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Keywords:

  • sponge bacteria;
  • collagenase;
  • metagenomics;
  • collagen

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Collagen is an important, extracellular structural protein for metazoans and provides a rich nutrient source for bacteria that possess collagen-degrading enzymes. In a symbiotic host system, collagen degradation could benefit the bacteria, but would be harmful for the eukaryotic host. Using a polyphasic approach, we investigated the presence of collagenolytic activity in the bacterial community hosted by the marine sponge Cymbastela concentrica. Functional screening for collagenase activity using metagenomic library clones (227 Mbp) and cultured isolates of sponge's bacterial community, as well as bioinformatic analysis of metagenomic shotgun-sequencing data (106 679 predicted genes) were used. The results show that the abundant members of the bacterial community contain very few genes encoding for collagenolytic enzymes, while some low-abundance sponge isolates possess collagenolytic activities. These findings indicate that collagen is not a preferred nutrient source for the majority of the members of the bacterial community associated with healthy C. concentrica, and that some low-abundance bacteria have collagenase activities that have the potential to harm the sponge through tissue degradation.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Collagen is the major component of extracellular matrices of all metazoan life and represents an important protein conferring integrity and the physical form of eukaryotic organisms (Harrington, 1996; Exposito et al., 2008). Sponges are among the oldest Metazoa and often contain collagen, which is either dispersed as thin fibrils or organized as bundles, termed spongin, in the intercellular matrix (Simpson, 1984; Brusca & Brusca, 1990). The expression of collagen is known to be essential for the development and structural integrity of sponges (Garrone et al., 1975; Shimizu & Yochizato, 1993; Krasko et al., 2000).

Sponges harbour specific bacterial communities in different cellular compartments, often for an extended period of time, and hence close associations between the microorganisms and the sponge host have been established (Taylor et al., 2007). Collagen is an essential and abundant part of the internal mesohyl structure of most sponges (and in particular the Demospongia), where many microorganisms reside. As a rich source of nitrogen and carbon, collagen could provide nutrients for the sponge-associated microorganisms, and this may potentially have implications for the structural integrity of the host. A few cases of sponge diseases have been attributed to the presence of bacterial pathogens (Gaino & Pronzato, 1989; Webster et al., 2002; Mukherjee et al., 2009) and collagenolytic enzymes have been speculated to lead to tissue necrosis in sponges.

Generally, bacterial collagenases, including the well-characterized enzymes from Clostridium sp. (Matsushita et al., 1994) and Vibrio sp. (Yu & Lee, 1999; Vaitkevicius et al., 2008), have been linked to pathogenicity and are regarded as virulence factors in human disease. They can cause tissue destruction and thus facilitate the invasion of a pathogen into internal parts of host tissue (Smith & Merkel, 1982; Harrington, 1996). The presence of collagenolytic bacteria in the marine environment is more widely distributed than previously thought (Dreisbach & Merkel, 1978; Takeuchi et al., 1992; Thomas et al., 2008). For example, Merkel et al. (1975) found that 44% of marine isolates obtained from coastal waters were capable of producing collagenolytic enzymes. It has also been found that marine bacteria utilize collagenolytic enzymes to obtain nutritional diversity, thus conferring them with a selective advantage (Harrington, 1996; Thomas et al., 2008).

Given the wide distribution of collagen-degrading activities in the marine environment and their potential negative impact on a eukaryotic host, we investigated the presence of collagenolytic enzymes in the bacterial community associated with a healthy sponge. We used a polyphasic approach that involved screening of a metagenomic library and cultured sponge isolates for the degradation of gelatin, a denatured form of collagen, as well as extensive bioinformatic analysis of bacterial metagenomic shotgun-sequencing data.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Strains and clone libraries

