Image_4_Epithelial Keratins Modulate cMet Expression and Signaling and Promote InlB-Mediated Listeria monocytogenes Infection of HeLa Cells.TIF

<p>The host cytoskeleton is a major target for bacterial pathogens during infection. In particular, pathogens usurp the actin cytoskeleton function to strongly adhere to the host cell surface, to induce plasma membrane remodeling allowing invasion and to spread from cell to cell and disseminate to the whole organism. Keratins are cytoskeletal proteins that are the major components of intermediate filaments in epithelial cells however, their role in bacterial infection has been disregarded. Here we investigate the role of the major epithelial keratins, keratins 8 and 18 (K8 and K18), in the cellular infection by Listeria monocytogenes. We found that K8 and K18 are required for successful InlB/cMet-dependent L. monocytogenes infection, but are dispensable for InlA/E-cadherin-mediated invasion. Both K8 and K18 accumulate at InlB-mediated internalization sites following actin recruitment and modulate actin dynamics at those sites. We also reveal the key role of K8 and K18 in HGF-induced signaling which occurs downstream the activation of cMet. Strikingly, we show here that K18, and at a less extent K8, controls the expression of cMet and other surface receptors such TfR and integrin β1, by promoting the stability of their corresponding transcripts. Together, our results reveal novel functions for major epithelial keratins in the modulation of actin dynamics at the bacterial entry sites and in the control of surface receptors mRNA stability and expression.</p

Image_2_Epithelial Keratins Modulate cMet Expression and Signaling and Promote InlB-Mediated Listeria monocytogenes Infection of HeLa Cells.TIF

<p>The host cytoskeleton is a major target for bacterial pathogens during infection. In particular, pathogens usurp the actin cytoskeleton function to strongly adhere to the host cell surface, to induce plasma membrane remodeling allowing invasion and to spread from cell to cell and disseminate to the whole organism. Keratins are cytoskeletal proteins that are the major components of intermediate filaments in epithelial cells however, their role in bacterial infection has been disregarded. Here we investigate the role of the major epithelial keratins, keratins 8 and 18 (K8 and K18), in the cellular infection by Listeria monocytogenes. We found that K8 and K18 are required for successful InlB/cMet-dependent L. monocytogenes infection, but are dispensable for InlA/E-cadherin-mediated invasion. Both K8 and K18 accumulate at InlB-mediated internalization sites following actin recruitment and modulate actin dynamics at those sites. We also reveal the key role of K8 and K18 in HGF-induced signaling which occurs downstream the activation of cMet. Strikingly, we show here that K18, and at a less extent K8, controls the expression of cMet and other surface receptors such TfR and integrin β1, by promoting the stability of their corresponding transcripts. Together, our results reveal novel functions for major epithelial keratins in the modulation of actin dynamics at the bacterial entry sites and in the control of surface receptors mRNA stability and expression.</p

APRc can process rOmpB <i>in vitro</i>.

<p>(A) rOmpB is proteolytically processed between the passenger and β-barrel domains through a yet unknown mechanism (<b>?</b>) and APRc was tested as the candidate enzyme to perform rOmpB preprotein processing <i>in vitro</i>. (B) Total membrane fractions of <i>E. coli</i> enriched in rOmpB were incubated with activated APRc soluble domain and the reaction products analyzed by Western blot with an anti-Histidine antibody. The integrity of rOmpB proprotein was confirmed in the absence of APRc whereas in the presence of the protease a product with approximately 35 kDa was observed, correlated with the disappearance of the full-length unprocessed form. (C) The integrity of recombinant rOmpB was further evaluated upon incubation with both activated APRc and the active site mutant form (D140A) for 16 h. The reaction products were then subjected to immunoblot analysis with anti-rOmpB MAb, confirming the disappearance of rOmpB in the presence of the active form of the enzyme. Molecular weight markers in kilodaltons (kDa) are shown on the left. Protein loading controls: Coomassie blue staining.</p

Recombinant full-length APRc accumulates in the outer membrane in <i>E. coli</i> and the soluble catalytic domain is exposed to the cell surface.

