Lactobacillus-derived extracellular vesicles enhance host immune responses against vancomycin-resistant enterococci
© The Author(s). 2017
Received: 4 November 2016
Accepted: 9 March 2017
Published: 14 March 2017
Probiotic bacteria are known to modulate host immune responses against various pathogens. Recently, extracellular vesicles (EVs) have emerged as potentially important mediators of host-pathogen interactions. In this study, we explored the role of L. plantarum derived EVs in modulating host responses to vancomycin-resistant Enterococcus faecium (VRE) using both Caenorhabditis elegans and human cells.
Our previous work has shown that probiotic conditioning C. elegans with L. acidophilus NCFM prolongs the survival of nematodes exposed to VRE. Similarly, L. plantarum WCFS1 derived extracellular vesicles (LDEVs) also significantly protected the worms against VRE infection. To dissect the molecular mechanisms of this EV-induced protection, we found that treatment of C. elegans with LDEVs significantly increased the transcription of host defense genes, cpr-1 and clec-60. Both cpr-1 and clec-60 have been previously reported to have protective roles against bacterial infections. Incubating human colon-derived Caco-2 cells with fluorescent dye-labeled LDEVs confirmed that LDEVs could be transported into the mammalian cells. Furthermore, LDEV uptake was associated with significant upregulation of CTSB, a human homologous gene of cpr-1, and REG3G, a human gene that has similar functions to clec-60.
We have found that EVs produced from L. plantarum WCFS1 up-regulate the expression of host defense genes and provide protective effects on hosts. Using probiotic-derived EVs instead of probiotic bacteria themselves, this study provides a new direction to treat antimicrobial resistant pathogens, such as VRE.
KeywordsLactobacillus Extracellular vesicles LDEVs Vancomycin-resistant enterococci VRE CTSB REG3G
Lactobacillus is a genus of Gram-positive facultative anaerobic bacteria . Considered as non-pathogenic and generally regarded as safe, lactobacilli have been widely used for fermentation and food production for centuries [2, 3]. The beneficial or probiotic effects of lactobacilli are under intense investigation with both laboratory and clinical studies [4–8], suggesting that administration of lactobacilli inhibit cytokine-induced apoptosis  and decreases the pathogenicity of various pathogens, such as E. coli  and VRE . However, the molecular mechanisms by which lactobacilli impact VRE are incompletely understood.
Lactobacilli may exert immunomodulatory effects using multiple mechanisms including binding directly to C-type lectin receptors (CLRs) or Toll-like receptors (TLRs), on the host cell surface [12, 13]. For example, administration of L. casei CRL 431 increased the expression of TLR2, TLR4, and TLR9 and improved the production and secretion of TNFα, IFNγ, and IL-10 in mice . Alternatively, lactobacilli may produce antimicrobial substances to inhibit the growth of various pathogens. For example, a bacteriocin produced by lactobacilli formed pores in the membranes of pathogens and thus caused leaking of target cells [14, 15]. More recently, studies have revealed that extracellular vesicles (EVs) and associated proteins from lactobacilli can also modulate the activity of immune cells and affect host innate and adaptive immune responses [16–18]. For example, EVs from lactobacilli were found to enhance cellular TLR2/1 and TLR4 responses while suppressing TLR2/6 signaling .
Extracellular vesicles (EVs) are nanometer-scale membrane-contained vesicles released in an evolutionally conserved manner by a wide range of cells [19, 20]. By facilitating the transfer of proteins, nucleic acids, and other molecules between cells [21, 22], EVs are associated with molecular transport, mediation of stress response and biofilm formation thus influencing their hosts [23, 24]. This EV-mediated interaction is likely prevalent in the gut as a major method of communication between bacteria and hosts, since a layer of mucin prevents direct physical contact between bacteria and host tissues . Another unique feature associated with EVs is their potential to mediate therapeutic molecule delivery without inducing adverse immune reactions .
