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Proteomic Characterization and Functional Analysis of Outer Membrane Vesicles of Francisella novicida Suggests Possible Role in Virulence and Use as a Vaccine Tony Pierson,†,‡ Demetrios Matrakas,†,‡ Yuka U. Taylor,‡ Ganiraju Manyam,|| Victor N. Morozov,§,# Weidong Zhou,^ and Monique L. van Hoek‡,§,* ‡
Department of Molecular and Microbiology, George Mason University, Manassas, Virginia 20110, United States National Center for Biodefense and Infectious Diseases, George Mason University, Manassas, Virginia 20110, United States Department of Bioinformatics & Computational Biology, The UT MD Anderson Cancer Center, Houston, Texas, United States ^ Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, Virginia 20110, United States # Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Pushchino, Moscow region 142290, Russia
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bS Supporting Information ABSTRACT: We have isolated and characterized outer membrane vesicles (OMVs) from Francisella. Transport of effector molecules through secretion systems is a major mechanism by which Francisella tularensis alters the extracellular proteome and interacts with the host during infection. Outer membrane vesicles produced by Francisella were examined using TEM and AFM and found to be 43-125 nm in size, representing another potential mechanism for altering the extracellular environment. A proteomic analysis (LC-MS/MS) of OMVs from F. novicida and F. philomiragia identified 416 (F. novicida) and 238 (F. philomiragia) different proteins, demonstrating that OMVs are an important contributor to the extracellular proteome. Many of the identified OMV proteins have a demonstrated role in Francisella pathogenesis. Biochemical assays demonstrated that Francisella OMVs possess acid phosphatase and hemolytic activities that may affect host cells during infection, and are cytotoxic toward murine macrophages in cell culture. OMVs have been previously used as a human vaccine against Neisseria meningitidis. We hypothesized that Francisella OMVs could be useful as a novel Francisella vaccine. Vaccinated BALB/C mice challenged with up to 50 LD50 of Francisella showed statistically significant protection when compared to control mice. In the context of these new findings, we discuss the relevance of OMVs in Francisella pathogenesis as well as their potential use as a vaccine. KEYWORDS: Francisella, tularemia, OMV, vesicle, virulence, vaccine, membrane
’ INTRODUCTION Outer membrane vesicles (OMVs) are 50 to 250 nm spherical bilayer structures discharged from the surface of many gramnegative bacteria during cell growth by a mechanism that has not fully been described.1-4 They have been identified from several human pathogens, including Escherichia coli,5 Pseudomonas aeruginosa,6 Helicobacter pylori,7 Vibrio cholera,8 Xanthamonas,9 Porphyromonas gingivalis,10 and Acinetobacter baumannii.11 The outer surfaces of OMVs are comprised of the normal constituents of the bacterial outer membrane from which they originate, while their lumen contains periplasmic and cytoplasmic components.12 It has been suggested that there may be a sorting mechanism segregating proteins into the OMV proteome.13,14 Several important questions about OMVs remain to be resolved, such as whether and how cytosolic or periplasmic proteins are incorporated into OMVs, and their physiological function.1-4 r 2010 American Chemical Society
OMVs contain active cellular enzymes in their membrane or lumen and play a role in bacterial virulence via several mechanisms, including periplasmic enzyme delivery, DNA transport, bacterial adherence, quorum sensing,4 and evasion of the immune system.2,15 We report the novel finding that Francisella tularensis subspecies novicida (F. novicida) produces OMVs. F. novicida is a model organism for study of the biothreat agent Francisella tularensis Schu S4 (F. tularensis). Francisella tularensis causes the disease tularemia in animals and humans.16 We also demonstrated that Francisella OMVs were produced by other members of the genus Francisella: F. philomiragia and F. tularensis Live Vaccine Strain (LVS). While not normally a human pathogen, Received: May 4, 2010 Published: December 07, 2010 954
