of NADH in the presence of benzoquinone (BQ) was monitored spectrophotometrically at 340 nm.

FIG 5

Structure of B. abortus WrpA. (A) WrpA tetramer with one monomer colored pink. Chloride ions and FMNs are colored green and orange, respectively. (B) Ribbon diagram of a WrpA monomer (from the P4₃2₁2 crystal form), with α-helices shown in cyan, β-strands in yellow, a chloride ion as a green sphere, and the FMN cofactor in orange. Missing regions of polypeptide are represented with dashed lines. (C) Zoomed view of the WrpA FMN binding site. The model is a surface-rendered model of WrpA (white) with the FMN cofactor from E. coli WrbA (FMN_Ec; PDB ID 3B6M) modeled at its expected position into the open B. abortus WrpA cavity. The sulfate ion from the P4₂22 crystal form is modeled as yellow sticks, and the chloride ion from the P4₃2₁2 crystal form is modeled as a green sphere. Chloride and sulfate ions from the two WrpA crystal forms overlap the terminal phosphate of FMN_Ec. The FMN cofactor (FMN_Ba; orange) from the FMN-soaked WrpA crystal structure is stacked between the W98 and Y80 side chains (red); Y80 hydrogen bonds with residues D92 and Q131 (cyan) are shown as dotted lines. (D) Zoomed views of B. abortus WrpA (WrpA_Ba) and E. coli WrbA (WrbA_Ec) active site cavities. The WrbA_Ec FMN cofactor (orange; PDB ID 3B6M) (33) is modeled into the WrbA_Ec and WrpA_Ba active sites to show the similarity in the physicochemical properties of these regions. (Left) Surface representation of active site hydrophobicity. (Right) Surface representation of the electrostatic potentials of the active sites. Missing regions of the WrpA_Ba structure described to participate in active site formation in E. coli WrbA were modeled using the SWISS-Model server (using the structure under PDB ID 3B6J as the template).

FIG 6

Comparison of B. abortus WrpA structure and that of classic WrbA family proteins. (A) WrpA monomer (transparent) with missing regions modeled using the E. coli WrbA_Ec structure (PDB ID 3B6J) as a template. (B) Conservation of WrpA and WrbA_Ec active sites. The WrbA_Ec FMN cofactor (orange) from PDB structure 3B6J (33) was modeled into the WrpA active site. (C) WrpA sequence alignment to WrbA-like proteins from E. coli (Ec) (33), Deinococcus radiodurans (Dr) (31), P. aeruginosa (Pa) (31), Sulfolobus tokodaii (St) (PDB ID 2ZKI), and Agrobacterium tumefaciens (At) (PDB ID 3D7N). The WrpA secondary structure is reported above the alignment, with the β-strands shown in yellow and α-helices shown in cyan. Unmodeled regions are delimited as indicated in panel A. Residues reported to interact with FMN and NADH in WrbA_Ec structures (PDB IDs 3B6J, 3B6K, and 3B6M) (33) are marked below the alignment iloop, insertion loop.