In this study, we selected L. plantarum WCFS1, a leading probiotic strain found in the gastrointestinal tract, due to its potency to inducing immunomodulatory effects . We found that L. plantarum WCFS1 produces EVs that are 30–200 nm in diameter. Proteomic analysis revealed that L. plantarum derived EV (LDEV) cargo was enriched with membrane-associated proteins. Using the experimental nematode C. elegans, LDEV treatment prolonged the survival rates of C. elegans under E. faecium (VRE) challenge. To investigate the underlying mechanisms, we found that the host defense genes, cpr-1 and clec-60, were significantly upregulated. LDEV treatment of human colonic cells lines also led to similar upregulation of CTSB (Cathepsin B) and REG3G (Regenerating islet-derived protein 3-gamma).
L. plantarum produces EVs
L. plantarum EVs are biofunctional and increase the survival of C. elegans
L. plantarum EVs up-regulate host defense genes, clec-60 and cpr-1 in a C. elegans model
L. plantarum EVs incubation led to LDEV cargo delivery and up-regulation of host defense genes, CTSB and REG3G in Caco-2 cells
The importance of EVs has been increasingly recognized. Virtually all kinds of cell types studied so far secret EVs, and they are also found in various bio-fluids [19, 20]. This phenomenon indicates that EVs are evolutionarily conserved and likely functionally important [21, 22]. Indeed, numerous studies on mammalian cell derived EVs have shown that EVs play important roles in intercellular communication and mediation of immunomodulatory response . However, EV-mediated interactions between host and bacterial pathogens are less explored. Limited studies suggest that pathogenic bacterial strains affect biofilm formation via EV pathways [23, 24]. A recent study on probiotic bacteria has also shown EVs from multiple Lactobacillus strains modulate host-microbe responses by regulating the TLR2 activity and phagocytosis . Here, we focused on L. plantarum, a gut-associated commensal bacteria often used in probiotic nutritional supplements. We found that L. plantarum WCFS1 produces functional EVs that enhance host defense gene expression and directly augments protection against VRE infections. These findings suggest LDEVs, at least partially, mediate the immunomodulatory properties of probiotic lactobacilli.
It is interesting to note that L. plantarum derived EVs up-regulate clec-60 and cpr-1, while the L. plantarum bacteria promote the expression of both genes and cpr-5. The shared upregulation of clec-60 and cpr-1 suggest that L. plantarum derived EVs retain much of the immunomodulatory effects of L. plantarum. This is probably because EVs have similar cargo contents as their parental bacteria . The different regulation observed with gene cpr-5, however, illustrates that bacterial EVs are not equal to the intact bacteria regarding the spectrum of induced immunomodulatory effects.
Our experiments using human Caco-2 cells confirmed biological activity of the LDEVs. Both REG3G , which is functionally similar to clec-6, and CTSB  (the human orthologue of cpr-1) are upregulated by LDEV treatment. REG3G is an intestinally secreted C-type lectin with potent bactericidal activity against Gram-positive bacteria . It also promotes the spatial segregation of microbiota and host in the intestine , thus decreasing the chance of bacterial colonization on the intestinal epithelial surfaces [38, 39]. CTSB, a cysteine proteinase involved in cell death and inflammation , is associated with antibacterial activity [41, 42]. Although it may involve autophagy , the exact mechanism of CTSB on bacterial pathogens is unclear.
This study provided a mechanistic insight as to how LDEVs enhance host immune response via upregulation of the two host genes, REG3G and CTSB.
In summary, our study revealed that EVs produced from L. plantarum up-regulate the expression of host defense genes, clec-60 and cpr-1, and provide protection against VRE infection in a C. elegans model. LDEV treatment of human colonic cells lines also led to similar upregulation of CTSB and REG3G. The findings of this study could be harnessed to design a new therapeutic treatment of antimicrobial resistant infections by using EVs derived from probiotic strains rather than the bacteria themselves.
Preparation of probiotic bacteria
Single colony inoculated L. plantarum WCFS1 (BAA-793, ATCC) was grown in de Man, Rogosa, and Sharpe (MRS) medium (Difco Laboratories) at 37 °C for 24 h.
