Abstract

In this section we will introduce the delivery mechanism of our interference system. Our priorities were to design something that could be safe, efficient and adaptable to the specific situation. We wanted to create a platform that could be used to address antibiotic resistant infections quickly and efficiently. At the same time, as our primary objective was to apply it in the human body, safety was of uttermost importance. To address these design considerations we thought of developing a phage delivering platform based on two engineered constructs that together allow the production of M13 phage particles bearing a genome of desire. The first construct is a plasmid, called phagemid, which incorporates the f1 intergenic region facilitating encapsidation within the M13 bacteriophage. This plasmid is further equipped with an origin of replication (ORI) and a selection marker, thereby enabling replication within host cells. The second plasmid, known as helper plasmid, serves as a repository for phage capsid proteins, in conjunction with an ORI and a selection marker. The absence of the encapsidation sequence within the helper plasmid allows for the production of phage particles carrying the phagemid. Consequently, upon infection of the target bacteria with the genetically modified phages, transduction of their genome is facilitated, while phage particle production within the host is hindered due to the absence of essential phage proteins. Thus, the phage system operates exclusively as a way for delivering the engineered DNA construct.

Introduction

In compassionate care, the employed bacteriophages are derived from environmental samples (i.e. from sewage systems) that have demonstrated in vitro lytic activity against the clinical isolate [1]. Therefore, up to now, phage particles are purified and administered without a precise characterization, and their potential clinical efficacy relies just on the assumption that their specific antibacterial activity in vitro could be reproducible also in vivo [1]; as a consequence, these therapies are usually personalized on the clinical case. However, in order for such an approach to reach everyday clinical practice, significantly higher levels of knowledge about what is being dispensed, as well as the standardization of the clinical protocol, are required. To this end, phage engineering has been proposed as a solution, allowing to thoroughly control phage particles production. In particular, a molecular engineering approach would allow the production of safer phage particles, purified from cellular debris and potential bacterial antigens, that currently represent one of the major issues [1, 2, 3].

In addition, the ability to produce engineered phage particles would make us capable of carrying a fully characterized genome of desire, containing gene editing tools or silencing systems that can interfere with the protein expression of pathogenic bacteria, namely the phagemid described lately. When co-transformed with the phagemid, the helper plasmid enables the production of viable phage particles carrying the desired genome [4,5](Figure 1).

Figure 1. The packaging cycle of a phagemid. This image shows how the co-transformation of a helper plasmid with a phagemid allows the production and the subsequent purification of phage particles with the desired genome [5]

Such an approach will result in the production of engineered bacteriophages that are no longer unknown and therefore suitable as clinical therapy. Nevertheless, many challenges still need to be faced, with a primary aim of promoting the widespread adoption of bacteriophage research, by developing innovative solutions that simplify exploration and implementation of novel and pragmatic approaches. Building on these foundations, the present work aims to lay the groundwork for an approach to bacteriophage engineering, based on the concepts of parts standardization with the scope of making research in this field more approachable.

Phagemid

In our pursuit of a phage delivery system capable of accommodating a customizable genome to facilitate the expression of a targeted silencing system in recipient cells, a construct with specific attributes was indispensable. This construct had to fulfill several criteria: it needed to be capable of packaging within bacteriophages, easily subject to modifications, and compliant with the BioBrick standard. To address these prerequisites, we started from a commercially available construct, pTZ19R (ThermoFisher Scientific). This particular construct possesses the ability to be encapsulated within M13 phage particles. This can be achieved either by co-transformation with a helper plasmid or by subjecting cells to infection by a helper phage. The basis of this ability lies in the presence of the f’ intergenic region within the plasmid, a sequence that encodes the encapsidation signal.

Hence, this plasmid can be denoted as a phagemid, a construct capable of being encapsulated when exposed to the viral proteins. However, when present solely within a host cell, it lacks the capability to independently generate viable phage particles. This fundamental characteristic makes it a secure conduit for delivering the interference system. Importantly, this delivery approach safeguards the recipient cell from harm and precludes exerting evolutionary pressure on bacteria. Such pressure could potentially lead to bacterial resistance against the phage system itself, something we clearly want to avoid.

While this construct (Figure2) exhibited several favorable attributes aligned with our objectives, certain notable issues necessitated resolution. Primarily, there existed an obstacle due to the presence of a multiple cloning site embedded within the LacZ gene. This site encompassed three restricted cut sites, a circumstance that could be readily rectified through targeted deletion, achieved using a primer pair that excludes this region. Another significant concern was the absence of the BioBrick RFC[10] prefix and suffix. This challenge was similarly addressed straightforwardly, utilizing PCR mutagenesis to seamlessly incorporate the two required sequences.