<p>(A) Full-length APRc was expressed in <i>E. coli</i> and total (TM) as well as outer membrane (OM) fractions were isolated and analyzed by Western blot with anti-APRc antibody (left panel). As a control for non-specific staining, peptide competition assays were performed by blocking the anti-APRc antibody with the immunizing peptide (right panel). One specific band with approximately 21 kDa was detected in the OM fraction. (B) The purity of OM fractions was confirmed by using OmpA and Lep proteins as internal markers for the outer and inner membranes of <i>E. coli</i>, respectively. Both proteins were present in TM faction, while only OmpA is detected in OM faction. (C) Flow cytometric analysis was carried out for recombinant APRc recognition at the surface of <i>E. coli</i> cells. PFA-fixed <i>E. coli</i> cells were incubated with anti-APRc and anti-RNA polymerase α (RNAPα mAb 4RA2,) followed by secondary detection using goat anti-rabbit IgG Alexa Fluor 488- and goat anti-mouse IgG R-PE-Cy5.5 conjugated secondary antibodies, respectively. Porous, permeabilized cells staining positive for RNAPα were excluded from the analysis by selective gating of this population. Fluorescence was detected on the remaining <i>E. coli</i> cells incubated with anti-APRc, thereby confirming the expression of recombinant APRc at the outer membrane and its exposure to extracellular milieu.</p

Protein sequence identity of each APRc homologue in relation to NP_360976 as well as the taxonomic group of analyzed rickettsial spp.

<p>(SFG: spotted fever group; TG: typhus group; TRG: transitional group; AG: ancestral group).</p

RC1339/APRc from <i>Rickettsia conorii</i> Is a Novel Aspartic Protease with Properties of Retropepsin-Like Enzymes

<div><p>Members of the species <i>Rickettsia</i> are obligate intracellular, gram-negative, arthropod-borne pathogens of humans and other mammals. The life-threatening character of diseases caused by many <i>Rickettsia</i> species and the lack of reliable protective vaccine against rickettsioses strengthens the importance of identifying new protein factors for the potential development of innovative therapeutic tools. Herein, we report the identification and characterization of a novel membrane-embedded retropepsin-like homologue, highly conserved in 55 <i>Rickettsia</i> genomes. Using <i>R. conorii</i> gene homologue RC1339 as our working model, we demonstrate that, despite the low overall sequence similarity to retropepsins, the gene product of <i>rc1339</i> APRc (for <u>A</u>spartic <u>P</u>rotease from <i><u>R</u>ickettsia <u>c</u>onorii</i>) is an active enzyme with features highly reminiscent of this family of aspartic proteases, such as autolytic activity impaired by mutation of the catalytic aspartate, accumulation in the dimeric form, optimal activity at pH 6, and inhibition by specific HIV-1 protease inhibitors. Moreover, specificity preferences determined by a high-throughput profiling approach confirmed common preferences between this novel rickettsial enzyme and other aspartic proteases, both retropepsins and pepsin-like. This is the first report on a retropepsin-like protease in gram-negative intracellular bacteria such as <i>Rickettsia</i>, contributing to the analysis of the evolutionary relationships between the two types of aspartic proteases. Additionally, we have also shown that APRc is transcribed and translated in <i>R. conorii</i> and <i>R. rickettsii</i> and is integrated into the outer membrane of both species. Finally, we demonstrated that APRc is sufficient to catalyze the <i>in vitro</i> processing of two conserved high molecular weight autotransporter adhesin/invasion proteins, Sca5/OmpB and Sca0/OmpA, thereby suggesting the participation of this enzyme in a relevant proteolytic pathway in rickettsial life-cycle. As a novel <i>bona fide</i> member of the retropepsin family of aspartic proteases, APRc emerges as an intriguing target for therapeutic intervention against fatal rickettsioses.</p></div

rAPRc activation product displays optimal activity at pH 6 and is strongly inhibited by specific HIV-1 PR inhibitors.

<p>The effect of pH, class-specific and HIV-1 PR specific inhibitors on the proteolytic activity of rAPRc activation product was evaluated using the synthetic fluorogenic substrate (MCA)Lys-Ala-Leu-Ile-Pro-Ser-Tyr-Lys-Trp-Ser-Lys(DNP). (A) Activity at different pH values. Activated rAPRc was incubated with the substrate at 37°C in buffers ranging between pH 4 and pH 9 containing 100 mM NaCl (50 mM sodium acetate pH 4.0, 5.0, 5.5 and 6.0 and 50 mM Tris-HCl pH 7.0, 8.0 and 9.0). (B) and (C) To test the effect different compounds, the protease was pre-incubated in the presence of each inhibitor for 10 minutes at room temperature in 50 mM sodium acetate pH 6.0 containing 100 mM NaCl before adding the substrate. The rate of substrate hydrolysis (RFU/sec) was monitored for 3 hours and the relative activity normalized by setting the maximum activity at 100%. The error bars represent standard deviation of the mean.</p

<i>RC1339/</i>APRc is expressed in <i>Rickettsia conorii</i> and <i>Rickettsia rickettsii</i> and accumulates at the outer membrane in both species.