Isolation of extracellular vesicles
Extracellular vesicle fractions were independently enriched from culture supernatants of L. plantarum WCFS1 and medium control. Supernatants from overnight cultures were generated by first centrifuging cultures at 1000 g for 10 min. All supernatants were then passed through a 0.22 μm filter to remove large particles and possible contaminants. EVs were isolated using an ExoQuick-TC™ (System Biosciences) kit per the manufacturer’s directions. Briefly, five parts of supernatant were mixed with one mL of ExoQuick-TC solution. The mixtures were incubated overnight at 4 °C and followed by two centrifugations at 1500 × g for 30 min and then 5 min, respectively. The supernatants were discarded, and the resulting pellets were resuspended in PBS to use directly in downstream experiments or placed in a −80 °C freezer for long-term storage. Mock EVs were isolated from sterile, uninoculated L. plantarum WCFS1 culture broth using the same EV isolation procedures.
LDEVs were fixed with 3% glutaraldehyde in 0.15 M sodium cacodylate buffer and then post-fixed in 1% osmium tetroxide (Electron Microscopy Sciences). Fixed samples were cut into 1.5 mm cubes and covered with a 3% agar solution. Samples were dehydrated through a graded series of acetone and embedded in Spurr epoxy resin (Ladd Research Industries). Ultra-thin sections were then prepared, retrieved onto 300-mesh thin bar copper grids, and contrasted with uranyl acetate and lead citrate. Sections were examined using a Morgagni 268-transmission electron microscope and images collected with an AMT Advantage 542 CCD camera system.
Nanoparticle tracking analysis (NTA)
The NTA analysis was carried out using a NanoSight™ NS500 (Malvern) and an automatic syringe pump system. This instrument generates a detailed analysis of the size distribution and concentration of nanoparticles. The analysis was performed on EVs suspended in PBS at 22 °C. Thirty of 30-s videos were recorded for each sample with camera shutter at 33 ms. Videos recorded for each sample were analyzed with NTA software (version 2.3). For this analysis, auto settings were used for blur, minimum track length, and expected particle size; detection threshold was set at 4 Multi.
Proteomic characterization of LDEVs was performed by liquid chromatography-tandem mass spectrometry (LC-MS/MS, nano-LC from LC Packings/Dionex, and Qstar XL from Applied Biosystems). LDEV samples were suspended in Novex® (Thermo Scientific) reducing sample buffer and heated for 10 min at 70 °C. Samples were run on Novex® 4-20% Tris-Glycine gradient gels and stained with SimplyBlue® SafeStain (Life Technologies) for 1 h followed by destaining with water. Gel bands were excised and digested with modified Trypsin (Promega). Tryptic digests were fractionated with a reversed-phase column and the column eluate introduced onto a Qstar XL mass spectrometer via ESI. Protein identifications were performed with ProteinPilot (Applied Biosystems) using the L. plantarum WCFS1 reference sequence database from UniProt and NCBI. To increase confidence, a further manual inspection was carried out to select the proteins associated with at least two unique as the potential candidates [44–46].
Gene ontology (GO) analysis
Protein candidates listed in Additional file 1: Table S1 were searched against UniProt, EBI, and GO databases. Visualization of GO analysis results was carried out in Excel.
Nematode and pathogenic bacteria
C. elegans Bristol N2 or fer-15;fem-1 was used in this study. C. elegans strains were routinely maintained on nematode growth medium (NGM) plates seeded with E. coli OP50 or HB101 using standard procedures . Clinically isolated Enterococcus faecium (vancomycin-resistant) C68  was grown at 37 °C using brain heart infusion (BHI; Difco) broth.
C. elegans killing assays
Solid killing assays were performed using published methods, with slight modifications . For positive control, 1x109 CFU L. plantarum bacteria were spread on an SK plate. LDEVs that were isolated from an equivalent number of L. plantarum, 1x109 CFU, were suspended in PBS and spread on an SK plate. For the negative control, the same volume of mock EVs was spread on SK plate. Each plate was dried for 3 h at room temperature before use. Forty to sixty C. elegans Bristol N2 were seeded into each plate after pre-incubating with L. plantarum WCFS1, LDEVs or controls for 24 h, followed by VRE challenge (a clinically isolated C68 E. faecium strain at 1x109 VRE/plate). After worms had been placed on the plates with the VRE, plates were then incubated at 25 °C and examined for viability at 24-h intervals for 15 days using a Nikon SMZ645 dissecting microscope. Worms were counted as alive or dead based on their response or lack of response to gentle touching with a platinum wire. For preventing hatching of examined adult worm, worms were treated with 5-fluorodeoxyuridine (50 μM) from L4 to end of assays.
