Inhibitory action of essential oils against proteases activity of Paenibacillus larvae, the etiological agent of American Foulbrood disease

María C. Pellegrini, Lucía Zalazar, Sandra R. Fuselli, Alejandra G. Ponce

Abstract


American foulbrood (AFB) is a disease affecting the larva of Apis mellifera. The etiological agent is Paenibacillus larvae, which releases metalloproteases involved in the degradation of larval tissues. Through quorum sensing (QS) mechanism, bacteria are able to activate specific genes such as virulence factors. The exoproteases regulation of P. larvae could be associated with QS. A promising mechanism of AFB control is to block QS mechanism with essential oils (EO). The aim of this study was to investigate the potential presence of QS signals in the regulation of P. larvae proteases and the effect of seven EOs on the exoproteases activity of P. larvae. From growth curves and evaluation of the presence of proteases by milk agar plates assay, it was observed protease activity during the late exponential phase of growth. Early production of protease activity (15 hours earlier than control) was observed when a low density culture was incubated with late exponential spent medium (SM) suggesting the presence of factor(s) inducing this activity. SM was obtained by the ultrafiltration of P. larvae cultures on late growth phase and was free of proteases. Proteolytic activity was quantified on P. larvae cultures in presence of sublethal concentration of EO by azocasein method. The EOs, except S. chilensis EO, reduced significantly protease activity (more than 50%). We report for the first time evidence on the possible role of QS on P. larvae and the antiproteolytic activity of EOs (except for S. chilensis) on exoproteases, an interesting therapeutic strategy to control AFB.

Keywords


virulence factor; proteases; spent medium; gram positive; bee infection

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References


Adams R, 2007. Identification of essential oils components by gas chromatography/mass spectrometry, 4th ed; Corporation Ap, editor. Illinois, USA.

Alippi A, 1991. A comparison of laboratory techniques for the detection of significant bacteria of the honey bee, Apis mellifera, in Argentina. J Apicult Res 30 (2): 75-80. https://doi.org/10.1080/00218839.1991.11101237

Antúnez K, Anido M, Schlapp G, Evans JD, Zunino P, 2009. Characterization of secreted proteases of Paenibacillus larvae, potential virulence factors involved in honeybee larval infection. J Invertebr Pathol 102 (2): 129-132. https://doi.org/10.1016/j.jip.2009.07.010

Antúnez K, Anido M, Evans JD, Zunino P, 2010. Secreted and immunogenic proteins produced by the honeybee bacterial pathogen, Paenibacillus larvae. Vet Microbiol 141 (3-4): 385-389. https://doi.org/10.1016/j.vetmic.2009.09.006

Antúnez K, Anido M, Arredondo D, Evans JD, Zunino P, 2011a. Paenibacillus larvae enolase as a virulence factor in honeybee larvae infection. Vet Microbiol 147 (1-2): 83-89. https://doi.org/10.1016/j.vetmic.2010.06.004

Antúnez K, Arredondo D, Anido M, Zunino P, 2011b. Metalloprotease production by Paenibacillus larvae during the infection of honeybee larvae. Microbiol 157 (5): 1474-1480. https://doi.org/10.1099/mic.0.044321-0

Bhardwaj AK, Vinothkumar K, Rajpara N, 2013. Bacterial quorum sensing inhibitors: Attractive alternatives for control of infectious pathogens showing multiple drug resistance. Recent Pat Antiinfect Drug Discov 8: 68-83. https://doi.org/10.2174/1574891X11308010012

Biswa P, Doble M, 2013. Production of acylated homoserine lactone by gram-positive bacteria isolated from marine water. FEMS Microbiol Lett 343 (1): 34-41. https://doi.org/10.1111/1574-6968.12123

Boyer M, Wisniewski-Dyé F, 2009. Cell-cell signalling in bacteria: not simply a matter of quorum. FEMS Microbiol Ecol 70: 1-19. https://doi.org/10.1111/j.1574-6941.2009.00745.x

Bradford MM, 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72 (1): 248-254. https://doi.org/10.1016/0003-2697(76)90527-3

Cámara M, Williams P, Hardman A, 2002. Controlling infection by tuning in and turning down the volume of bacterial small-talk. Lancet Infect Dis 2: 667-676. https://doi.org/10.1016/S1473-3099(02)00447-4

Camiletti BX, Asensio CM, Gadban LC, Pecci MPG, Conles MY, Lucini EI, 2016. Essential oils and their combinations with iprodione fungicide as potential antifungal agents against withe rot (Sclerotium cepivorum Berk) in garlic (Allium sativum L.) crops. Ind Crops Prod 85: 117-124. https://doi.org/10.1016/j.indcrop.2016.02.053

Castillo-Juárez I, Maeda T, Mandujano-Tinoco EA, Tomás M, Pérez-Eretza B, García-Contreras SJ, Wood TK, García-Contreras R, 2015. Role of quorum sensing in bacterial infections. World J Clin Cases 3: 575. https://doi.org/10.12998/wjcc.v3.i7.575

Chamorro ER, Ballerini G, Sequeira AF, Velasco GA, Zalazar MF, 2008. Chemical composition of essential oil from Tagetes minuta L. leaves and flowers. J Arg Chem Soc 96 (1): 80-86.

