HK022 bacteriophage Integrase mediated RMCE as a potential tool for human gene therapy.

HK022 coliphage site-specific recombinase Integrase (Int) can catalyze integrative site-specific recombination and recombinase-mediated cassette exchange (RMCE) reactions in mammalian cell cultures. Owing to the promiscuity of the 7 bp overlap sequence in its att sites, active 'attB' sites flanking human deleterious mutations were previously identified that may serve as substrates for RMCE reactions for future potential gene therapy. However, the wild type Int proved inefficient in catalyzing such RMCE reactions. To address this low efficiency, variants of Int were constructed and examined by integrative site-specific recombination and RMCE assays in human cells using native 'attB' sites. As a proof of concept, various Int derivatives have demonstrated successful RMCE reactions using a pair of native 'attB' sites that were inserted as a substrate into the human genome. Moreover, successful RMCE reactions were demonstrated in native locations of the human CTNS and DMD genes whose mutations are responsible for Cystinosis and Duchene Muscular Dystrophy diseases, respectively. This work provides a steppingstone for potential downstream therapeutic applications.

Plasmid-DNA Delivery by Covalently Functionalized PEI-SPIONs as a Potential 'Magnetofection' Agent.

Nanoformulations for delivering nucleotides into cells as vaccinations as well as treatment of various diseases have recently gained great attention. Applying such formulations for a local treatment strategy, e.g., for cancer therapy, is still a challenge, for which improved delivery concepts are needed. Hence, this work focuses on the synthesis of superparamagnetic iron oxide nanoparticles (SPIONs) for a prospective "magnetofection" application. By functionalizing SPIONs with an active catechol ester (CafPFP), polyethyleneimine (PEI) was covalently bound to their surface while preserving the desired nanosized particle properties with a hydrodynamic size of 86 nm. When complexed with plasmid-DNA (pDNA) up to a weight ratio of 2.5% pDNA/Fe, no significant changes in particle properties were observed, while 95% of the added pDNA was strongly bound to the SPION surface. The transfection in A375-M cells for 48 h with low amounts (10 ng) of pDNA, which carried a green fluorescent protein (GFP) sequence, resulted in a transfection efficiency of 3.5%. This value was found to be almost 3× higher compared to Lipofectamine (1.2%) for such low pDNA amounts. The pDNA-SPION system did not show cytotoxic effects on cells for the tested particle concentrations and incubation times. Through the possibility of additional covalent functionalization of the SPION surface as well as the PEI layer, Caf-PEI-SPIONs might be a promising candidate as a magnetofection agent in future.

An Extended Cyclic Di-GMP Network in the Predatory Bacterium Bdellovibrio bacteriovorus.

UNLABELLED: Over the course of the last 3 decades the role of the second messenger cyclic di-GMP (c-di-GMP) as a master regulator of bacterial physiology was determined. Although the control over c-di-GMP levels via synthesis and breakdown and the allosteric regulation of c-di-GMP over receptor proteins (effectors) and riboswitches have been extensively studied, relatively few effectors have been identified and most are of unknown functions. The obligate predatory bacterium Bdellovibrio bacteriovorus has a peculiar dimorphic life cycle, in which a phenotypic transition from a free-living attack phase (AP) to a sessile, intracellular predatory growth phase (GP) is tightly regulated by specific c-di-GMP diguanylate cyclases. B. bacteriovorus also bears one of the largest complement of defined effectors, almost none of known functions, suggesting that additional proteins may be involved in c-di-GMP signaling. In order to uncover novel c-di-GMP effectors, a c-di-GMP capture-compound mass-spectroscopy experiment was performed on wild-type AP and host-independent (HI) mutant cultures, the latter serving as a proxy for wild-type GP cells. Eighty-four proteins were identified as candidate c-di-GMP binders. Of these proteins, 65 did not include any recognized c-di-GMP binding site, and 3 carried known unorthodox binding sites. Putative functions could be assigned to 59 proteins. These proteins are included in metabolic pathways, regulatory circuits, cell transport, and motility, thereby creating a potentially large c-di-GMP network. False candidate effectors may include members of protein complexes, as well as proteins binding nucleotides or other cofactors that were, respectively, carried over or unspecifically interacted with the capture compound during the pulldown. Of the 84 candidates, 62 were found to specifically bind the c-di-GMP capture compound in AP or in HI cultures, suggesting c-di-GMP control over the whole-cell cycle of the bacterium. High affinity and specificity to c-di-GMP binding were confirmed using microscale thermophoresis with a hypothetical protein bearing a PilZ domain, an acyl coenzyme A dehydrogenase, and a two-component system response regulator, indicating that additional c-di-GMP binding candidates may be bona fide novel effectors. IMPORTANCE: In this study, 84 putative c-di-GMP binding proteins were identified in B. bacteriovorus, an obligate predatory bacterium whose lifestyle and reproduction are dependent on c-di-GMP signaling, using a c-di-GMP capture compound precipitation approach. This predicted complement covers metabolic, energy, transport, motility and regulatory pathways, and most of it is phase specific, i.e., 62 candidates bind the capture compound at defined modes of B. bacteriovorus lifestyle. Three of the putative binders further demonstrated specificity and high affinity to c-di-GMP via microscale thermophoresis, lending support for the presence of additional bona fide c-di-GMP effectors among the pulled-down protein repertoire.

