Diversity of Culturable Actinobacteria Producing Protease Inhibitors from The
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bioRxiv preprint doi: https://doi.org/10.1101/2020.02.14.949438; this version posted February 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Diversity of culturable actinobacteria producing protease inhibitors from the 2 intertidal zones of Maharashtra, India 3 4 Neha Shintre1, Ulfat Baig2, Anagha Pund2, Rajashree Patwardhan3, Vaijayanti 5 Tamhane4, Neelima Deshpande1 6 1Department of Microbiology, M.E.S. Abasaheb Garware College, Karve Road, Pune 7 411004, Maharashtra, India. 8 2Indian Institute of Science Education and Research, Pune (IISER-P), Dr. Homi Bhabha 9 Road, Pashan, Pune 411008, Maharashtra, India. 10 3Department of Microbiology, Haribhai V. Desai College of Commerce, Arts and Science, 11 Pune – 411002, Maharashtra, India H. V. Desai College, Pune 12 4Institute of Bioinformatics and Biotechnology, Savitribai Phule Pune University, Pune – 13 411007, Maharashtra, India 14 15 Corresponding authors: V. Tamhane (email: [email protected],) and 16 N. Deshpande (email: [email protected] ) 17 18 Keywords: Marine actinobacteria; Molecular phylogeny; Protease inhibitors; chemical 19 ecology 20 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.14.949438; this version posted February 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 21 ABSTRACT 22 Phylogenetic diversity of culturable actinobacteria isolated from the intertidal regions of west 23 coast of Maharashtra, India was studied using 16S rRNA gene sequencing. Total of 140 24 actinobacterial isolates were obtained, which belonged to 14 genera, 10 families and 65 putative 25 species with Streptomyces being the most dominant genus followed by Nocardiopsis and 26 Micromonospora. They were screened for production of extracellular protease inhibitors (PI) 27 against three pure proteases viz. chymotrypsin, trypsin, subtilisin and one crude extracellular 28 protease from Pseudomonas sp. 80% of the isolates showed protease inhibition activity against 29 at least one of the proteases, majority of them belonged to genus Streptomyes. Actinobacterial 30 diversity from two sites Ade and Harnai with varying degrees of anthropological pressures 31 showed that more species diversity was obtained from site with minimal human disturbance i.e 32 Ade, however, site with higher human disturbance, i.e. Harnai contained higher percentage of 33 PI producing strains. Understanding association of PI production and abundance of 34 extracellular proteases due to organic load in the marine environments can give interesting 35 insights into the chemical ecology of microbial community in the intertidal region. 36 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.14.949438; this version posted February 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 37 INTRODUCTION 38 Proteases are enzymes that catalyse proteolytic reactions and are involved in variety of 39 biological processes like digestion, cell signalling or tumour formation (Groll et al., 2002; 40 Koblinski et al., 2000; Overall and Blobel, 2007; Roberts et al., 2012). They are ubiquitously 41 present across different plant, animal and microbial taxa. Two of the many roles of these 42 enzymes in bacteria is to aid in bacterial pathogenesis (Ingmer and Brøndsted, 2009; Maeda, 43 1996) and bacterial predation (Martin, 2002) where they secrete hydrolytic enzymes in order 44 to degrade prey species in the vicinity (Martin, 2002). Protease inhibitors bind reversibly or 45 irreversibly with the enzymes, rendering them inactive. Hence, protease inhibitors (PIs) play a 46 crucial role as defence molecules in intracellular or extracellular environment. They are 47 classified based on either the type of protease they inhibit (e.g. serine protease inhibitor, aspartic 48 protease inhibitor) or based on the mechanism of action (e.g. reversible or irreversible enzyme 49 inhibitors). Various plants, animals and microorganisms produce PIs in order to deter pests and 50 predators or secrete them as toxins for self-defence (Habib and Fazili, 2007; Hartl et al., 2011; 51 Mourão and Schwartz, 2013). Also, PIs have high commercial potential since they are used as 52 drugs against viral, bacterial and other parasitic infections (Karthik et al., 2014; Liu et al., 2012; 53 Sreedharan and Bhaskara Rao, 2017; Umezawa, 1976). 