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. 2011 Aug;13(8):1261-74.
doi: 10.1111/j.1462-5822.2011.01618.x. Epub 2011 Jun 24.

In search of Brucella abortus type IV secretion substrates: screening and identification of four proteins translocated into host cells through VirB system

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In search of Brucella abortus type IV secretion substrates: screening and identification of four proteins translocated into host cells through VirB system

María Inés Marchesini et al. Cell Microbiol. 2011 Aug.

Abstract

Type IV secretion systems (T4SS) are specialized protein complexes used by many bacterial pathogens for the delivery of effector molecules that subvert varied host cellular processes. Brucella spp. are facultative intracellular pathogens capable of survival and replication inside mammalian cells. Brucella T4SS (VirB) is essential to subvert lysosome fusion and to create an organelle permissive for replication. One possible role for VirB is to translocate effector proteins that modulate host cellular functions for the biogenesis of the replicative organelle. We hypothesized that proteins with eukaryotic domains or protein-protein interaction domains, among others, would be good candidates for modulation of host cell functions. To identify these candidates, we performed an in silico screen looking for proteins with distinctive features. Translocation of 84 potential substrates was assayed using adenylate cyclase reporter. By this approach, we identified six proteins that are delivered to the eukaryotic cytoplasm upon infection of macrophage-like cells and we could determine that four of them, encoded by genes BAB1_1043, BAB1_2005, BAB1_1275 and BAB2_0123, require a functional T4SS for their delivery. We confirmed VirB-mediated translocation of one of the substrates by immunofluorescence confocal microscopy, and we found that the N-terminal 25 amino acids are required for its delivery into cells.

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Figures

Figure 1
Figure 1
Flow chart of the bioinformatic screening performed to identify B. abortus putative effector proteins (BPEs).
Figure 2
Figure 2. BPE translocation into J774.A1 cells
Intracellular cAMP levels in J774.A1 cells infected with B. abortus strains expressing 10 BPE-CyaA hybrid proteins were measured after a 5 h infection. Controls included strains expressing VirB2-CyaA, VirB6-CyaA, VirB7-CyaA or FlgE-CyaA hybrid proteins as well as a wild-type strain containing no plasmid (WT) or a strain containing the CyaA domain alone (pDCyaA). Intracellular cAMP levels were also quantified in non-infected cells. cAMP levels are representative data from four independent experiments.
Figure 3
Figure 3. VirB-dependent translocation of BPEs into J774.A1 cells
(A) Intracellular cAMP levels in J774.A1 cells infected with isogenic strains with a functional (WT) or nonfunctional VirB system (virB10 and virB11) expressing BPE-CyaA fusion proteins were measured after a 5 h infection. Controls included wild type strain (WT) and virB mutants (virB10 and virB11) either containing no plasmids, containing the CyaA domain alone (pDCyaA), or VirB2-CyaA fusion protein. Intracellular cAMP levels were also quantified in non-infected cells. Mean and SD are shown for one representative out of three independent experiments. (B) BPE-CyaA fusion protein levels of the indicated B. abortus strains were determined by immunoblot analysis with anti-CyaA antibodies.
Figure 4
Figure 4. BPE123 recruitment to the Brucella containing vacuole (BCV) is a VirB dependent process
Representative confocal micrographs of mouse BMDM infected with wild-type B. abortus (A) or a virB10 mutant strain (B) both expressing BPE123-3xFLAG (MOI 20:1). At 5 h p.i. cells were fixed and processed for immunostaining as described in materials and methods. Arrows indicate the location of BPE123 in the proximity of a wild-type BCV. (C) Percentage of FLAG positive BCVs scored during the time course of infection (mean± SD, n=3 independent experiments) (D) Intracellular cAMP levels in J774.A1 cells infected with B. abortus strain expressing BPE123-CyaA hybrid protein measured after 2, 5 and 21 h p.i. A strain containing the CyaA domain alone (pDCyaA) was included as a control. Intracellular cAMP levels were also quantified in non-infected cells at 21 h p.i. Mean and SD are shown for one representative out of three independent experiments. (E) BPE123-3xFLAG protein levels of the indicated B. abortus strains were determined by immunoblot analysis with anti-FLAG monoclonal antibody.
Figure 5
Figure 5. The N-terminal 25 amino acids of BPE123 are essential for translocation into host cells
(A and B) J774.A1 cells were infected with B. abortus strains expressing full length and N-terminal truncations of BPE123-CyaA (a–e) as well as CyaA-BPE123 hybrid protein (f). Protein translocation was measured by determining the intracellular cAMP levels in J774.A1 cells infected for 5 h with B. abortus strains harboring the indicated plasmids. The wild type strain (2308) expressing the CyaA domain alone (pDCyaA) was included as a control. Intracellular cAMP was also quantified in non-infected cells. Mean and SD are shown for one representative out of three independent experiments. (C) Full length and N-terminal truncations of BPE123-CyaA (a–e) as well as CyaA-BPE123 (f) hybrid protein levels were determined by immunoblot analysis with anti-CyaA antibodies. (D) Representative confocal micrographs of mouse BMDM infected with wild-type B. abortus expressing full length (left panel) or truncated (right panel) BPE123-3xFLAG (MOI 20:1). At 5 h p.i. cells were fixed and processed for immunostaining as described in materials and methods. Arrows indicate the location of full length BPE123 in the proximity of a BCV. (D) Full length (lane 1) or truncated (lane 2) BPE123-3xFLAG protein levels of the indicated B. abortus strains were determined by immunoblot analysis with anti-FLAG monoclonal antibody.
Figure 6
Figure 6. Amino acid sequence analysis of identified VirB substrates
(A) Schematic representation of BPE proteins translocated across B. abortus VirB system. The number of amino acids is indicated on the right. cNMP: cyclic Nucleotide Mono-Phosphate; Vertical bars represent transmembrane domains; APO: apolipoprotein; SP: Sec Signal Peptide; (B) Percentage of identity between B. abortus 2308 VirB translocated BPEs and their orthologs in representative Brucella species and the phylogenetically related species O. anthropi and O. intermedium. (C) B. abortus VirB translocated substrates C-terminal 30 amino acids. Positively charged amino acids are shaded.

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