Synthetic biology approach to reconstituting the ubiquitylation cascade in bacteria.

Covalent modification of proteins with ubiquitin (Ub) is widely implicated in the control of protein function and fate. Over 100 deubiquitylating enzymes rapidly reverse this modification, posing challenges to the biochemical and biophysical characterization of ubiquitylated proteins. We circumvented this limitation with a synthetic biology approach of reconstructing the entire eukaryotic Ub cascade in bacteria. Co-expression of affinity-tagged substrates and Ub with E1, E2 and E3 enzymes allows efficient purification of ubiquitylated proteins in milligram quantity. Contrary to in-vitro assays that lead to spurious modification of several lysine residues of Rpn10 (regulatory proteasomal non-ATPase subunit), the reconstituted system faithfully recapitulates its monoubiquitylation on lysine 84 that is observed in vivo. Mass spectrometry revealed the ubiquitylation sites on the Mind bomb E3 ligase and the Ub receptors Rpn10 and Vps9. Förster resonance energy transfer (FRET) analyses of ubiquitylated Vps9 purified from bacteria revealed that although ubiquitylation occurs on the Vps9-GEF domain, it does not affect the guanine nucleotide exchanging factor (GEF) activity in vitro. Finally, we demonstrated that ubiquitylated Vps9 assumes a closed structure, which blocks additional Ub binding. Characterization of several ubiquitylated proteins demonstrated the integrity, specificity and fidelity of the system, and revealed new biological findings.

Efficient Flp-Int HK022 dual RMCE in mammalian cells.

Recombinase-mediated cassette exchange, or RMCE, is a clean approach of gene delivery into a desired chromosomal location, as it is able to insert only the required sequences, leaving behind the unwanted ones. RMCE can be mediated by a single site-specific DNA recombinase or by two recombinases with different target specificities (dual RMCE). Recently, using the Flp-Cre recombinase pair, dual RMCE proved to be efficient, provided the relative ratio of the enzymes during the reaction is optimal. In the present report, we analyzed how the efficiency of dual RMCE mediated by the Flp-Int (HK022) pair depends on the variable input of the recombinases-the amount of the recombinase expression vectors added at transfection-and on the order of the addition of these vectors: sequential or simultaneous. We found that both in the sequential and the simultaneous modes, the efficiency of dual RMCE was critically dependent on the absolute and the relative concentrations of the Flp and Int expression vectors. Under optimal conditions, the efficiency of 'simultaneous' dual RMCE reached ∼12% of the transfected cells. Our results underline the importance of fine-tuning the reaction conditions for achieving the highest levels of dual RMCE.

Arm site independence of coliphage HK022 integrase in human cells.

The integrase encoded by the lambdoid phage HK022 (Int-HK022) resembles its coliphage λ counterpart (Int-λ) in the roles of the cognate DNA arm binding sites and in controlling the direction of the reaction. We show here that within mammalian cells, Int-HK022 does not exhibit such a control. Rather, Int-HK022 recombined between all ten possible pairwise att site combinations, including attB × attB that was more effective than the conventional integrative attP × attB reaction. We further show that Int-HK022 depends on the accessory integration host factor (IHF) protein considerably less than Int-λ and exhibits stronger binding affinity to the att core. These differences explain why wild-type Int-HK022 is active in mammalian cells whereas Int-λ is active there only as an IHF-independent mutant.