Figure2. Plasmid map of pTZ19R

Through these essential modifications, we successfully constructed a novel standard backbone, deposited as BBa_K4727003 in the registry. This backbone has demonstrated its ability to maintain a high copy number within E. coli cells (replicon pMB1) and to effectively express the encoded protein. Critically, when coexisting with a helper plasmid or helper phage, this construct is capable of being encapsulated within M13 phage particles. This achievement leads to the creation of engineered bacteriophages carrying a customized genomic payload.

As a consequence, this construct emerges as a viable solution to the essential need for comprehensive characterization in propelling phage therapy towards routine clinical implementation. Importantly, its safety profile is well-established, devoid of significant threats to patient well-being. Furthermore, its inability to propagate within host cells due to the absence of phage protein encoding genes, ensures the prevention of unintended dissemination of engineered constructs. Lastly, its adaptability is a remarkable feature - it can be easily tailored to the specifics of individual cases, thus presenting a versatile and readily adjustable platform tailored to combat diverse pathogens.

Helper plasmid

Currently, the production of engineered M13 phage particles through a helper plasmid involves constructs composed of a replication origin (ORI) that facilitates replication within E. coli, an antibiotic selection marker that allows for the selection of transformed cells, and the genes encoding the M13 phage capsid proteins. These constructs are typically obtained by removing the encapsidation sequence from the M13 phage genome and replacing it with ORI and the selection marker. Of particular interest to us was the M13cp plasmid, built by Chasteen et al.[6]. However, this plasmid is not compatible with the BioBrick standard as it carries two PstI restriction sites flanking the ORI, furthermore, it lacks the BioBrick prefix and suffix. Thus, the region bearing the ORI and the chloramphenicol resistance was replaced with the standard pSB3K3 backbone, which carries the same ORI as the original helper plasmid, particularly p15a, and utilizes kanamycin resistance as the selection marker, compatible with the marker carried by the M13 phagemid BBa_K4727003. In this way we propose the new plasmid backbone BBa_K4727002, that allows for the production of engineered phage particles.

Tropism variation

Furthermore we wanted to explore the possibility of varying the tropism of the bacteriophage. Viral tropism is defined as the ability of a virus to target a specific molecular pattern which is characteristic of a cell or a tissue. Bacteriophages are capable of infecting bacteria only, and they do so by recognizing peculiar molecules exposed on the cell surface[1] (i.e. protein structures as pili, flagella, porins, or efflux pumps [1]. Otherwise, sugars in lipopolysaccharides have been reported [1]).

The interactions of the phage are highly specific and rely on proteins exposed on its surface. This renders the phage's ability to discriminate against the bacteria it can infect, and in some cases even among different strains, exceptionally selective. If, on one hand, it is a great advantage to have an organism able to target bacteria in such a specific manner, on the other it poses many limits in terms of spectrum of application. These considerations prompted an exploration of potential strategies for engineering host phage range. Despite the presence of examples in the existing literature and by past iGEM teams, none of them proved suitable for the intended purposes due to variations in the bacteriophages utilized [7,8]. Moreover, while tropism variation was explored in some head-tail phages [7, 8], as the Zurich iGEM team, there are few examples for filamentous phages, such as the Aix-Marseille 2017 iGEM team.

This purpose could have been achieved in two opposites, yet complementary, ways. The first one uses a random approach, inspired by antibody specific engineering, that allows mutagenesis of regions involved in target recognition, followed by a screening against the desired host(s) [8], this was the approach developed and exploited by the 2019 Zurich iGEM team. They developed a system to introduce random mutations in the tropism determinant proteins of T7 bacteriophages and to select them against the putative target. On one hand this methodology is far more time consuming, on the other it can result in a broader extension of the tropism as many more possibilities are explored. The second method can be otherwise described as a rational approach, using synthetic biology to modulate phage host ranges by swapping tropism determinants between different phage species[7]. This allows a higher confidence over the possible results nevertheless it requires a deeper knowledge of the tropism determinants. These two different approaches, even if applied to profoundly different families of bacteriophages (i.e. T4, T3, and T7) [7,8], represented the guidelines of our research. Keeping in mind these considerations the rational design of parts was chosen, as it allows easy swap between alternative genes to vary the host range of the system.