The marine demosponge Cymbastela concentrica was collected by SCUBA diving from Bare Island (Thomas et al., 2010) and washed twice (5 min each time with agitation at 200 r.p.m.) in calcium- and magnesium-free seawater (L-1: 25 g NaCl, 0.8 g KCl, 1 g Na2SO4, 0.04 g NaHCO3) to remove planktonic or loosely associated microorganisms. A culture collection was obtained by plating serially diluted and homogenized sponge samples onto marine broth 2216 (MB; Becton, Dickinson and Company, Sparks, MD), supplemented with 1.5% agar. MB has been shown to recover similar amounts of bacteria from sponge samples as other medium types (including those that contain sponge extracts) (Olson et al., 2000) and is therefore likely to represent the generally culturable bacteria from C. concentrica. Plates were incubated at room temperature for 5 days and pure cultures were obtained by restreaking colonies onto fresh agar. The phylogenetic identity of the bacterial isolates was assessed by PCR amplification and sequencing of the 16S rRNA gene using universal primers (27F and 1492R) (Lane, 1991). Metagenomic libraries of the bacterial community associated with C. concentrica were previously constructed from two specimens in Yung et al. (2009). The libraries contained a total of 6500 inserts (average size of 35 kb) cloned in the fosmid vector pCC1FOS and hosted in Escherichia coli Epi300. All sponge samples used in this and our previous studies, which yielded metagenomic fosmid libraries and shotgun-sequencing datasets (Yung et al., 2009; Thomas et al., 2010), showed no signs of tissue damage and were healthy specimens.

Activity screening for gelatinolytic enzymes

Colonies were stabbed onto 96-well microtitre plates containing in each well 180 μL of MB for marine isolates, or LB10 (L-1:10 g tryptone, 5 g yeast extract, 10 g NaCl; pH 7.5) supplemented with 12.5 μg mL−1 chloramphenicol and 0.01% w/v arabinose for E. coli clones, both solidified with 12% gelatin (Oxoid, Adelaide, Australia). Colonies were grown at 25 °C for 5 days and then cooled at 4 °C for 3 h before checking for liquefaction by adding 3 μL of the 6 × gel loading dye (Fermentas Inc., Glen Burnie, MD) to each well. Evidence of liquefaction was established if the dye diffused rapidly (within 5 s) through the well and sank to the bottom. The Pseudoalteromonas tunicata D2 wild-type strain (Holmström et al., 1998) and a genomic library of P. tunicata DNA, which was constructed by Burke et al. (2007) and which used the same fosmid vector and host strain as the metagenomic library described above, were used as positive controls.

Assay of collagenolytic activity using Azocoll

Cultures exhibiting activities on the solidified gelatin were subjected to a further assay using Azocoll, an insoluble, ground collagen, to which an azo-dye is attached. The assay was conducted in triplicates. Strains were grown for approximately 48 h at room temperature in MB and bacterial cells were harvested by centrifugation at 8000 g for 10 min. Cell pellets were resuspended in the Azocoll substrate at a concentration of 5 × 108 CFU mL−1, supplemented with a final concentration of 1 mM CaCl2. To prepare the substrate, 2.0 mg mL−1 of Azocoll (Sigma, St. Louis, MO) was washed twice using 0.01 M phosphate-buffered saline (pH 7.4) as described in Jiang et al. (2007). The tubes were incubated at room temperature with shaking at 90 r.p.m. for 24 h before centrifugation for 5 min to remove the undegraded Azocoll. Supernatants were taken for the measurement of OD520 nm. Escherichia coli Epi 300 pCC1FOS and Pseudomonas aeruginosa PAO1 strain were used as a negative and a positive control, respectively.

Analysis of collagenase genes in sponge shotgun metagenome data

The shotgun metagenome-sequencing data (92.6 Mbp of unique sequence) of the bacterial community associated with two C. concentrica specimen (BBAY04 and BBAY15) described in Thomas et al. (2010) were searched for genes that were annotated as collagenase/matrix proteinase-related genes. Searches were performed on KEGG (Kanehisa & Goto, 2000), COG (Tatusov et al., 2003), Swiss-Prot (Boeckmann et al., 2003) and TIGRFAM (Haft et al., 2003) annotations using the keywords: ‘collagenase’, ‘Zn-dependent aminopeptidase’, ‘metalloproteinase’, ‘matrixin’ and ‘matrix proteinase’. The results were checked manually and matches that had an e-value lower than 1 × 10−20 in at least one annotation were regarded as putative collagenase protein sequences. In addition, collagenase-related proteins were retrieved from NCBI's protein sequence database and the curated Swiss-Prot database (Boeckmann et al., 2003) using the keywords: ‘gelatinase’, ‘microbial collagenase’ and ‘matrix proteinase’ as well as proteins with the M9 peptidase and peptidase U32 conserved domains (which are domains in collagenases). Those database sequences were searched against the C. concentrica protein dataset with blastp (Altschul et al., 1990). Sequences with low percentage coverage (<50%) and percentage identity (<20%) were excluded from further analysis. The putative collagenase sequences were searched against the curated and non-curated database in Swiss-Prot and aligned using clustalw with default parameters (Thompson et al., 1994).

