PNA decreases protein production and <i>in vitro</i> and <i>in vivo</i> infection by SFG <i>R</i>. <i>montanensis</i>.

<p>A) Western blot of Opti-prep purified, serially diluted <i>R</i>. <i>montanensis</i> treated with <i>rickA</i> PNA and quantitative densitometry of the RickA expression as a percent of undiluted RickA. B) Adherence percentages and C) Infection percentages of L929 cells by <i>R</i>. <i>montanensis</i> treated with PNA designed to <i>rickA</i> at 24 and 48 hours. Infection by <i>rickA</i> PNA-treated <i>R</i>. <i>montanensis</i> is reduced 88% (p = 0.00006) at 24 hours and 80% (p = 0.005) at 48-hours post-infection. There is no statistically significant change in adherence with PNA treatment. <i>In vitro</i> experiments were repeated twice and performed in duplicate. Error bars represent standard deviation. D) Unfed <i>D</i>. <i>variabilis</i> adult ticks were injected with <i>rickA</i> PNA-treated (n = 11) or non-targeting control PNA-2-treated (n = 10) rickettsia. Rickettsial burden was measured using qPCR. Genomic copies for <i>gltA</i> were normalized to genomic copies for <i>actin</i>. Closed circles represent individual ticks and the closed horizontal bars represent the mean. Rickettsial burden in ticks infected with <i>rickA</i> PNA-treated <i>R</i>. <i>montanensis</i> is reduced 90% compared to the control (p = 0.004). <i>In vivo</i> experiments were repeated twice with 10 biological replicates per treatment.</p

PNA decreases rOmpB protein production and <i>in vitro</i> infection by TG <i>R</i>. <i>typhi</i>.

<p>A) Western blot of Opti-prep purified, serially diluted <i>rOmpB</i> PNA-treated <i>R</i>. <i>typhi</i> and quantitative densitometry of the rOmpB expression as a percent of undiluted rOmpB. Adherence percentages of Vero cells by <i>rOmpB</i> or non-targetng control PNA-treated <i>R</i>. <i>typhi</i>. No statistical significance found between treatment groups. C) Infection percentages of Vero cells by <i>rOmpB</i> or non-targeting control PNA-treated <i>R</i>. <i>typhi</i>. Infection by <i>rOmpB</i> PNA treated <i>R</i>. <i>typhi</i> is reduced 56% (p = 0.02) compared to non-targeting control PNA. <i>In vitro</i> experiments were repeated twice and performed in duplicate. Error bars represent standard deviation.</p

PNA is specific for designed target.

<p>A) <i>rickA</i> PNA target with putative Shine Dalgarno region in red and start codon in bold B) Western blot of <i>in vitro</i> translation assay supplemented with PNA complementary to the cloned region of <i>rickA</i>, no PNA, or non-targeting control-1 PNA. <i>In vitro</i> translation reactions contained vectors coding both truncated RickA and the control CALML3, or CALML3 alone. C) <i>rOmpB</i> PNA target with start codon in bold D) Dot blot confirming biotinylation of <i>rOmpB</i> and non-targeting-2 PNA. E) Nylon membrane probed to detect biotinylated PNA-ssRNA pairs following <i>rOmpB</i> PNA target RNA denaturation and hybridization to biotinylated PNA, incubated with increasing ratios of unlabeled PNA for competitive binding assay. Incubations with either no PNA or biotinylated non-targeting-2 PNA serve as a control.</p

Phospholipase A activity assay of recombinant proteins.

<p>The phospholipase A (PLA) activity was determined via calculation of fluorescent emission/µg recombinant protein/min (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003399#s4" target="_blank">Materials and Methods</a>). Error bars represent standard errors of the means. <b>A</b>) The PLA activity of recombinant proteins Pat2 or Pat1 increased significantly (P<0.05[two-tail t-test]) in presence of Vero76 cell lysate compared to that in absence of Vero76 cell lysate. The PLA activity of Pat1 was found to be significantly different (P<0.05[two-tail t-test]) from that of mutant Pat1-SD or in presence of 0.5 µM MAPF (PLA₂ inhibitor). <b>B</b>) PLA activity assay of recombinant proteins: Pat2 and Pat1 in presence of bovine liver superoxide dismutase (SOD) as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003399#s4" target="_blank">materials and methods</a>.</p

Effect of anti-Pat1 and anti-Pat2 pretreatment on <i>R. typhi</i> phagosome escape by IFA.

