Brucella NpeA is a secreted Type IV effector containing an N-WASP-binding short linear motif that promotes niche formation

Abstract

The modulation of actin polymerization is a common theme among microbial pathogens. Even though microorganisms show a wide repertoire of strategies to subvert the activity of actin, most of them converge in the ones that activate nucleating factors, such as the Arp2/3 complex. Brucella spp. are intracellular pathogens capable of establishing chronic infections in their hosts. The ability to subvert the host cell response is dependent on the capacity of the bacterium to attach, invade, avoid degradation in the phagocytic compartment, replicate in an endoplasmic reticulum-derived compartment and egress. Even though a significant number of mechanisms deployed by Brucella in these different phases have been identified and characterized, none of them have been described to target actin as a cellular component. In this manuscript, we describe the identification of a novel virulence factor (NpeA) that promotes niche formation. NpeA harbors a short linear motif (SLiM) present within an amphipathic alpha helix that has been described to bind the GTPase-binding domain (GBD) of N-WASP and stabilizes the autoinhibited state. Our results show that NpeA is secreted in a Type IV secretion system-dependent manner and that deletion of the gene diminishes the intracellular replication capacity of the bacterium. In vitro and ex vivo experiments demonstrate that NpeA binds N-WASP and that the short linear motif is required for the biological activity of the protein.IMPORTANCEThe modulation of actin-binding effectors that regulate the activity of this fundamental cellular protein is a common theme among bacterial pathogens. The neural Wiskott-Aldrich syndrome protein (N-WASP) is a protein that several pathogens target to hijack actin dynamics. The highly adapted intracellular bacterium Brucella has evolved a wide repertoire of virulence factors that modulate many activities of the host cell to establish successful intracellular replication niches, but, to date, no effector proteins have been implicated in the modulation of actin dynamics. We present here the identification of a virulence factor that harbors a short linear motif (SLiM) present within an amphipathic alpha helix that has been described to bind the GTPase-binding domain (GBD) of N-WASP stabilizing its autoinhibited state. We demonstrate that this protein is a Type IV secretion effector that targets N-WASP-promoting intracellular survival and niche formation.

Keywords: Brucella; N-WASP; Type IV effector; bab2_0195 gene.

Fig 1

Identification of bab2_0195 as a candidate effector. (A) Disorder propensity plot of Bab2_0195. The blue line indicates the threshold above which a polypeptide region is considered intrinsically disordered. (B) Schematic representation of Bab2_0195 showing the signal peptide at the N-terminus of the protein (SP/TM) as well as the predicted lipidated Cysteine 23. The inset shows the region where the actin regulatory ELM:LIG_GBD_Chelix_1 SLiM candidate is present with an amino acid sequence matching the motif regular expression. Below is the regular expression for the SLiM as defined in the ELM entry (41). To the right, a schematic representation of N-WASP in its autoinhibited state with the GBD domain (yellow) interacting with a region (helix in red) that is displaced by the LIG_GBD_Chelix_1 motif present in Bab2_0195. (C) Amino acid sequence alignment of the region of Bab2_0195 that harbors the N-WASP-binding SLiM with the Brucella species that have the protein. The species marked with an asterisk are former Ochrobactrum spp.

Fig 2

Bab2_0195 codes for a protein that promotes niche formation. (A) Intracellular replication in HeLa cells of the B. abortus wild type, B. abortus Δbab2_0195, and B. abortus Δbab2_0195 (Bab2_0195) strains. (B) Quantification by immunofluorescence microscopy of the number of niches per 100 HeLa cells at 48 h post-infection with the B. abortus wild type, B. abortus Δbab2_0195, B. abortus Δbab2_0195 (Bab2_0195), and B. abortus (Bab2_0195) strains. The mean and standard deviation for 100 measurements per strain are shown. ***P < 0.001, one-way ANOVA Tukey post-hoc test. (C) Representative immunofluorescence microscopy images of the quantification are shown in B. Green, Brucella; red, phalloidin. Three hundred cells per experiment were counted.