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

In order to investigate the presence of collagenase activities in the bacterial community associated with the sponge C. concentrica, we firstly established a high-throughput screen for fosmid clone libraries in E. coli (see Materials and methods). We used gelatin, a denatured form of collagen, as an initial screening substrate, as it can solidify a growth medium and its degradation can therefore be easily detected. A screen of 900 fosmid clones containing genomic DNA of P. tunicata, an organism known to produce collagenase, identified three positive E. coli fosmids (data not shown). Sequencing of these fosmids revealed three different genes, two of which encoded proteins that have previously been annotated as collagenases (Thomas et al., 2008). The sequences have pair-wise sequence identities of <5%, indicating that our screen can detect a large variety of expressed gelatinolytic enzymes. Using the same procedure to screen 6500 metagenomic clones (227 Mbp), which covered the dominant groups of bacteria in the sponge (Yung et al., 2009), did not reveal any gelatinolytic activity, suggesting that the collagenase proteins are either not encoded by the genomes of bacteria contained in the library or that they are poorly expressed.

To further investigate the collagenolytic/gelatinolytic potential in the sponge's bacterial community, a comprehensive and manually supported analysis of available shotgun-sequencing data was performed. One gene in the sponge metagenome dataset (BBAY15; Thomas et al., 2010) could be confidently classified as collagenase. The protein sequence (ID=1108814257276_ORF001, 352 amino acids) had a blastpe-value of 4 × 10−91, 49% identity and 100% coverage with the collagenase precursor PrtC protein (334 amino acid long) in Porphyromonas gingivalis (Kato et al., 1992). Sequence alignment of this protein sequence against PrtC indicated that it contains the signature pattern of the peptidase U32 family: E-x-F-x(2)-G-[SA]-[LIVM]-C-x(4)-G-x-C-x-[LIVM]-S (Fig. 1) (Kato et al., 1992).

image

Figure 1. clustalw alignment of the amino acid sequence of the 1108814257276 ORF001 and the experimentally (with enzymatic activity) confirmed sequences (PrtC) from Swiss-Prot database. P59916 and P33437 are the PrtC proteins of the collagenase precursor from Porphyromonas gingivalis (Bacteriodes gingivalis). ‘*’, Column of the alignment contains identical amino acid residues in all sequences. ‘:’, Column of alignment contains conserved substitutions of amino acids. ‘.’, Column of alignment contains semi-conserved substitutions. ‘-’, Gaps in the amino acid sequence. Blank, column of the alignment contains dissimilar amino acids. Box, the signature sequence for the Peptidase U32 family.

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Our previous study on the bacterial community of C. concentrica has identified 14 phylotypes that account for 89% (±2%) of the total diversity in three 16S rRNA gene libraries (1981 sequences in total) (Thomas et al., 2010). The 319.6 Mbp of metagenomic information analysed here through screening and similarity searches is equivalent to 80 bacterial genomes (assuming an average genome size of 4 Mbp) and is therefore likely to cover those dominant phylotypes on average at least 5.5-fold. The presence of only one gene encoding for a collagenase in the 106 679 predicted genes of metagenomic database of C. concentrica (Thomas et al., 2010), in addition to the lack of expressed gelatinolytic activity, strongly suggest that the dominant members of the bacterial community within the sponge contain very few proteins related to collagenases.

