Letters in Applied Microbiology ISSN 0266-8254
ORIGINAL ARTICLE
Aeromonas piscicola AH-3 expresses an extracellular collagenase with cytotoxic properties s2 and A.S. Duarte1, E. Cavaleiro1, C. Pereira1,2, S. Merino2, A.C. Esteves1, E.P. Duarte3, J.M. Toma 1 A.C. Correia 1 Department of Biology & CESAM, University of Aveiro, Aveiro, Portugal 2 Departamento de Microbiologıa, Facultad de Biologıa, Universidad de Barcelona, Barcelona, Spain 3 Centre for Neurosciences and Cell Biology & Department of Zoology, University of Coimbra, Coimbra, Portugal
Significance and Impact of the Study: Collagenases play a central role in processes where collagen digestion is needed, for example host invasion by pathogenic micro-organisms. We identified a new collagenase from Aeromonas using an integrated in silico/in vitro strategy. This enzyme is able to bind and cleave collagen, contributes for AH-3 cytotoxicity and shares low similarity with known bacterial collagenases. This is the first report of an enzyme belonging to the gluzincin subfamily of the M9 family of peptidases in Aeromonas. This study increases the current knowledge on collagenolytic enzymes bringing new perspectives for biotechnology/medical purposes.
Keywords AH-3, collagen interaction, cytotoxicity, metalloprotease, microbial collagenase. Correspondence Ana Sofia Duarte, Department of Biology & CESAM, University of Aveiro, 3810-193 Aveiro, Portugal. E-mail:
[email protected] 2014/1890: received 12 September 2014, revised 10 November 2014 and accepted 26 November 2014 doi:10.1111/lam.12373
Abstract The aim of this study was to investigate the presence and the phenotypic expression of a gene coding for a putative collagenase. This gene (AHA_0517) was identified in Aeromonas hydrophila ATCC 7966 genome and named colAh. We constructed and characterized an Aeromonas piscicola AH-3::colAh knockout mutant. Collagenolytic activity of the wild-type and mutant strains was determined, demonstrating that colAh encodes for a collagenase. ColAh– collagen interaction was assayed by Far-Western blot, and cytopathic effects were investigated in Vero cells. We demonstrated that ColAh is a gluzincin metallopeptidase (approx. 100 kDa), able to cleave and physically interact with collagen, that contributes for Aeromonas collagenolytic activity and cytotoxicity. ColAh possess the consensus HEXXH sequence and a glutamic acid as the third zinc binding positioned downstream the HEXXH motif, but has low sequence similarity and distinct domain architecture to the well-known clostridial collagenases. In addition, these results highlight the importance of exploring new microbial collagenases that may have significant relevance for the health and biotechnological industries.
Introduction During the past decades, substantiating evidence has been gathered supporting the hypothesis that growth and proliferation of pathogenic bacteria depend on the action of proteolytic enzymes (Harrington 1996; Watanabe 2004). Collagenases are involved in the degradation of extracellular matrixes of animal cells, due to their ability to digest native and denatured collagen (Duarte et al. 2014). Collagenases and other collagen-degrading enzymes have been implicated in the virulence of many pathogenic bacteria Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
(Lawson and Meyer 1992; Takeuchi et al. 1992; Matsushita et al. 1999; Mukherjee et al. 2009). The genus Aeromonas includes Gram-negative, facultative anaerobes, present in aquatic environments, food and soil (Kingombe et al. 1999; Chac on et al. 2003; Carvalho et al. 2012). Species of this genus have been described as pathogens of humans, fish, invertebrates and insects (Seshadri et al. 2006; Reith et al. 2008; Castro et al. 2010; Janda and Abbott 2010; Li et al. 2011; Parker and Shaw 2011) and occasionally as symbionts of leeches and fish (Janda and Abbott 2010; Silver et al. 2011). 1
ColAh: Aeromonas collagenase
In particular, Aeromonas hydrophila is associated with gastroenteritis, wound diseases, soft tissue and burn infections, and sepsis, with lethal course in humans (Janda and Abbott 2010). Virulence of Aer. hydrophila seems to involve several extracellular molecules including enterotoxins, hemolysins, elastases and other proteases (Kingombe et al. 1999; Chac on et al. 2003; Seshadri et al. 2006; Reith et al. 2008; Li et al. 2011; Parker and Shaw 2011). Nevertheless, data on Aeromonas collagenases are scarce: until now, only one report describes a collagenase in Aeromonas veronii. This enzyme is involved in the progression of bacterial colonization and infection (Han et al. 2008). Several studies have demonstrated the ability of AH-3 —previously Aer. hydrophila and now