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Laterosporulin10: a novel defensin like Class IId bacteriocin from Brevibacillus sp.
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strain SKDU10 with inhibitory activity against microbial pathogens
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Piyush Baindara, Nisha Singh, Manish Ranjan, Nayudu Nallabelli, Vasvi Chaudhry, Geeta
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Lal Pathania, Nidhi Sharma, Ashwani Kumar, Prabhu B. Patil and Suresh Korpole*
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CSIR-Institute of Microbial Technology, Sector 39A, Chandigarh-160036, India
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*To whom correspondence be addressed: Suresh Korpole, MTCC and Gene Bank, CSIR-
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Institute of Microbial Technology, Sector 39A, Chandigarh-160036, India. Phone +91-172-
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6665159 email: [email protected]
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EMBL accession number for 16S rRNA gene sequence of strain is HF545882 and the
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genome sequence is LSSO00000000
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Abstract:
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Bacteriocins are antimicrobial peptides (AMPs) produced by bacteria to acquire survival
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benefits during competitive interactions between inter and intra species in complex
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ecosystems. In this study, an AMP producing soil bacterial strain designated as SKDU10 was
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isolated and identified as a member of the genus Brevibacillus. The AMP produced by strain
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SKDU10 was identified as a class IId bacteriocin with 57.6% homology to laterosporulin, a
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defensin like class IId bacteriocin. However, substantial differences were observed in
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antimicrobial activity spectrum of this bacteriocin named as laterosporulin10 when compared
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to laterosporulin. Laterosporulin10 effectively inhibited the growth of Staphylococcus aureus
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and Mycobacterium tuberculosis (Mtb H37Rv) with LD50 values of 4.0 µM and 0.5 µM,
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respectively. Further, laterosporulin10 inhibited the growth of Mtb H37Rv strain at about 20
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times lesser MIC value compared to S. aureus MTCC 1430 or M. smegmatis mc2155 in vitro
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and ex vivo. Electron micrographic studies along with membrane potential analysis using
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FACS analyses revealed that the laterosporulin10 is a membrane permeabilizing peptide.
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Interestingly, laterosporulin10 was able to efficiently kill Mtb H37Rv strain residing inside
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the macrophages and did not show haemolysis up to 40 µM concentration.
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Keywords: Brevibacillus, Bacteriocin, Laterosporulin10, M. tuberculosis, MALDI, S. aureus
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Introduction
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Low molecular weight antimicrobial peptides (AMPs) produced by bacteria are usually called
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as bacteriocins. However, bacteriocins have been categorized as ribosomally synthesized and
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posttranslationally modified peptides (RiPPs) in a recent classification (Arnison et al., 2013).
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Bacteriocins with unique features have been reported from a multitude of bacteria that are
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classified into various classes based on their structure and functional characteristics
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(Klaenhammer, 1993b). While class I bacteriocins contains antimicrobial peptides with
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extensive posttranslational modifications, class II includes unmodified peptides which are
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subdivided into various subclasses. Bacteriocins are being considered as potential alternative
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to antibiotics to treat diverse pathogenic bacteria (Cotter et al., 2013) and a significant step
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along this direction is the exploration of the antimicrobial peptides (AMPs) for
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antimycobacterial activity (Gutsmann T, 2016). AMPs are being considered as an important
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and promising antimicrobial candidates (Semvua et al., 2014) due to their unique
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mechanism(s) of action and their capability to treat drug-resistant bacteria (Baindara et al.,
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2015; Fickers, 2013). Notably, AMPs display low toxicity towards mammalian cells (Grosset
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& Leventis, 2014; Yew & Chi Chu Leung, 2006) and show potent bactericidal activity
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against pathogens as well as potential pathogens. There are about 200 bacteriocins reported
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with diverse amino acid sequence are available at different bacteriocin database including
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www.bactibase.pfba-lab-tun.org. Though AMPs are predominantly reported from the strains
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belonging to genera Lactobacillus and Bacillus (Teixeira et al., 2013; Zhao et al., 2012;
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Klaenhammer, 1993a), members from other genera like Paenibacillus and Brevibacillus were
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also found to produce bacteriocins (Baindara et al., 2015; Singh et al., 2012). Accordingly,
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members of the genus Brevibacillus, specifically, strains of species B. laterosporus are
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known for their beneficial functions (Ruiu et al., 2014) and antimicrobial substance
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production. Moreover, it is known as a bio-control agent for nematodes and also used to treat
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fungal disease (Oliveira et al., 2004). Besides the production of antimicrobials such as
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bacteriocins, antibiotics and lipopeptide antibiotics, members of B. laterosporus also produce
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thrombin inhibitors and anti-tumour agents (Kamiyama et al., 1994; Umezawa & Takeuchi,
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1987). In fact, the whole genome sequencing of this bacterial species revealed it’s potential to
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produce diverse antimicrobial peptides, polyketides and toxins (Van Belkum et al., 2011;
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Shida et al., 1996; Smirnova et al., 1996). Recently, we have characterized a class IId
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bacteriocin from a B. laterosporus strain GI-9 and named as laterosporulin. This bacteriocin
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displayed structural homology and sequence similarity with various human defensins (Singh
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et al., 2014). The laterosporulin produced by B. laterosporus strain GI-9 displayed broad
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range of antibacterial activity through membrane permeabilization ((Singh et al., 2014;
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Carrillo et al., 2003). However, it is important to test the ability of bacteriocins including the
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lantibiotics for their efficiency to inhibit the growth of M. tuberculosis (Donaghy, 2010; Piper
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et al., 2012). Therefore, in the present study, we report characterization and antimicrobial
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activity of laterosporulin10 against various potentially pathogenic and pathogenic bacteria.
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Materials and methods
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Bacterial strain and identification. Antimicrobial substance producing strain SKDU10 was
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isolated from a rhizosphere soil sample. Phenotypic characteristics used for strain
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identification were determined as mentioned in Bergey’s manual of systematic bacteriology.
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For genotypic identification, the 16S rRNA gene was amplified and sequenced as described
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earlier (Sharma et al., 2012). The sequence (accession no. HF545882) was compared with
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other close relatives by phylogenetic analysis (Theodore et al., 2014). Indicator strains used
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in this study including Staphylococcus aureus (MTCC 1430), Bacillus subtilis (MTCC 121),
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Pseudomonas aeruginosa (MTCC 1934), Vibrio cholerae (MTCC 3904), Escherichia coli
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(MTCC 1610), Candida albicans (MTCC 1637), Saccharomyces cerevisiae (MTCC 170),
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Fusarium oxysporum (MTCC 2773) and Asperigillus niger (MTCC 281) were obtained from
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Microbial Type Culture Collection and Gene Bank (MTCC), Chandigarh, India. While all
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bacterial indicator strains were grown on nutrient agar (NA, Himedia, India) medium, fungal
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indicator strains were grown on potato dextrose agar (PDA, Himedia, India) at 30°C under
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aerobic conditions. All strains maintained as -70°C glycerol stocks.
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Antimicrobial peptide production and activity assay. A growth curve was established up
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to 30 h to test the antimicrobial peptide production at different growth phases using nutrient
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broth medium (NB, Himedia, India). To test the effect of different carbon and nitrogen
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sources on antimicrobial production by strain SKDU10, a 0.5% concentration of different
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substrates like glucose, lactose, yeast extract, peptone and beef extract were added to the
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minimal medium (composition (g/l): Na2HPO4.2H2O, 7.9; KH2PO4, 3.0; NaCl, 0.5; NH4Cl,
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1.0; pH 7.2). Antimicrobial production ability of the strain was measured as diameter of
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inhibition zone by performing an antimicrobial bioassay using the cell free fermented broth
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(CFB). Strain SKDU10 was grown in minimal medium containing different carbon and
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nitrogen sources for 48 h and CFB obtained was tested by well diffusion assay. The CFB
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obtained by growing strain SKDU10 in NB (pH 7.0) was also used to test the activity against
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indicator strains including S. aureus (MTCC 1430), B. subtilis (MTCC 121), P. aeruginosa
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(MTCC 1934), V. cholerae (MTCC 3904), E. coli (MTCC 1610), C. albicans (MTCC 1637),
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S. cerevisiae (MTCC 170), F. oxysporum (MTCC 2773) and A. niger (MTCC 281). Bacterial
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indicator strains were grown to obtain OD600 of 0.2 and subsequently 50 µl of culture aliquots
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(final concentration, ̴ 104-105 CFU/ml) were used for well diffusion assay. All NA plates for
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antimicrobial activity assay were incubated at 30°C for overnight in aerobic conditions.
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Purification of bacteriocin. The bacteriocin was extracted from cell free supernatant (CFS)
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by using 2% (w/v) of activated Diaion HP-20 as mentioned earlier (Singh et al., 2012). This
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crude extract was purified by gel filtration chromatography using a manually packed
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sephadex G-50 (GE healthcare, USA) column. The peptide eluted with 50 mM NaCl with a
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flow rate of 1.0 ml/min and fractions showing antimicrobial activity were collected, pooled
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and concentrated. Subsequently, the extract was dialyzed through Float-A-Lyzer G2 (MWCO
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0.5-1 kD; Spectrum laboratories, USA) to remove excess salt. Desalted peptides were finally
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purified by HPLC (1260 Infinity, Agilent Technologies, USA) using a reverse phased, semi-
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preparative C18 column (250 mm x 10 mm x 150 Å, Venusil, agela technologies) as
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described earlier (Baindara et al., 2015). The concentration of purified peptide was
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determined using a Pierce BCA protein assay kit (Thermo Scientific, Waltham, MA, USA).