Site-specific recombination in the cyanobacterium Anabaena sp. strain PCC 7120 catalyzed by the integrase of coliphage HK022.

The integrase (Int) of the lambda-like coliphage HK022 catalyzes the site-specific integration and excision of the phage DNA into and from the chromosome of its host, Escherichia coli. Int recognizes two different pairs of recombining sites attP x attB and attL x attR for integration and excision, respectively. This system was adapted to the cyanobacterium Anabaena sp. strain PCC 7120 as a potential tool for site-specific gene manipulations in the cyanobacterium. Two plasmids were consecutively cointroduced by conjugation into Anabaena cells, one plasmid that expresses HK022 Int recombinase and the other plasmid that carries the excision substrate P(glnA)-attL-T1/T2-attR-lacZ, where T1/T2 are the strong transcription terminators of rrnB, to prevent expression of the lacZ reporter under the constitutive promoter P(glnA). The Int-catalyzed site-specific recombination reaction was monitored by the expression of lacZ emanating as a result of T1/T2 excision. Int catalyzed the site-specific excision reaction in Anabaena cells when its substrate was located either on the plasmid or on the chromosome with no need to supply an accessory protein, such as integration host factor and excisionase (Xis), which are indispensable for this reaction in its host, E. coli.

SPIONs functionalized with small peptides for binding of lipopolysaccharide, a pathophysiologically relevant microbial product.

Systemic inflammation such as sepsis represents an acute life-threatening condition, to which often no timely remedy can be found. A promising strategy may be to functionalize magnetic nanoparticles with specific peptides, derived from the binding motives of agglutinating salivary proteins, that allow immobilization of pathogens. In this work, superparamagnetic iron oxide nanoparticles with stable polycondensed aminoalkylsilane layer were developed, to which the heterobifunctional linkers N-succinimidyl 3-(2-pyridyldithio)-propanoate (SDPD) and N-succinimidyl bromoacetate (SBA) were bound. These linkers were further chemoselectively reacted with the thiol group of singularly present cysteines of selected peptides. The resulting functional nanoparticles underwent a detailed physicochemical characterization. The biocompatibility of the primarily coated aminoalkylsilane particles was also investigated. To test the pathogen-binding efficacy of the particles, the lipopolysaccharide-immobilization capacity of the peptide-coated particles was compared with free peptides. Here, one particle-bound peptide species succeeded in capturing 90% of the toxin, whereas the degree of immobilization of the toxin with a system that varied in the sequence of the peptide dropped to 35%. With these promising results, we hope to develop extracorporeal magnetic clearance systems for removing pathogens from the human body in order to accelerate diagnosis and alleviate acute disease conditions such as sepsis.

Molecular analysis of recombinase-mediated cassette exchange reactions catalyzed by integrase of coliphage HK022.

The integrase (Int) protein of coliphage HK022 can catalyze in Escherichia coli as well as in in vitro integrative and excisive recombinase-mediated cassette exchange reactions between plasmids as substrates. Atomic force microscopy images have revealed that in the protein-DNA complexes that are formed, the plasmid substrates are connected via one and not two pairs of attachment sites. This observation, together with the elucidation of intermediate co-integrates between the two circular plasmids, suggest that a sequential mechanism of the RMCE reaction is possible.

Human genomic site-specific recombination catalyzed by coliphage HK022 integrase.

It has been previously demonstrated that the wild type integrase (Int) protein of coliphage HK022 can catalyze site-specific recombination in human cells between attachment (att) sites that were placed on extrachromosomal plasmids. In the present report it is shown that Int can catalyze the site-specific recombination reactions in a human cell culture on the chromosomal level. These include integrative (attP x attB) as well as excisive (attL x attR) reactions each in two configurations. In the cis configuration both sites are on the same chromosome, in the trans configuration one site is on a chromosome and the other on an episome. The reactions in cis were observed without any selection force, using the green fluorescent protein (GFP) as a reporter. The reactions in trans could be detected only when a selection force was applied, using the hygromycin-resistant (Hyg(R)) phenotype as a selective marker. All reactions were catalyzed without the need to supply any of the accessory proteins that are required by Int in its Escherichia coli host. The versatility of the att sites may be an advantage in the utilization of Int to integrate plasmid DNA into the genome, followed by a partial exclusion of the integrated plasmid.