known as Aeromonas piscicola—to adhere and to invade host cells mediated by the expression of a high number of virulence determinants (Merino et al. 1992; Beaz-Hidalgo et al. 2009; Vilches et al. 2009; Molero et al. 2011). To investigate the function of the putative collagenase in AH-3, we detected the gene in Aer. piscicola and constructed an AH-3 knockout mutant (AH-3::colAh). Phenotypic characterization of the mutant and the wild-type strains included cytotoxicity evaluation and assessment of collagenolytic activity and enzyme–substrate physical interaction. Results and discussion An open reading frame (AHA_0517) of 2748 bp encoding a 915 amino acids protein is annotated as a putative collagenase in the genome of Aer. hydrophila ATCC 7966T (accession number NC_008570). To characterize the role of this putative collagenase, we designed and constructed a knockout mutant by disrupting the AHA_0517 locus in AH-3 by homologous recombination. Using specific primers, we amplified by PCR a fragment of 904 bp from the genomic DNA of Aer. piscicola AH-3 (accession number JQ639076). The nucleotide sequence of the amplicon shared 88% similarity with a region with the same length from AHA_0517. We hypothesized that the genome of Aer. piscicola AH-3, similarly to Aer. hydrophila ATCC 7966T, also contains the putative collagenase gene (colAh). To investigate this hypothesis, we performed mutagenesis of the genome targeted to the colAh region (Figure S1). The 904-bp amplicon was inserted in the suicide plasmid pFS100 to provide homologous recombination with the genome, giving rise to the plasmid pFS-colAh. By triparental mating, using Escherichia coli MC1061 as donor, the construct was introduced into Aer. piscicola AH-3. Sequence analysis of the mutant confirmed the integration into the chromosomal DNA. 2
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As seen by zymography, Aer. piscicola AH-3 and the AH-3::colAh knockout mutant express several extracellular gelatinases (Fig. 1a). Nevertheless, the mutant strain lacks a gelatinase with an apparent molecular mass of approx. 100 kDa (Fig. 1a), corresponding to the molecular mass of the product of the AHA_0517 gene. The extracellular collagenolytic activities of the mutant and of the wild-type strains were quantified by hydrolysis of FALGPA substrate, a collagenase-specific synthetic peptide (Van Wart and Steinbrink 1981). Results show that AH-3::colAh cell-free supernatant (CFS) has a significant lower collagenolytic activity (P < 0001) when compared to the wild-type strain (Fig. 1b), confirming that the AHA_0517 gene is responsible for collagenolytic activity. Both PMSF and 1,10-phenanthroline caused an inhibition of collagenolytic activity of AH-3 wild type and AH3::colAh. This inhibition pattern suggests the presence of metallopeptidases and of serine peptidases. Taken together, these results favour the hypothesis of the presence of a gene in the genome of Aer. piscicola AH-3 encoding an enzyme (ColAh) with relevant contribution for the extracellular collagenolytic activity displayed by strain. The mutant strain still exhibited partial collagenolytic activity on FALGPA hydrolysis assays; this is an indication that other collagenolytic enzymes may also be present in the extracellular medium. The interaction between ColAh and type I collagen was assessed by Far-Western blot (Fig. 1c). No protein–collagen interactions were detected in colAh-deficient mutant CFS, but in the wild strain, one band, with an apparent molecular weight of 100 kDa, was detected confirming that ColAh is able to physically interact with collagen. This interaction suggests the presence of a collagen-binding domain on ColAh. This domain shares functional— but not structural—similarity with the well-known collagen recruitment domains, such as the CBD or the PKDlike domains in clostridial collagenases (Eckhard et al. 2011, 2013; Duarte et al. 2014). AH-3 CFS is highly cytotoxic, inducing the loss of 941 % Vero cells’ viability (Fig. 2). This verotoxic effect was significantly reduced (P < 0001 at 1 : 4 CFS dilution) for the AH-3::colAh mutant CFS, which promoted the loss of 857% cell viability. Regarding ColAh collagenolytic activity and its cytotoxic effects, the results suggest that ColAh may play a role in AH-3 infection mechanism, degrading host collagen-rich matrices and favouring bacterial penetration and migration in the host tissues. SMART analysis of ColAh sequence shows the existence of a signal peptide at the N-terminal of ColAh (with 23amino acid residues) and a presumable active site localized at the C-terminal half of the core protein containing an HEXXH motif (Fig. 3a). The HEXXH motif is a metal-binding site (Bode et al. 1993; Rawlings and Barrett Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
