Legionella pneumophila Secretes a Mitochondrial Carrier Protein during Infection

The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionella nucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms

Transport of LncP across five membranes.

<p>Unlike regular bacterial inner membrane proteins with alpha-helical transmembrane segments (non-Dot/Icm effectors) (red), LncP (blue) avoids the YidC and SecYEG machinery in the bacterial inner membrane and is instead loaded into the T4SS for secretion across both the inner and outer bacterial membrane and across the vacuolar membrane. Similar to endogenous carrier proteins, LncP is then presumably recognized by Hsp70 and Hsp90 chaperones in the host cell cytosol and delivered to the TOM complex via interactions with the Tom70 receptor. The protein is then translocated across the outer mitochondrial membrane and interacts with the Tim9/10 chaperones in the intermembrane space to be assembled into the mitochondrial inner membrane by the TIM22 complex. There, the transport activity of LncP would impact on nucleotide homeostasis between the mitochondrial matrix and host cell cytosol.</p

LncP is a nucleotide carrier with unique properties.

<p>(A) Liposomes reconstituted with LncP were preloaded internally with various substrates (concentration, 10 mM). Transport was started by the addition of 0.2 mM [³H]ATP and terminated after 2 min. Values are means ± S.D. of at least three independent experiments. α-OG, α-oxoglutarate; Pi, phosphate; PPi, pyrophosphate. (B) Proteoliposomes were preloaded internally with 10 mM ATP and transport was initiated by adding 0.2 mM [³H]ATP. The reaction time was 2 min. Thiol reagents were added 2 min before the labeled substrate; the other inhibitors were added together with the labeled substrate. The final concentrations of the inhibitors were 20 mM (PLP, pyridoxal-5′-phosphate; BAT, bathophenanthroline), 0.2 mM (<i>p</i>-HMB, <i>p</i>-hydroxymercuribenzoate; MER, mersalyl), 1 mM (NEM, <i>N</i>-ethylmaleimide), 0.2% (TAN, tannic acid), 0.2 mM (BrCP, bromcresol purple), 25 µM (HgCl₂, mercuric chloride) and 10 µM (BKA, bongkrekic acid; CAT, carboxyatractyloside). The extent of inhibition (%) from representative experiments is given. (C) Uptake of [³H]ATP (▪, □) and [³H]GTP (•, ○) into liposomes reconstituted with LncP. 1 mM [³H]ATP or [³H]GTP was added to proteoliposomes containing 10 mM ATP or GTP, respectively (exchange, filled shapes), or 10 mM NaCl and no substrate (uniport, open shapes). Similar results were obtained in three independent experiments. (D) Efflux of [³H]ATP from LncP proteoliposomes. The internal substrate (2 mM ATP) was labeled by carrier-mediated exchange equilibration. After removal of the external substrate by Sephadex G-75, the efflux of [³H]ATP was started by adding buffer A alone (filled circles), 5 mM ATP, 20 mM pyridoxal-5′-phosphate and 10 mM bathophenanthroline in buffer A (filled squares), 5 mM ATP in buffer A (open squares) or 5 mM phosphate (open triangles). Similar results were obtained in three independent experiments.</p

A mitochondrial carrier protein in <i>Legionella</i>.

<p>(A) Sequence alignment of LncP from <i>L. pneumophila</i> and Llo1924 from <i>L. longbeachae</i> with the ADP/ATP carrier from <i>Bos taurus</i>. Amino acid residues are colored red (hydrophobic), blue (acidic), magenta (basic), green (polar) and the six predicted transmembrane segments shown. Conservation is seen through the predicted transmembrane segments and in the three-fold repeated signature motif (labeled SM1a-SM1b, SM2a-SM2b, SM3a-SM3b), all of which are characteristic of all members of the mitochondrial carrier protein family <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002459#ppat.1002459-Palmieri2" target="_blank">[40]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002459#ppat.1002459-Palmieri3" target="_blank">[41]</a>. (B) The three-dimensional structure of the ADP/ATP carrier from <i>B. taurus</i> (PDB: 1OKC), with the three-fold repeated signature motif color-coded as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002459#ppat-1002459-g001" target="_blank">Figure 1A</a>. The folded protein has a “height” of 46 Å and the maximum “width” dimension is 41 Å.</p

LncP is transported to the mitochondrial inner membrane.

