Dispersing biofilms with engineered enzymatic bacteriophage

Izvorni članek: Lu, T., et al, Dispersing biofilms with engineered enzymatic bacteriophage, PNAS, 2007.<ref>[1]</ref>

Introduction

When faced with certain challenges in various living habitats, bacteria have the ability to form biofilms, or organized aggregates of microorganisms living within an extracellular polymeric matrix (EPM) <ref name="jamal">https://www.ncbi.nlm.nih.gov/pubmed/29042186</ref>. The complex EPM is formed by heterogeneous extracellular polymeric substances (EPS), which is occupied mostly by water (97%) and other macromolecules in lower concentrations (proteins (~2%), polysaccharides (1-2%); nucleic acids (<1%) and ions (bound and free)) <ref name="jamal" />. This organized communities are formed on either biological or non-biological surfaces, and allow bacterium to surpass harsh environmental conditions, such as UV exposure, metal toxicity, acid exposure, dehydration and salinity, phagocytosis and several antibiotics and antimicrobial agents <ref name="dva">https://www.ncbi.nlm.nih.gov/pubmed/15040259</ref>. The ability to surpass latter conditions represent a challenge in the medical, industrial and food branches, as biofilm formation accounts for over 65% of microbial infections, and over 80% of chronic infections based on the statistics from the National Institutes of Health <ref name="dva" />. Due to the concerning numbers, and the rise of antibiotic resistance, a novel and effective treatment for bacterial biofilms is necessary. The main target in biofilm degradation is disruption of the EPM, more precisely to target the EPS and mechanism involved in EPS production and secretion (DNase, exopolysaccharides, protein components, cGMP/cAMP levels, signal and secretion pathways) <ref>[2]</ref>. In the following paper, we will show a potential treatment with synthetically engineered bacteriophages that possess the enzymatic ability to disperse bacterial biofilms.

Bacteriophages and biofilms

The use of bacteriophages against bacterial infections is not a novelty method, as it is dates from the early 20th century. With the growing knowledge of engineering and manipulating biological organisms, and the highly annotated phage genome, makes bacteriophages prime candidates for targeting biofilms. Bacteriophages are viruses that infect and replicate within bacteria, and in comparison with antibiotics and other antimicrobial agents, possess the ability to penetrate biofilms, and disperse biofilms by various proposed mechanisms <ref>[3]</ref> <ref>[4]</ref>. As mentioned above, enzymatic targeting exopolysaccharides with EPS-degrading is one of the possible strategies for targeting biofilms <ref name="tri" />. The challenge lies in isolating a natural phage that is both specific for the bacteria to be targeted and expresses a relevant EPS-degrading enzyme. The solution lies in designing an artificial biofilm-degrading bacteriophage that express a specific EPS-degrading enzyme.

Dispersin B

While studying the strain A. actinomycetemcomitans which causes periodontal disease in adolescents and its biofilm formation and degradation properties for disease spreading, researchers have come upon a novel specific biofilm-releasing glycoside hydrolase <ref name="tri">https://www.ncbi.nlm.nih.gov/pubmed/15878175</ref>. This novel protein disperin B or DspB has the ability to degrade an important EPS polysaccharide adhesin known as β-1,6-N-acetyl-D-glucosamine <ref name="tri" />. N-acetyl-D-glucosamine residues form various polymeric structures with their linear β-1,6-linkages, such as PIA and PNAG (abbreviations)<ref name="tri" />. By hydrolyzing polymers, the protein disrupts the formation of the biofilm matrix and allows adherent cells to be released <ref name="tri" />. From a structural point of view, disperin B consist of a single domain with intertwining α/β structures <ref name="tri" />. The protein is a member of the 20 β-hexosaminidases family (GH-20), and has a highly conserved acidic active site (D183, E184, E332) which cleaves terminal monosaccharide residues from the non-reducing end of the polymers <ref name="tri" />.

Design of enzymatically active bacteriophage

Once a suitable protein for biofilm removal that covers a wide specter was found, the design of engineered bacteriophages could commence. The idea is based on exploiting the lytic phage life cycle, which is based on hijacking the cell machinery, and synthesizing components of the phage genome <ref name="cet">https://www.ncbi.nlm.nih.gov/pubmed/12776216</ref>. Once all sufficient components are available, the phages reassembles inside the cell, causing the cell to burst and releasing its component in to the local environment <ref name="cet" />. This two-pronged attack strategy, would exploit the enzyme to remove bacterial biofilm, and the phage infections to lyse cells, while achieving high concentrations of the enzyme and lytic phage. The backbone of the design is based on using an E.coli specific lytic T7 phage, which was modified in a way that it had a few of nonessential gene deletions. The T7 phage had one of the first completely sequenced genomes (40-kb) that codes for 55 proteins <ref name="pet">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2525648/</ref>. The T7 is widely used in molecular biology and has various traits that make this strain suitable for phage experiments <ref name="pet" />. The biofilm removing T7 phage was design to express DspB under the strong control of T7 ϕ10 promoter intracellularly during the infection, so that it could be released in to the environment upon cell lysis. The experiment was focused on strains that possess the F-plasmid, as it enhances biofilm maturation, and forms more thick biofilms, making them a more appealing group. Bacterial strains that contain the F-plasmid can disrupt efficient T7 replication. To tackle this problem, they inserted a 1.2 gene from T3 phage so the phage would not be limited only to strains that lack the F-plasmid, but to widen the spectrum of target <ref name="set">https://www.ncbi.nlm.nih.gov/pmc/articles/PMC208987/</ref>. Gene 1.2 is an inhibitor of the host dGTPase, which is involved in the process of DNA replication <ref name="set" />. A control was designed by cloning an S-tag into the T7 genome, to assure quality results. The phages were named T7DspB and T7Control respectively.