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(a)
1
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80·0 (c) kDa 50·9
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150 100 75
37·2
50 29·2
MW (kDa) (b) 100
80 Collagenolytic activity (%)
Figure 1 Collagenase detection: (a) Gelatin zymography of extracellular enzymatic activity of Aeromonashydrophila strain AH-3 (lane 1) and AH-3 mutant (AH-3::colAh; lane 2). The collagenase expected migration position is indicated by a black arrow. (b) FALGPA extracellular hydrolytic activity AH-3 (grey bar) and AH-3 mutant (white bar). Effect of 2 mmol l1 PMSF, 10 mmol l1 1,10-phenanthroline and thermal denaturation (100°C) on the collagenolytic activity of CFS. Data are expressed in percentage of clostridial collagenase activity (mean standard error; n = 3). Statistical significance of mutant AH-3 CFS collagenolytic activity was determined using Student’s t test. One-way ANOVA, followed by a Dunnett’s multiple comparison test, was used to determine the statistical significance of inhibitors of AH-3 (*P < 005, **P < 001 and ***P< 0001) or mutant AH-3 CFS (#P < 005, ##P < 001 and ###P < 0001). (c) Far-Western blot of AH-3 collagenase. CFS from AH-3 (lane 1), AH-3 mutant (lane 2) and collagen type I (positive control) (3) were subjected to SDS. After electrophoresis, proteins were transferred to a nitrocellulose membrane and probed with collagen type I. Bound proteins were detected by chemiluminescence using an anti-collagen type I antibody. ( ) AH-3 and ( ) AH-3 mutant.
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1995; Wu and Chen 2011; Duarte et al. 2014), usually found at the N-terminal half of the core protein of bacterial collagenases. This HEXXH sequence and the glutamate positioned 33–35 residues downstream the HEXXH motif (Fig. 3b) were reported as the third zinc-binding ligand of ColH collagenase (Clostridium histolyticum) and are characteristic of the subfamily of gluzincin (Hooper 1994) of the MEROPS M9 family of peptidases. This locates ColAh in the gluzincin subfamily of metallopeptidases. The catalytic domain of ColAh (Met603-Phe871), predicted by SMART analysis, has a sequence identity of 51% Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
CFS
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CFS+Phe
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CFS+100°C
(67% similarity), 47% (66% similarity) and 50% (65% similarity), respectively, with the sequences of the catalytic domains of Vibrio, Shewanella and Myxococcus collagenases (Fig. 3b), suggesting that ColAh may have a distinct specificity and/or a different mechanism of collagen digestion. Modelling of amino acid sequences into 3D structures, although surrounded by controversy, has gained increased attention as it allows to predict protein structure and function. 3D model of ColAh was made by I-TASSER server utilities. This tool generates high-quality predictions of 3D structure and biological function of protein molecules from their amino acid sequences. 3
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120 110
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Figure 2 Evaluation of verotoxicity: Cytotoxicity of AH-3; AH-3:: colAh, Escherichia coli BL25 (negative control) and Aeromonas hydrophila ATCC 7966 (positive control) CFSs. Cytotoxicity was analysed for differences with two-way ANOVA, followed by a Bonferroni post-test, using a significance level of 001 (**) or 0001 (***). Data are presented as mean standard error of two independent experiments performed in quadruplicate. (h) AH-3; ( ) AH-3::colAh; ( ) Aer. hydrophila ATCC 7966 and ( ) E. coli BL21
Catalytic and noncatalytic domains of ColAh appear to be independently organized, suggesting some flexibility during macromolecular substrate recognition and catalysis (Fig. 3c). To degrade fibrillar collagen—collagen in tissues —collagenases must interact with insoluble collagen fibril and then unwind the triple helix on tropocollagen to expose the scissile peptide bond (Philominathan et al. 2009; Duarte et al. 2014). Analysis of ColAh showed the presence of two internal repeats of approx. 120 residues (RPT1 and RPT2; Fig. 3c). These repeated regions contribute to the typical high molecular mass of collagenolytic enzymes and are suggested to participate in the recognition of the macromolecular substrate (Ghuysen et al. 1994; Philominathan et al. 2009). RPT1 sequence of ColAh share no homology to the well-known bacterial collagen-binding domains, but its relative position in the overall sequence (Fig. 3c) and predicted secondary structure suggest an eventual participation as a collagen-binding domain. As shown in Fig. 3c-2, the predicted three-dimensional structure of the RPT1 sequence estimates an 189- A-wide cleft. The predicted width of ColAh RPT1 cleft is compatible with the diameter of collagen triple helix (15 A), similarly to the collagen-binding domain of clostridial collagenases (Eckhard et al. 2009, 2011, 2013; Philominathan et al. 2009). Although the computational approach supports the ColAh–collagen physical interaction and collagenolysis, obtained by in vitro studies, it is vital that ITASSER data find experimental validation. Experimental 4