Anti-cancer binary system activated by bacteriophage HK022 integrase.

The binary system presented in this work is based on the bacteriophage HK022 integrase recombinase that activates the expression of a silenced Diphtheria toxin gene, both controlled by the cancer specific hTERT promoter. Using a lung cancer mice model, assays of different apoptotic and anti-apoptotic factors have demonstrated that the Integrase based binary system is highly specific towards cancer cells and more efficient compared to the conventional mono system whose toxin is directly expressed under hTERT. In a mice survival test, this binary system demonstrated longer persistence compared to the untreated and the mono treated ones. The reason underlying the advantage of this binary system over the mono system seems to be an overexpression of various hTERT suppressing factors induced by the mono system.

Tomato yellow leaf curl virus confronts host degradation by sheltering in small/midsized protein aggregates.

Tomato yellow leaf curl virus (TYLCV) is a begomovirus transmitted by the whitefly Bemisia tabaci to tomato and other crops. TYLCV proteins are endangered by the host defenses. We have analyzed the capacity of the tomato plant and of the whitefly insect vector to degrade the six proteins encoded by the TYLCV genome. Tomato and whitefly demonstrated the highest proteolytic activity in the fractions containing soluble proteins, less-in large protein aggregates; a significant decrease of TYLCV proteolysis was detected in the intermediate-sized aggregates. All the six TYLCV proteins were differently targeted by the cytoplasmic and nuclear degradation machineries (proteases, ubiquitin 26S proteasome, autophagy). TYLCV could confront host degradation by sheltering in small/midsized aggregates, where viral proteins are less exposed to proteolysis. Indeed, TYLCV proteins were localized in aggregates of various sizes in both host organisms. This is the first study comparing degradation machinery in plant and insect hosts targeting all TYLCV proteins.

Tomato yellow leaf curl virus infection mitigates the heat stress response of plants grown at high temperatures.

Cultured tomatoes are often exposed to a combination of extreme heat and infection with Tomato yellow leaf curl virus (TYLCV). This stress combination leads to intense disease symptoms and yield losses. The response of TYLCV-susceptible and resistant tomatoes to heat stress together with viral infection was compared. The plant heat-stress response was undermined in TYLCV infected plants. The decline correlated with the down-regulation of heat shock transcription factors (HSFs) HSFA2 and HSFB1, and consequently, of HSF-regulated genes Hsp17, Apx1, Apx2 and Hsp90. We proposed that the weakened heat stress response was due to the decreased capacity of HSFA2 to translocate into the nuclei of infected cells. All the six TYLCV proteins were able to interact with tomato HSFA2 in vitro, moreover, coat protein developed complexes with HSFA2 in nuclei. Capturing of HSFA2 by viral proteins could suppress the transcriptional activation of heat stress response genes. Application of both heat and TYLCV stresses was accompanied by the development of intracellular large protein aggregates containing TYLCV proteins and DNA. The maintenance of cellular chaperones in the aggregated state, even after recovery from heat stress, prevents the circulation of free soluble chaperones, causing an additional decrease in stress response efficiency.

Cancer-specific binary expression system activated in mice by bacteriophage HK022 Integrase.

Binary systems based on site-specific recombination have been used for tumor specific transcription targeting of suicide genes in animal models. In these binary systems a site specific recombinase or integrase that is expressed from a tumor specific promoter drives tumor specific expression of a cytotoxic gene. In the present study we developed a new cancer specific binary expression system activated by the Integrase (Int) of the lambdoid phage HK022. We demonstrate the validity of this system by the specific expression of a luciferase (luc) reporter in human embryonic kidney 293T (HEK293T) cells and in a lung cancer mouse model. Due to the absence viral vectors and of cytotoxicity the Int based binary system offers advantages over previously described counterparts and may therefore be developed into a safer cancer cell killing system.