<p>Rickettsiae were incubated with 2 µg of affinity purified anti-Pat1, anti-Pat2 or pre-immune IgG (PI) for 30 min on ice. Pretreated rickettsiae were added onto Vero76 monolayer followed by incubation at 34°C and 5% CO₂. At 30 min postinfection, the cells were fixed with 4% paraformaldehyde and immunolabeled with anti-LAMP-1 (at 1∶100 dilution) mouse monoclonal antibodies (Abcam Inc) and anti-<i>R. typhi</i> rat serum (at 1∶500 dilution) as primary antibodies. The anti-rat-Alexa Fluor-488 (green) and anti-mouse-Alexa Fluor-594 (red) were used as secondary antibodies. The cell nuclei were stained with DAPI (blue). Samples were viewed under a LSM5DUO confocal microscope and images were processed using ZEN imaging and analysis software. (<b>A</b>) Representative micrograph of anti-Pat1 (panels a to c), anti-Pat2 (panels d to f) or pre-immune IgG (panels g to i) treated rickettsiae that were infected of Vero76 cells. The white arrows show rickettsiae in LAMP-1 positive phagosome, with the square head arrow showing rickettsiae escaping or escaped from the phagosome. Scale Bar = 5 µm. (<b>B</b>) Quantitation of percent rickettsiae in LAMP-1 positive phagosome was determined as percent LAMP-1 positive <i>R. typhi</i> by scoring 100 bacteria for each treatment. Each treatment was repeated four times. Error bars represent standard error of the means. Percent LAMP-1 positive <i>R.typhi</i> for anti-Pat1 or anti-Pat2 pretreatment was found to be significantly different (P<0.05[two-tail t-test]) from that of PI.</p

Effect of anti-Pat1 and anti-Pat2 antibody pretreatment on <i>R. typhi</i> infectivity of Vero76 cells by IFA.

<p>Rickettsiae were treated with 2 µg of affinity purified anti-Pat1, anti-Pat2 or pre-immune IgG (PI) for 30 min on ice, followed by incubation for infection of Vero76 for 18 hour at 34°C and 5% CO₂. The infected cells were labeled for IFA and % infectivity was determined as described (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003399#s4" target="_blank">Materials and Methods</a>). Infectivity (%) was determined in % host cells infected by <i>R. typhi</i> pretreated with anti-Pat1 or anti-Pat2 antibody with respect to that with PI. Error bars represent standard errors of the means. Infectivity (%) of anti-Pat1 or anti-Pat2 antibody pretreated <i>R. typhi</i> was found to be significantly different (P<0.05[two-tail t-test]) from that of PI.</p

Effect of anti-Pat1 and anti-Pat2 pretreatment on <i>R. typhi</i> infection by plaque assay.

<p>Rickettsiae were treated with affinity purified anti-Pat1, anti-Pat2 or pre-immune IgG (PI) for 30 min on ice, followed by incubation for infection into Vero76 for 1 hour at 34°C and 5% CO₂. <i>R. typhi</i> infected cells were left unwashed (<b>A</b>) or washed (<b>B</b>) before addition of agar for plaque assay as described (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003399#s4" target="_blank">Materials and Methods</a>). The effect of antibody treatment on <i>R. typhi</i> was determined in percent (%) plaque formation by antibody treated rickettsiae with respect to that by no treatment. Error bars represent standard errors of the means. In both Panel <b>A</b> and <b>B</b>, percent (%) plaque formation by antibody pretreated <i>R. typhi</i> were found to be significantly different (P<0.05[two-tail t-test]) from that by PI.</p

Secretion of Pat1 and Pat2 into host cells.