characterization of 3D structure of ColAh will be conducted in the future. The expression of extracellular collagenases by bacteria may be related either to virulence or to nutrition, but in both cases, the activity of these enzymes is dependent on the capacity of these proteins to adhere and hydrolyse collagens. Han and co-workers (Han et al. 2008) have identified a gene involved in Aer. veronii pathogenesis (corresponding to AHA_1043 in the genome of Aer. hydrophila ATCC 7966T). This gene codes for an enzyme belonging to the U32 peptidase family. Unlike bacterial collagenases (M9 family), U32-peptidases do not possess the zinc-binding motif but, taking into account their function in degrading collagen matrices, it is expected that these peptidases may have similar physiological and pathological roles, already demonstrated for the true collagenases belonging to M9 family (Duarte et al. 2014). Further studies are necessary to understand the relative role of these distinct collagenolytic enzymes in bacterial pathogenesis, namely in Aeromonas. We have confirmed that Aer. piscicola AH-3 secretes a 100-kDa active collagenase belonging to the MEROPS peptidase family M9 (PF01752), here named as ColAh. It was possible to confirm that the enzyme hydrolyses and physically interacts with collagen and that it shares the typical motifs of gluzincins. Although we have shown that the ColAh knockout mutant is less cytotoxic than the wild-type strain, further studies are needed to demonstrate the involvement of this enzyme in infection processes by Aer. piscicola AH-3. As recently reviewed (Duarte et al. 2014), although most bacterial collagenases are still uncharacterized, their industrial applications are extensive. These enzymes have been used in the food technology (Zhao et al., 2012), tannery and meat industries (Dettmer et al., 2011; Kanth et al., 2008) in the preparation of cells (Suphatharaprateep et al., 2011; Takagi et al., 2010), and in the production of pharmaceutical compounds (Sakai et al. 1998) and cosmetics (Demina 2009) or even in the (bio)restoration of frescoes (Ranalli et al., 2005). The most important area of application of bacterial collagenases is the health industry: debridement of wounds and burns (Ramundo and Gray 2008), cancer genetic therapy or electro-genetic therapy (Cemazar et al. 2012; Kato et al. 2012), the treatment of lumbar disc herniation (Chu 1987; Wu et al. 2009) and also the treatment of chronic total occlusions (Strauss et al. 2003). Currently, bacterial collagenases are accepted as therapy in several human diseases (Jordan 2008; Bayat 2010; Thomas and Bayat 2010), showing significant results in the treatment of Dupuytren’s disease (DD) and Peyronie’s disease (PD), until recently, were generally treated by invasive surgical methods. Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
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(a)
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Figure 3 Protein alignment studies: (a) Comparison of ColAh (Aeromonas spp.) with other bacterial collagenases. SP—Signal peptide; RPT and PKD domains—repeated sequences (generally associated with protein–protein interactions); PPC domain (bacterial prepeptidase C-terminal domain). (b) Multiple sequence alignment of the catalytic centre of nine microbial collagenases from AH-3, Aeromonas salmonicida (A449), Aeromonas hydrophila (ATCC 7966), Myxococcus xanthus (DK 1622), Shewanella piezotolerans WP3, Burkholderia pseudomallei (Pakistan 9), Vibrio parahaemolyticus (K5030), Clostridium histolyticum and Clostridium perfringens. Collagenases’ amino acid sequence identity and similarity values are indicated. (c) Ribbon diagram of ColAh. The signal peptide is shown in red, catalytic domain in pink, GG-motif in light green and zinc ligands in teal (c1). The repeated sequences are shown in green and in blue (c2). It is predicted that this region is where triple helical collagens binds. Protein structure and function was predicted using I-TASSER* and the image was prepared using the Pymol (http://www.pymol.org).
