Tat–Dependent Translocation of an F420–Binding Protein of Mycobacterium tuberculosis

F420 is a unique cofactor present in a restricted range of microorganisms, including mycobacteria. It has been proposed that F420 has an important role in the oxidoreductive reactions of Mycobacterium tuberculosis, possibly associated with anaerobic survival and persistence. The protein encoded by Rv0132c has a predicted N–terminal signal sequence and is annotated as an F420–dependent glucose-6-phosphate dehydrogenase. Here we show that Rv0132c protein does not have the annotated activity. It does, however, co–purify with F420 during expression experiments in M. smegmatis. We also show that the Rv0132c–F420 complex is a substrate for the Tat pathway, which mediates translocation of the complex across the cytoplasmic membrane, where Rv0132c is anchored to the cell envelope. This is the first report of any F420–binding protein being a substrate for the Tat pathway and of the presence of F420 outside of the cytosol in any F420–producing microorganism. The Rv0132c protein and its Tat export sequence are essentially invariant in the Mycobacterium tuberculosis complex. Taken together, these results show that current understanding of F420 biology in mycobacteria should be expanded to include activities occurring in the extra-cytoplasmic cell envelope

Identification of a Lactate-Quinone Oxidoreductase in Staphylococcus aureus that is Essential for Virulence

Staphylococcus aureus is an important human pathogen commonly infecting nearly every host tissue. The ability of S. aureus to resist innate immunity is critical to its success as a pathogen, including its propensity to grow in the presence of host nitric oxide (NO·). Upon exogenous NO· exposure, S. aureus immediately excretes copious amounts of L-lactate to maintain redox balance. However, after prolonged NO·-exposure, S. aureus reassimilates L-lactate specifically and in this work, we identify the enzyme responsible for this L-lactate-consumption as a L-lactate-quinone oxidoreductase (Lqo, SACOL2623). Originally annotated as Mqo2 and thought to oxidize malate, we show that this enzyme exhibits no affinity for malate but reacts specifically with L-lactate (KM = ∼330 μM). In addition to its requirement for reassimilation of L-lactate during NO·-stress, Lqo is also critical to respiratory growth on L-lactate as a sole carbon source. Moreover, Δlqo mutants exhibit attenuation in a murine model of sepsis, particularly in their ability to cause myocarditis. Interestingly, this cardiac-specific attenuation is completely abrogated in mice unable to synthesize inflammatory NO· (iNOS−/−). We demonstrate that S. aureus NO·-resistance is highly dependent on the availability of a glycolytic carbon sources. However, S. aureus can utilize the combination of peptides and L-lactate as carbon sources during NO·-stress in an Lqo-dependent fashion. Murine cardiac tissue has markedly high levels of L-lactate in comparison to renal or hepatic tissue consistent with the NO·-dependent requirement for Lqo in S. aureus myocarditis. Thus, Lqo provides S. aureus with yet another means of replicating in the presence of host NO·

Structural comparison of Rv0132c with FGD1.

<p>(A) Amino acid sequence alignment. The secondary structure elements for FGD1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045003#pone.0045003-Bashiri2" target="_blank">[5]</a> are shown above the sequence. FGD1 residues that hydrogen bond with F₄₂₀ or the phosphate group of glucose-6-phosphate are indicated below the sequence by F and asterisk, respectively. The twin arginines in the Tat motif and the critical cysteine residue in the lipobox motif are shown in red in the Rv0132c signal sequence. (B) Superposition of the FGD1 (orange) crystal structure on the modeled Rv0132c (cyan). The F₄₂₀ cofactor (green) bound to FGD1 is shown in stick representation. Replacement of helix α₉ with a smaller loop extends the active site cavity in Rv0132c. For details of FGD1 structure see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045003#pone.0045003-Bashiri2" target="_blank">[5]</a>.</p

Rv0132c export is Tat dependent.

<p>Equalized whole cell lysates (WCL) from wild type (WT) and <i>ΔtatC M. smegmatis</i> expressing Rv0132c-HA were fractionated to generate cell wall (CW), cytoplasmic membrane (CM), and soluble (SOL) fractions. Fractions were separated by SDS-PAGE and proteins were detected with an anti-HA antibody. Native GroEL was detected as a cytoplasmic control. Rv0132c-HA was exported to the CW and CM fractions in wild type <i>M. smegmatis</i>, but it was not exported in the absence of a functional Tat pathway.</p

Functional assay of Rv0132c–Δ38 protein.

