Species composition of the mosquito larvae collected from used tires in Vietnam (Higa et al. unpublished data).

<p>Species composition of the mosquito larvae collected from used tires in Vietnam (Higa et al. unpublished data).</p

Annual pyrethroid use for dengue vector control in 2007 and the sum of annual pyrethroid use (1998–2002) for malaria vector control in Vietnam.

<p>The blanks in the map indicate that no data were available.</p

Susceptibility distribution of mosquito larvae collected from used tires in Vietnam.

<p>Susceptibility distribution of mosquito larvae collected from used tires in Vietnam.</p

The date and location of the mosquito collection from used tires in Vietnam.

<p>The bars in the right map indicate the relative altitude at each collection point (The highest bar indicates 1563 m).</p

A Redox Regulatory System Critical for Mycobacterial Survival in Macrophages and Biofilm Development

<div><p>Survival of <i>M</i>. <i>tuberculosis</i> in host macrophages requires the eukaryotic-type protein kinase G, PknG, but the underlying mechanism has remained unknown. Here, we show that PknG is an integral component of a novel <i>r</i>edox <i>ho</i>meostati<i>c</i><i>s</i>ystem, RHOCS, which includes the ribosomal protein L13 and RenU, a Nudix hydrolase encoded by a gene adjacent to <i>pknG</i>. Studies in <i>M</i>. <i>smegmatis</i> showed that PknG expression is uniquely induced by NADH, which plays a key role in metabolism and redox homeostasis. <i>In vitro</i>, RenU hydrolyses FAD, ADP-ribose and NADH, but not NAD+. Absence of RHOCS activities <i>in vivo</i> causes NADH and FAD accumulation, and increased susceptibility to oxidative stress. We show that PknG phosphorylates L13 and promotes its cytoplasmic association with RenU, and the phosphorylated L13 accelerates the RenU-catalyzed NADH hydrolysis. Importantly, interruption of RHOCS leads to impaired mycobacterial biofilms and reduced survival of <i>M</i>. <i>tuberculosis</i> in macrophages. Thus, RHOCS represents a checkpoint in the developmental program required for mycobacterial growth in these environments.</p></div

PknG-catalyzed phosphorylation of L13 at a mycobacterial specific site, T11, is required for mycobacterial biofilm growth.

<p>(<b>A</b>) Sequence alignment of the N-terminal 20 amino acids of L13 proteins from different bacteria. Residues marked with asterisks (T11, T12, and S14) are potential targets of phosphorylation by PknG. (<b>B</b>) Phosphorylation of L13 and its mutants by PknG. In L13(3A), all three residues (T11, T12, and S14) were mutated to alanine. Inhibition was achieved by pre-incubation of PknG in 1 mM AX20017 (AX). (<b>C</b>) Chromatograms confirming wild type and mutant alleles of <i>rplM</i> on <i>M</i>. <i>smegmatis</i> chromosomes. The chromosomal loci were amplified from genomic DNA of <i>M</i>. <i>smegmatis</i> strains by primers that anneal to DNA sequences outside the regions homologous to the allelic exchange substrates, followed by cloning and sequencing. (<b>D</b>) Biofilm of <i>M</i>. <i>smegmatis</i> strains. Similar to <i>Ms</i>Δ<i>pknG</i> and <i>Ms</i>Δ<i>renU</i>, <i>Ms</i>.<i>L13(T11A)</i> exhibited defective biofilm growth, while biofilm of <i>Ms</i>.<i>L13(T11E)</i> was largely identical to wild type. <i>In trans</i> expression of an allele encoding L13(T11E) restored biofilm growth to <i>Ms</i>.<i>L13(T11A)</i> strain. (<b>E</b>) Quantitation of biofilm growth of <i>M</i>. <i>smegmatis</i> strains. The biofilm biomass was harvested and quantified by determining total protein per plate. Error bars represent standard deviations of biological triplicates. Statistical significances of differences were analyzed using Students <i>t</i>-test; ns, not significant difference. (<b>F</b>) Biofilm of <i>Mtb</i> strains. Similar to <i>Mtb</i>Δ<i>pknG</i> and <i>Mtb</i>Δ<i>renU</i>, <i>Mtb</i>.<i>L13(T11A)</i> exhibited defective biofilm growth while <i>in trans</i> expression of an allele encoding L13(T11E) restored its biofilm growth. Addition of PknG inhibitor AX20017 (+AX) had no effect on the biofilm of the complemented strain. (<b>G</b>) Quantitation of biofilm growth of <i>Mtb</i> strains. The biofilm biomass was harvested and quantified by determining total protein per plate. Error bars represent standard deviations of biological triplicates. Statistical significances of differences were analyzed using Students <i>t</i>-test; ns, not significant difference.</p

L13, a ribosomal protein associated with RenU, is phosphorylated by PknG.