The identification of novel enzymes involved in bacterial infection mechanisms may lead to the development of specific inhibition therapies. Also, the identification of new bacterial collagenases has an enormous biotechnological potential: the characterization of Aeromonas collagenases surely deserves more attention. Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
Materials and methods Bacterial strains, plasmids and growth conditions The bacterial strains and plasmids used in this study are listed in Table 1. Aeromonas strains were grown in tryptic 5
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Table 1 Bacterial strains and plasmids Strain or plasmid
Relevant characteristics
Source or reference
Escherichia coli DH5a MC1061 Aeromonas piscicola AH-3 AH-405 AH-3::colAh Plasmids pRK2073 pGEMâ-T Easy pFS100 pFS-colAh
F endA1 hsdR17 (rk mk+) supE44 thi-1 recA1 relA1 gyr-A96 Φ80lacZDM15 thi thr1 leu6 proA2 his4 argE2 lacY1 galK2 ara14 xyl5 supE44, kpir O34, wild type AH-3, spontaneous RifR AH-3 colAh insertion mutant with pFS100, KmR Helper plasmid, SpcR PCR cloning vector, ApR pGP704 suicide plasmid, kpir-dependent, KmR pFS100 with an internal fragment of colAh, KmR
Hanahan (1983) Rubir es et al. (1997) Merino et al. (1992) Merino et al. (1992) This study Ditta et al. (1985) Promega Rubir es et al. (1997) This study
Table 2 Primers used Primer pair
Sequence (50 –30 )
Annealing temperature, °C
Amplicon size, bp
A-COL-F1 A-COL-R1 A3-COL-F1 A3-COL-R1
50 -GGAAGGGGACAAGACCATCA-30 50 -CGTTGTTGAGCAGGAACAG-30 50 -AGAGAGCCGAGTGCTCAAT-30 50 -GCATCGCTGTAGTCACTGG-30
60
904
58
636
soy broth (TSB) or on tryptic soy agar (TSA). Escherichia coli strains were grown on Luria-Bertani Miller broth (LB-Miller) and LB-Miller agar (LA). DNA amplification, plasmid and mutant construction Primers A-COL-F1/A-COL-R1 (Table 2) were designed according to the sequence of the putative collagenase gene from Aer. hydrophila ATCC 7966 genome (accession number NC_008570). They were used to amplify a 904bp DNA fragment of colAh (accession number JQ639076) from Aer. piscicola, formerly Aer. hydrophila AH-3 (BeazHidalgo et al. 2009). The following amplification program was used: one cycle at 94°C for 5 min, followed by 40 cycles of 94°C for 1 min, 60 °C for 1 min and 72°C for 4 min and a final extension at 72°C for 30 min. Amplicons were purified, ligated into the plasmid pGEMâ-T Easy and transformed into E. coli DH5a. Transformants were selected on LA containing 100 lg ml1 ampicillin. The plasmid construction was purified, and the insertion was confirmed by sequencing with vector-specific primers M13/SP6 (Promega, Madison, Wisconsin, USA). Primers for mutant construction are indicated in Table 2 (A3-COL-F1/A3-COL-R1) and were used to amplify a 636-bp internal fragment of colAh from Aer. hydrophila AH-3 genomic DNA. The PCR product was purified and cloned into pGEMâ-T Easy as described above, digested with EcoRI, ligated into the kpir replication-dependent suicide plasmid pFS100 (Hanahan 1983; 6
Rubires et al. 1997) and electroporated into E. coli MC1061 (kpir). Transformants were grown in LA containing 50 lg ml1 kanamycin, at 30°C. To obtain the knockout mutant, AH-3::colAh, triparental mating with the mobilizing strain HB101/pRK2073 was used to transfer the plasmid pFS-colAh from E. coli MC1061 to Aer. hydrophila AH-405 (a spontaneous AH-3 rifampicinresistant mutant) (Ditta et al. 1985; Merino et al. 1992). Transconjugants, selected on plates containing 50 lg ml1 kanamycin and 100 lg ml1 rifampicin at 30°C, contained the mobilized plasmid integrated onto the chromosome by homologous recombination, leading to two incomplete copies of the colAh gene (Figure S1). Plasmid integration was verified by Southern blot using middle stringency conditions (homology 75–100%) in 20% formamide hybridization buffer at 62°C, following the manufacturer’s recommendations (Roche-Diagnostic). A probe for colAh gene was generated by PCR using wildtype AH-3 as template DNA and primers A1-COL-F1/A1COL-R1 (Table 2) and PCR conditions described above were used, except that PCR DIG Labelling Mix (Roche) was included in the reaction mixture instead of dNTPs. Zymography analysis After 18-h incubation of each strain (wild centrifuged at 8000 g cell-free supernatants