54 Marine intercostal community harbours a variety of sedentary invertebrates and 55 microorganisms. These organisms rely primarily on chemicals for self-defence (Engel et al., 56 2002; Pawlik, 1993). Additionally, as stated by Hay et al. the “biggest challenge for marine 57 organisms is to obtain lunch without becoming lunch” (Hay, 2009). Protease inhibitors can 58 play a major role in these situations since they can inhibit the action of digestive proteases 59 present in the surroundings. Literature reports suggest that PIs are indeed produced by marine 60 intercostal communities (Covaleda et al., 2012; Karthik and Kirthi, 2015; Karthik et al., 2014; 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.14.949438; this version posted February 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 61 Mourão and Schwartz, 2013) Considering these facts, it might be possible to detect PIs 62 synthesised by marine microorganisms for their defence. 63 Intertidal zones are exposed to air at low tides and are submerged at high tides. Thus they 64 experience varying dry and wet periods, fluctuating temperatures and atmospheric pressure. 65 Intertidal life forms adapt to these constantly changing environment by developing unique 66 features. Their adaptations for survival in dynamic environments might also increase the 67 chances of finding unique set of metabolites from them. Moreover, it is expected that marine 68 microorganisms will have different set of defence molecules than their terrestrial counterparts 69 (Mourão and Schwartz, 2013; Xie et al., 2018). 70 Actinobacteria is one of the largest phyla in domain bacteria. These are gram positive organisms 71 with high guanine to cytosine ratio (Trujillo, 2016). They at large are known to produce 72 commercially important diverse metabolites. Some reports show that the marine environment 73 has become a prime source for discovery of novel actinobacteria and novel natural products 74 produced by them (Maldonado et al., 2005; Ward and Bora, 2006). There are few recent reports 75 of PI production by marine actinobacteria (Karthik et al., 2014; Sreedharan and Bhaskara Rao, 76 2017; Sun et al., 2014) yet, to our knowledge, there are limited reports on PIs from west coast 77 of India. Thus, in the current study, marine actinobacteria from intertidal zone were screened 78 for presence of PIs. 79 It has been reported that sponges and bacteria are two main sources of enzyme inhibitors from 80 marine environments (Ruocco et al., 2017). Some reports also suggest that many a times, 81 metabolites obtained from sponges are actually produced by the bacterial symbionts (Haygood 82 et al., 1999; Mehbub et al., 2014). Therefore, in the current study sponge, sediment and water 83 samples were collected from the coast of Maharashtra and Goa for isolation of actinobacteria. 84 Obtained isolates were subjected to molecular identification using 16S rRNA gene sequencing 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.14.949438; this version posted February 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 85 and isolates were screened for presence of protease inhibitors. Data was analysed to check 86 correlations between inhibitor molecules and environmental parameters and their probable role 87 as defence molecules. 88 MATERIALS AND METHODS 89 90 Sampling 91 Around 60 samples comprising of sponge, sediment and sea water were collected from the 92 intertidal rock pools from seven different locations along the coast of Ratnagiri district from 93 Velas, Kelshi, Ade, Anjarle, Harnai, Murud and Aare Ware. (17°57'04.9"N 73°01'43.0"E to 94 17°04'35.9"N 73°17'17.5"E). From Ade and Harnai, sampling was carried out in different 95 seasons viz. pre monsoon (Mar, Apr, May), Monsoon (Early Sep), post-monsoon (Oct) and 96 winter season (Dec, Jan, Feb). Whereas, a single sampling was done from rest of the sites. Sub- 97 tidal sampling of sediment was also done in a single event at a place off shore to Goa 98 (15°21'08.4"N 73°46'41.8"E). The depths of collection for this sampling were 9m, 11m, 12m 99 and 16m. (Figure 1). 100 Small tissue samples of sponge were collected without damaging the sponge colonies or their 101 habitat. Samples were rinsed in sterile Poor Ravan Saline (PRS) broth to remove loosely bound 102 particles and debris and were stored in sterile collection tubes containing the same medium. 103 Sediment and water samples in the vicinity of sponge were collected in sterile collection tubes. 104 All the samples were transported to the laboratories in ice box and were processed fresh for 105 microbial isolations. 106 Sample Processing, Selective Isolation and Culture Maintenance 107 All the samples were given heat treatment at 60°C for 15 mins to reduce the load of non- 108 sporulating bacteria. Sponge samples were homogenised in Poor Ravan Saline (PRS) medium 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.14.949438; this version posted February 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 109 (Watve et al., 2000) and diluted serially with 10 fold dilutions up to 10-5.