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The quantified peptide between 0-16 µM concentrations was used for further studies
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including determination of minimum inhibitory concentration (MIC), molecular mass and N-
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terminal sequencing as mentioned below. This peptide was also used for an in-gel activity
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assay against S. aureus MTCC 1430 as described earlier (Baindara et al., 2013).
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Mass spectrometry analysis of bacteriocin. Matrix-assisted laser desorption ionization
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(MALDI) mass spectrometry (AB SCIEX, 5800 TOF/TOF, USA) was used primarily to
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determine the molecular weight of AMP. The peptide was re-suspended in methanol and 4 μl
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of this solution was mixed with equal amount of matrix (CHCA, 10 mg/ml). From this
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mixture solution 1.0 µl was spotted onto the MALDI stainless steel sample plate and allowed
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to air dry prior to the MALDI analysis (Mandal et al., 2009). The spectra were recorded in
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the positive ion linear mode.
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N-terminal sequencing and analysis. Upon separation on Tricine-SDS-PAGE (16% with 6
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M urea), the peptide was transferred on to PVDF membrane (Bio-Rad, USA), rinsed with
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Milli-Q water and stained with amido black (Sigma, USA) for 2-3 min. The membrane was
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processed for N-terminal sequencing as mentioned earlier (Singh et al., 2012).
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Minimum inhibitory concentration (MIC) and killing kinetics. Lowest concentration that
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inhibited 90% of growth was considered as MIC and was determined using microtiter plate
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dilution assay. Protein concentration was estimated using BCA kit (Thermo Scientific, USA)
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as described by manufacturers and confirmed with extension coefficient of the peptide
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sequence. Lysozyme protein standards (Thermo Scientific, USA) were used as references for
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estimation of protein concentration. Purified AMP used in the range of 0-16 µM
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concentrations to determine the MIC values against various indicator strains. To determine
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killing kinetics of bacteriocin, indicator strain S. aureus MTCC 1430 (~2x104 cells) was
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treated with bacteriocin of different concentrations including 4, 8 and 20 µM in a time-
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dependent manner (for 30, 60 and 90 min). Upon incubation, cells were pelleted by
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centrifugation (8000 g) and washed with PBS (Gibco, USA), subsequently, serially diluted
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and plated different dilutions on NA plates. Untreated cells were used as a positive control
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and were processed along with treated cells for CFU counts. The experiment was performed
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in triplicate and repeated as three individual sets then analysed for final results (Singh et al.,
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2014).
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MIC determination against Mtb using microplate alamar blue assay. Mtb H37Ra, Mtb
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H37Rv, M. smegmatis mc2155 (Msemg) were cultured in Middlebrook 7H9 media
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(supplemented with 10% O-ADC along with 0.05 % Tween80) for 48 and 16 h duration (for
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Mtb and Msemg strains, respectively) to obtain an OD600 of 0.2 and used to set up the
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Microplate Alamar Blue Assay (MABA) (Pettit et al., 2005). A 100 µl of culture was added
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to each well of polypropylene 96 well plate (Eppendorf, USA) followed by serial dilution of
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the laterosporulin10 from 10-0.0015 µg/ml to determine the MIC. The plate was incubated
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for 24 h (for M. smegmatis mc2155 strain) and 72 h (for Mtb H37Ra and Mtb H37Rv strains)
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at 37°C. After the incubation 50 µl of 0.02 % resazurin was added to each well and the plate
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was monitored from 12 to 24 h for the change in colour. Rifampicin was used as a positive
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control. The absorbance at 600 nm was measured using ELISA reader (Thermo Scientific,
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USA) and % inhibition was calculated. The experiments were performed in triplicates and
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repeated three times at the least.
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Determination of fractional inhibitory concentration (FIC). As rifampicin is one of the
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frontline drug for Mtb H37Rv, to test further whether a combination of rifampicin and
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laterosporulin10 could reduce the MIC (MIC of rifampicin is 0.025 µM) values of rifampicin
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against Mtb H37Rv, MABA was performed. Varying concentrations of rifampicin
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(0.003125-0.2 µM) and laterosporulin10 (0.031-4.0 µM) were used individually or in
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combination to check the additive effect of laterosporulin10 and rifampicin. Next, to
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distinguish between additive and synergistic effect of laterosporulin10 with rifampicin, we
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have determined the ∑FIC values (Chaturvedi et al., 2011). The ∑FIC was calculated by
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using the following formula: ∑FIC = FICA + FICB. Wherein FICA equals the MIC of drug A
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in combination/MIC of drug A alone, and FICB equals the MIC of drug B in
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combination/MIC of drug B alone. Laterosporulin10 considered as drug A while rifampicin
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as drug B. The interpretation of ∑FIC values were as follows: ≤0.5, synergistic; >0.5 to <4.0,
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indifferent (no antagonism); and ≥4.0, antagonistic.
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Sensitivity of AMP to temperature, pH and proteolytic enzymes. The sensitivity of
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purified AMP toward pH, temperature and hydrolytic enzymes was confirmed by performing
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agar well diffusion assay. To determine the pH stability, aliquots of purified peptide (1
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mg/ml) were adjusted to pH 2.0 to 12.0 with an increment of 2 pH units (using 10 mM HCl
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or NaOH) followed by incubation at room temperature for 4 h, and the leftover activity was
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measured upon neutralizing the sample to pH 7.0. For the thermal stability assay, aliquots of
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purified peptide (1 mg/ml) were exposed to 60, 80 and 100°C for 30 min and 121°C for 15
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min and used for antimicrobial activity assay. Similarly, proteolytic enzymes such as trypsin
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and proteinase K were used at three different concentrations (0.1, 1.0 and 5.0 mg/ml) to
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ensure their effect on peptide (1 mg/ml). The enzyme solutions were prepared in 50 mM
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phosphate buffer (pH 7.0). All reactions were performed at 37°C for 6 h followed by
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deactivation of enzyme by heating the solution in boiling water for 5 min before performing
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the activity (Baindara et al., 2015). Antimicrobial activity of treated peptide (100 µl of 1
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mg/ml peptide) was performed by agar well diffusion assay using S. aureus MTCC 1430 as
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indicator strain on NA medium.
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Survival of Mtb H37Rv strain in macrophages. RAW 264.7 murine macrophages were
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seeded in 6 well plate (~ 5X105 cells) and infected with Mtb H37Rv strain (1:10 MOI) for
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three hours (Kumar et al., 2008). The cells were then treated with 100 µg/ml gentamycin for
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45 min to remove extracellular bacteria. The cells were independently stimulated with 0.025
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and 0.125 µM of rifampicin or 0.5, 2.5 and 5 µM of laterosporulin10. After 48 h, cells were
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lysed using 0.06 % SDS in PBS, diluted serially and plated on 7H11 agar plates.
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Mammalian cell toxicity. Cultures of RAW 264.7 murine macrophage were subjected to
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MTT
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technologies, USA) assay to determine the number of surviving cells after treatment with
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laterosporulin10. The cells were plated in 96-well BD Falcon vessels (5-10X103 cells/well)
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using DMEM medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco,
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USA) and 1% penicillin-streptomycin cocktail (Sigma, USA). Cultures were incubated under
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standard conditions (37°C / 5% CO2). After 24 h, the growth medium was replaced with fresh
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medium containing various concentrations of laterosporulin10 (1-40 µM). Medium without
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peptide was used as a negative control and 1% triton X-100 was used as positive control. The
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plates were incubated for additional 24 h and then each well added with 20 µl of MTT
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solution (5 mg/ml in PBS), and plates were incubated for 3 h at 37°C. Subsequently, the MTT
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containing medium was removed and 50 µl of DMSO was added to each well. To assess the
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percentage of live cells in samples, the absorbance (590 nm) was assessed as described earlier
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(Baindara et al., 2015).
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide,
Invitrogen,
life
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Haemolysis assay. Rabbit (New Zealand white) blood sample was collected in test tubes
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containing EDTA and processed as mentioned earlier (Singh et al., 2014). Positive control
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used was 1% Triton X-100 (G biosciences USA) in water and sterile PBS as a negative
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control. After incubation at 37°C, all treated samples were centrifuged at 1500 rpm for 5 min
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and supernatants were transferred into a fresh 96 well plate to examine erythrocyte lysis using
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spectrophotometer (Thermo fisher Scientific, USA) at a wavelength of 541 nm (Smolarczyk
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et al., 2010).
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ATP determination assay. Actively growing cultures (one ml) of S. aureus MTCC 1430 (16
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h culture) and Mtb H37Rv strain (0.2 OD600 48 h grown culture) were treated with different
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concentrations of laterosporulin10 (0.5, 2.5 µM for Mtb H37Rv and 4, 8, 20 µM for S. aureus
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MTCC 1430) in a time-dependent manner (10, 60 min for Mtb H37Rv and 30, 60, 90and 120
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min for S. aureus). After incubation, cells were separated by centrifugation (8000 g) and
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washed with PBS (Gibco, USA) twice. The cells recovered from the pellets were resuspended
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in 50 µl of lysis buffer (Promega, USA), mixed gently for 1 min and centrifuged (8000 g).