Charney J, Tomarelli RM, 1947. A colonmetric method for the determination of the proteolytic activity of duodenal juice. World J Biol Chem 171: 501-505.

Choi SC, Zhang C, Moon S, Oh YS, 2014. Inhibitory effects of 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF) on acyl-homoserine lactone-mediated virulence factor production and biofilm formation in Pseudomonas aeruginosa PAO1. J Microbiol 52 (9):734-742 https://doi.org/10.1007/s12275-014-4060-x

Chu YY, Nega M, Wölfle M, Plener L, Grond S, Jung K, Götz F, 2013. A new class of quorum quenching molecules from Staphylococcus species affects communication and growth of gram-negative bacteria. PLoS Pathog 9 (9): e1003654. https://doi.org/10.1371/journal.ppat.1003654

Costa DCM, Vermelho AB, Almeida CA, De Souza Dias EP, Cedrola SML, De Fátima Arrigoni-Blank M, Blank AF, Alviano CS, Alviano DS, 2014. Inhibitory effect of linalool-rich essential oil from Lippia alba on the peptidase and keratinase activities of dermatophytes. J Enzyme Inhib Med Chem 29: 12-17. https://doi.org/10.3109/14756366.2012.743537

da Silva MFR, Bezerra-Silva PC, de Lira CS, de Lima Albuquerque BN, Agra Neto AC, Pontual EV, Maciel JR, Paiva PMG, Navarro DMAF, 2016. Composition and biological activities of the essential oil of Piper corcovadensis (Miq.) C. DC (Piperaceae). Exp Parasitol 165: 64-70. https://doi.org/10.1016/j.exppara.2016.03.017

Demo M, Oliva MDML, López ML, Zunino MP, Zygadlo JA, 2005. Antimicrobial activity of essential oils obtained from aromatic plants of Argentina. Pharm Biol 43: 129-134. https://doi.org/10.1080/13880200590919438

Duschatzky CB, Possetto ML, Talarico LB, García CC, Michis F, Almeida NV, De Lampasona MP, Schuff C, Damonte EB, 2005. Evaluation of chemical and antiviral properties of essential oils from South American plants. Antivir Chem Chemother 16: 247-251. https://doi.org/10.1177/095632020501600404

Eguaras MJ, Fuselli S, Gende L, Fritz R, Ruffinengo SR, Clemente G, Gonzalez A, Bailac PN, Ponzi MI, 2005. An in vitro evaluation of Tagetes minuta essential oil for the control of the honeybee pathogens Paenibacillus larvae and Ascosphaera apis, and the parasitic mite Varroa destructor. J Essent Oil Res 17: 336-340. https://doi.org/10.1080/10412905.2005.9698924

Fuqua C, Parsek MR, Greenberg EP, 2001. Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing. Annu Rev Genet 35: 439-468. https://doi.org/10.1146/annurev.genet.35.102401.090913

Fuselli SR, De La Rosa SBG, Eguaras MJ, Fritz R, 2008. Susceptibility of the honeybee bacterial pathogen Paenibacillus larvae to essential oils distilled from exotic and indigenous Argentinean plants. J Essent Oil Res 20: 464-470.

Gala V, Desai K, 2014. Plant based quorum sensing inhibitors of Pseudomonas aeruginosa. Int J Pharm Pharm Sci 6: 20-25.

Gastaldi B, Assef Y, Van Baren C, Di Leo Lira P, Retta D, Bandoni AL, SB González, 2016. Antioxidant activity in teas, tinctures and essential oils of native species from Patagonia Argentina. Revista Cubana de Plantas Medicinales 21: 51-62.

