Engineered Escherichia coli Silver-Binding Periplasmic Protein That Promotes Silver Tolerance

This is the published version. Copyright © 2012, American Society for Microbiology. All Rights Reserved.Silver toxicity is a problem that microorganisms face in medical and environmental settings. Through exposure to silver compounds, some bacteria have adapted to growth in high concentrations of silver ions. Such adapted microbes may be dangerous as pathogens but, alternatively, could be potentially useful in nanomaterial-manufacturing applications. While naturally adapted isolates typically utilize efflux pumps to achieve metal resistance, we have engineered a silver-tolerant Escherichia coli strain by the use of a simple silver-binding peptide motif. A silver-binding peptide, AgBP2, was identified from a combinatorial display library and fused to the C terminus of the E. coli maltose-binding protein (MBP) to yield a silver-binding protein exhibiting nanomolar affinity for the metal. Growth experiments performed in the presence of silver nitrate showed that cells secreting MBP-AgBP2 into the periplasm exhibited silver tolerance in a batch culture, while those expressing a cytoplasmic version of the fusion protein or MBP alone did not. Transmission electron microscopy analysis of silver-tolerant cells revealed the presence of electron-dense silver nanoparticles. This is the first report of a specifically engineered metal-binding peptide exhibiting a strong in vivo phenotype, pointing toward a novel ability to manipulate bacterial interactions with heavy metals by the use of short and simple peptide motifs. Engineered metal-ion-tolerant microorganisms such as this E. coli strain could potentially be used in applications ranging from remediation to interrogation of biomolecule-metal interactions in vivo

Targeting Hepatitis B Virus with Zinc Finger Nucleases

Despite an existing effective vaccine, hepatitis B virus (HBV) remains a major public health concern. There are effective suppressive therapies for HBV, but they remain expensive and inaccessible to many, and not all patients respond well. Furthermore, HBV can persist as genomic covalently closed circular DNA (cccDNA) that remains in hepatocytes even during otherwise effective therapy and facilitates rebound in patients after treatment has stopped. Therefore, the need for an effective treatment that targets active and persistent HBV infections remains. As a novel approach to treat HBV, we have targeted the HBV genome for disruption to prevent viral reactivation and replication. We generated 3 zinc finger nucleases (ZFNs) that target sequences within the HBV polymerase, core and X genes. Upon the formation of ZFN-induced DNA double strand breaks (DSB), imprecise repair by non-homologous end joining leads to mutations that inactivate HBV genes. We delivered HBV-specific ZFNs using self-complementary adeno-associated virus (scAAV) vectors and tested their anti-HBV activity in HepAD38 cells. HBV-ZFNs efficiently disrupted HBV target sites by inducing site-specific mutations. Cytotoxicity was seen with one of the ZFNs. scAAV-mediated delivery of a ZFN targeting HBV polymerase resulted in complete inhibition of HBV DNA replication and production of infectious HBV virions in HepAD38 cells. This effect was sustained for at least 2 weeks following only a single treatment. Furthermore, high specificity was observed for all ZFNs, as negligible off-target cleavage was seen via high-throughput sequencing of 7 closely matched potential off-target sites. These results show that HBV-targeted ZFNs can efficiently inhibit active HBV replication and suppress the cellular template for HBV persistence, making them promising candidates for eradication therapy

scAAV-ZFN induced cytotoxicity.

<p>(<b><i>a</i></b>) HepAD38 cells were transduced with scAAV2 vectors expressing GFP and mCherry reporter genes, ZFN pairs 1, 2 or 3, or mismatched ZFN pairs at a total MOI of 10000 genomes/cell, or all 3 ZFN pairs at a total MOI of 10000 (low) and 30000 (high) genomes/cell. At 48 hours post transduction, cell viability was measured by MTT assay and reported as percent of control. (<b><i>b</i></b>) HepAD38 cells transduced with scAAV2 vectors expressing individual ZFN half sites at a MOI of 5000 genomes/cell were also analyzed. (<b><i>c</i></b>) Untreated cells and cells treated with scAAV2 vectors expressing reporter genes, ZFN pairs 1, 2 or 3 or a mismatch ZFN pair were monitored for cell viability at 3, 5, 7 and 14 days post transduction. eGFP – enhanced green fluorescent protein; ZFN – zinc finger nuclease.</p

Levels of HBV DNA present in ZFN-treated HepAD38 cells and levels of secreted infectious HBV.

<p>(<b><i>a</i></b>) For experiments shown in panels <b><i>b</i></b> and <b><i>c</i></b>, HepAD38 cells in the presence of dox were transduced with ZFN- or control-expressing scAAV2 vectors (total MOI 10000 genomes/cell) and 3 days later dox was removed from culture medium to enable HBV replication. Cells were left in culture for a further 7 days before HBV genomic levels were quantified in cells (<b><i>b</i></b>) and infectious HBV levels were quantified in supernatants (<b><i>c</i></b>) by ddPCR. (<b><i>d–e</i></b>) HepAD38 cells were passaged every two to three days over the course of 14 days following treatment with ZFN- or control-expressing scAAV vectors and monitored for cellular (<b><i>d</i></b>) and supernatant (<b><i>e</i></b>) HBV levels by ddPCR. ddPCR – droplet digital polymerase chain reaction; dox – doxycycline; eGFP – enhanced green fluorescent protein; scAAV – self-complementary adeno-associated virus; UC – untreated control; ZFN – zinc finger nuclease. *p≤0.05.</p

Summary of HBV-ZFN-induced mutagenesis in HepAD38 cells.

<p>*% indel  =  (1−(1−(a+b)/(a+b+c))∧0.5)*100; where a and b =  cut bands, c =  uncut band.</p

HBV-ZFN target sites.

<p>(<b><i>a</i></b>) HBV rcDNA genome map showing HBV ORFs and ZFN target site locations. (<b><i>b</i></b>) HBV target site sequence heterogeneity for HBV-ZFN pairs 1–3 across 3847 complete HBV genotype A–H sequences found in Genbank. For each ZFN pair, the target sequence, consensus sequence logo plot and nucleotide Rate4Site (R4S) scores are shown. ZFN spacer nucleotides are highlighted in red and divergent nucleotides between the ZFN target site and the consensus sequence are bold and underlined. Rate4Site scores are graded from low (white) to high (black) sequence heterogeneity. Single nucleotide polymorphisms present in the HepAD38 genomic HBV sequence are shown above each target site in blue. ORF – open reading frame; R4S – Rate4Site; ZFN – zinc finger nuclease.</p

SMRT sequencing results showing on-target and off-target activity of HBV-ZFNs.

<p>LQ, low quality; TS, target site; *Significance between Z1 and Z3 (p<0.01), Z1 and Z2 (p<0.001), Z2 and Z3 (p<0.001).</p