Culture and EV treatment of cell lines
Caco-2 (HTB-37, ATCC), a human colon carcinoma cell line, was maintained in Eagle’s Minimum Essential Medium (EMEM) supplemented with 20% fetal bovine serum (FBS) and was used to test LDEV’s effect on mammalian cells. LDEVs were labeled by Exo-Green (System Biosciences) according to manufacturer’s instruction. Briefly, 500 μl of LDEVs suspended in PBS was mixed with 50 μl stain. After 10 min 37 °C incubation and precipitation by ExoQuick-TC, the labeled LDEVs was re-suspended in PBS and added to Caco-2 cells. At 24 h post-incubation, the culture wells were rinsed twice with PBS to remove residual fluorescent dyes. The cells were then examined by fluorescent microscopy (Olympus IX-70). A control experiment using mock EV was also carried out in parallel.
Caco-2 viability assay
(3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) or MTT assay (Sigma) was used to measure Caco-2 cellular proliferation rate after LDEV treatment. All procedures were performed according to the manufacture’s instruction.
RNA isolation and qPCR
Total RNA from C. elegans and Caco-2 cells was extracted using TRIzol® (Thermo Scientific) by following standard protocols. The concentrations of all RNA samples were determined by spectrophotometry. 1 μg of total RNAs was used for reverse transcription and PCR, which was carried out on a Mastercycler® gradient 5331 (Eppendorf, Westbury, NY) by using Maxima® First Strand cDNA Synthesis Kit (Thermo Scientific). Primers were designed by using PrimerQuest online tools available at http://www.idtdna.com/Primerquest/Home/Index. Primer sequences are provided in Additional file 2: Table S2. Real-time PCR was performed on Mastercycler® ep realplex (Eppendorf). All reactions were performed in 96-well plates with the following reagents in a final volume of 20 μl: 1 μl of primers (5 μM each for forward and reverse) and 2X Maxima® SYBR Green qPCR Master Mix from Thermo Scientific. 10 ng of cDNA was added to this mixture. Triplicate reactions of the target and housekeeping genes were performed simultaneously for each cDNA template analyzed. The PCR reaction consisted of an initial enzyme activation step at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. A cycle threshold value (Ct) value was obtained for each sample, and triplicate sample values were averaged. The 2-ΔΔCt method was used to calculate relative expression of each target gene. The control genes snb-1  and ACTB  were used to normalize the gene expression data from C. elegans or Caco-2 cells respectively.
The log-rank test was used to determine the difference in C. elegans survival rates. Differences in qPCR results were determined by using the Student’s t-test. A P < 0.05 in all experiments was considered statistically significant. Statistical analysis and graphing were performed with Prism version 6.05 (GraphPad).
Liquid chromatography–tandem mass spectrometry
L. plantarum derived extracellular vesicles
Quantitative polymerase chain reaction
Regenerating islet-derived protein 3 gamma
Tumor necrosis factor alpha
We thank Carol A. Ayala at the Division of Core Research Laboratories of Rhode Island Hospital for helping with electron microscopy. We thank Mark S. Dooner and Yan Cheng at the Division of Hematology/Oncology of Rhode Island Hospital for helping with NanoSight analyses. We also thank Dr. Louis Rice for the kind gift of VRE C68.
This work was supported by a University Medicine Foundation Research Fund to M. Li and P30GM110759, R01HD072693, K24HD080539 to B. Ramratnam.
Availability of data and materials
All data generated during this study are included in this published article and its supplementary information files. Moreover, the reader can contact the corresponding author to get needed information.
