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, 4 (6), e6096

Bacterial Ortholog of Mammalian Translocator Protein (TSPO) With Virulence Regulating Activity

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Bacterial Ortholog of Mammalian Translocator Protein (TSPO) With Virulence Regulating Activity

Annelise Chapalain et al. PLoS One.

Abstract

The translocator protein (TSPO), previously designated as peripheral-type benzodiazepine receptor, is a protein mainly located in the outer mitochondrial membrane of eukaryotic cells. TSPO is implicated in major physiological functions and functionally associated with other proteins such as the voltage-dependent anionic channel, also designated as mitochondrial porin. Surprisingly, a TSPO-related protein was identified in the photosynthetic bacterium Rhodobacter sphaeroides but it was initially considered as a relict of evolution. In the present study we cloned a tspO gene in Pseudomonas fluorescens MF37, a non-photosynthetic eubacterium and we used bioinformatics tools to identify TSPO in the genome of 97 other bacteria. P. fluorescens TSPO was recognized by antibodies against mouse protein and by PK 11195, an artificial ligand of mitochondrial TSPO. As in eukaryotes, bacterial TSPO appears functionally organized as a dimer and the apparent Kd for PK 11195 is in the same range than for its eukaryotic counterpart. When P. fluorescens MF37 was treated with PK 11195 (10(-5) M) adhesion to living or artificial surfaces and biofilm formation activity were increased. Conversely, the apoptotic potential of bacteria on eukaryotic cells was significantly reduced. This effect of PK11195 was abolished in a mutant of P. fluorescens MF37 deficient for its major outer membrane porin, OprF. The present results demonstrate the existence of a bacterial TSPO that shares common structural and functional characteristics with its mammalian counterpart. This protein, apparently involved in adhesion and virulence, reveals the existence of a possible new inter kingdom signalling system and suggests that the human microbiome should be involuntarily exposed to the evolutionary pressure of benzodiazepines and related molecules. This discovery also represents a promising opportunity for the development of alternative antibacterial strategies.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Comparison of the putative topological organization of bacterial and mitochondrial TSPO.
(a) Pseudomonas fluorescens MF37 bactTSPO. (b) BactTSPO from Rhodobacter sphaeroides 2.4.1 recalculated from Yeliseev and Kaplan . (c) Mitochondrial TSPO . P. fluorescens MF37 bactTSPO has a typical five transmembrane helix structure, a larger L1 intra-cytoplasmic loop, and an extended cytoplasmic C-terminal end. In contrast, the extracellular N-terminal end is absent, and a peptidoglycane binding domain signature is present between the L2 loop and H3 transmembrane domain.
Figure 2
Figure 2. Identification of tspO genes in fluorescent Pseudomonas.
PCR amplification of tspO-encoding sequences in the biovar V strains Pseudomonas fluorescens MF37 and MF0, the clinical biovar II strain Pseudomonas fluorescens MFY70, the biovar B strain Pseudomonas putida MFN3597, and the biovar I strains, Pseudomonas fluorescens MFY162 and MFN1032. The biovar II strain, Pseudomonas fluorescens MFY63, lacks tspO-related sequences. Std: Molecular mass standards.
Figure 3
Figure 3. Amino acid sequence cladogram showing the relationships between bacterial TSPO.
Bacterial names, accession numbers, and taxonomic groups are indicated for each branch. Bold characters indicate γ-proteobacteria, and the black arrow indicates the position of TSPO expressed in human mitochondria. The two dotted boxes indicate fluorescent Pseudomonas strains containing a bactTSPO gene (P. fluorescens and P. syringae). The degree of statistical support for branches was determined with 1000 bootstrap replicates. All branches showed less than 30% divergence.
Figure 4
Figure 4. Comparison of tspO genomic environments among Pseudomonas.
Physical maps of the regions upstream and downstream of tspO are shown for P. fluorescens strains MF37, SBW25, and Pf0-1 as well as P. syringae strains 1448A (pathovar phaseolicola), DC3000 (pv. tomato), and B728a (pv. syringae). ORF: non-identified putative protein, ¤ Data obtained from Blast, * Protein putative function. Black areas represent intergenic regions.
Figure 5
Figure 5. Expression of TSPO in fluorescent Pseudomonas.
(a) Western blot analysis of total proteins extracts from the biovars V Pseudomonas fluorescens MF37 and MF0, the clinical strains of Pseudomonas fluorescens MFY70, MFY162, and MFN1032, and Pseudomonas putida MFN3597. (b) Alignment of the fragments obtained by ESI-MS/MS analysis of the TSPO immunoreactive bands extracted from MF37 and showing 92% recovery with the theoretical sequence of the protein. Std: Molecular mass standards.
Figure 6
Figure 6. Binding of PK 11195 to bacterial TSPO of Pseudomonas fluorescens MF37.
(a) Demonstration of the binding of [3H] PK 11195 at the position of the TSPO-immunoreactive band visualized by western blots in P. fluorescens MF37 extracts. (b) Scatchard plot showing the binding characteristics of PK 11195 to TSPO of P. fluorescens MF37. The maximal binding potential (Bmax) is 0.087 pmole.µ g−1 bacterial protein and the Kd is 0.92 nM.
Figure 7
Figure 7. Effect of PK 11195 on Pseudomonas fluorescens MF37 adhesion to glass or eukaryotic cells, and on biofilm formation on PVC.
Results are expressed as percentages of the values measured in the absence of treatment (100%). These changes were not associated with variations in the global surface polarity of bacteria. ***P<0.001.
Figure 8
Figure 8. Effect of PK 11195 on the cytotoxicity of Pseudomonas fluorescens MF37 and its OprF-deficient (373) and complemented OprF-deficient (373O) mutants.
(a) Effect of PK 11195 (10−5 M) on the apoptotic-like activity of the bacteria determined by measuring nitrite production (NO2 ) resulting from NO biosynthesis by eukaryotic cells. (b) Effect of PK 11195 (10−5 M) on necrotic-like effect of P. fluorescens MF37, 373 and 373O determined by lactate dehydrogenase (LDH) activity released by eukaryotic cells. ***P<0.001. NS: not significant.

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