in LB-Miller at 37°C, 5 ml culture type and knockout mutant) was for 20 min at 4°C to obtain the (CFSs). CFSs were filtered (020-
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lm-pore-size filter, Orange Scientific) prior to use and stored at 4°C until use for no longer than 24 h. For zymography analysis, CFSs were incubated at 25°C for 10 min with sample buffer (100 mmol l1 Tris-HCl, pH 88; 4% SDS; 20% glycerol) in a 1 : 1 ratio (v:v). Proteins were separated by electrophoresis at 4°C in gelatine–polyacrylamide gels (Sarmento et al. 2009). After electrophoresis, proteins were renatured in Triton X-100 [25 % (v/v)] for 30 min at room temperature. The gels were then incubated at 30°C for 16 h in reaction buffer (50 mmol l1 Tris, 5 mmol l1 NaCl, 10 mmol l1 CaCl2, 0001 mmol l1 ZnCl2, pH 76). Afterwards, the gels were stained [1% Coomassie Blue R-250 (SigmaAldrich, Madrid, Spain), 50% ethanol, 10% acetic acid] and distained in 25% ethanol and 10% acetic acid. Gelatinolytic activity was detected by the presence of clear bands on a blue background. Collagenolytic activity Collagenase activity was measured by hydrolysis of the synthetic peptide FALGPA (2-furanacryloyl-Leu-Gly-ProAla) (Van Wart and Steinbrink 1981). The reaction mixture consisted of 1% FALGPA (F5135, Sigma) (v/v) in 50 mmol l1 Tricine, 400 mmol l1 NaCl, 10 mmol l1 CaCl2, 002% NaN3, pH 75, according to Van Wart and Steinbrink (1981). CFSs were prepared as described for zymography and incubated FALGPA at 25°C for 24 h. The absorbance of at least three independent experiments was determined at 345 nm in a NanoDrop (Thermo Scientific). The Cl. histolyticum collagenase (Sigma) was prepared in cold deionized water (10 mg ml1) and used as positive control in a final concentration of 500 nmol l1, according to the manufacturer’s instructions (Sigma). Values are expressed in percentage: a decrease in absorbance of approx. 0500 OD345 unit corresponds to complete hydrolysis (100%) of FALGPA substrate by clostridial collagenase. The influence of protease inhibitors on collagenolytic activity was assessed [1,10-phenanthroline (10 mmol l1) and PMSF (2 mmol l1)]. Thermo-inactivation of collagenolytic activity of CFSs was carried out at 100°C for 5 min. Far-Western blot analysis CFSs were prepared as described for zymography and fractionated on a 10 % SDS-PAGE. Proteins were transferred onto nitrocellulose membranes that were subsequently incubated with human collagen type I (250 lg ml1 in 100 mmol l1 sodium phosphate buffer, pH 74). Membranes were blocked with skim milk in TBS-T [10 mmol l1 Tris-HCl at pH 80, 150 mmol l1 NaCl, 05% (v/v) Tween] and afterwards were incubated Letters in Applied Microbiology © 2014 The Society for Applied Microbiology
overnight with anti-collagen type I primary antibody (Novus Biologicals, UK). Detection was achieved using a horseradish peroxidase-linked secondary antibody (ECL kit, GE Healthcare) according to the manufacturer’s instructions. Resazurin-based cytotoxicity assay CFSs were prepared as described for zymography. Vero cell growth in tissue culture flasks was performed as described previously (Ammerman et al. 2008; Cruz et al. 2013). Afterwards, 100 ll of a suspension of Vero cells in DMEM (Dulbecco’s modified Eagle medium, Gibco) supplemented with 10% FBS (Foetal Bovine Serum, Gibco) was distributed into a 96-well tissue culture plate (2 9 104 cells per well) and incubated for 24 h (80% confluent monolayer) at 37°C in 5% CO2 atmosphere. Serial dilutions [1 : 4; 1 : 16 (v:v)] of CFS in Phosphate Buffered Saline (PBS) were made, and an aliquot of 100 ll of filtered supernatants was