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Journal of Proteome Research subspecies of F. philomiragia are important fish pathogens, and this organism is widely distributed around the globe.17,18 Francisella uses Type I and Type VI secretion systems,19,20 a Type II secretion system via the Type IV pilus system,21 as well as a Sec-dependent system22 to secrete effector molecules and virulence factors. OMVs may represent a “vesicle-mediated secretion” system employed by Francisella to deliver cargo to the extracellular environment.23,24 Once released, OMVs either fuse to or are taken up by targeted cells, providing a method by which bacteria can affect other cells at a distance.14,25 Our knowledge of virulence mechanisms for some human pathogens is fairly extensive; for others, such as the genus Francisella, the virulence mechanisms are not fully understood. This knowledge gap limits our ability to develop new prophylaxes, therapeutics and vaccines, which are needed in order to protect against potential attacks by weaponized Francisella.26 A detailed analysis of Francisella OMVs will lead to a greater understanding of Francisella pathogenesis, ultimately leading to the development of a novel vaccine. Currently, there is no FDA approved Francisella vaccine. Many different strategies have been employed in an attempt to develop a safe and effective tularemia vaccine.27 To date, none is without flaws, and the development of a better vaccine to protect against Francisella is still a critical need.28,29 The Live Vaccine Strain (LVS) provides adequate protection against ulceroglandular tularemia, but in limited human trials it failed to provide full protection against aerosolized Schu S4.30 In addition, there are concerns that the LVS vaccine could undergo reversion to a more virulent form.31 Several subunit vaccines have also been evaluated in animal models, with limited success.32 Immunostimulating complexes (ISCOMs) derived from LVS offer some protection against subsequent challenge with F. tularensis subspecies holarctica, but not against Schu S4.27,33 An outer membrane protein-based vaccine offered partial protection against subsequent pulmonary Francisella challenge.34 We feel that by improving the antigen presentation to both humoral and cellular immune system (through use of OMVs), more complete protection could be obtained. Outer membrane vesicles (OMVs) present antigenic proteins in their native context, stimulating both a T and B cell response. An OMV derived Neisseria meningitidis (N. meningitidis) vaccine given to mice has previously been shown to stimulate humoral and cell mediated immunity without the addition of exogenous, synthetic adjuvants.35 The N. meningitidis vaccine, combined with an adjuvant, safely protects toddlers and adolescents against systemic serogroup B meningococcal disease, and its mass implementation halted a decade long meningitidis epidemic in New Zealand. The success of the Neisseria vaccine in humans suggests that an OMV vaccine with clinical utility against Francisella is possible.36 Our OMV preparations differ from the Neisseria membrane-vesicle preparation as we do not use detergent to prepare our Francisella OMVs; instead we collect them as they are naturally produced. A Francisella OMV vaccine may be superior to other vaccine strategies for tularemia because OMVs are isolated in their natural context from Francisella bacteria. Using OMVs may stimulate an immune response more similar to the response to live Francisella bacteria than subunit vaccines. Although the LPS of most gram negative bacteria acts as an endotoxin,37 Francisella LPS is 1000 times less endotoxic than E. coli,38 thus eliminating the need to genetically or biochemically alter Francisella OMVs to reduce endotoxin toxicity. Because OMV preparations are
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sterile, there are no intact bacteria left to be a source of infection, as there might be in a live vaccine strain. We characterized the protein content of OMVs by massspectrometry. Many of the proteins identified in OMVs have also been described as known or proposed virulence factors for Francisella.39 The biochemical activity of the purified OMVs40 was measured and their vaccine potential was determined. Our hypothesis is that Francisella derived OMVs delivered intranasally (IN) would protect mice against subsequent IN Francisella challenge. To test our hypothesis, we used a BALB/C mouseFrancisella infection model41 to show the protection generated by vaccination with Francisella derived OMVs against subsequent bacterial challenge. The development of a Francisella vaccine strategy utilizing the natural antigenic and adjuvant activity of OMVs is promising. Such a vaccine could potentially elicit the immune stimulation of live cell vaccines without the fear of vaccine reversion and potential infection that is possible with live cell vaccines. Taken together, our data advances the understanding of the pathogenicity of an important pathogen and may lead to the development of a protective vaccine against this potentially lethal biothreat agent.