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The supernatant was collected and 10 µl of the dilution was added to standard reaction
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solution (90 µl) from the ATP determination kit (Invitrogen, Molecular Probes, USA).
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Oxyluciferin concentration was determined after measuring luminescence in white plate
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using a Lumat LB 9501 luminometer (Promega, USA). The protein content was assayed
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using BCA kit (Thermo Scientific, USA) and sample luminescence was expressed as
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RLU/mg of protein. Data from three separate measurements were used to calculate average
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values.
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NAD/NADH, NADP/NADPH assay. One ml of 0.2 OD600 cultures of Mtb H37Rv strain
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were treated with 0.5 µM of laterosporulin10 in a time dependent manner (10 and 60 min).
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After incubation, cells were pellet down at 10,000 rpm for 5 min and the pellet was
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resuspended in one ml lysis solution (0.2 N NaOH and 1% dodecyltrimethylammonium
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bromide, DTAB) followed by cell lysis using OMNI bead ruptor (5 cycles, 45 sec each). The
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lysate was filtered and taken out from BSL3. The protein was estimated using the BCA
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method (kit procured from Sigma, USA). Once the protein was estimated the lysate was used
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for estimation of NAD/NADH (NAD/NADH-Glo™ assay kit, Promega, USA),
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NADP/NADPH (NADP/NADPH-Glo™ assay kit, Promega, USA) as per the manufacturer’s
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protocol.
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Preparation of cells for SEM. Since a high cell density is needed to observe EM images, the
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Mtb H37Rv cultures of mid exponential growth phase (of about 108 to 106 CFU/ml) were
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centrifuged at 8000 rpm for 10 min and cells washed with PBS two times. Finally, cell pellet
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was diluted with PBS to obtain cell density of 108 CFU/ml. Subsequently, the cells were
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treated with different concentrations (0.5 and 1.0 µM) of laterosporulin10 in a time dependent
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manner (10 and 30 min) and incubated at 37°C (Hartmann et al., 2010). Untreated controls
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were prepared by diluting cell pellet in PBS to the cell density of 108 CFU/ml. Mtb H37Rv
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cells were immobilized on polylysine (0.1% wt/vol aqueous solution, Sigma, USA) coated
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cover slips and washed thrice with ice cold PBS then fixed in modified Karnovskys fixative
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(4% paraformaldehyde, 1% glutaraldehyde and 0.2 M sodium cacodylate buffer, pH 7.4) for
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2 h and dehydrated in graded ethanol (30-100%). Ethanol dehydrated samples were freeze
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dried and processed further for scanning electron microscopy utilizing tertiary butyl alcohol
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as an intermediate fluid. Cover slips were placed on aluminium stubs using silver paint and
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sputter coated with gold. Cells were then observed and photographed using a S-260, Leica
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Cambridge scanning electron microscope (Raje et al., 2006).
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Preparation of cells for TEM. Mid exponential growth cultures of S. aureus MTCC 1430
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and Mtb H37Rv strain were centrifuged at 8000 g for 10 min, cells were collected and
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washed with PBS (Gibco, USA) two times. Cell pellet obtained after final washing was
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diluted with PBS to obtain a cell density of approximately 108 CFU/ml and treated with
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different concentrations of laterosporulin10 (0.5, 1.0 µM for Mtb H37Rv strain and 4, 8 µM
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for S. aureus MTCC 1430) in a time dependent manner (10 and 30 min) at 37°C. After the
269
treatment, cell pellet was washed with PBS and fixed in modified Karnovskys fixative. Cells
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were further fixed in 1% osmium tetroxide (Sigma-Aldrich, USA) in 100 mM PBS and
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embedded in 2% agarose. After making blocks with ACM I and ACM II (Sigma-Aldrich,
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USA) sectioning (70 nm) was performed using Ultramicrotome (Leica EM UC7, USA).
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Subsequently, cells were stained with 0.1% (w/v) PTA (sodium phosphate tungstate, Sigma)
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on a carbon coated copper grid (300 mess, Polysciences, USA) and observed under a JEOL
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JEM 2100, 200-kV transmission electron microscope (TEM) at a resolution of 0.2 - 0.5 µM
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(Raje et al., 2006). Untreated cells were used as control.
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FACS analysis by flow cyotometry. Cell membrane integrity was determined by using
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propidium iodide (Invitrogen, USA). Propidium iodide (PI) is a nucleic acid stain that binds
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to DNA by intercalating between the bases and is membrane impermeant that is excluded
280
from viable cells (Arndt-Jovin & Jovin, 1989). S. aureus MTCC 1430 cells of mid
281
exponential growth phase were centrifuged at 8000 rpm for 10 min and cell pellet was
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washed three times with PBS (GIBCO’s PBS without Ca2+ and Mg2+ ions). Cell pellet was
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diluted with PBS to obtain a cell density of 106 CFU/ml and subsequently treated with 4 µM
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of laterosporulin10 in a time dependent manner (5, 10 and 30 min) and incubated at 37°C.
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Upon incubation at mentioned time intervals, the treated cells were pelleted down, washed
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with PBS three times and were loaded with 5 µl of PI (1 mg/ml). After 15 min of incubation
287
with PI cells were centrifuged at 8000 rpm for 10 min and washed with PBS three times.
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Untreated cells in PBS were used as control. Subsequently, the treated cell pellet was diluted
289
with PBS and analysed immediately using flow cytometer (BD acuri, USA).
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PEGylation. To determine the number of free cysteine in AMP, the maleimide PEG
291
(MalPEG, 5kDa) (Sigma-aldrich, USA) molecule was reacted in different states (native,
12
292
reduced, unfolded & reduced unfolded) of the HPLC purified peptide. 1 mM TCEP (Thermo
293
scientific, USA) at 25°C for 1 h used as reducing agent and 6 M Urea (Sigma-Aldrich, USA)
294
at 40°C for 1 h used for unfolding of native peptide (Yoneyama et al., 2009).
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Whole genome sequencing, assembly and annotation. The genomic DNA was isolated by
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using DNA extraction kits (Zymo Research, USA) and quality was assessed using agarose gel
297
electrophoresis, Qubit and Nanodrop. The input of 1 µg of genomic DNA from each sample
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was used. Standard protocol for the Nextera XT DNA sample preparation kit was used for
299
library construction. Purified fragmented DNA was used as a template for a limited cycle
300
PCR using Nextera primers and index adaptors. Cluster generation and sequencing of
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libraries were performed on the Illumina MiSeq platform (Illumina, San Diego, USA) with a
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2x250 paired-end run. The paired-end raw-reads containing FASTQ files were assembled
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using CLC Genomics Workbench software version 7.0.3.
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In silico identification of bacteriocin genes in SKDU10. A detailed in silico analysis of the
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bacteriocin biosynthetic gene clusters was evaluated in genome of strain SKDU10 along with
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genomes of other closely related strains of B. laterosporus, who’s partial or complete
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genomes are available at NCBI Genome database. Initially, the assembled genome was
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uploaded for annotation to the Rapid Annotation using Subsystems Technology (RAST)
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server (Aziz et al., 2008). To fish out the putative bacteriocin encoding ORF from draft
310
genome sequence, a homology search was carried out using Tblastn function against draft
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genome sequence of SKDU10 as mentioned earlier (Singh et al., 2012). ClustalW was used
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to carry out alignment of nucleotide and amino acid sequences.
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Statistical analysis. All comparisons were based on the mean ± standard deviation of the
314
mean (SD). Parametric data was analysed using one-way analysis of variance (ANOVA) with
315
Bonferroni post-test method for comparison between groups. Column statistics for non-
316
parametric data was analysed by D'Agostino & Pearson omnibus normality test along with
13
317
one sample t test. Results considered as significant when p<0.05 in all experiments. All
318
experiments have been done independently three times in triplicate.
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Results
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Characterization of bacterial strain SKDU10. In an effort to look for the antimicrobial
322
producing strains from complex environments, we have isolated few strains with inhibitory
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zones in their colony periphery. One of the strain designated as SKDU10 was studied in
324
detail for its ability to inhibit the growth of other bacterial indicator strains. Before
325
proceeding
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characterization of strain SKDU10 revealed it as a Gram-positive, rod shape, endospore
327
forming bacteria with the ability to grow under aerobic and anaerobic conditions. Both, 16S
328
rRNA and rpoB gene sequences showed high identity with strain of B. laterosporus DSM 25.
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However, it displayed less than 97% similarity with other species of the genus Brevibacillus.
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Strain SKDU10 formed a distinct cluster with B. laterosporus DSM 25 in a neighbour joining
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phylogenetic tree constructed using 16S rRNA (Supplementary Fig. S1) and rpoB gene
332
sequences. Strain SKDU10 displayed differences with B. laterosporus DSM 25 in phenotypic
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properties like growth at pH 5 and 4% NaCl, casein hydrolysis and utilization of sugars like
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mannose. The antimicrobial substance production by strain SKDU10 in NB was initiated
335
towards late-logarithmic phase (Supplementary Fig. S2) similar observations were also found
336
while grown on minimal medium with different substrates.