To further explore the presence of collagenases, we generated a collection of 40 bacterial isolates (comprising a total of 19 unique phylotypes) from the sponge and showed through 16S rRNA gene sequencing that they covered 17 distinct genera within the classes Alpha-, Gammaproteobacteria, Flavobacterales and Bacilli (see Supporting Information, Table S1). We screened this collection for gelatinolytic activity and found seven positive isolates. Their 16S rRNA gene sequences showed that they belonged to four unique phylotypes (Table 1). All three isolates from the Vibrio-related phylotype showed gelatinolytic activity, while two isolates of the Zobellia-related phylotype were positive. For the latter phylotype, we also found six isolates in our culture collection that had no gelatinolytic activity, indicating a strain-level variation. The collagenolytic activity of strains representing the four species was assessed by their ability to degrade Azocoll, demonstrating that all, except for the Zobellia sp.-related strain, were capable of degrading (azo-dye impregnated) collagen (Fig. 2). These four organisms, as well as the other isolates, were regarded as low-abundance members of the sponge community, as they were not present in the 16S rRNA gene sequence database, the shotgun-sequencing dataset and the fosmid library from C. concentrica (Yung et al., 2009; Thomas et al., 2010). While not representing the major bacterial community in C. concentrica, it is noteworthy that the collagenase-producing sponge isolates identified in this study are phylogenetically closely related to taxa of known pathogens. For example, isolate I's 16S rRNA gene sequence is 99% identical to those of Vibrio crassostreae, which has been reported a pathogen of oysters (Faury et al., 2004) and Vibrio splendidus, which causes disease in turbot larvae. Other Vibrio and Bacillus species have also been reported to contain collagenase genes with potential roles in disease (Dreisbach & Merkel, 1978; Smith & Merkel, 1982; Mäkinen & Mäkinen, 1987; Lund & Granum, 1999).

Table 1.   Sponge isolates identified to exhibit gelatinase activity
Isolate namesSimilarity scoreOrganism with the closest match; accession numberClassColony morphology
  1. Similarity scores (according to the Ribosomal Database Project database) of the 16S rRNA gene sequence to the closest cultured representatives, taxonomy, and the respective colony morphologies are presented.

D0.957Zobellia sp. KMM 3665; AB084262FlavobacteralesYellow, mucoid
I1Vibrio crassostreae; AJ582809GammaproteobacteriaWhite, opaque
R1Bacillus pumilus; EU236743BacilliWhite, dry
S0.999Shewanella sp. MJ5323; DQ531951GammaproteobacteriaPeach-pink, wet
image

Figure 2.  Bar chart of the OD measurements of the Azocoll assay of each bacterial isolate (Table 1) that were identified to liquefy 12% solid gelatin. Escherichia coli (EC) and Pseudomonas aeruginosa PAO1 (PA) strains were used as negative and positive controls, respectively, for the assay in marine broth. D, relative of Zobellia sp. KMM 3665; I, relative of Vibrio crassostreae; R, relative of Bacillus pumilus; S, relative of Shewanella sp. MJ5323 (see also Table 1).

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Our results indicate that collagenase activity is not a dominant feature of the abundant bacteria in C. concentrica and that hence collagen might not be a preferred nutrient source. The identification of low-abundance bacteria with collagenase activity, however, raises the possibility that collagen in the sponge mesohyl could undergo degradation, potentially leading to tissue destruction. The aetiology of sponge diseases is often difficult to identify and only in a few cases have tissue disintegration and sponge disease been attributed to the presence of bacterial pathogens. For example, an alphaproteobacterium (strain NW4327) producing collagenolytic enzyme was identified as the primary causative agent of necrosis in the sponge tissue of Rhopaloeides odorabile (Webster et al., 2002; Mukherjee et al., 2009). Potential spongin-fibre invading bacterial pathogens have also been found in the sponge Spongia officinalis (Gaino & Pronzato, 1989) and Ianthella basta (Cervino et al., 2006). Given the general involvement of collagenases in tissue destruction, it is likely that bacteria capable of producing collagenases are being selected against and will not become dominant members in a healthy sponge. This could explain the low abundance of such bacteria in C. concentrica, which we have never observed to be diseased in the field. Nevertheless, environmental stresses imposed onto sponges are likely to alter the abundance of specific members of the bacterial community, as has been demonstrated in response to increased temperature for the sponge R. odorabile (Webster et al., 2008). This may provide an opportunity for pathogenic bacteria, including low-abundance, collagenase-producing organisms, to degrade the sponge tissue and obtain nutrients.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This work was supported by the Australian Research Council, the Betty and Gordon Moore Foundation and the Centre for Marine Bio-Innovation.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Table S1. Bacterial isolate collection from the sponge Cymbastela concentrica.

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FilenameFormatSizeDescription
FML_2306_sm_tables1.doc47KSupporting info item

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.