<p>(<b>A</b>) Representative <i>in vitro</i> phosphorylation of RenU.6H preparations purified from <i>M</i>. <i>smegmatis</i> (left) or <i>E</i>. <i>coli</i> (right) by purified PknG. PI, phosphatase inhibitors. (<b>B</b>) <i>In vitro</i> phosphorylation of corresponding fractions eluted from ion exchange columns by PknG. Numbers indicate the NaCl concentrations used in elution buffer. Samples loaded to the ion exchange columns were obtained from an immobilized Cobalt affinity chromatography of <i>M</i>. <i>smegmatis</i> RenU.6H (+) cell lysates or control lysates (-). (<b>C</b>) <i>In vitro</i> phosphorylation of purified 6H.L13 or 6H.SmpB by PknG. (<b>D</b>) Co-purification of L13 from <i>M</i>. <i>smegmatis</i> lysates by exogenous RenU.6H. Another recombinant 6H-tagged protein (6H.SHMT) was used as a control. Blots were detected by Anti-L13 or Anti-6H antibodies. (<b>E</b>) <i>In vitro</i> phosphorylation of recombinant or native L13 protein associated with RenU by PknG kinase activity. (<b>F</b>) <i>In vitro</i> phosphorylation of purified 6H.L13 by <i>M</i>. <i>smegmatis</i> cell lysates, followed by pull-down using Nickel-agarose beads.</p

PknG kinase activity is required for biofilm growth in mycobacteria.

<p>(<b>A</b>) Role of <i>pknG</i> in <i>M</i>. <i>smegmatis</i> planktonic growth. Wild type <i>M</i>. <i>smegmatis</i> mc²155 (red filled circles), its derived <i>Ms</i>Δ<i>pknG</i> mutant (blue filled triangles), and the complemented strain <i>Ms</i>Δ<i>pknG</i>/<i>pknG</i> (green filled squares) were grown in 7H9 medium supplemented with 0.2% glucose with shaking at 200 r.p.m. and 37°C. Growth was assessed by measuring optical absorbance at 600 nm. Error bars represent standard deviation of biological triplicates. Differences between wild type and <i>Ms</i>Δ<i>pknG</i> in stationary phase are not significant. (<b>B</b>) Role of <i>pknG</i> in <i>Mtb</i> planktonic growth. Wild type <i>Mtb</i> H37Rv (open circles) and its derived <i>Mtb</i>Δ<i>pknG</i> mutant (open triangles) were grown in 7H9-OADC medium with 0.2% glucose (blue) or 1% glucose (red). Cultures were shaken at 200 r.p.m. and 37°C. Growth was assessed by measuring optical absorbance at 600 nm. Error bars represent standard deviation of biological triplicates. Differences between wild type and <i>Mtb</i>Δ<i>pknG</i> in stationary phase (5–11 hours) are statistically significant (two-tailed <i>t</i>-test, p<0.05). (<b>C</b>) <i>pknG</i> is required for <i>M</i>. <i>smegmatis</i> biofilm growth. Wild type <i>M</i>. <i>smegmatis</i>, <i>Ms</i>Δ<i>pknG</i>, and the mutant strains complemented with the <i>M</i>. <i>smegmatis</i> (<i>Ms-pknG</i>) or <i>M</i>. <i>tuberculosis</i> (<i>Mtb-pknG</i>) gene. Pictures were taken after 7 days of static growth at 30°C. Shown images are representatives of biological triplicates. (<b>D</b>) <i>pknG</i> is required for <i>Mtb</i> biofilm growth. Wild type <i>Mtb</i> H37Rv, <i>Mtb</i>Δ<i>pknG</i>, and the complemented strain were assayed as previously described [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004839#ppat.1004839.ref010" target="_blank">10</a>]. Pictures were taken after 6 weeks of growth at a static humidified condition of 37°C and 5% CO₂. Shown images are representatives of biological triplicates. (<b>E</b>) Quantitation of biofilm growth of <i>Mtb</i> strains. Biofilms were harvested and quantified as described in Experimental Procedures. Error bars represent standard deviation of biological triplicates (*, p<0.0001; ns, not significant difference between wild type H37Rv and the complemented strain). (<b>F</b>) PknG kinase activity is required for <i>Mtb</i> biofilm growth. Wild type <i>Mtb</i> H37Rv, <i>Mtb</i>Δ<i>pknG</i>, and the complemented strain were assayed in the absence (-) or presence (+) of 1 mM AX20017, a specific inhibitor of PknG. Pictures were taken after 6 weeks of growth at static humidified condition of 37°C and 5% CO₂. Shown images are representatives of biological triplicates.</p

Both <i>pknG</i> and its adjacent gene <i>renU</i> are each required for oxidative stress resistance.