Determinants that target the integrase of phage HK022 into the mammalian nucleus.

The integrase (Int) protein of coliphage HK022 catalyzes the site-specific integration and excision of the phage into and from its Escherichia coli host chromosome. Int expressed from a plasmid in COS1 monkey cells is localized in the nucleus, as is a fusion protein between Int and the green fluorescent protein (GFP). Mutation analysis of the GFP-Int fusion has revealed in Int two regions of positively charged amino acid residues that cooperate in the nuclear localization. One region harbors residues Arg90 and Arg93. The other, which spans residues 307-340 belongs to the catalytic domain of Int, is rich in basic residues and is strongly conserved within the Int protein family. Being localized in the nucleus renders Int of HK022 as a potential recombinase for site-specific gene manipulations in mammals.

Protein-protein interaction between monomers of coliphage HK022 excisionase.

Excisionase (Xis) is an accessory protein that is required for the site-specific excision reaction of the coliphages HK022 and lambda. Xis binds in a strong cooperative manner to two tandem binding sites (X1 and X2) located on the P arm of the attachment (att) sites on the phage genome. As a result of crosslinking experiments in vivo and in vitro of Xis-overexpressing cells, by gel filtration of purified Xis and by FRET analyses we show that Xis monomers of HK022 interact and form dimers that are not dependent on the single Cys residue of the protein and on the presence of DNA. The formation of the dimers may explain the strong binding cooperativity of Xis to its sites on DNA.

Activity of coliphage HK022 excisionase (Xis) in the absence of DNA binding.

A mutated excisionase (Xis) protein of coliphage HK022 whose single Cys residue was replaced by Ser does not bind to its two tandem binding sites (X1, X2) on the P arm of attR. Despite its DNA-binding inability the protein showed 30% excision activity of the wild type Xis both in vitro and in vivo. This partial activity is attributed to the interaction of Xis with integrase that is retained in the mutant protein. This protein-protein interaction occurs in the absence of DNA binding.

Progressive aggregation of Tomato yellow leaf curl virus coat protein in systemically infected tomato plants, susceptible and resistant to the virus.

Tomato yellow leaf curl virus (TYLCV) coat protein (CP) accumulated in tomato leaves during infection. The CP was immuno-detected in the phloem associated cells. At the early stages of infection, punctate signals were detected in the cytoplasm, while in the later stages aggregates of increasing size were localized in cytoplasm and nuclei. Sedimentation of protein extracts through sucrose gradients confirmed that progress of infection was accompanied by the formation of CP aggregates of increasing size. Genomic ssDNA was found in the cytoplasm and in the nucleus, while the dsDNA replicative form was exclusively associated with the nucleus. CP-DNA complexes were detected by immuno-capture PCR in nuclear and cytoplasmic large aggregates. Nuclear aggregates contained infectious particles transmissible to test plants by whiteflies. In contrast to susceptible tomatoes, the formation of large CP aggregates in resistant plants was delayed. By experimentally changing the level of resistance/susceptibility of plants, we showed that maintenance of midsized CP aggregates was associated with resistance, while large aggregates where characteristic of susceptibility. We propose that sequestering of virus CP into midsized aggregates and retarding the formation of large insoluble aggregates containing infectious particles is part of the response of resistant plants to TYLCV.

High efficiency of a sequential recombinase-mediated cassette exchange reaction in Escherichia coli.

A comparison between the efficiency of recombinase-mediated cassette exchange (RMCE) reactions catalyzed in Escherichia coli by the site-specific recombinases Flp of yeast and Int of coliphage HK022 has revealed that an Flp-catalyzed RMCE reaction is more efficient than an Int-HK022 catalyzed reaction. In contrast, an RMCE reaction with 1 pair of frt sites and 1 pair of att sites catalyzed in the presence of both recombinases is very inefficient. However, the same reaction catalyzed by each recombinase individually supplied in a sequential order is very efficient, regardless of the order. Atomic force microscopy images of Flp with its DNA substrates show that only 1 pair of recombination sites forms a synaptic complex with the recombinase. The results suggest that the RMCE reaction is sequential.