337
Purification and characterization of antimicrobial substance. The antimicrobial substance
338
was recovered with hydrophobic interaction chromatography using activated Diaion HP-20
339
beads as crude extract which was purified subsequently by size exclusion chromatography
340
and finally using semi-preparative RP-HPLC (Fig. 1A). While absorption wavelength and
341
HPLC elution profile indicated the antimicrobial substance to be a peptide, the gel filtration
with
characterization
of
antimicrobial
substance,
the
morphological
14
342
elution profile showed a single peak with low molecular weight (Fig. 1B). Accordingly, a
343
single band observed in gel electrophoresis that displayed antimicrobial activity during the in-
344
gel activity assay using S. aureus (MTCC 1430) as a reference strain (Fig. 1C). The mass of
345
peptide was confirmed as 6061.37 Da by MALDI analysis (Fig. 1D) that differed with
346
laterosporulin, a class IId defensin like bacteriocin produced by another strain of B.
347
laterosporus designated GI-9 and therefore, it was named as laterosporuin10. Antimicrobial
348
activity of the purified laterosporulin10 was not affected upon exposure to 121°C for 15 min
349
(Supplementary Fig. S3A). Similarly, it was found to be stable between pH 2.0 and 12.0
350
(Supplementary Fig. S3B). Studies on tolerance of the peptide to proteolytic enzymes did not
351
show any decrease in inhibition activity of laterosporulin10 upon incubation for 6 h with
352
trypsin and proteinase K (Supplementary Fig. S3C, D).
353
Minimum inhibitory concentration and killing kinetics. The quantified laterosporulin10
354
was used to determine the minimum inhibitory concentrations required for various indicator
355
strains. It displayed effective inhibition activity towards Gram-positive indicator strains as the
356
LD50 values showed 4 to 6 µM concentrations against S. aureus (MTCC 1430) and B. subtilis
357
(MTCC 121) respectively, but the growth of Gram-negative indicator strains was not affected
358
even at a concentration of 16 µM (Fig. 2A). Furthermore, the MIC for E. coli was found to be
359
~100 µM. But, the bactericidal kinetic studies obtained for laterosporulin10 revealed its
360
effectiveness against cells of S. aureus as more than 90% of bacterial load reduced within 30
361
min of treatment at 4 µM concentration of the bacteriocin (Fig. 2B). There was no reduction
362
in CFU count observed for negative control incubated without any bacteriocin. Maximum
363
cell death was observed within 30 min of treatment for different concentrations of the
364
laterosporulin10.
365
Laterosporulin10 disrupts ATP homeostasis in S. aureus MTCC 1430. As membrane is
366
the target for most of the bacteriocins, we have tested whether the laterosporulin10 acts by
15
367
disrupting the cell membrane of sensitive strains like S. aureus MTCC 1430. Since
368
membrane disintegration often result in depletion of intracellular ATP, we determined the
369
change in ATP content upon treatment of S. aureus MTCC 1430 cells with latersporulin10.
370
The log phase cultures of S. aureus MTCC 1430 were treated with laterosporulin10 at 4, 8
371
and 20 µM for 30, 60, 90 and 120 min and subsequently their ATP content was determined.
372
We found that treatment of S. aureus MTCC 1430 cells with 4 and 8 µM of laterosporulin10
373
lead to the disruption of ATP homeostasis within 90 min, whereas treatment with 20 µM
374
resulted in disruption of the ATP homeostasis as early as in 30 min (Fig. 2C). Accordingly,
375
reduction in ATP levels supports that laterosporulin10 involves membrane associated
376
mechanism for bacterial killing. Further, loss of membrane integrity induced by
377
laterosporulin10 was also evaluated by using nucleic acid stain PI and the fluorescence was
378
detected after treatment of 10, 30 and 60 min of time intervals. The group without bacteriocin
379
was used as a negative control where only 0.3% cells showed PI uptake. Results confirmed
380
that the membrane was significantly compromised in the presence of laterosporulin10 as
381
intake of PI was observed for 39.9% of total cell population within 30 min of treatment (Fig.
382
3).
383
Laterosporulin10 found active against Mtb H37Rv strain. The MIC of laterosporulin10
384
against the Mtb H37Rv strain was measured using MABA, which utilizes a redox-indicator
385
dye resazurin that fluoresces and changes colour upon reduction (blue to pink) due to cell
386
growth. Therefore, while change of the colour from blue to pink indicates cell growth, blue
387
colour shows bacterial killing or inhibition of bacterial growth. We observed that
388
laterosporulin10 was highly efficient in killing Mtb H37Rv strain with a LD50 of 0.5µM (Fig.
389
4). Since this LD50 was 8 fold lower than the LD50 obtained for S. aureus MTCC 1430 (4
390
µM), we have analysed if the laterosporulin10 kills M. smegmatis mc2155 strain, a
391
saprophytic mycobacterial species, with similar efficiency. To our surprise laterosporulin10,
16
392
revealed an MIC of 45 µM against the M. smegmatis mc2155 (Supplementary Fig. S4). Thus
393
the MIC for M. smegmatis mc2155 was about 50 folds higher than what observed for Mtb
394
H37Rv strain. These findings are very interesting as the laterosporulin10 has species-specific
395
inhibitory activity against the Mtb H37Rv strain. Killing of Mtb H37Rv strain was also
396
confirmed through plating and determination of CFU in the presence or absence of
397
laterosporulin10. Since rifampicin is one of the frontline drug for Mtb H37Rv, to analyse
398
whether a combination of laterosporulin10 with rifampicin could reduce the MIC of
399
rifampicin (MIC of rifampicin is 0.025µM) against Mtb H37Rv strain, MABA was
400
performed, wherein varying concentrations of rifampicin (0.003125 - 0.2 µM) and
401
laterosporulin10 (0.31 - 4.0 µM) were used alone or in combination. Interestingly, the
402
treatment in combination led to four-fold decrease (0.00625 µM) in the MIC of rifampicin in
403
the presence of 0.25 µM of laterosporulin10 (Fig. 5). To distinguish between additive and
404
synergistic effect of laterosporulin10 and rifampicin, we determined FIC values. Strikingly,
405
the effect of laterosporulin10 and rifampicin revealed as synergistic (∑FIC = 0.275).
406
Furthermore, we have analysed whether laterosporulin10 is capable of killing Mtb H37Rv
407
strain in macrophages. Towards this, we infected RAW 264.7 murine macrophages with Mtb
408
H37Rv at a MOI of 1:10 and then treated them with 0.5 and 2.5 µM of laterosporulin10 and
409
rifampicin independently for 48 h. Upon incubation, the macrophage cells were lysed, and the
410
lysates were plated on 7H11 agar for monitoring the CFU of Mtb H37Rv strain. We observed
411
that laterosporulin10 killed intracellular Mtb H37Rv strain at both the concentrations, though
412
the killing was significantly higher at 2.5 µM (Fig. 6). These experiments suggest that the
413
laterosporulin10 can enter the phagosomes and efficiently kill the intracellular Mtb H37Rv
414
strain.
415
Laterosporulin10 disrupts ATP, NAD/NADH and NADP/NADPH homeostasis in Mtb
416
H37Rv strain. Since laterosporulin10 kills Mtb H37Rv strain very efficiently by disrupting
17
417
the cell membrane as shown by the electron microscopy experiments, we determined the
418
ability of laterosporulin10 to disrupt ATP, NAD/NADH and NADP/NADPH homoeostasis in
419
Mtb H37Rv strain. Towards this, log phase cultures of Mtb H37Rv strain treated with
420
laterosporulin10 for 10, 30 and 60 min with 0.5 and 2.5 µM, and the lysates were measured to
421
determine the ATP level and NAD/NADH. Results showed that in contrast to S. aureus
422
MTCC 1430 that required 90 min for the disruption of ATP homeostasis upon treatment with
423
4 or 8 µM, laterosporulin10 efficiently disrupted Mtb H37Rv ATP homeostasis in less than
424
10 minutes at lower concentrations (Fig. 7A). These findings were further supported by the
425
observation that the NAD/NADH and NADP/NADPH ratio was also disrupted upon
426
treatment with laterosporulin10 (Fig. 7B and C).
427
Laterosporulin10 causes alterations in bacterial cell membrane. We have observed that
428
the laterosporulin10 was capable of inhibiting bacterial growth within an hour. Therefore, to
429
capture interactions of the laterosporulin10 with cell membrane, we performed scanning and
430
transmission electron microscopy (SEM and TEM, respectively). Since the laterosporulin10
431
showed activity primarily against Gram-positive bacteria, cells of S. aureus MTCC 1430
432
treated with laterosporulin10 were observed through electron microscopy. While TEM
433
images of control S. aureus MTCC 1430 cells showed smooth and intact surface, cells treated
434
with 4 µM laterosporulin10 for a duration of 10 and 30 min displayed significant alterations
435
in cell morphology, including formation of cell clumps along with debris material (Fig. 8A).
436
The results of SEM analysis of Mtb H37Rv strain treated with 0.5 µM laterospourlin10 for
437
duration of 10 and 30 min also showed cells clumping with cell debris material (Fig. 8B). In
438
accordance TEM micrographs revealed disintegration of cell membrane and subsequent total
439
cell lysis of Mtb H37Rv strain (Fig. 8C) suggesting that the laterosporulin10 acts against S.
440
aureus MTCC 1430 and Mtb H37Rv strain by disruption of their cell membrane.