<p>(<b>A</b>) Alignment of the <i>pknG</i> loci from <i>Mtb</i> and <i>M</i>. <i>smegmatis</i>. <i>renU</i> (previously annotated as <i>mutT3</i>) shares the same intergenic region with the operon encoding <i>pknG</i>. Bar, 1kb. (<b>B</b>) Both <i>pknG</i> and <i>renU</i> are each required for <i>M</i>. <i>smegmatis</i> resistance to H₂O₂ (left) and diamide (right). Wild type <i>M</i>. <i>smegmatis</i> (circles), <i>Ms</i>Δ<i>pknG</i> (triangles) and <i>Ms</i>Δ<i>renU</i> (squares) were grown in 7H9 medium. At the indicated times (arrows), 10mM H₂O₂ or 15mM diamide was added. Growth was estimated through optical absorbance at 600 nm (OD_600nm). Error bars represent standard deviation of biological triplicates. (<b>C</b>) <i>pknG</i> and <i>renU</i> are each required for <i>Mtb</i> resistance to H₂O₂ (left) and diamide (right). Wild type <i>Mtb</i> (circles or striped bars), <i>Mtb</i>Δ<i>pknG</i> (triangles or black filled bars) and <i>Mtb</i>Δ<i>renU</i> (squares or grey filled bars) of were grown in 7H9-OADC medium. At the indicated times (arrows), 20 mM H₂O₂ or 10 mM diamide was added. Growth was estimated through measuring optical absorbance at 600 nm (OD_600nm, top) or determining colony forming units (CFU, bottom) by serial dilution plating. Error bars represent standard deviation of biological triplicates.</p

<i>renU</i> encodes a Nudix hydrolase required for biofilm growth.

<p>(<b>A</b>) Relative Nudix hydrolase activity of RenU on a substrate panel (left). Nucleoside diphosphate derivatives (NDPX) are preferred substrates compared to nucleoside triphosphates (NTP). A catalytically-inactive mutant of RenU (RenU^DEAD) protein, in which 3 glutamate residues (E74, E77, and E78) in the Nudix box were mutated to alanines, exhibits no phosphatase activity towards the preferred substrates (right). (<b>B</b>) Kinetics studies of Nudix hydrolase activity of RenU on the three NDPXs as preferred substrates ADP-ribose, FAD, and NADH. (<b>C</b>) Rate of RenU catalytic activity on NADH compared to its oxidative form NAD⁺. Fit curve is shown for NADH. (<b>D</b>) The Nudix hydrolase activity of RenU is required for <i>M</i>. <i>smegmatis</i> biofilm growth. Wild type <i>M</i>. <i>smegmatis</i>, <i>Ms</i>Δ<i>renU</i>, and the mutant strains completed with wild type RenU or RenU^DEAD were assayed for biofilm growth. Whereas <i>renU</i> fully restored biofilm growth to <i>Ms</i>Δ<i>renU</i>, <i>renUDEAD</i> failed to complement the mutant. Shown images are representatives of biological triplicates. (<b>E</b>) The Nudix hydrolase activity of RenU is required for <i>Mtb</i> biofilm growth. Wild type <i>Mtb</i> H37Rv, <i>Mtb</i>Δ<i>renU</i>, and the mutant strains completed with wild type RenU or RenU^DEAD were was assayed for biofilm growth. Whereas <i>renU</i> fully restored biofilm growth to <i>Mtb</i>Δ<i>renU</i>, <i>renUDEAD</i> failed to complement the mutant. Shown images are representatives of biological duplicates. (<b>F</b>) Quantitation of biofilm growth of <i>Mtb</i> strains. The biofilm biomass was harvested and estimated by determining total protein per plate. Error bars represent standard deviation of biological triplicates. Statistical significances of differences were analyzed using Students <i>t</i>-test; ns, not significant difference.</p