Fig 3

NpeA is a Type IV secretion substrate with a periplasmic intermediary. (A) Confocal immunofluorescence microscopy images of HeLa cells infected with the B. abortus wild type or B. abortus ΔvirB10 strains expressing NpeA-3×Flag. Cells were infected and fixed 2 h post-infection. Red, Brucella; green, Flag; white, phalloidin. The lower panels show blow-up images of the NpeA-Flag staining in the B. abortus and B. abortus ΔvirB10 strains. Arrows indicate the positive staining for Flag. (B) Quantification of the secretion of NpeA-3×Flag in the B. abortus wild type or B. abortus ΔvirB10 strains as determined by the number of Flag-positive BCVs. Inset: western blot showing the expression of NpeA-3×Flag in both strains. (C) Western blot analysis of total membranes or soluble fraction (supernatant) prepared with the B. abortus (NpeA-3×Flag) strain. NpeA-3×Flag, Flag staining; Omp19, outer membrane protein 19. (D) Western-blot analysis of the subcellular localization of NpeA-3×Flag fusion performed with the B. abortus (NpeA-3×Flag) strain. NpeA-3×Flag, Flag staining; Omp16, outer membrane protein 16; GroEL, cytoplasmic chaperonin.

Fig 4

NpeA interacts with N-WASP. (A) Schematic representation of the recombinant proteins used in the experiments shown in panels B and C. (B) In vitro interaction assays. Western blot analysis of a pull-down assay was performed with recombinant NpeA-His and MBP-N-WASP proteins, and the resin-bound fraction was developed with anti-MBP and anti-6×His antibodies. As a negative control MBP alone was used. Input: prior to the pull-down assay, 20 µL of each sample was removed for analysis. (C) Ex vivo interaction assays. NpeA-His was immobilized on a metal-chelating affinity column, washed, and total cell extracts from HeLa cells injected. After extensive washing, the proteins retained were eluted and analyzed by western Western blot with anti-His and anti-N-WASP antibodies. As a negative control, a column not preloaded with NpeA-His was used.

Fig 5

The short linear motif in NpeA is necessary for the intracellular activity of the protein. Intracellular CFUs in HeLa infection assays at 4 h post-infection with the B. abortus wild type, B. abortus ΔnpeA, B. abortus ΔnpeA (NpeA), and B. abortus ΔnpeA (NpeA_ΔSLiM) strains. ***P < 0.001, one-way ANOVA (post-hoc Tukey test).

Fig 6

Endogenous N-WASP is recruited to Brucella during infection in an NpeA and SLiM-dependent manner. (A–D) HeLa cells were infected for 60 min with either B. abortus wild type, B. abortus ΔnpeA, B. abortus expressing Flag-tagged NpeA [B. abortus (NpeA)] or B. abortus expressing a NpeA with the short linear motif deleted (ΔSLiM), then fixed and triple stained for Brucella (green), N-WASP (red), and F-actin (blue). Fluorescent cells were analyzed by confocal microscopy. Representative panels of B. abortus (NpeA) (A and B) and B. abortus ΔnpeA (C and D) infected cells. Note the presence of N-WASP spots associated with bacteria when NpeA is overexpressed (A and B) but not when it is deleted (C and D) (yellow arrows). Bar = 10 µm. (E) High magnifications of single bacteria, either overexpressing [B. abortus (NpeA)] or lacking (B. abortus ΔnpeA) NpeA. Note the tight association of an N-WASP spot with the external side of a bacteria overexpressing NpeA. Plot profiles of each fluorescent signal are shown on the right. The peaks of the green signal correspond to the bacterial cell wall. (F) Quantification of overlapping events between N-WASP spots and bacteria. Data are expressed as the percentage of positive bacteria per HeLa cell. The horizontal line represents the mean of each data set. Statistical significance was analyzed using the nonparametric Kruskal-Wallis test followed by a Dunn’s post test (***P = 0.0001). (G) Left panel, western blot showing the expression of NpeA_ΔSLiM (Flag-tagged) in B. abortus. Right panel, secretion of NpeA_ΔSLiM (Flag-tagged) in infected HeLa cells at 2 h post-infection as performed in Fig. 3. Red, Brucella; green, Flag; blue, phalloidin.