18
441
Laterosporulin10
442
laterosporulin10 has demonstrated anti-mycobacterial activity against intracellular Mtb
443
H37Rv strain, it was important to find its effect on mammalian cells. Towards this, we
444
assayed whether laterosporulin10 has any haemolytic activity. Fresh blood drawn from New
445
Zealand white rabbit was incubated with different concentrations of laterosporulin10 (1-100
446
µM) for 24 h. Notably, haemolysis was not observed up to 20 µM concentration of
447
bacteriocin and less than 20% haemolysis was observed with 100 µM of laterosporulin10
448
(Fig. 3D). Since the concentration used was several fold higher compared to the MIC against
449
Mtb H37Rv strain and S. aureus MTCC 1430, this bacteriocin could be potentially used in
450
animals for further study on its antimycobacterial potential. In an attempt to test the toxicity
451
of laterosporulin10 against nucleated cells, we used RAW 264.7 (murine macrophage).
452
Remarkably, laterosporulin10 did not show any cytotoxic effect up to 30 µM (Supplementary
453
Fig. S5), which is a significantly higher concentration than the MIC50 observed for different
454
bacterial strains tested, including Mtb H37Rv strain. More than 80% cell viability was
455
observed in three independent experiments.
456
Genetic characterization of putative laterosporulin10 biosynthetic gene cluster. In order
457
to identify the amino acid composition of the laterosporulin10 and its identity with other
458
bacteriocins, we have performed N-terminal amino acid sequencing and obtained a partial
459
sequence composed of ACVNQCPDAIDR. The 12 amino acids N-terminal sequence
460
displayed identity with only laterosporulin. However, this partial sequence was used to
461
identify the biosynthetic gene encoding the bacteriocin from the draft genome sequence of
462
strain SKDU10 along with other genes involved in biosynthesis (Fig. 9A). The identified
463
ORF contained 165 nucleotides with a conserved Shine Dalgarno sequence, 8 bp upstream of
464
this ORF encoding (Fig. 9B). Computational translation of this ORF results in a protein of 54
465
amino acids with conserved motif PDAI, six cysteine amino acids at conserved positions with
is
a
non-haemolytic
and
non-cytotoxic
bacteriocin.
Since
19
466
probability of their involvement in disulphide bond formation as observed in laterosporulin.
467
However, the peptide composition differed with the amino acid composition of the
468
laterosporulin. Though the biosynthetic cluster of this novel bacteriocin contained identical
469
transcriptional regulator, ABC transporter and dehydrogenase gene as observed in
470
laterosporulin, it contained higher number of cationic amino acids and displayed low
471
similarity (57.6%) with laterosporulin and represent a novel class IId bacteriocin. Overall, the
472
comparative studies suggested that laterosporulin10 shares homology with the antimicrobial
473
peptides belonging to beta defensin family of mammals as observed in laterosporulin.
474
All cysteine are paired in laterosporulin10. Laterosporulin10 contained six cysteins with
475
conserved position as observed for laterosporulin (Fig. 9A) and the mass obtained was 6 Da
476
lesser to theoritcal mass of the peptide sequence deduced from biosynthetic gene. The mass
477
difference observed was attributed to the formation of 3 disulfide bonds as found in
478
laterosporulin. The involvement of six cysteine molecules in disulfide bond formation was
479
confirmed by a PEGylation experiment, wherein electrophoretic mobility of native and
480
reduced, PEGylated laterosporulin10 molecules showed addition of PEG molecules in
481
structured and unstructured conditions (Supplementary Fig. S6A). Additionally, increase in
482
the molecular mass of laterosporulin10 under reduced conditions (Supplementary Fig. S6B)
483
also confirmed the intramolecular disulfide bond formation in laterosporulin10.
484
485
Discussion
486
Emergence of resistance against predominant antimicrobials confers a significant threat to the
487
control of communicable diseases. These observations are more relevant for treatment of TB
488
that requires administration of multiple drugs for prolonged duration and where multidrug
489
resistance is commonly observed. In the light of emerging drug resistance, genome
490
sequencing has revolutionized our approach to the problem of understanding multi drug
20
491
resistance in pathogens and discovery of novel therapeutic agents. Therefore, approach for
492
identification of novel bacteriocin producing genes is a powerful approach to identify novel
493
therapeutic agents. Both broad and narrow spectrum bacteriocins are often produced by
494
bacterial strains to compete with other microbes including different strains of the same
495
species. In the recent past, antimicrobial peptides (Singh et al., 2012; Zhao et al., 2012) and
496
lipopeptides (Desjardine et al., 2007) were isolated and characterized from strains of B.
497
laterosporus with broad-spectrum antimicrobial activity. In the present study, a bacteriocin
498
producing isolate was characterized to understand its ability to produce bacteriocin that
499
inhibited the growth of S. aureus and other Gram-positive bacteria. However, among the
500
various conventional indicator strains laterosporulin10 effectively inhibited S. aureus (Fig.
501
2A), a bacterial strain that is considered to be a surrogate bacteria for the prediction of
502
antimicrobial peptide activity against Mtb H37Rv strain (Ramón-García et al., 2013).
503
Therefore, we have selected laterosporulin10 to test the antimicrobial activity against
504
pathogenic M. tuberculosis H37Rv strain.
505
Though amino acid sequence analysis of the purified bacteriocin showed similarity with the
506
laterosporulin (Singh et al., 2012), it significantly differed in antimicrobial spectrum as well
507
as amino acid composition. As observed for acidocinA, a bacteriocin isolated from
508
Lactobacillus salivarius (Stern et al., 2006) that displayed activity only against Gram-
509
positive bacteria, the natural replacement of amino acids in laterosporulin10 was also found
510
to be less efficient against Gram-negative bacteria in comparison to laterosporulin. Growth
511
inhibitory activity assays showed that laterosporulin10 exhibited strong anti-mycobacterial
512
activity. The amino acid composition analysis of laterosporulin10 showed the predominance
513
of hydrophobic amino acids, indicating the significance of hydrophobic property to establish
514
their interaction with bacterial cytoplasmic membrane. It is known in literature that the Arg-
515
rich cationic antimicrobial peptides exhibit activity towards both Gram-positive and Gram-
21
516
negative bacteria (Nakatsuji & Gallo, 2014). Bioinformatics analysis suggested that the C-
517
terminal was the active region of bacteriocin for antimicrobial activity, therefore we speculate
518
that the change in the C-terminal amino acid sequence is the primary reason for weak or no
519
activity of laterosporulin10 against Gram-negative indicator strains. Here, it is important to
520
note that replacement of Serine (a polar amino acid) residue with neutral Proline residue in
521
laterosporulin10 might be another reason for weak or no activity of this bacteriocin towards
522
Gram-negative strains. Interestingly, in comparison to other anti-mycobacterial drugs such as
523
rifampicin and pyrazinamide, this bacteriocin is more specific in targeting the Mtb H37Rv
524
strain. Thus, it fulfils the need for identification of species-specific bacteriocins which is an
525
important step in the direction of generating M. tuberculosis specific drugs. Earlier, NisinA
526
has been bioengineered to create variants with species-specific activity against M.
527
tuberculosis (Carroll et al., 2010a). Among other bacteriocins studied, lacticin 3147,
528
produced by Lactococcus lactis subsp. lactis DPC3147 was found to have higher bactericidal
529
activity against Mtb H37Rv compared to Mycobacterium avium subsp. paratuberculosis
530
09890 and Mycobacterium kansasii (Carroll et al., 2010b). However, such identification is
531
only a first step towards creation of an efficient antimycobacterial drug and was never
532
compared with combinatorial drug therapy. Rifampicin is one of the frontline drugs for M.
533
tuberculosis, but its long term usage and the dosage are related to interaction with
534
antiretroviral drugs (Semuva et al., 2015), causes hepatotoxicity (Yew & Leung, 2006) and
535
many other adverse effects (Grosset & Leventis, 2013). Many of the adverse effects could be
536
controlled through use of lower concentration of rifampicin (Fresard et al., 2011) and due to
537
these reasons, drugs that synergizes with rifampicin are desired to be included in the
538
treatment regimen. However, earlier it was demonstrated that different bacteriocins isolated
539
from B. circulans, P. polymyxa, L. salivarius, S. cricetus
540
antimycobacterial activity but they could not inhibit mycobacterial growth inside the
and E. faecalis possess
22
541
macrophage cells (Sosunov et al., 2007). In this study we have shown that the
542
laterosporulin10 is capable of killing Mtb H37Rv strain residing in the phagosomes of murine
543
macrophages. Nevertheless, the mechanism(s) utilized by lateropsorulin10 to penetrate the
544
cell membrane to target intracellular Mtb H37Rv remains unknown and are beyond the scope
545
of this manuscript. As ATP balance in bacterial cells maintained in mesosomes, the reduction
546
in ATP content is attributed to membrane disintegration (Kaplan et al., 2011; Lee et al.,
547
2015). In fact, laterosporulin10 is nontoxic to the macrophage cells at higher concentrations
548
unlike other bacteriocins (Sosunov et al., 2007). The underlying mechanism which ensures
549
that laterosporulin10 targets only the bacterial cells and not the mammalian cells are yet to be
550
understood. A distinguishing component of this study is the identification of genetic locus
551
responsible for the biosynthesis of laterosporulin10. These finding will further help in
552
identifying more natural variants or in creation of recombinant variants of laterosporulin10 or
553
bioengineering of the laterosporulin10 for improving its efficacy against Mtb as done earlier
554
for Nisin A (Carroll et al., 2010a).