ML, GJN, EM and BR conceived and designed the study. ML, KL, and MH designed and performed the experiments. ML, KL, GJN, EM, and BR wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
Ethics approval and consent to participate
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
- Makarova K, Slesarev A, Wolf Y, Sorokin A, Mirkin B, Koonin E, Pavlov A, Pavlova N, Karamychev V, Polouchine N, et al. Comparative genomics of the lactic acid bacteria. Proc Natl Acad Sci U S A. 2006;103(42):15611–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Adams MR, Marteau P. On the safety of lactic acid bacteria from food. Int J Food Microbiol. 1995;27(2–3):263–4.View ArticlePubMedGoogle Scholar
- Stiles ME, Holzapfel WH. Lactic acid bacteria of foods and their current taxonomy. Int J Food Microbiol. 1997;36(1):1–29.View ArticlePubMedGoogle Scholar
- Dobrogosz WJ, Peacock TJ, Hassan HM. Evolution of the probiotic concept from conception to validation and acceptance in medical science. Adv Appl Microbiol. 2010;72:1–41.View ArticlePubMedGoogle Scholar
- Schlee M, Harder J, Koten B, Stange EF, Wehkamp J, Fellermann K. Probiotic lactobacilli and VSL#3 induce enterocyte beta-defensin 2. Clin Exp Immunol. 2008;151(3):528–35.View ArticlePubMedPubMed CentralGoogle Scholar
- Bongaerts GP, Severijnen RS. The beneficial, antimicrobial effect of probiotics. Med Hypotheses. 2001;56(2):174–7.View ArticlePubMedGoogle Scholar
- Kaur IP, Chopra K, Saini A. Probiotics: potential pharmaceutical applications. Eur J Pharm Sci. 2002;15(1):1–9.View ArticlePubMedGoogle Scholar
- Kim YG, Ohta T, Takahashi T, Kushiro A, Nomoto K, Yokokura T, Okada N, Danbara H. Probiotic Lactobacillus casei activates innate immunity via NF-kappaB and p38 MAP kinase signaling pathways. Microbes Infect. 2006;8(4):994–1005.View ArticlePubMedGoogle Scholar
- Yan F, Polk DB. Probiotic bacterium prevents cytokine-induced apoptosis in intestinal epithelial cells. J Biol Chem. 2002;277(52):50959–65.View ArticlePubMedPubMed CentralGoogle Scholar
- Medellin-Pena MJ, Griffiths MW. Effect of molecules secreted by Lactobacillus acidophilus strain La-5 on Escherichia coli O157:H7 colonization. Appl Environ Microbiol. 2009;75(4):1165–72.View ArticlePubMedGoogle Scholar
- Manley KJ, Fraenkel MB, Mayall BC, Power DA. Probiotic treatment of vancomycin-resistant enterococci: a randomised controlled trial. Med J Aust. 2007;186(9):454–7.PubMedGoogle Scholar
- Castillo NA, Perdigon G, de Moreno de Leblanc A. Oral administration of a probiotic Lactobacillus modulates cytokine production and TLR expression improving the immune response against Salmonella enterica serovar Typhimurium infection in mice. BMC Microbiol. 2011;11:177.View ArticlePubMedPubMed CentralGoogle Scholar
- Walker WA. Mechanisms of action of probiotics. Clin Infect Dis. 2008;46 Suppl 2:S87–91. discussion S144-151.View ArticlePubMedGoogle Scholar
- Ng SC, Hart AL, Kamm MA, Stagg AJ, Knight SC. Mechanisms of action of probiotics: recent advances. Inflamm Bowel Dis. 2009;15(2):300–10.View ArticlePubMedGoogle Scholar
- Todorov SD. Bacteriocins from Lactobacillus plantarum - production, genetic organization and mode of action: producao, organizacao genetica e modo de acao. Braz J Microbiol. 2009;40(2):209–21.View ArticlePubMedPubMed CentralGoogle Scholar
- Corthesy B, Gaskins HR, Mercenier A. Cross-talk between probiotic bacteria and the host immune system. J Nutr. 2007;137(3 Suppl 2):781S–90.PubMedGoogle Scholar
