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

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 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

Which Way In? The RalF Arf-GEF Orchestrates <i>Rickettsia</i> Host Cell Invasion

<div><p>Bacterial Sec7-domain-containing proteins (RalF) are known only from species of <i>Legionella</i> and <i>Rickettsia</i>, which have facultative and obligate intracellular lifestyles, respectively. <i>L</i>. <i>pneumophila</i> RalF, a type IV secretion system (T4SS) effector, is a guanine nucleotide exchange factor (GEF) of ADP-ribosylation factors (Arfs), activating and recruiting host Arf1 to the <i>Legionella</i>-containing vacuole. In contrast, previous <i>in vitro</i> studies showed <i>R</i>. <i>prowazekii</i> (Typhus Group) RalF is a functional Arf-GEF that localizes to the host plasma membrane and interacts with the actin cytoskeleton via a unique C-terminal domain. As RalF is differentially encoded across <i>Rickettsia</i> species (e.g., pseudogenized in all Spotted Fever Group species), it may function in lineage-specific biology and pathogenicity. Herein, we demonstrate RalF of <i>R</i>. <i>typhi</i> (Typhus Group) interacts with the <i>Rickettsia</i> T4SS coupling protein (RvhD4) via its proximal C-terminal sequence. RalF is expressed early during infection, with its inactivation via antibody blocking significantly reducing <i>R</i>. <i>typhi</i> host cell invasion. For <i>R</i>. <i>typhi</i> and <i>R</i>. <i>felis</i> (Transitional Group), RalF ectopic expression revealed subcellular localization with the host plasma membrane and actin cytoskeleton. Remarkably, <i>R</i>. <i>bellii</i> (Ancestral Group) RalF showed perinuclear localization reminiscent of ectopically expressed <i>Legionella</i> RalF, for which it shares several structural features. For <i>R</i>. <i>typhi</i>, RalF co-localization with Arf6 and PI(4,5)P₂ at entry foci on the host plasma membrane was determined to be critical for invasion. Thus, we propose recruitment of PI(4,5)P₂ at entry foci, mediated by RalF activation of Arf6, initiates actin remodeling and ultimately facilitates bacterial invasion. Collectively, our characterization of RalF as an invasin suggests that, despite carrying a similar Arf-GEF unknown from other bacteria, different intracellular lifestyles across <i>Rickettsia</i> and <i>Legionella</i> species have driven divergent roles for RalF during infection. Furthermore, our identification of lineage-specific Arf-GEF utilization across some rickettsial species illustrates different pathogenicity factors that define diverse agents of rickettsial diseases.</p></div

Model for the variable pathways utilized by divergent <i>Rickettsia</i> species for host cell entry.

<p>General pathways for Typhus Group (TG, left) and Spotted Fever Group (SFG, right) rickettsiae species are inferred primarily from previous work on SFG rickettsiae species <i>R</i>. <i>conorii</i> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005115#ppat.1005115.ref026" target="_blank">26</a>] and <i>R</i>. <i>parkeri</i> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005115#ppat.1005115.ref108" target="_blank">108</a>] or data from the present study (<i>R</i>. <i>typhi</i>). At center, a conserved proximal hub of the pathway commences with Sca5 binding to host receptor Ku70 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005115#ppat.1005115.ref110" target="_blank">110</a>], which triggers a host-signaling cascade (gray box) involving c-Cbl-mediated ubiquitination of Ku70, Rho-family GTPases Cdc42 and Rac1, phosphoinositide 3-kinase (PI3K) activity, and activation of tyrosine kinases (e.g., c-Src, FAK and p-TK) and their phosphorylated targets. The divergent distal arms of this pathway involve recruitment of factors for activating the actin nucleating complex (Arp2/3), which leads to host actin polymerization, extensive membrane ruffling and filopodia formation, and bacterial internalization in a clathrin and calveolin dependent process. For SFG rickettsiae, the WAVE complex recruits Arp2/3, with its activation via an unknown nucleation promoting factor (either host or bacterial; e.g., RickA). While these processes remain to be characterized for TG rickettsiae, our work suggests that secreted RalF recruits the GTPase Arf6, precipitating an accumulation of PI(4,5)P₂ that modulates the activities of a range of actin-associated host proteins (green star). Additional bacterial proteins, some of which are known to facilitate host cell entry, have white lettering with colored boxed backgrounds. Known pathways for protein secretion and host cell receptor-binding, as recently reviewed [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005115#ppat.1005115.ref045" target="_blank">45</a>], are shown with solid black lines; all other modeled pathways (shown with dashed lines) are either inferred by homology (e.g., Sca1 of TG rickettsiae as an adhesin based on characterization for Sca1 of <i>R</i>. <i>conorii</i> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005115#ppat.1005115.ref036" target="_blank">36</a>]) or estimated based on <i>in silico</i> analyses (e.g., Sca3 of TG rickettsiae as a putative analog to the α2β1 integrin-binding Sca0 of <i>R</i>. <i>conorii</i> [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005115#ppat.1005115.ref035" target="_blank">35</a>]). A phylogenomics analysis across select <i>Rickettsia</i> species (bottom, left) illustrates the genomic variation underlying all of the bacterial components of the models. Adapted from our recent report on the <i>Rickettsia</i> secretome [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005115#ppat.1005115.ref045" target="_blank">45</a>]. Red, ancestral group (AG); blue, transitional group (TRG); aquamarine, TG; brown, SFG.</p