<p>The FGD activity was assessed for Rv0132c–Δ38 protein and <i>Mtb</i>–FGD1 as a positive control. <i>Mtb</i>–FGD1 shows a decrease at 420 nm absorbance (green and red lines), whereas Rv0132c–Δ38 protein indicated no change in the absorbance (yellow and blue lines). The same results were observed using various concentrations of Rv0132c–Δ38 protein in the presence of different concentrations of glucose-6-phpsphate. The graph shows assays containing 1 µM of each enzyme, 25 µM F₄₂₀ with 0.1 mM (green and yellow lines) and 1 mM (red and blue lines) glucose-6-phosphate.</p

Molecular structure of cofactor F₄₂₀.

<p>(A) Schematic representation of cofactor F₄₂₀, where <i>n</i> varies from 2–9 in different microorganisms. (B) Mass spectrometry analysis of cofactor F₄₂₀ bound to the purified Rv0132c–Δ38 protein showing the population of species differing in the number of glutamate residues in the poly-Glu tail.</p

Immunoelectron microscopy of the <i>M. tuberculosis</i> H37Ra cells using anti–Rv0132c antiserum.

<p>Electron micrographs are shown in which thin cryo–sectioned <i>Mtb</i> cells are (A) treated with preimmune serum, and (B) treated with anti-Rv0132c antisera at a dilution of 1/200. The gold particles (indicated by arrowheads) are present mainly on the periphery of the cells in panel B, but are absent from the control panel (A).</p

Subcellular localization of Rv0132c protein.

<p>(A) Western blots of <i>M. tuberculosis</i> H37Rv subcellular fractions using 1/25000 dilution of anti–Rv0132c antiserum. Clear signals are found for the WCL, CW and CM fractions, but not for the SOL fraction. (B) Western blots of Triton X–114 treated fractions. The signal is present only in the DET fraction. WCL: whole cell lysate; CW: cell wall; CM: cytoplasmic membrane; SOL: soluble; AQU: aqueous fraction from Triton X–114 treatment; DET: detergent–enriched fraction from Triton X–114 treatment. In both panels recombinant Rv0132c–Δ38 protein (REC) is used as a positive control (0.7 µg).</p

Export of an Rv0132cSS–'BlaC¹ fusion protein is dependent on the Tat pathway.

1<p>'BlaC = truncated BlaC lacking its native signal sequence.</p>2<p>All strains were resistant to 20 µg/mL kanamycin due to the vector resistance marker. The presence (+) or absence (−) of carbenicillin resistance was determined by colony growth on LB–agar plates plus 20 µg/mL kanamycin and 50 µg/mL carbenicillin for <i>M. smegmatis</i> and 7AGT plates plus 20 µg/mL kanamycin and 50 µg/mL carbenicillin for <i>M. tuberculosis</i>. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045003#s2" target="_blank">materials and methods</a> for additional experimental details.</p>3<p>Carbenicillin resistance was determined after 4–7 days.</p>4<p>Carbenicillin resistance was determined after 21 days.</p

Tat–Dependent Translocation of an F₄₂₀–Binding Protein of <em>Mycobacterium tuberculosis</em>

<div><p>F₄₂₀ is a unique cofactor present in a restricted range of microorganisms, including mycobacteria. It has been proposed that F₄₂₀ has an important role in the oxidoreductive reactions of <em>Mycobacterium tuberculosis</em>, possibly associated with anaerobic survival and persistence. The protein encoded by Rv0132c has a predicted N–terminal signal sequence and is annotated as an F₄₂₀–dependent glucose-6-phosphate dehydrogenase. Here we show that Rv0132c protein does not have the annotated activity. It does, however, co–purify with F₄₂₀ during expression experiments in <em>M. smegmatis</em>. We also show that the Rv0132c–F₄₂₀ complex is a substrate for the Tat pathway, which mediates translocation of the complex across the cytoplasmic membrane, where Rv0132c is anchored to the cell envelope. This is the first report of any F₄₂₀–binding protein being a substrate for the Tat pathway and of the presence of F₄₂₀ outside of the cytosol in any F₄₂₀–producing microorganism. The Rv0132c protein and its Tat export sequence are essentially invariant in the <em>Mycobacterium tuberculosis</em> complex. Taken together, these results show that current understanding of F₄₂₀ biology in mycobacteria should be expanded to include activities occurring in the extra-cytoplasmic cell envelope.</p> </div