555
In agreement with most of the bacteriocins that target the cell wall components (Cotter et al.,
556
2013; Desjardine et al., 2007; Nilsen et al., 2003), disrupt membranes or alter membrane
557
permeability (Van Belkum et al., 1991; Ma et al., 2015; Maftah et al., 1993), microscopic
558
studies revealed that laterosporulin10 also acts on cell membrane of the S. aureus and Mtb
559
H37Rv strain (Fig. 8) by disrupting the cellular metabolic homeostasis. The activity is varied
560
based on the amino acid composition. Laterosporulin10 altered the membrane of Mtb H37Rv
561
strain, which is covered with thick layer of lipid layer with properties of bipolar lipid
562
membrane. The disruption of the Mtb H37Rv strain membrane was also demonstrated by
563
alterations in the ATP levels, NAD/NADH and NADP/NADPH ratio which precede the
564
death of Mtb H37Rv cells. While few studies showed direct evidence on interaction or
565
involvement of AMP with different cell wall components (Martínez et al., 2008a), other
23
566
studies speculated on the activity of bacteriocins or interaction of the bacteriocins with cell
567
wall/cell membrane components (Martínez et al., 2008b; Yoneyama et al., 2011). On the
568
other hand, hemolysis assay suggested that laterosporulin10 had no effect on RBCs as no
569
haemolysis was observed even at 10 times higher concentrations to the Gram-positive strains
570
or about 100 fold molar excess of LD50 value of Mtb H37Rv strain. Most importantly, this
571
bacteriocin is not toxic to mammalian cells and could kill the intracellular Mtb H37Rv strain,
572
suggesting the potential of such compounds to treat drug-resistant tuberculosis. These new
573
forms of peptides with improved antimicrobial activity and low cytotoxicity towards
574
eukaryotic cells could be developed to use as therapeutic agents or in food preservation.
575
24
576
Acknowledgements: Financial assistance from the Department of Biotechnology (grant no.
577
DBT/In-Bz/2013-16/16/SO-R1) and Council of Scientific and Industrial Research (CSIR)-
578
Network project (BSC-119) are duly acknowledged. Dr. Vishakha Grover (Dr. H. S. Judge
579
Institute of Dental Sciences and Hospital, Punjab University, India) and Dr. Pradip Kumar
580
Singh (University of Maryland, USA) for useful discussions. We would like to thank Mrs.
581
Sharanjeet Kaur and Dr. Santi Mandal (Vidyasagar University, West Bengal, India) for their
582
help in MALDI analysis of the peptide, Mrs. Paramjeet Kaur for her help in N-terminal
583
sequencing of the peptide and Mr. Randeep and Mr. Anil Theophilus for their help in electron
584
microscopy.
585
25
586
References
587
588
Arndt-Jovin, D. J. & Jovin, T. M. (1989). Fluorescence labeling and microscopy of DNA.
Methods Cell Biol.
589
590
591
592
Arnison, P. G., Bibb, M. J., Bierbaum, G., Bowers, A. a., Bugni, T. S., Bulaj, G.,
Camarero, J. a., Campopiano, D. J., Challis, G. L. & other authors. (2013).
Ribosomally synthesized and post-translationally modified peptide natural products:
overview and recommendations for a universal nomenclature 108–160.
593
594
595
Aziz, R. K., Bartels, D., Best, A. a, DeJongh, M., Disz, T., Edwards, R. a, Formsma, K.,
Gerdes, S., Glass, E. M. & other authors. (2008). The RAST Server: rapid annotations
using subsystems technology. BMC Genomics 9, 75.
596
597
598
599
Baindara, P., Mandal, S. M., Chawla, N., Singh, P. K., Pinnaka, A. K. & Korpole, S.
(2013). Characterization of two antimicrobial peptides produced by a halotolerant
Bacillus subtilis strain SK.DU.4 isolated from a rhizosphere soil sample. AMB Express
3, 2. AMB Express.
600
601
602
603
Baindara, P., Chaudhry, V., Mittal, G., Liao, L. M., Matos, C. O., Khathri, N., Franco,
O. L., Patil, P. B. & Korpole, S. (2015). Characterization of Antimicrobial Peptide,
Penisin, a Class Ia Novel Lantibiotic from a Paenibacillus sp. Strain A3. Antimicrob
Agents Chemother.
604
605
606
607
Van Belkum, M. J., Kok, J., Venema, G., Holo, H., Nes, I. F., Konings, W. N. & Abee, T.
(1991). The bacteriocin lactococcin A specifically increases permeability of lactococcal
cytoplasmic membranes in a voltage-independent, protein-mediated manner. J Bacteriol
173, 7934–7941.
608
609
Van Belkum, M. J., Martin-Visscher, L. a. & Vederas, J. C. (2011). Structure and
genetics of circular bacteriocins. Trends Microbiol 19, 411–418. Elsevier Ltd.
610
611
612
Carrillo, C., Teruel, J. A., Aranda, F. J. & Ortiz, A. (2003). Molecular mechanism of
membrane permeabilization by the peptide antibiotic surfactin. Biochim Biophys Acta Biomembr 1611, 91–97.
613
614
615
Carroll, J., Field, D., O’Connor, P. M., Cotter, P. D., Coffey, A., Hill, C., Ross, R. P. &
O’Mahony, J. (2010a). Gene encoded antimicrobial peptides, a template for the design
of novel anti-mycobacterial drugs. Bioeng Bugs 1, 408–412.
616
617
618
619
Carroll, J., Draper, L. a, O’Connor, P. M., Coffey, A., Hill, C., Ross, R. P., Cotter, P. D.
& O’Mahony, J. (2010b). Comparison of the activities of the lantibiotics nisin and
lacticin 3147 against clinically significant mycobacteria. Int J Antimicrob Agents 36,
132–6.
620
621
622
Chaturvedi, V., Ramani, R., Andes, D., Diekema, D. J., Pfaller, M. A., Ghannoum, M.
A., Knapp, C., Lockhart, S. R., Ostrosky-Zeichner, L. & other authors. (2011).
Multilaboratory testing of two-drug combinations of antifungals against Candida
26
623
624
albicans, Candida glabrata, and Candida parapsilosis. Antimicrob Agents Chemother 55,
1543–1548.
625
626
Cotter, P. D., Ross, R. P. & Hill, C. (2013). Bacteriocins - a viable alternative to antibiotics?
Nat Rev Microbiol 11, 95–105. Nature Publishing Group.
627
628
629
630
Desjardine, K., Pereira, A., Wright, H., Matainaho, T., Kelly, M. & Andersen, R. J.
(2007). Tauramamide, a lipopeptide antibiotic produced in culture by Brevibacillus
laterosporus isolated from a marine habitat: Structure elucidation and synthesis. J Nat
Prod 70, 1850–1853.
631
632
Donaghy, J. (2010). Lantibiotics as prospective antimycobacterial agents. Bioeng Bugs 1,
437–9.
633
634
Fickers, P. (2013). Characterization of amylolysin, a novel lantibiotic from Bacillus
amyloliquefaciens GA1. PLoS One 8, e83037.
635
636
637
Fresard, I., Bridevaux, P.-O., Rochat, T. & Janssens, J.-P. (2011). Adverse effects and
adherence to treatment of rifampicin 4 months vs isoniazid 6 months for latent
tuberculosis: a retrospective analysis. Swiss Med Wkly 141, w13240.
638
639
Grosset, J. & Leventis, S. (2014). Adverse effects of rifampin. Rev Infect Dis 5 Suppl 3,
S440–S450.
640
641
Grosset, J. & Leventis, S. (2013). Adverse effects of rifampin. Rev Infect Dis 5 Suppl 3,
S440–50.
642
Gutsmann T. (2016). Biochim Biophys Acta. Biochim Biophys Acta 5, 1034–43.
643
644
645
646
Hartmann, M., Berditsch, M., Hawecker, J., Ardakani, M. F., Gerthsen, D. & Ulrich, A.
S. (2010). Damage of the bacterial cell envelope by antimicrobial peptides gramicidin S
and PGLa as revealed by transmission and scanning electron microscopy. Antimicrob
Agents Chemother 54, 3132–42.
647
648
649
Kamiyama, T., Umino, T., Nakamura, Y., Itezono, Y., Sawairi, S., Satoh, T. & Yokose,
K. (1994). Bacithrocins A, B and C, novel thrombin inhibitors. J Antibiot (Tokyo) 47,
959–68.
650
651
652
Kaplan, C. W., Sim, J. H., Shah, K. R., Kolesnikova-Kaplan, A., Shi, W. & Eckert, R.
(2011). Selective membrane disruption: Mode of action of C16G2, a specifically
targeted antimicrobial peptide. Antimicrob Agents Chemother 55, 3446–3452.
653
654
Klaenhammer, T. R. (1993a). Genetics of bacteriocins produced by lactic acid bacteria.
FEMS Microbiol Rev 12, 39–85.
655
656
Klaenhammer, T. R. (1993b). Genetics of bacteriocins produced by lactic acid bacteria.
FEMS Microbiol Rev 12, 39–85.