’ METHODS Bacterial Species
F. tularensis LVS (live vaccine strain, ATCC 29684), F. tularensis subsp. novicida (ATCC 15482) (F. novicida) and F. philomiragia (ATCC 25015) were grown in Trypticase Soy Broth plus 0.1% cysteine (TSB-C), 0.1% L-cysteine HCl (Fisher Scientific) brain heart infusion broth (Cysteine Brain Heart Infusion Broth, C-BHI) (Becton-Dickinson) or Chamberlains medium (CM), a chemically defined medium,42 and were grown at 37 C in a shaker incubator for 36-48 h. The bacterial stocks used were frozen in 20% glycerol solution and stored at -80 C. Bacteria were grown on Chocolate Agar II (Becton-Dickinson) plates at 37 C for 24-48 h, depending on species. Cell Lines
The murine macrophage (J774A.1, ATCC TIB-67) cell line was grown with Dulbecco’s Minimal Essential Media (DMEM) plus 10% Fetal Bovine Serum at 37 C and 5% CO2, following manufacturer’s directions. The human lung epithelial cell line (A549, ATCC CCL-185) was grown in Ham’s F-12 Media supplemented with 10% Fetal Bovine Serum at 37 C and 5% CO2, following manufacturer’s directions. Both cell lines were maintained at low passage numbers. Purification of OMVs from Francisella Species
Fifty microliters (∼109 cfu/ml) of each Francisella strain (FtLVS, Fn, Fp) was inoculated into 350 mL of either Chamberlain’s media, C-BHI or TSB-C as indicated at 37 C with constant shaking at 200 rpm for 48 h. Growth curves were obtained using optical density readings at 600 nm every 4 h until bacteria reached stationary phase (data not shown). OMVs were isolated using a modification of the method by Kadurugamuwa and Beveridge.40 Briefly, cells were removed from suspension by centrifugation at 6000 g. The supernatant was collected and sequentially filtered through a 0.45 μm, and 0.0.22 μm pore sized cellulose acetate membrane filters to remove cells and debris. Filtered supernatants were then ultracentrifuged for 2 h at 125 000 g (Ti-70 rotor, Beckman Instruments, Inc.), at 4 C. The supernatant was discarded, and the subsequent pellet was
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Cells were then incubated with 10 μg total protein (in 10 μL) of F. novicida OMVs isolated from bacteria grown in TSB-C. After 18 h, the Ham’s F-12 growth media was removed, and cells were washed in 1 PBS, followed by fixation for 15 min with 3.5% (wt/vol) paraformaldehyde, followed by thorough washing. Cells were then permeabilized for 10 min with 0.25% (v/v) Triton-X 100 in PBS to allow internalization of antibody. After 10 min, cells were thoroughly washed and a drop of Image-it Signal Enhancer (Invitrogen 126933) was added to each well to reduce nonspecific binding. After 30 min, cells were washed and blocked for an additional 30 min in 1% BSA (wt/vol). After washing, 1:500 dilution of anti-Francisella primary antibody (Tetracore TC7005) was applied for 1 h. Cells were thoroughly washed. Cells were then incubated in the dark with a 1:5000 dilution of AlexaFluor 568 secondary antibody (Invitrogen, A-11036, emission at 603 nM) containing the actin-stain phalloidin: Oregon Green 488 (emission at 519 nM) (5 μL of 6.6 μM phalloidin/mL secondary Ab) (Invitrogen 07466). After 30 min, cells were thoroughly washed to remove unbound stain, and Molecular Gold AntiFade Reagent with Dapi (Pierce 46190, excitation 350-405 nM, emission 470 nM) was used to mount the coverslip. Images were obtained using Nikon Eclipse 90i confocal laser scanning microscopy with three lasers: 405, 488, and 561 nM, using a 40x oil immersion objective.
washed with 50 mM HEPES buffer (pH 6.8). Next, samples were centrifuged for an additional 30 min at 125 000 g to repellet the OMVs. Finally, OMVs were resuspended with 100 μL 1 PBS (pH 7.2), and protein concentration was determined by protein assay (Pierce BCA Protein Assay). Initially, we measured OMV production at 4 h intervals, during which we determined that 44 h was the optimal growth period for OMV production. Subsequent Francisella cultures were grown for 44 h before OMVs were harvested. OMV aliquots (100 μL) were plated onto Chocolate II agar to check for sterility, and the remaining sample was stored at -80 C until ready for use. LPS Western Blotting
Fifteen μg total protein of OMV sample isolated from bacteria grown in TSB-C were separated using 4-6% NuPAGE gel run at 120 V for 90 min. Protein was transferred to a PVDF membrane (at 100 V, 30-40 mA, 1 h) and blocked in bovine serum albumin to prevent nonspecific binding. The membrane was then incubated with anti-F. novicida LPS antibody (1:5000) (IPA009, IPAFnov8.2, raw ascites) in 3% BSA in 1 phosphate buffered-saline with 0.1% Tween-20 (PBS-T). After washing, the membrane was treated with an HRP labeled goat antimouse antibody (1:2000-1:5000) (secondary antibody) (Chemicon) and an Opti-4CN kit from BioRad was used to visulaize bands. Transmission Electron Microscopy (TEM)
Whole bacteria were grown in C-BHI overnight with gentle shaking (100 rpm) and were subjected to negative staining and TEM as unfixed samples. One-hundred microliters of a filter sterilized 4.2% (wt/vol) glutaraldehyde/phosphate buffer solution was added to 100 μL of OMVs to make a 2.1% glutaraldehyde solution. OMV samples were incubated for 5-10 min and stored at 4 C until shipped for negative staining and transmission electron microscopy. Twenty microliters of samples were placed onto carbon and Formvar-coated nickel grids, then stained with 2% aqueous uranyl acetate for negative staining, rinsed, and examined with a JEOL 100CXII transmission electron microscope (TEM) at the Advanced Microscopy Facility, University of Virginia (Charlottesville, VA).