<p>(A) RT-PCR analysis of <i>RC1339/</i>APRc expression on rickettsial spp.. The housekeeping gene <i>hrtA</i> (17 kDa surface antigen) was used as a control. The negative control for the cDNA synthesis lacking reverse transcriptase is identified by (RTase -). Rickettsial species are identified on the top and the gene names are shown on the left side of the agarose gel. (B) A whole cell lysate from <i>R. rickettsii</i> (1) and insoluble (2) and soluble (3) fractions from <i>R. conorii</i> extracts were isolated and then subjected to Western Blot analysis with anti-APRc antibody. A specific band with approximately 21 kDa was detected. (C) Whole cell lysates (WCL), inner (IM) and outer membrane (OM) fractions from sarkosyl treatment of <i>R. rickettsii</i> and <i>R. conorii</i> extracts were isolated and then subjected to Western Blot analysis with anti-APRc and anti-rOmpB antibody. APRc shares the same localization of rOmpB, an internal marker for outer membrane of <i>Rickettsia</i> spp. Molecular mass markers in kilodaltons (kDa) are shown on the left. (D) For flow cytometric analysis of APRc expression in <i>R. conorii</i>, fixed bacteria were queried for deposition of anti-APRc (orange trace), negative control lacking primay antibody (blue trace), or the positive control anti-OmpB (red trace), a known rickettsial surface protein. After incubation with fluorescent secondary antibody, both anti-APRc and anti-OmpB detected on the bacterial surface (increased fluorescence), indicating accessibility of these target proteins to exogenously applied antibody.</p

APRc can process rOmpA <i>in vitro</i>.

<p>Total membrane fractions of <i>E. coli</i> enriched in rOmpA were incubated with both activated APRc and the active site mutant form (D140A) for 16 h. The reaction products were then subjected to immunoblot analysis with anti-rOmpA Ab, confirming the disappearance of rOmpA in the presence of the active form of the enzyme. Molecular weight markers in kilodaltons (kDa) are shown on the left. Protein loading controls: Coomassie blue staining.</p

The recombinant soluble catalytic domain of APRc displays autoprocessing activity dependent on the catalytic aspartate residue.

<p>(A) The soluble catalytic domain (amino acids 87–231) was fused to GST and produced in <i>E. coli</i>. Upon purification, the auto-activation of rGST-APRc_87–231 was performed <i>in vitro</i> in 0.1 M sodium acetate buffer pH 6 at 37°C for 48 h and monitored by SDS-PAGE stained with Coomassie blue. rGST-APRc_87–231 undergoes multi-step auto-activation processing, resulting in the formation of the activated form APRc_105–231 with ∼14.2 kDa (left panel). Mutation of the active site aspartic acid by alanine in this fusion construct [rGST-APRc(D140A)_87–231] clearly impaired the auto-catalytic activity of the protease (right panel). (B) Schematic representation of full-length APRc domain organization. APRc is predicted to comprise three transmembrane domains (TM 1–3) at the N terminus and the soluble catalytic domain at the C terminus. The three auto-cleavage sites (shown in A) identified by Edman degradation are highlighted by order of cleavage (1–3). (C) Activity of wt rGST-APRc_87–231 towards oxidized insulin B chain was tested over activation time. Samples from each time point evaluated in panel A were incubated with oxidized insulin B chain. Reaction products were then evaluated by RP-HPLC showing that substrate cleavage (appearance of four major peaks) was concomitant with appearance of the final activation product. T0, T12, T24, T36 and T48, correspond to the different time points of activation (hours) tested, as shown in A. (D) rGST-APRc_87–231 auto-processing ability during expression was evaluated in total lysates of <i>E. coli</i> cells expressing wt rGST-APRc_87–231 or the correspondent active site mutant rGST-APRc(D140A)_87–231 over a time-course of 3 h and subsequently subjected to Western blot analysis with anti-APRc antibody. A band with approximately 15 kDa was only detected for the wt construct. Incubation and expression time course in hours (h) are indicated above gels and the molecular weight markers in kilodaltons (kDa) are shown on the left.</p