657
658
Kumar, A., Deshane, J. S., Crossman, D. K., Bolisetty, S., Yan, B. S., Kramnik, I.,
Agarwal, A. & Steyn, A. J. C. (2008). Heme oxygenase-1-derived carbon monoxide
27
659
660
induces the Mycobacterium tuberculosis dormancy regulon. J Biol Chem 283, 18032–
18039.
661
662
663
664
Lee, Y.-S., Lee, D.-Y., Kim, Y. B., Lee, S.-W., Cha, S.-W., Park, H.-W., Kim, G.-S.,
Kwon, D.-Y., Lee, M.-H. & Han, S.-H. (2015). The Mechanism Underlying the
Antibacterial Activity of Shikonin against Methicillin-Resistant Staphylococcus aureus.
Evidence-Based Complement Altern Med 2015, 1–9.
665
666
667
Ma, S., Zhao, Y., Xia, X., Dong, X., Ge, W. & Li, H. (2015). Effects of Streptococcus
sanguinis Bacteriocin on Cell Surface Hydrophobicity, Membrane Permeability, and
Ultrastructure of Candida Thallus. Biomed Res Int 1–8.
668
669
670
671
Maftah, a., Renault, D., Vignoles, C., Hechard, Y., Bressollier, P., Ratinaud, M. H.,
Cenatiempo, Y. & Julien, R. (1993). Membrane permeabilization of Listeria
monocytogenes and mitochondria by the bacteriocin mesentericin Y105. J Bacteriol
175, 3232–3235.
672
673
674
Mandal, S. M., Dey, S., Mandal, M., Sarkar, S., Maria-Neto, S. & Franco, O. L. (2009).
Identification and structural insights of three novel antimicrobial peptides isolated from
green coconut water. Peptides 30, 633–637.
675
676
677
Martínez, B., Böttiger, T., Schneider, T., Rodriguez, A., Sahl, H. G. & Wiedemann, I.
(2008a). Specific interaction of the unmodified bacteriocin lactococcin 972 with the cell
wall precursor lipid II. In Appl Environ Microbiol, pp. 4666–4670.
678
679
680
Martínez, B., Böttiger, T., Schneider, T., Rodríguez, A., Sahl, H.-G. & Wiedemann, I.
(2008b). Specific interaction of the unmodified bacteriocin Lactococcin 972 with the
cell wall precursor lipid II. Appl Environ Microbiol 74, 4666–70.
681
682
Nakatsuji, T. & Gallo, R. L. (2014). Dermatological therapy by topical application of nonpathogenic bacteria. J Invest Dermatol 134, 11–4. Nature Publishing Group.
683
684
Nilsen, T., Nes, I. F. & Holo, H. (2003). Enterolysin A, a cell wall-degrading bacteriocin
from Enterococcus faecalis LMG 2333. Appl Environ Microbiol 69, 2975–2984.
685
686
687
Oliveira, E. J. De, Rabinovitch, L., Monnerat, R. G., Konovaloff, L., Passos, J., Zahner,
V. & Icrobiol, A. P. P. L. E. N. M. (2004). Molecular Characterization of Brevibacillus
laterosporus and Its Potential Use in Biological Control 70, 6657–6664.
688
689
690
Pettit, R. K., Weber, C. A., Kean, M. J., Hoffmann, H., Pettit, G. R., Tan, R., Franks, K.
S. & Horton, M. L. (2005). Microplate alamar blue assay for Staphylococcus
epidermidis biofilm susceptibility testing. Antimicrob Agents Chemother 49, 2612–2617.
691
692
693
Piper, C., Casey, P. G., Hill, C., Cotter, P. D. & Ross, R. P. (2012). The Lantibiotic
Lacticin 3147 Prevents Systemic Spread of Staphylococcus aureus in a Murine Infection
Model. Int J Microbiol 2012, 806230.
694
695
Raje, M., Dhiman, R., Majumdar, S., Dass, T., Dikshit, K. L. & Kaur, R. (2006).
Charged nylon membrane substrate for convenient and versatile high resolution
28
696
697
microscopic analysis of Escherichia coli & mammalian cells in suspension culture.
Cytotechnology 51, 111–7.
698
699
700
701
Ramón-García, S., Mikut, R., Ng, C., Ruden, S., Volkmer, R., Reischl, M., Hilpert, K. &
Thompson, C. J. (2013). Targeting mycobacterium tuberculosis and other microbial
pathogens using improved synthetic antibacterial peptides. Antimicrob Agents
Chemother 57, 2295–2303.
702
703
704
Ruiu, L., Satta, a & Floris, I. (2014). Administration of Brevibacillus laterosporus spores as
a poultry feed additive to inhibit house fly development in feces: a new eco-sustainable
concept. Poult Sci 93, 519–26.
705
706
707
708
Semvua, H. H., Kibiki, G. S., Kisanga, E. R., Boeree, M. J., Burger, D. M. & Aarnoutse,
R. (2014). Pharmacological interactions between rifampicin and antiretroviral drugs:
challenges and research priorities for resource-limited settings. Ther Drug Monit 255,
22–32.
709
710
Sharma, V., Singh, P. K., Midha, S., Ranjan, M., Korpole, S. & Patil, P. B. (2012).
Genome sequence of Brevibacillus laterosporus strain GI-9. J Bacteriol.
711
712
713
Shida, O., Takagi, H., Kadowaki, K. & Komagata, K. (1996). Proposal for two new
genera, Brevibacillus gen. nov. and Aneurinibacillus gen. nov. Int J Syst Bacteriol 46,
939–946.
714
715
716
Singh, P. K., Chittpurna, Ashish, Sharma, V., Patil, P. B. & Korpole, S. (2012).
Identification, purification and characterization of laterosporulin, a novel bacteriocin
produced by Brevibacillus sp. strain GI-9. PLoS One 7.
717
718
719
Singh, P. K., Solanki, V., Sharma, S., Thakur, K. G., Krishnan, B. & Korpole, S. (2014).
The intramolecular disulfide-stapled structure of laterosporulin, a class IId bacteriocin,
conceals a human defensin-like structural module. FEBS J 1–12.
720
721
722
Smirnova, T. A., Minenkova, I. B., Orlova, M. V., Lecadet, M. M. & Azizbekyan, R. R.
(1996). The crystal-forming strains of Bacillus laterosporus. Res Microbiol 147, 343–
350.
723
724
725
Smolarczyk, R., Cichoń, T., Kamysz, W., Głowala-Kosińska, M., Szydło, A., Szultka, L.,
Sieroń, A. L. & Szala, S. (2010). Anticancer effects of CAMEL peptide. Lab Invest 90,
940–952.
726
727
728
Sosunov, V., Mischenko, V., Eruslanov, B., Svetoch, E., Shakina, Y., Stern, N., Majorov,
K., Sorokoumova, G., Selishcheva, A. & Apt, A. (2007). Antimycobacterial activity of
bacteriocins and their complexes with liposomes. J Antimicrob Chemother 59, 919–925.
729
730
731
732
733
Stern, N. J., Svetoch, E. a, Eruslanov, B. V, Perelygin, V. V, Mitsevich, E. V, Mitsevich,
I. P., Pokhilenko, V. D., Levchuk, V. P., Svetoch, O. E. & Seal, B. S. (2006).
Isolation of a Lactobacillus salivarius strain and purification of its bacteriocin, which is
inhibitory to Campylobacter jejuni in the chicken gastrointestinal system. Antimicrob
Agents Chemother 50, 3111–6.
29
734
735
736
Teixeira, M. L., Rosa, A. D. & Brandelli, A. (2013). Characterization of an antimicrobial
peptide produced by Bacillus subtilis subsp. spizezinii showing inhibitory activity
towards Haemophilus parasuis. Microbiol (United Kingdom) 159, 980–988.
737
738
739
740
Theodore, C. M., Stamps, B. W., King, J. B., Price, L. S. L., Powell, D. R., Stevenson, B.
S. & Cichewicz, R. H. (2014). Genomic and metabolomic insights into the natural
product biosynthetic diversity of a feral-hog-associated Brevibacillus laterosporus strain.
PLoS One 9, 3–12.
741
742
Umezawa, K. & Takeuchi, T. (1987). Spergualin: a new antitumour antibiotic. Biomed
Pharmacother 41, 227–232.
743
744
Yew, W. W. & Chi Chu Leung. (2006). Antituberculosis drugs and hepatotoxicity.
Respirology 11, 699–707.
745
746
747
748
Yoneyama, F., Imura, Y., Ohno, K., Zendo, T., Nakayama, J., Matsuzaki, K. &
Sonomoto, K. (2009). Peptide-lipid huge toroidal pore, a new antimicrobial mechanism
mediated by a lactococcal bacteriocin, lacticin Q. Antimicrob Agents Chemother 53,
3211–7.
749
750
751
752
Yoneyama, F., Ohno, K., Imura, Y., Li, M., Zendo, T., Nakayama, J., Matsuzaki, K. &
Sonomoto, K. (2011). Lacticin Q-mediated selective toxicity depending on
physicochemical features of membrane components. Antimicrob Agents Chemother 55,
2446–2450.
753
754
755
Zhao, J., Guo, L., Zeng, H., Yang, X., Yuan, J., Shi, H., Xiong, Y., Chen, M., Han, L. &
Qiu, D. (2012). Purification and characterization of a novel antimicrobial peptide from
Brevibacillus laterosporus strain A60. Peptides 33, 206–211. Elsevier Inc.