M13 tropism

As stated above, there are few examples of tropism variation in filamentous bacteriophages, for this reason a possible approach in M13 bacteriophage is proposed in this work. M13 uses a minor protein (Gp3) of its capsule to interact with the pilus (F’ factor) of Escherichia coli [9,10]. Subsequent publications also demonstrated how this interaction is mediated and takes place, involving not only the protein of the pili but also another protein of E. coli outer membrane, TolA9. On the virus side, the tropism of filamentous phages is determined by a protein encoded in the gene 3, referred as Gene Product 3 (Gp3)[10]. Gp3, a 406 amino acids-long protein[9,10] that sits in three to five copies on one end of the capsid[10], being responsible not only for the interaction with the outer structures of the bacteria but also for the termination of the virion assembly[9,10]. Once correctly folded it forms three functionally distinguished domains (N1, N2 and the C-terminal domain CTD), each of which is involved in the infective process[9,10]. The CTD anchors Gp3 to the phage by interacting with other phage coat proteins, whereas the N1 and N2 domains are responsible for the interaction with the bacteria[10]. The three-dimensional structures of the N1 and N2 domains have been determined by circular dichroism (CD) spectrum analysis and X-ray diffraction[11,12], whereas the CTD has not yet been characterized. Given N1 and N2 domains structure, it has been possible to determine the functions of the respective interacting parts. Even though N2 is in the middle of the amino acid sequence, the 3D structure reveals how it is located on one end of the protein and is responsible for the interaction with the F’ factor[10]. The positioning of the domain hides N1 which includes the N-terminus of the protein, but it becomes available after the first contact with the pili resulting in N1 domain interaction with the TolA protein[9]. TolA is a 421 amino acids-long protein, located in the periplasmic space of E. coli[13]; it anchors itself to the inner membrane with the N-terminus, spanning through the periplasm with a long α-helix ending with the C terminal domain (TolAIII)[13]. It has been demonstrated that TolAIII is functional in the infection process of M13 [9,13] thanks to its high affinity interaction (1-1,9μM) with N1 [9].

For this reason, two approaches to tropism variation will be discussed, the first revolving around the interaction of Gp3 with TolA and its homologues, whereas the second focuses on other “gene 3” homologues of the one encoded in M13 bacteriophage.

An approach to tropism engineering

Many benefits come with standardization of the helper plasmid, in particular the ability to easily interchange the parts present between the prefix and suffix of the vector. For this reason, we wanted to take advantage of this strategy to address the problem posed by tropism variation in bacteriophages. As described above, tropism in the M13 bacteriophage is determined by Gp3 protein, encoded by gene 3 [10]. Based on this knowledge, we decided to adopt a rational design strategy for new genes that can modify tropism. The rationale behind this choice primarily lies in the reduced time required by this approach and the increased predictability of potential outcomes. Such advantages would not be achievable through a random gene mutation approach, although it might enable a broader exploration of tropism variants. It is remarkable to note that the work made by the Zurich iGEM team in 2019 could represent a viable method to further extend the ideas we developed. Coupling the rational design with a random mutagenesis approach and an efficient selection method can take to the next step phage research in tropism variation.

Exploit TolA homologues

The rational design of alternative genes took two paths, both founded on literature research, past works from iGEM teams and the use of computational tools. The first path, leading to the generation of "Gp3_cut" (part BBa_K4727004), was based on the fundamental work of DA Marvin [14], which suggested that mutants lacking the N2 domain can infect bacteria without pilus with a 100-fold higher efficiency [14], as N2 masks the interaction of N1 with the co-receptor TolA. Building on this, the idea was to design a gene with a deleted N2 domain, drawing from the findings of Nilsson et al.[10], who precisely delineated the amino acid regions corresponding to individual domains. Therefore, an alternative gene was designed for tropism that could ideally enable E. coli infection in the absence of a pilus, thus extending the tropism. This gene, named "Gp3_cut," was obtained by in silico deletion of the region between residues 87 and 256, corresponding to the N2 domain [10]. However, considering this protein's ability to interact with the TolA protein, specifically the TolAIII domain [9,10], it was hypothesized that this same modified gene could also interact with homologous TolA proteins. Through bioinformatic analysis, using the BLASTp tool [15], a surface protein of Klebsiella pneumoniae, called "cell envelope integrity protein TolA" (protein ID: MBS4159696.1), was identified, sharing 100% similarity with the TolAIII domain of E. coli (residues 305-421 [13]). This suggests that such modification could potentially allow the engineered bacteriophage's entry into K. pneumoniae as well.