Genersch E, Forsgren E, Pentikäinen J, Ashiralieva A, Rauch S, Kilwinski J, Fries I, 2006. Reclassification of Paenibacillus larvae subsp. pulvifaciens and Paenibacillus larvae subsp. larvae as Paenibacillus larvae without subspecies differentiation. Int J Syst Evol Micr 56 (3): 501-511. https://doi.org/10.1099/ijs.0.63928-0

Ghosh R, Tiwary BK, Kumar A, Chakraborty R, 2014. Guava leaf extract inhibits quorum-sensing and Chromobacterium violaceum induced lysis of human hepatoma cells: Whole transcriptome analysis reveals differential gene expression. PLoS ONE 9 (9): e107703. https://doi.org/10.1371/journal.pone.0107703

Gillij YG, Gleiser RM, Zygadlo JA, 2008. Mosquito repellent activity of essential oils of aromatic plants growing in Argentina. Bioresour Technol 99: 2507-2515. https://doi.org/10.1016/j.biortech.2007.04.066

González MJ, Marioli JM, 2010. Antibacterial activity of water extracts and essential oils of various aromatic plants against Paenibacillus larvae, the causative agent of American Foulbrood. J Invertebr Pathol 104: 209-213. https://doi.org/10.1016/j.jip.2010.04.005

Henke JM, Bassler BL, 2004. Bacterial social engagements. Trends Cell Biol 14 (11): 648-656. https://doi.org/10.1016/j.tcb.2004.09.012

Hentzer M, Wu H, Andersen JB, Riedel K, Rasmussen TB, Bagge N, Kumar N, Schembri MA, Song Z, Kristoffersen P, et al., 2003. Attenuation of Pseudomonas aeruginosa virulence by quorum sensing inhibitors. EMBO J 22 (15): 3803-3815. https://doi.org/10.1093/emboj/cdg366

Inouye K, Kusano M, Hashida Y, Minoda M, Yasukawa K, 2007. Engineering, expression, purification, and production of recombinant thermolysin. Biotechnol Annu Rev 13: 43-64. https://doi.org/10.1016/S1387-2656(07)13003-9

Khan M, Ahmad S, Zahin M, Hasan S, Husain FM, Ahmad I, 2009. Inhibition of quorum sensing regulated bacterial functions by plant essential oils with special reference to clove oil. Lett Appl Microbiol 49: 354-360. https://doi.org/10.1111/j.1472-765X.2009.02666.x

Kleerebezem M, Quadri LEN, Kuipers OP, 1997. Quorum sensing by peptid pheromones and two-component signal-transduction systems in gram-positive bacteria. Mol Micro 24 (5): 895-904. https://doi.org/10.1046/j.1365-2958.1997.4251782.x

Laemmli UK, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227 (5259): 680-685. https://doi.org/10.1038/227680a0

Larrán S, Ringuelet JA, Carranza MR, Henning CP, Ré MS, Cerimele EL, Urrutia MI, 2001. In vitro fungistatic effect of essential oils against Ascosphaera apis. J Essent Oil Res 13: 122-124. https://doi.org/10.1080/10412905.2001.9699633

López SB, López ML, Aragón LM, Tereschuk ML, Slanis AC, Feresin GE, Zygadlo JA, Tapia AA, 2011. Composition and anti-insect activity of essential oils from Tagetes L species (Asteraceae, Helenieae) on Ceratitis capitata Wiedemann and Triatoma infestans Klug. J Agric Food Chem 59: 5286-5292. https://doi.org/10.1021/jf104966b

Mojarab-Mahboubkar M, Sendi JJ, Aliakbar A, 2015. Effect of Artemisia annua L. essential oil on toxicity, enzyme activities, and energy reserves of cotton bollworm Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae). J Plant Prot Res 55: 371-377. https://doi.org/10.1515/jppr-2015-0049

Monnet V, Gardan R, 2015. Quorum-sensing regulators in gram-positive bacteria: 'cherchez le peptide. Mol Microbiol 97 (2): 181-118. https://doi.org/10.1111/mmi.13060

Musthafa KS, Voravuthikunchai SP, 2016. Eugenyl acetate inhibits growth and virulence factors of drug-resistant. Flavour Fragr J 31: 448-454. https://doi.org/10.1002/ffj.3336

Neuendorf S, Hedtke K, Tangen G, Genersch E, 2004. Biochemical characterization of different genotypes of Paenibacillus larvae subsp. larvae, a honey bee bacterial pathogen. Microbiology 150 (7): 2381-2390. https://doi.org/10.1099/mic.0.27125-0

Novick RP, Ross HF, Projan SJ, Kornblum J, Kreiswirth B, Moghazehs S, 1993. Synthesis of staphylococcal virulence factors is controlled by a regulatory RNA molecule. EMBO J 12 (10): 3967.

Olivero-Verbel J, Barreto-Maya A, Bertel-Sevilla A, Stashenko EE, 2014. Composition, anti-quorum sensing and antimicrobial activity of essential oils from Lippia alba. Braz J Microbiol 45: 759-767. https://doi.org/10.1590/S1517-83822014000300001

Otero Casal AM, Crego AM, Bernardez Hermida MI, Fabregas Casal J, 2005. Quórum sensing: El lenguaje de las bacterias. Ed. Acribia SA, Zaragoza, Spain.