756
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758
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Figures
760
Figure 1: Purification, molecular weight determination and in-gel activity assay of
761
laterosporulin10. (A) Reverse phase HPLC profile of laterosporulin10 (inset shows the
762
antimicrobial activity against S. aureus MTCC 1430). (B) Molecular weight determination on
763
Shodex 802 HPLC column. Laterosporulin10, elution profile suggested it as a monomer. (C)
764
Tricine-SDS-PAGE and in-gel activity assay of the purified laterosporulin10. Molecular
765
weight marker along with laterosporulin10 (L1), portion of the SDS-PAGE gel overlaid with
766
0.8% soft agar containing S. aureus MTCC 1430, (L2) showing zone of clearance. All
767
electrophoresis and activity assays done with purified peptide dissolved in PBS. (D) MALDI
768
TOF analysis of the purified peptide from B. laterosporus strain SKDU10 shows the mass
769
(m/z) of 6.0 kDa.
770
Figure 2: Determination of minimum inhibitory concentrations (MICs), killing kinetics,
771
amount of ATP released and hemolysis activities of for laterosporulin10. (A) MICs against
772
Gram-positive and Gram-negative bacteria. (B) Laterosporulin10 mediated killing kinetics of
773
S. aureus MTCC 1430 (n = 3). Symbols represent the viable cell counts of S. aureus at
774
different time intervals along with 4 µM, 8 µM and 20 µM of laterosporulin10. (C) Amount
775
of ATP released from S. aureus (represented by symbols) after treatment with 4 µM, 8 µM
776
and 20 µM of laterosporulin10 at different time intervals. Error bars represents SD. All
777
groups were compared with the untreated control for statistical significance. P<0.05 is
778
considered as significant. (D) Hemolysis assay of laterosporulin10 using rabbit RBCs. All
779
experiments performed in triplicates Bars show SD. P values are indicated above each bar
780
(significance was at a P level of <0.05). Purified peptide, bacterial cells and RBC samples
781
were prepared in PBS.
782
Figure 3: Determination of membrane permeabilizing properties of laterosporulin10 by
783
FACS analysis of S. aureus MTCC 1430. Bacterial cells treated with 4 µM of
31
784
laterosporulin10 in a time dependent manner. Percentage of bacterial cells in upper right hand
785
corner shown the increased uptake of propidium iodide with increased time interval (i)
786
bacterial cells without treatment serves as control, (ii), (iii) and (iv) shown the results after 5,
787
10 and 30 min treatment of laterosporulin10. Bacterial cells and peptide samples were
788
prepared in PBS.
789
Figure 4: Determination of anti-mycobacterial effect of laterosporulin10 by microtiter plate
790
alamar blue assay (n = 3). Rifampicin was used as positive control. Purified peptide and
791
bacterial cell samples were prepared in PBS. Error bars show SD. *, P < 0.05 in comparison
792
to the positive control, indicated above each bar. Microtiter plate assay picture shown above
793
the graph.
794
Figure 5: Combination of laterosporulin10 with rifampicin reduces the MIC of rifampicin.
795
MABA was performed using Mtb H37Rv strain where varying concentrations of rifampicin
796
(0.003125–0.2 µM) and laterosporulin10 (0.31-4.0 µM) were used in combination and alone.
797
Addition of laterosporulin10 (0.25 µM) led to four-fold (0.025 µM) decrease in the MIC of
798
rifampicin against Mtb H37Rv. Heatmap was generated using the gene-e software.
799
Figure 6: Effect of laterosporulin10 on survival of intracellular Mtb H37Rv. Bars represent
800
the log CFU/ml after treatment with 0.025, 0.125 µM of rifampicin and 0.5, 2.5 µM of
801
laterosporulin10. Mtb H37Rv cells without treatment served as negative control while Mtb
802
H37Rv treated with 0.025 and 0.125 µM, MIC values of rifampicin served as positive
803
control. Bacterial cells and peptide samples were prepared in PBS. Error bars represent SD. *,
804
P < 0.05 in comparison to the negative control and indicated above bars.
805
Figure 7: Determination of dinucleotide homeostasis disruption ability of laterosporulin10 in
806
Mtb H37Rv (A) ATP release assay. Amount of ATP released represented by bars after
807
treatment with 0.5 and 2.5 µM of laterosporulin10 (10 and 60 min for both). (B)
808
NAD/NADH assay. Ration of NAD/NADH represented by bars after treatment with 0.5 µM
32
809
of laterosporulin10 (10 and 60 min). (C) Ration of NADP/NADPH represented by bars after
810
treatment with 0.5 µM of laterosporulin10 (10 and 60 min). Untreated cells used as control.
811
Bacterial cells and peptide samples were prepared in PBS. Error bars show SD. *, P < 0.05 in
812
comparison to the untreated controls, indicated above bars.
813
Figure 8: Determination of bactericidal effect of laterosporulin10 on S. aureus MTCC 1430
814
and Mtb-H37Rv strain upon 30 min incubation (A) Transmission electron microscopy of S.
815
aureus without laterosporulin10 treatment (i), with 4 µM (ii) and 8 µM (iii) laterosporulin10.
816
(B) Scanning electron microscopy of Mtb H37Rv without laterosporulin10 treatment (i), with
817
0.5 µM (ii) and 1.0 µM (iii) laterosporulin10. (C) Transmission electron microscopy of Mtb
818
H37Rv (sections) without laterosporulin10 treatment (i), with 0.5 µM (ii) and 1.0 µM (iii)
819
laterosporulin10. Bacterial cells and peptide samples were prepared in PBS.
820
Figure 9: The laterosporulin gene clusters of B. laterosporus strain SKDU10 and GI-9 (A)
821
Organization, comparison and sequence alignment of laterosporulin10 biosynthetic gene
822
cluster and amino acid sequence with laterosporulin. ORFs with different functions are
823
colored differently, ORF encoding structural gene depicting in yellow showing amino acid
824
sequence alignment of LS10 and LS inside dotted lines. One side of laterosporulin is flanked
825
by a transcriptional regulator ORF shown in orange color and other side with ORF involved
826
in transportation of mature peptide is shown in light blue color, a dehydrogenase in purple
827
and hypothetical protein are shown in light brown color. First amino acid of the peptide
828
sequence is methionine which cleaved during maturation (shown by dotted arrow). (B)
829
Nucleotide sequence of the putative ORF encoding the laterosporulin10 structural gene along
830
with putative start and stop codon and ribosome binding site (RBS).
831
832
833
33
834
Supplementary Figure S1: Phylogenetic tree of B. laterosporus SKDU10 based on 16S
835
rRNA gene sequence.
836
Supplementary Figure S2: Growth phase-dependent bacteriocin production by B.
837
laterosporus SKDU10. Filled circles indicate bacteriocin activity as determined by inhibition
838
zone assay while crossed lines represent bacterial growth as measured by absorbance at 600
839
nm.
840
Supplementary Figure S3: Temperature, pH and protease test of laterosporulin10 (100 µl of
841
1mg/ml) against S. aureus MTCC 1430 grown on nutrient agar. (A) Agar well diffusion assay
842
of laterosporulin10 after treatment at different temperatures for 30 min. laterosporulin10 at
843
37°C serves as control. (B) Agar well diffusion of laterosporulin10 at different pH range.
844
Laterosporulin10 at 7.0 serves as control. (C) Agar well diffusion assay of laterosporulin10
845
after 1 h treatment with trypsin. Untreated laterosporulin10 serves as control at different
846
concentrations of trypsin. (D) Agar well diffusion assay of laterosporulin10 after 6 h
847
treatment with proteinase K. Untreated laterosporulin10 serves as control at different
848
concentrations of proteinase K.
849
Supplementary Figure S4: Determination of MICs of laterosporulin10 against M.
850
smegmatis by microtiter plate alamar blue assay (n = 3). Rifampicin was used as positive
851
control. Purified peptide and bacterial cell samples were prepared in PBS. Error bars show
852
SD. *, P < 0.05 in comparison to the positive control, indicated above each bar. Microtiter
853
plate assay picture shown above the graph.
854
Supplementary Figure S5: Cytotoxic effect of laterosporulin10 on RAW 264.7 (murine
855
macrophage) cells in the MTT assay. Cells were maintained in a humidified CO2 incubator at
856
37°C, and different concentrations (1-30 µM) of laterosporulin10 were added after 24 h. A
857
purified peptide sample was used for treatment in the growth medium used to grow cell lines.
34
858
The percentage of cell viability was calculated as described in Methods. Results shown are
859
means of SD in three independent experiments performed in triplicates.
860
Supplementary Figure S6: Determination the presence of disulfide bonds. (A) PEGylation
861
assay of purified laterosporulin10 with MPEG (maleimide PEG, 5 kDa) in presence of TCEP
862
(tris(2-carboxyethyl)phosphine) to access the presence of disulfide bonds. Same assay has
863
been done in presence of denaturing agent, Urea. MPEG; maleimide PEG, 5 kDa, T; tris(2-
864
carboxyethyl)phosphine, U ; urea. (B) MALDI TOF Molecular weight determination of
865
laterosporulin10 in reduced conditions showing increase of 6 Da in molecular weight which
866
is correspond to formation of three disulfide bonds in native form of laterosporulin10.
35