Characterization of enzymatically active bacteriophage

The first task was to determine if T7DspB was more effective against biofilms than T7Control. Effectiveness of modified phages was also compared to wild-type T3 and T7 phages to determine if modified phages possess an edge over natural occurring ones. To measure effectiveness, crystal violet (CV) methods was used, which is based on staining attached cells (in our case bacterial cells/biofilms) with crystal violet dye, which binds to proteins and DNA <ref name="ket">https://www.ncbi.nlm.nih.gov/pubmed/27037069/</ref>. Those cells that undergo cell death lose their adherence, which shows as reducing amount of CV staining in a culture <ref name="ket" />. Absorbance (A600) measurement after a 24 h treatment period had shown that T7DspB phage removes biofilms with more efficiency than wild-type and especially T7Control. To confirm primary results, additional test such as sonication were ran, to obtain viable cell counts (CFU per peg) for bacteria surviving in biofilms after treatment. The results show consistency with CV, and confirm that T7DspB shows much more promising signs of biofilm removal than other tested phages, especially in comparison with T7Control. As mentioned above, the idea is based on a two-pronged attack strategy. Promising results were obtained for enzymatic activity of DspB, but also we have to ensure that phages sustain sufficient replication. PFU counts from microtiter plate wells and biofilms (after sonication) show that PFU counts were significantly higher in plates than biofilms, and that overall PFU surpassed initial 10³ PFU by a few orders of magnitude which confirms efficient phage multiplication.

Time courses and dose-response for enzymatically active bacteriophage treatment

Experiments to optimize the time-course and dose-response for both enzymatic activity and phage replication were carried out. Time-course results shown that after a 24h period of treatment, T7DspB had biofilms cell densities of two magnitudes lower than T7Control and 99,9% in comparison with untreated biofilms. Further on, time dependence on phage replication was tested, and showed that both T7Control and T7DspB began to multiply rapidly after inoculation in a similar fashion. Dosage response showed that T7DspB had lower cell densities at starting inoculation levels (PFU 10²) than T7Control, while higher inoculation concentrations showed even more efficiency against removing biofilms. Phage dosage tested exhibited phage multiplication within the biofilm.

Discussion

The following experiments have shown that enzymatically modified phage shows greater efficiency in biofilm removal than natural accruing phages. Future improvements to this design may include directed evolution for optimal enzyme activity, delaying cell lysis or using multiple phage promoters to allow for increased enzyme production, targeting multiple biofilm EPS components with different proteins as well as targeting multi-species biofilm with a mixture of different species-specific engineered enzymatically active phage, and combination therapy with antibiotics and phage to improve the efficacy of both types of treatment. This strategy allows opens a possibility of establishing a library of biofilm dispersing phage. The upside of this method is that it does not need to deliver, express and purify large enzyme concentrations to the site of infection. This type of phage therapy should be looked into as additional therapy for treating bacterial biofilms in various industries, but not before several challenges are overcome. Firstly, a properly designed clinical trial is needed, which will tackle the problems such as phage development resistance, immunogenicity in humans, body clearance, release of toxins after cell lysis and phage specificity. Once all the challenges are overcome, phage therapy against biofilms will be considered as our first line of defense.

References

[1]. Lu, T., et al., Dispersing biofilms with engineered enzymatic bacteriophage, PNAS, 2007.

[2]. Jamal, M., et al., Bacterial biofilm and associated infections, Journal of the Chinese Medical Association, 2017.

[3]. Hall-Stoodley, L., et al., Bacterial biofilms: From the natural environment to infectious disease, Nature Reviews, 2004.

[4]. Koo, H., et al., Targeting microbial biofilms: current and prospective therapeutic strategies, Nature Reviews, 2017.

[5]. Hu, B., et al., The bacteriophage t7 virion undergoes extensive structural remodeling during infection, Science, 2013.

[6]. Harper, D., et al., Bacteriophages and Biofilms, Antibiotics, 2014.

[7]. Ramasubbu, N., et al., Structural Analysis of Dispersin B, a Biofilm-releasing Glycoside Hydrolase from the Periodontopathogen Actinobacillus actinomycetemcomitans, Journal of Molecular Biology, 2005.

[8]. Campbell, A., The future of bacteriophage biology, Nature Reviews, 2003.

[9]. Serwer, P., et al., Evidence for bacteriophage T7 tail extension during DNA injection, BMC Research Notes, 2008.

[10]. Schmitt, C.K., et al., Genes 1.2 and 10 of bacteriophages T3 and T7 determine the permeability lesions observed in infected cells of Escherichia coli expressing the F plasmid gene pifA, Journal of Bacteriology, 1991.

[11]. Feoktistova, M., et al., Crystal Violet Assay for Determining Viability of Cultured Cells, Cold Spring Harbor Protocols, 2018.

Abbreviations

extracellular polymeric matrix (EPM)

extracellular polymeric substances (EPS)

partially deacetylated Poly-β-1,6-N-Acetyl-glucosamine(PIA)

Poly-β-1,6-N-Acetyl-glucosamine (PNAG)

crystal violet (CV)