Paggi RA, Martone CB, Fuqua C, De Castro RE, 2003. Detection of quorum sensing signals in the haloalkaliphilic archaeon Natronococcus occultus. FEMS Microbiol Lett 221 (1): 49-52. https://doi.org/10.1016/S0378-1097(03)00174-5

Palacios SM, Bertoni A, Rossi Y, Santander R, Urzúa A, 2009. Insecticidal activity of essential oils from native medicinal plants of Central Argentina against the house fly, Musca domestica (L.). Parasitol Res 106: 207-212. https://doi.org/10.1007/s00436-009-1651-2

Pellegrini MC, Alvarez MV, Ponce AG, Cugnata NM, De Piano FG, Fuselli SR, 2014. Anti-quorum sensing and antimicrobial activity of aromatic species from South America. J Essent Oil Res 26: 458-465. https://doi.org/10.1080/10412905.2014.947387

Rasmussen TB, Givskov M, 2006. Quorum-sensing inhibitors as anti-pathogenic drugs. Int J Med Microbiol 296 (2): 149-161. https://doi.org/10.1016/j.ijmm.2006.02.005

Ruffinengo S, Eguaras M, Floris I, Faverin C, Bailac P, Ponzi M, 2005. LD50 and repellent effects of essential oils from Argentinian wild plant species on Varroa destructor. J Econ Entomol 98: 651-655. https://doi.org/10.1603/0022-0493-98.3.651

Ruffinengo SR, Maggi M, Fuselli S, Floris I, Clemente G, Firpo NH, Bailac PN, Ponzi MI, 2006. Laboratory evaluation of Heterothalamus alienus essential oil against different pests of Apis mellifera. J Essent Oil Res 18: 704-707. https://doi.org/10.1080/10412905.2006.9699211

Selje N, Hoffmann EM, Muetzel S, Ningrat R, Wallace RJ, Becker K, 2007. Results of a screening programme to identify plants or plant extracts that inhibit ruminal protein degradation. Br J Nutr 98: 45-53. https://doi.org/10.1017/S0007114507472506

Sivamani P, Singaravelu G, Thiagarajan V, Jayalakshmi T, Kumar GR, 2012. Comparative molecular docking analysis of essential oil constituents as elastase inhibitors. Bioinformation 8: 457. https://doi.org/10.6026/97320630008457

Tang K, Zhang XH, 2014. Quorum quenching agents: Resources for antivirulence therapy. Mar Drugs 12: 3245-3282. https://doi.org/10.3390/md12063245

Tran L, Naik A, Koronkiewicz B, Peethambaran B, 2014. Epigallocatechin gallate inhibits biofilm production and attenuates virulent factors of Pseudomonas aeruginosa and Pseudomonas fluorescences. J Nat Remedies 14 (1): 106-111.

Vila R, Mundina M, Tomi F, Furlán R, Zacchino S, Casanova J, Cañigueral S, 2002. Composition and antifungal activity of the essential oil of Solidago chilensis. Planta Med 68: 164-167. https://doi.org/10.1055/s-2002-20253

Waters CM, Bassler BL, 2005. Quorum sensing: cell-to-cell communication in bacteria. Annu Rev Cell Dev Biol 21: 319-346. https://doi.org/10.1146/annurev.cellbio.21.012704.131001

Weng L, Zhang Y, Yang Y, Wang L, 2014. Isolation of the autoinducer-quenching strain that inhibits lasR in Pseudomonas aeruginosa. Int J Mol Sci 15 (4): 6328-6342. https://doi.org/10.3390/ijms15046328

Williams P, 2007. Quorum sensing, communication and cross-kingdom signalling in the bacterial world. Microbiology 153: 3923-3938. https://doi.org/10.1099/mic.0.2007/012856-0

Williams P, Winzer K, Chan W, Cámara M, 2007. Look who's talking: communication and quorum sensing in the bacterial world. Philos T R Soc London B Biol Sci 362: 1119-1134. https://doi.org/10.1098/rstb.2007.2039

Zhang LH, 2003. Quorum quenching and proactive host defense. Trends Plant Sci 8: 238-244. https://doi.org/10.1016/S1360-1385(03)00063-3

Zygadlo JA, Guzman CA, Grosso NR, 1994. Antifungal properties of the leaf oils of Tagetes minuta L. and T. filifolia Lag. J Essent Oil Res 6: 617-621. https://doi.org/10.1080/10412905.1994.9699353




DOI: 10.5424/sjar/2017154-10785