<i>R</i>. <i>typhi</i> RalF_Rt interacts with RvhD4 and is expressed early during host cell invasion.

<p>(A) Bacterial two-hybrid (B2H) assay reveals an interaction between RalF_Rt and RvhD4. <i>ralF</i>_<i>RtFL</i> and <i>ralF</i>_{<i>RtΔT4S</i>} were cloned into pTRG (prey) and <i>rvhD4</i> was cloned into pBT (bait) of the B2H system. Constructed bait and prey plasmids were co-transformed into BacterioMatch II reporter electro-competent cells. Transformants were screened on non-selective plate (left) and positive interactions were identified on dual selective screening plate (right). The amino acid sequence deleted from <i>ralF</i>_RtΔT4S (positively charged residues are colored blue) is shown at bottom. (B) Quantification of bacterial growth in the B2H assay described in panel A. Percent growth of CFUs of reporter cells harboring recombinant plasmids on dual selective screening medium was calculated relative to CFUs obtained on non-selective medium. Error bars represent mean ± SD of three independent experiments (Student’s two-sided t-test). (C) <i>R</i>. <i>typhi</i> RvhD4 exhibits ATPase activity. A series dilution of purified RvhD4 in assay buffer was incubated with reagent for 30 min at 21°C. The inorganic phosphate (Pi) released from ATP was quantified by measuring absorbance at OD 620 nm. As a negative control, a non-related <i>R</i>. <i>typhi</i> protein (RT0600) was assayed. Error bars represent mean ± SD of three independent experiments. * p = 0.01, **** p<0.0001; Student’s two-sided t-test. (D) Protein immunoblot of recombinant RvhD4 (~64 kDa) used in ATPase activity assays described in panel C. (E) RalF_Rt is surface exposed. Purified <i>R</i>. <i>typhi</i> was treated with 400 μg/mL or 800 μg/mL Proteinase K or in buffer alone for 1 hr. Lysates were resolved and immunoblotted for RalF or the <i>R</i>. <i>typhi</i> cytoplasmic control protein, elongation factor Ts (EF-Ts). Densitometry was performed using ImageJ and the intensity of RalF was normalized to EF-Ts. Representative image from two independent experiments is shown. Intensity of RalF normalized to EF-Ts and relative to untreated control is shown below the immunoblots. (F) RalF is expressed during early infection. HeLa cells infected with <i>R</i>. <i>typhi</i> for 10 and 30 min were fixed and <i>R</i>. <i>typhi</i> and RalF detected with rat anti-<i>R</i>. <i>typhi</i> (red) and affinity purified rabbit anti-RalF_Rt (green) antibodies, respectively. DAPI (blue) is shown in the merged image. Boxed regions are enlarged to show detail. Pre-immune (PI) cells were treated with rabbit PI serum in place of anti-RalF_Rt antibody. (Scale bar: 10 μm). (G) Anti-RalF_Rt IgG and Fab fragments inhibit <i>R</i>. <i>typhi</i> host cell infection. HeLa cells were infected with partially purified <i>R</i>. <i>typhi</i> pre-absorbed for 30 min with 20μg PI IgG serum, anti-RalF_Rt IgG, PI Fab fragments or anti-RalF_Rt Fab fragments. Cells were fixed 2 hrs post infection and <i>R</i>. <i>typhi</i> and the cell membrane detected with anti-<i>R</i>. <i>typhi</i> serum and Alexa Fluor 594 wheat germ agglutinin, respectively. The number of <i>R</i>. <i>typhi</i> per host cell was counted for 100 individual host cells in three independent experiments and normalized to PI serum. Error bars represent mean ± SD (Student’s two-sided t-test).</p

PI(4,5)P₂ interacts with RalF_Rt and mediates <i>R</i>. <i>typhi</i> infection.