<p>Cells were fixed with 4% paraformaldehyde at 24 h postinfection and immunolabeled with anti-Pat1 (panels A to C for <i>R. typhi</i> infected and panel D for uninfected) or anti-Pat2 (panels F to H for <i>R. typhi</i> infected and panel I for uninfected) rabbit antibodies (at 1∶200 dilution) and anti-<i>R. typhi</i> rat serum (at 1∶500 dilution) as primary antibodies. <i>R. typhi</i> infected Vero76 cells were also labeled similarly with rabbit pre-immune serum (at 1∶200 dilution) and anti-<i>R. typhi</i> rat serum (at 1∶500 dilution) shown in panel E. The anti-rat-Alexa Fluor-594 (red) and anti-rabbit-Alexa Fluor-488 (green) antibodies were used as secondary antibodies. The cell nuclei were stained with DAPI (blue). Samples were viewed under a LSM5DUO confocal microscope and images were processed using ZEN imaging and analysis software. The white arrows showed punctate structure indicating translocation of Pat1 or Pat2 from rickettsiae into host cell cytoplasm. Scale Bar = 5 µm.</p

<i>Rickettsia typhi</i> Possesses Phospholipase A₂ Enzymes that Are Involved in Infection of Host Cells

<div><p>The long-standing proposal that phospholipase A₂ (PLA₂) enzymes are involved in rickettsial infection of host cells has been given support by the recent characterization of a patatin phospholipase (Pat2) with PLA₂ activity from the pathogens <i>Rickettsia prowazekii</i> and <i>R. typhi</i>. However, <i>pat2</i> is not encoded in all <i>Rickettsia</i> genomes; yet another uncharacterized patatin (Pat1) is indeed ubiquitous. Here, evolutionary analysis of both patatins across 46 <i>Rickettsia</i> genomes revealed 1) <i>pat1</i> and <i>pat2</i> loci are syntenic across all genomes, 2) both Pat1 and Pat2 do not contain predicted Sec-dependent signal sequences, 3) <i>pat2</i> has been pseudogenized multiple times in rickettsial evolution, and 4) ubiquitous <i>pat1</i> forms two divergent groups (<i>pat1A</i> and <i>pat1B</i>) with strong evidence for recombination between <i>pat1B</i> and plasmid-encoded homologs. In light of these findings, we extended the characterization of <i>R. typhi</i> Pat1 and Pat2 proteins and determined their role in the infection process. As previously demonstrated for Pat2, we determined that 1) Pat1 is expressed and secreted into the host cytoplasm during <i>R. typhi</i> infection, 2) expression of recombinant Pat1 is cytotoxic to yeast cells, 3) recombinant Pat1 possesses PLA₂ activity that requires a host cofactor, and 4) both Pat1 cytotoxicity and PLA₂ activity were reduced by PLA₂ inhibitors and abolished by site-directed mutagenesis of catalytic Ser/Asp residues. To ascertain the role of Pat1 and Pat2 in <i>R. typhi</i> infection, antibodies to both proteins were used to pretreat rickettsiae. Subsequent invasion and plaque assays both indicated a significant decrease in <i>R. typhi</i> infection compared to that by pre-immune IgG. Furthermore, antibody-pretreatment of <i>R. typhi</i> blocked/delayed phagosomal escapes. Together, these data suggest both enzymes are involved early in the infection process. Collectively, our study suggests that <i>R. typhi</i> utilizes two evolutionary divergent patatin phospholipases to support its intracellular life cycle, a mechanism distinguishing it from other rickettsial species.</p></div

Translocation of Pat1 during <i>R. typhi</i> infection of Vero76 cells by Western blotting.

<p><i>R. typhi</i> infected or uninfected Vero76 grown for 48 hr were fractionated by 0.1% Triton X-100 treatment into pellet containing intact rickettsia with host cell debris and supernatant containing rickettsial secreted protein with host soluble proteins and was probed with rabbit anti-Pat1 antibody, rabbit anti-Pat2 antibody (as positive control for <i>R. typhi</i> secreted protein), rabbit anti-rOmpB antibody (as control for <i>R. typhi</i> surface protein), rabbit anti-EF-Ts antibody (as control for <i>R. typhi</i> cytoplasmic protein) or mouse anti-GAPDH monoclonal antibody (as control for host cytoplasmic protein). <b>Lane1</b>: pellet of uninfected Vero76; <b>Lane2</b>: supernatant of uninfected Vero76; <b>Lane3</b>: pellet of <i>R. typhi</i> infected Vero76; <b>Lane4</b>: supernatant of <i>R. typhi</i> infected Vero76. The size of the expected protein bands is shown on the right.</p