Hemolysis Assay
Hemolytic activity was adapted from a method used by Bergmann et al.44 A 2% horse erythrocyte suspension (Hemasource Inc.) in 1 PBS (Cellgro) was used for this assay. 50 μL of OMV sample (64 μg/μL) isolated from bacteria grown in TSB-C were added to 50 μL of erythrocyte suspension, and the sample incubated at 37 C for 60 min. Controls included the 2% erythrocyte solution alone with PBS, and 2% erythrocyte solution with 50 μL of ddH2O for complete lysis. Unlysed erythrocytes were removed by centrifugation for 2 min at 1000 g. Then, absorbance of the supernatants was read at 540 nm. The formula to calculate % hemolysis was as follows: ((Absorbance of sample - Absorbance of no hemolysis)/(Absorbance of total hemolysis - Absorbance of no hemolysis) 100).
Atomic Force Microscopy (AFM)
A piece of mica was attached onto a glass slide, and the upper layer of mica was removed using scotch tape. Then, 1-2 μL of bacterial or OMV samples were placed on mica glass slides (Polyscience, Inc.), air-dried, rinsed with ddH2O and then airdried again. The samples were viewed by the Nano-R2 Atomic Force Microscope (AFM)43 to determine size of vesicles and to visualize the vesicles shedding from the bacterial surface. Scanning was performed in the tapping mode with a resonance frequency of 300-350 kHz in air-dried over silica-gel. The size and shape of OMVs from Francisella spp. were measured using the software provided with the AFM. Due to the drying of the sample on the mica surface before AFM is performed, the OMVs have assumed a flattened circular or elliptic shape, much like a water balloon sitting on a flat surface. We calculated the volume of the OMVs from their measured lateral sizes and the height using the formula, V = (4/3)πabc, where a and b are lateral semimajor axes and c is half the measured height of each vesicle, and then determined what diameter perfect sphere would have that same volume.
Acid Phosphatase (Acp) Activity
The method used to detect acid phosphatase activity was adapted from Bergmann et al.44 50 μL of OMVs (86 μg/μL) isolated from bacteria grown in TSB-C were added to 50 μL of 0.1 M sodium acetate buffer (pH 5.5) containing 10 mM p-nitrophenylphosphate (p-NPP). The suspension was incubated for 1 h at 37 C, and the reaction was stopped with 50 μL of 3 N sodium hydroxide (NaOH). The production of p-nitrophenol (pNP) was monitored at 405 nm. Potato acid phosphatase (lyophilized powder, 3.0-10.0 units/mg solid, P1146, Sigma) was used as a positive control and pNPP reagent was the negative control. The acid phosphatase activity was calculated by plotting a standard curve and using y = mx þ b to determine the number of Units (U) of AcpA in the OMVs compared the Units of phosphatase activity in the control Potato acid phosphatase. Phospholipase C Activity
The method used to detect phospholipase C activity was adapted from Bergmann et al.44 50 μL of OMV sample (66 μg/μL) isolated from bacteria grown in TSB-C was added to 50 μL of 0.25 M PBS buffer, pH 7.2, 1.0 μM ZnCl2, 10 mM O-(4nitrophenylphosphoryl) choline (p-NPPC) reagent in a microtiter
Visualizing OMVs with Confocal Microscopy
A549 cells were grown to 95% confluence in a chambered slide containing Ham’s F-12 growth media at 37 C with 5% CO2. 956
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plate. Following incubation for 1 h at 37 C, 50 μL of 3N NaOH was added to stop the reaction. Sample absorbancies were read at 405 nm. The positive control for the experiment was phospholipase C from Clostridium perfringens (Type I, lyophilized powder, 10-50 units/mg protein, P7633, Sigma) and the negative control was PBS alone. The phospholipase C activity was calculated by plotting a standard curve and using y = mx þ b to determine the number of units (U) of PLC in the OMVs by comparing to the units of activity of the control phospholipase C.
100% B in an additional 5 min. The mass spectrometer was operated in a data-dependent mode in which each full MS scan was followed by five MS/MS with dynamic exclusion. Tandem mass spectra were searched against the NCBI Francisella philomiragia subsp. philomiragia ATCC 25017 and/or Francisella tularensis subsp. novicida (strain U112) using SEQUEST (Bioworks software from ThermoFisher, version 3.3.1) with full tryptic cleavage constraints, static cysteine alkylation by iodoacetamide, and variable methionine oxidation. Confident peptide identifications were determined using stringent filter criteria “Xcorr versus charge 1.9, 2.2, 3.5 for 1þ, 2þ, 3þ ions; ΔCn > 0.1; ranked top #1; probability of randomized identification of peptide