- van Bergenhenegouwen J, Kraneveld AD, Rutten L, Kettelarij N, Garssen J, Vos AP. Extracellular vesicles modulate host-microbe responses by altering TLR2 activity and phagocytosis. PLoS One. 2014;9(2):e89121.View ArticlePubMedPubMed CentralGoogle Scholar
- Ruiz L, Hevia A, Bernardo D, Margolles A, Sanchez B. Extracellular molecular effectors mediating probiotic attributes. FEMS Microbiol Lett. 2014;359(1):1–11.View ArticlePubMedGoogle Scholar
- Raposo G, Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends. J Cell Biol. 2013;200(4):373–83.View ArticlePubMedPubMed CentralGoogle Scholar
- Harding CV, Heuser JE, Stahl PD. Exosomes: looking back three decades and into the future. J Cell Biol. 2013;200(4):367–71.View ArticlePubMedPubMed CentralGoogle Scholar
- Valadi H, Ekström K, Bossios A, Sjöstrand M, Lee JJ, Lötvall JO. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol. 2007;9(6):654–9.View ArticlePubMedGoogle Scholar
- Colombo M, Raposo G, Thery C. Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol. 2014;30:255–89.View ArticlePubMedGoogle Scholar
- Yanez-Mo M, Siljander PR, Andreu Z, Zavec AB, Borras FE, Buzas EI, Buzas K, Casal E, Cappello F, Carvalho J, et al. Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles. 2015;4:27066.View ArticlePubMedGoogle Scholar
- Kuehn MJ, Kesty NC. Bacterial outer membrane vesicles and the host-pathogen interaction. Genes Dev. 2005;19(22):2645–55.View ArticlePubMedGoogle Scholar
- Artis D. Epithelial-cell recognition of commensal bacteria and maintenance of immune homeostasis in the gut. Nat Rev Immunol. 2008;8(6):411–20.View ArticlePubMedGoogle Scholar
- Marcus ME, Leonard JN. FedExosomes: engineering therapeutic biological nanoparticles that truly deliver. Pharmaceuticals. 2013;6(5):659–80.View ArticlePubMedPubMed CentralGoogle Scholar
- Paolillo R, Romano Carratelli C, Sorrentino S, Mazzola N, Rizzo A. Immunomodulatory effects of Lactobacillus plantarum on human colon cancer cells. Int Immunopharmacol. 2009;9(11):1265–71.View ArticlePubMedGoogle Scholar
- Caradec J, Kharmate G, Hosseini-Beheshti E, Adomat H, Gleave M, Guns E. Reproducibility and efficiency of serum-derived exosome extraction methods. Clin Biochem. 2014;47(13–14):1286–92.View ArticlePubMedGoogle Scholar
- Kaparakis-Liaskos M, Ferrero RL. Immune modulation by bacterial outer membrane vesicles. Nat Rev Immunol. 2015;15(6):375–87.View ArticlePubMedGoogle Scholar
- Kulp A, Kuehn MJ. Biological functions and biogenesis of secreted bacterial outer membrane vesicles. Annu Rev Microbiol. 2010;64:163–84.View ArticlePubMedPubMed CentralGoogle Scholar
- Kim Y, Mylonakis E. Caenorhabditis elegans immune conditioning with the probiotic bacterium Lactobacillus acidophilus strain NCFM enhances gram-positive immune responses. Infect Immun. 2012;80(7):2500–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Shivers RP, Pagano DJ, Kooistra T, Richardson CE, Reddy KC, Whitney JK, Kamanzi O, Matsumoto K, Hisamoto N, Kim DH. Phosphorylation of the conserved transcription factor ATF-7 by PMK-1 p38 MAPK regulates innate immunity in Caenorhabditis elegans. PLoS Genet. 2010;6(4):e1000892.View ArticlePubMedPubMed CentralGoogle Scholar
- Hidalgo IJ, Raub TJ, Borchardt RT. Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial permeability. Gastroenterology. 1989;96(3):736–49.View ArticlePubMedGoogle Scholar
- Hentze H, Lin XY, Choi MS, Porter AG. Critical role for cathepsin B in mediating caspase-1-dependent interleukin-18 maturation and caspase-1-independent necrosis triggered by the microbial toxin nigericin. Cell Death Differ. 2003;10(9):956–68.View ArticlePubMedGoogle Scholar