<p>(A) HeLa cells were transformed to express LncP-GFP or a control plasmid. The LncP-GFP cells were co-stained with tetramethylrhodamine methyl ester (TMRM) and viewed by confocal microscopy. The merge shows the mitochondrial localization of LncP-GFP (B) Mitochondria (50 µg protein) from wild-type yeast cells were incubated with [³⁵S]-labeled LncP. After the indicated time at 25°C, mitochondria were isolated, treated with Proteinase K to degrade protein that had not been imported, and analyzed by SDS-PAGE and fluorography. “T” represents non-Proteinase K treated control. “-Δψ_m” indicates a sample where the mitochondria were pre-incubated with inhibitors and uncouplers to deplete the transmembrane potential (see Methods) (C) Mitochondria (100 µg protein) from wild-type cells were incubated with [³⁵S]-labeled LncP. After 20 minutes at 25°C, mitochondria were isolated, extracted with 0.1 M Na2CO3 and the membrane-containing pellet (“Pel”) and extracted proteins in the supernatant (“S/N”) analyzed by SDS-PAGE and fluorography and immunoblot against a known membrane embedded protein (Tim23) and a non-membrane embedded protein matrix localized protein (F₁β). A sample of mitochondria prior to extraction and representing the total amount (“Tot.”) is shown for comparison. The right-hand panel shows the percentage distribution of LncP in the pellet and supernatant fractions after 5 repeat experiments ± standard error. (D) Mitochondria (50 µg protein) from wild-type and Tim10 depleted (<i>tim10</i>↓) yeast cells were resuspended in isotonic import buffer and incubated with [³⁵S]-labeled LncP and PiC. After the indicated time at 25°C, mitochondria were isolated, solubilized in digitonin and analyzed by BN-PAGE and fluorography. Asterisk indicates bands formed by folded carrier proteins. The lower asterisk represents the folded monomer and the upper asterisk represents assembled carrier dimers (Stage V) (E) Mitochondria (50 µg protein) from wild-type yeast or from <i>tim10</i>↓ yeast depleted of Tim10 were incubated with [³⁵S]-labeled LncP or PiC. After the indicated time at 25°C, the mitochondria were treated with Proteinase K and then analysed by SDS-PAGE and fluorography. “-Δψ_m” indicates a sample where the mitochondria were pre-incubated with inhibitors and uncouplers to deplete the transmembrane potential (see Methods). (F) Control western blots with mitochondria isolated from wild-type and <i>tim10</i>↓ cells respectively showing that Tim10 has been selectively depleted. (G) The localization of LncP within mitochondria after import was determined using a sequential proteolysis assay. After import of [³⁵S]-labeled LncP at 25°C for 20 minutes, mitochondria were treated with hypotonic buffer to induce mitoplasting, or Triton-X-100 to rupture both membranes and Proteinase K (50 µg/mL) as indicated (see Methods). “L” is lysate only without mitochondria to show size of unimported protein. The control proteins, the inner membrane embedded protein (Tim23) and a non-membrane embedded protein matrix localized protein (F₁β) were detected by immunoblot on the same membrane.</p

Mutant <i>L. pneumophila</i> lacking LncP replicate proficiently in host cells.

<p>Two independent mutants of <i>L. pneumophila</i> 130b lacking LncP (<i>lncP-</i>3 and <i>lncP-</i>4) were tested, along with a <i>dotA</i> mutant lacking the Dot/Icm T4SS. Replication of <i>L. pneumophila</i> 130b (•),<i>lncP-</i>3 (Δ),<i>lncP-</i>4 (▿) and <i>dotA</i> (□) within the macrophage cell-line THP-1 (A) and <i>A. castellanii</i> (B) is shown. Results are expressed as the log₁₀CFU of viable bacteria present in the extracellular medium (and associated with cells for THP-1) at specific time points after inoculation, mean ± standard deviation of at least three independent experiments from duplicate wells.</p

LncP is translocated into macrophages by the Dot/Icm T4SS.

<p>(A) THP-1 macrophages were left uninfected or infected with derivatives of <i>L. pneumophila</i> 130b carrying the pEC34 vector or expressing the indicated Cya hybrid proteins. Following infection for 1 hour, macrophages were lysed and total intracellular cAMP was measure by ELISA. Results are expressed as fmol cAMP and are the mean ± standard deviation of three independent experiments, each performed in duplicate. Note Cya-LncP_ΔPTRKR is a truncated protein lacking the C-terminal residues (PTRKR) of LncP. (B) Immortalized macrophages from C57BL/6 mice were infected with derivatives of <i>L. pneumophila</i> 130b for 5 h as indicated. Bacteria were visualized using anti-<i>Legionella</i> antibodies (blue) 4HA-LncP was visualized with antibodies to HA (green). Prior to fixation, cells were stained with MitoTracker Red. Cells were viewed by confocal microscopy under a 100× objective. White scale bars represent 5 µm. (C) Immortalized macrophages from C57BL/6 mice were infected with derivatives of <i>L. pneumophila</i> 130b for 30 min, 1 h, 2 h or 3 h as indicated, stained as above, and viewed by confocal microscopy under a 100× objective. White scale bars represent 5 µm. Arrows indicate LncP at the poles of the bacterial cell.</p