added to each well. The microtiter plates were incubated at 37°C in 5% CO2 for 48 h. After cell treatment, the medium was removed by aspiration and 50 ll of DMEM with 10% resazurin (01 mg ml1 in PBS) was directly added to each well. The microtiter plates were incubated at 37°C in 5% CO2 until reduction of resazurin (Al-Nasiry et al. 2007). The absorbance was read at 570 and 600 nm wavelength in a microtiter plate spectrophotometer (Infinite 200, Tecan i-control). The CFS preparations that induced cytopathic effect at least up to 1 : 16 dilution in 50% or more cells were recorded as a cytotoxic positive result as described before (Sha et al. 2002; Ghatak et al. 2006). Aeromonas hydrophila ATCC 7966 and E. coli BL21 (DE3) were used as positive and negative controls, respectively. Each sample was tested in two independent experiments performed in quadruplicate. Data analysis Cytotoxicity and activity data were expressed as means of at least 3 replicates standard error. Statistical significance of differences of collagenolytic activity was determined using Student’s t-tests or by one-way analysis of variance (ANOVA), followed by a Dunnett’s multiple comparison test. Cytotoxicity was analysed for differences with two-way ANOVA, followed by a Bonferroni post-test, using a significance level of 001 or 0001. DNA sequencing and analysis Sequencing reactions were carried out using the ABI Prism dye terminator cycle sequencing kit (Perkin Elmer). The DNA sequence was translated in all six frames, and 7
ColAh: Aeromonas collagenase
the deduced amino acid sequences of all open reading frames (ORFs) were compared with sequences from the nonredundant GenBank and EMBL databases using the BLAST (Altschul et al. 1997) tool. ClustalW was used for multiple sequence alignments (Figure S2 Thompson et al. 1994). Protein structure and function prediction The deduced amino acid sequence of colAh gene was analysed using the Simple Modular Architecture Research Tool (SMART; Letunic et al. 2008). The 3D model of the ColAh (residues 1–915) was predicted using ab initio modelling. In this study, the I-TASSER method (Ambrish et al. 2010), a protein structure modelling approach based on an algorithm consisting of consecutive steps of threading, fragment assembly and iteration, was used to obtain structure with the lowest energy. Function insights were derived by matching the predicted models with protein function databases. Images were produced using PyMOL (DeLano 2002). Nucleotide sequence accession number The nucleotide sequence of colAh from Aer. piscicola AH-3 was deposited in the GenBank under the accession number JQ639076. Acknowledgements This work was supported by European Funds through COMPETE and by National Funds through the Portuguese Science Foundation (FCT) within project PEst-C/ MAR/LA0017/2013. The author also wish to acknowledge FCT for grants to AC Esteves, AS Duarte and E Cavaleiro (FCT; BPD/38008/2007, BPD/46290/2008 and BD/47502/ 2008). Part of this work was also supported by Plan Nacional de I+D+I and FIS grants (Ministerio de Educaci on, Ciencia y Deporte and Ministerio de Sanidad, Spain) and by Generalitat de Catalunya (Centre de Referencia en Biotecnologia). Conflict of Interest No conflict declared. References Al-Nasiry, S., Geusens, N., Hanssens, M., Luyten, C. and Pijnenborg, R. (2007) The use of Alamar Blue assay for quantitative analysis of viability, migration and invasion of choriocarcinoma cells. Hum Reprod 22, 1304–1309.
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Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1. Directed mutagenesis by homologous recombination of AH-3. Figure S2. Alignment of deduced amino acid sequences of the ColAh from Aeromonas hydrophila (AH-3) with collagenase family protein from Aer. salmonicida (GENE ID: 4997592 ASA_3723) and putative collagenase from Aer. hydrophila (GENE ID: 4487009 AHA_0517).
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