<p>(A) RalF_RtCTD co-localizes with PI(4,5)P₂. HeLa cells transfected with pEYFP-C1 empty vector, GFP-C1-PLCδ-PH (a PI(4,5)P2 biosensor), EYFP–RalF_RtCTD, or EYFP–RalF_RbCTD were treated with 5 μM ionomycin alone, with Ca²⁺, or with Ca²⁺ and EGTA. Nuclei were stained with DAPI (blue). (Scale bar: 10 μm). (B) PI(4,5)P₂ is recruited during <i>R</i>. <i>typhi</i> infection. HeLa cells transfected with GFP-C1-PLCδ-PH (green) were infected with <i>R</i>. <i>typhi</i> (MOI ~100:1) for indicated times. <i>R</i>. <i>typhi</i> was detected with rat anti-<i>R</i>. <i>typhi</i> serum and Alexa Fluor 594 anti-rat antibody (red). Nuclei were stained with DAPI (blue). Boxed regions are enlarged to show detail (inset). (Scale bar: 1 μm). (C) RalF localizes to PI(4,5)P₂-enriched regions of the plasma membrane during <i>R</i>. <i>typhi</i> infection. HeLa cells transfected with GFP-C1-PLCδ-PH (green) were infected with <i>R</i>. <i>typhi</i> (MOI ~100:1) for indicated times. RalF_Rt was detected with rabbit anti-RalF_Rt and Alexa Fluor 594 anti-rabbit antibodies (red). Nuclei were stained with DAPI (blue). Boxed regions are enlarged to show detail (inset). (Scale bar: 1 μm). (D) Ionomycin and Ca²⁺ treatment decreases <i>R</i>. <i>typhi</i> infection. HeLa cells treated with 5 μM ionomycin and Ca²⁺ or no treatment were infected with <i>R</i>. <i>typhi</i> (MOI ~100:1) for 2 hrs. <i>R</i>. <i>typhi</i> was detected with rat anti-<i>R</i>. <i>typhi</i> serum and Alexa Fluor 488 anti-rat antibody. Cell membrane was stained with Alexa Fluor 594 wheat germ agglutinin. The number of infected host cells was counted, with percent infection of three independent experiments (100 host cells counted for each) plotted. Error bars represent mean ± SD (Student’s two-sided t-test).</p

Subcellular localization of rickettsial RalF proteins to host membranes.

<p>HeLa cells expressing YFP tagged RalF proteins (green, described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005115#ppat.1005115.g002" target="_blank">Fig 2</a>) were fixed and stained with Alexa Fluor 594 wheat germ agglutinin (WGA) to detect the plasma membrane (left) or anti-PDI antibody to detect the endoplasmic reticulum (right). DAPI (blue) is shown in the merged image. (Scale bar: 10 μm).</p

Schematic of <i>R</i>. <i>typhi</i> entry.

<p><i>R</i>. <i>typhi</i> entry has been broken down into five conceptual stages: binding (1); extension of pseudopodia (2); membrane fusion and internalization (3); formation of early endosome (4); bacterial escape from endosome (5). Schematic is a representation of micrographs from Figs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005115#ppat.1005115.g005" target="_blank">5B, 5C</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005115#ppat.1005115.g006" target="_blank">6C</a>. Inset depicts hypothetical recruitment and activation of PIP5K via RalF_Rt-activated Arf6, which results in PI(4,5)P₂ enrichment and actin rearrangement to facilitate for <i>R</i>. <i>typhi</i> entry.</p

RalF subcellular localization and actin filament disruption mediated by the SCD and VPR.

<p>HeLa cells transfected with YFP tagged constructs (green, described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005115#ppat.1005115.g002" target="_blank">Fig 2B</a>) were stained with Alexa Fluor 594 phalloidin to detect actin (red). DAPI (blue) is shown in the merged image. Cytoplasmic (C) and membrane (M) localization was confirmed via membrane fractionation of HEK293T cells Lipofectamine 2000 transfected with the indicated plasmids followed by immunoblotting. Immunoblot primary antibodies: 1, rabbit anti-GFP (Life Technologies); 2, membrane marker rabbit anti-Calnexin (Abcam); 3, cytoplasmic marker mouse anti-GAPDH (Abcam). Rt, <i>R</i>. <i>typhi</i>; Rf, <i>R</i>. <i>felis</i>; Rm, <i>R</i>. <i>montanensis</i>; Rb, <i>R</i>. <i>bellii</i>. (Scale bar: 10 μm).</p