- Brandl K, Plitas G, Mihu CN, Ubeda C, Jia T, Fleisher M, Schnabl B, DeMatteo RP, Pamer EG. Vancomycin-resistant enterococci exploit antibiotic-induced innate immune deficits. Nature. 2008;455(7214):804–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Vaishnava S, Yamamoto M, Severson KM, Ruhn KA, Yu X, Koren O, Ley R, Wakeland EK, Hooper LV. The antibacterial lectin RegIIIgamma promotes the spatial segregation of microbiota and host in the intestine. Science. 2011;334(6053):255–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Mort JS, Buttle DJ. Cathepsin B. Int J Biochem Cell Biol. 1997;29(5):715–20.View ArticlePubMedGoogle Scholar
- Gallo RL, Hooper LV. Epithelial antimicrobial defence of the skin and intestine. Nat Rev Immunol. 2012;12(7):503–16.View ArticlePubMedPubMed CentralGoogle Scholar
- Mukherjee S, Hooper LV. Antimicrobial defense of the intestine. Immunity. 2015;42(1):28–39.View ArticlePubMedGoogle Scholar
- Reiser J, Adair B, Reinheckel T. Specialized roles for cysteine cathepsins in health and disease. J Clin Invest. 2010;120(10):3421–31.View ArticlePubMedPubMed CentralGoogle Scholar
- Lawrence CP, Kadioglu A, Yang AL, Coward WR, Chow SC. The cathepsin B inhibitor, z-FA-FMK, inhibits human T cell proliferation in vitro and modulates host response to pneumococcal infection in vivo. J Immunol. 2006;177(6):3827–36.View ArticlePubMedGoogle Scholar
- Flannagan RS, Cosio G, Grinstein S. Antimicrobial mechanisms of phagocytes and bacterial evasion strategies. Nat Rev Microbiol. 2009;7(5):355–66.View ArticlePubMedGoogle Scholar
- Amer AO, Swanson MS. Autophagy is an immediate macrophage response to Legionella pneumophila. Cell Microbiol. 2005;7(6):765–78.View ArticlePubMedPubMed CentralGoogle Scholar
- Gupta N, Bark SJ, Lu WD, Taupenot L, O’Connor DT, Pevzner P, Hook V. Mass spectrometry-based neuropeptidomics of secretory vesicles from human adrenal medullary pheochromocytoma reveals novel peptide products of prohormone processing. J Proteome Res. 2010;9(10):5065–75.View ArticlePubMedPubMed CentralGoogle Scholar
- Li M, Aliotta JM, Asara JM, Tucker L, Quesenberry P, Lally M, Ramratnam B. Quantitative proteomic analysis of exosomes from HIV-1-infected lymphocytic cells. Proteomics. 2012;12(13):2203–11.View ArticlePubMedGoogle Scholar
- Li M, Ramratnam B. Proteomic characterization of exosomes from HIV-1-infected cells. Methods Mol Biol. 2016;1354:311–26.View ArticlePubMedPubMed CentralGoogle Scholar
- Breger J, Fuchs BB, Aperis G, Moy TI, Ausubel FM, Mylonakis E. Antifungal chemical compounds identified using a C. elegans pathogenicity assay. PLoS Pathog. 2007;3(2):e18.View ArticlePubMedPubMed CentralGoogle Scholar
- Carias LL, Rudin SD, Donskey CJ, Rice LB. Genetic linkage and cotransfer of a novel, vanB-containing transposon (Tn5382) and a low-affinity penicillin-binding protein 5 gene in a clinical vancomycin-resistant Enterococcus faecium isolate. J Bacteriol. 1998;180(17):4426–34.PubMedPubMed CentralGoogle Scholar
- Irazoqui JE, Ng A, Xavier RJ, Ausubel FM. Role for beta-catenin and HOX transcription factors in Caenorhabditis elegans and mammalian host epithelial-pathogen interactions. Proc Natl Acad Sci U S A. 2008;105(45):17469–74.View ArticlePubMedPubMed CentralGoogle Scholar
- Li M, Tucker LD, Asara JM, Cheruiyot CK, Lu H, Wu ZJ, Newstein MC, Dooner MS, Friedman J, Lally MA, et al. Stem-loop binding protein is a multifaceted cellular regulator of HIV-1 replication. J Clin Invest. 2016;126(8):3117–29.View ArticlePubMedPubMed CentralGoogle Scholar