Gp3 homologues

The second path that was taken, which led to the generation of “Gp3_Pf3” (part BBa_K4727005), stem from the research of photogenically closed bacteriophages and the work of Aix-Marseille 2017 iGEM team. This choice follows the idea that proteins of evolutionary related phages maintain a certain grade of similarity thus being still able to interact between each other and correctly assemble the capsid. M13 bacteriophage is part of the Inoviridae family, that includes other filamentous phages, among them the Pf3 bacteriophage was identified as it is capable of infecting Pseudomonas aeruginosa. Given the relevance of this pathogen in the AMR phenomenon [1] it was considered as an interesting possibility in attempting tropism variation. As in all filamentous phages the tropism determinant protein is annotated as “Gene 3 product”[10], the coding sequence was identified by analyzing the deposited genome in the NCBI databank (ID: NC_074763), and isolated.

In silico design of expression cassettes

Once the putative genes for tropism variation were identified, it was necessary to design appropriate expression cassettes that could be inserted into the helper plasmid. To accomplish this, preliminary considerations were made. Firstly, the metabolic burden imposed by this plasmid is significant, as it expresses a total of 12 proteins (11 from the phage capsid, plus the selection marker), which significantly prolong colony growth (Figure 3). Secondly, our goal is to preferentially express alternative tropism genes and consequently incorporate them into the phage, as opposed to the wild-type gene 3 product, which is normally expressed. Only in this way can phage particles with altered tropism be obtained.

Figure 3. Metabolic burden of pSB3KM13. In the figure, E. coli TOP10 colonies bearing pSB3KM13 after different incubation times are reported. (a) After overnight incubation (16 hours), few and small colonies are visible on the plate. (b) After 24 hours the colonies were bigger and in a higher number. (c) After 36 hours, the colonies were clearly visible and could be easily isolated.

The aforementioned compromise stems from an essential consideration. The phage genome inserted into the standard helper plasmid is inherently complex, comprising multiple overlapping sequences, with promoters and terminators placed on-frame within coding sequences. This complexity, along with the lack of precise genome annotation, prevents the silencing, given current knowledge, of the naturally expressed gene 3, as doing so would risk disrupting other fundamental regulatory sequences required for the expression of the remaining genes. Consequently, the expected result is a mixed population of phages with two different tropisms: the natural one resulting from Gp3 expression and an altered one conferred by the gene placed between the prefix and suffix.

Given these premises, in constructing the expression cassettes, a strong but inducible promoter was chosen. This choice allows the metabolic burden to be limited, if necessary, while, when fully induced, enabling high expression levels of the alternative gene so that it can be preferentially incorporated into the phage particles. The selected promoter was pL-lac0-1 (part BBa_J428041) that combines the strength of the pL promoter, with the possibility to regulate it, derived from the lacO binding site. Indeed, if the cell expresses the lacI gene at high levels, it becomes feasible to induce the expression of the gene regulated by this promoter through the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to the culture medium. Moreover, by employing varying concentrations of the inducer, it is possible to finely adjust the expression level, thus creating a controllable system that is in line with the requirement of promoting cell vitality. Similarly, the choice of the ribosome binding site (RBS) fell on a strong element to ensure that, upon transcript production, it is efficiently translated by ribosomes. This decision aligns with the aim of favoring the production and incorporation of this gene product over the gene 3 of phage M13. Hence, the RBS chosen was part BBa_B0034.

Finally, the terminator was selected based on its strength and efficiency, with a preference for an intrinsic terminator, independent from the expression of cellular proteins. Therefore, the T1 terminator of E. coli was opted (part BBa_B0010).

Synthetic biology is based not only on the standardization of parts but, as the name suggests, on the synthesis of DNA, made possible by the development of new technologies. For this reason, the sequences designed as described above were synthesized in vitro by IDT Technologies. However, before sending the sequences, it was deemed necessary to perform the codon optimization for the two gene sequences. Codon optimization refers to the modification of base triplets that encode the same amino acid, in a way that optimizes the genetic code according to the characteristic frequency of codon usage in the target organism. It is used in genetic engineering when expressing heterologous proteins in an organism to achieve ideal expression efficiency. In the case under consideration, codon optimization was performed at two levels: the first aimed to harmonize the genetic code to favor the expression of the encoded protein in E. coli, and the second to optimize the sequence for synthesis, thus avoiding the presence of repeated sequences and hairpins.

The first step was carried out using a bioinformatic tool known as CODONATOR 3000, developed by the Sydney Australia iGEM team in 2019 (Ofner et al.). This software can provide four different results, each optimizing codons based on different rules. In this case, the optimization was chosen according to the relative proportion in which the starting codon is translated into the codon of the target species (specifically E. coli) based on the most similar frequency of occurrence, considering the bias in amino acid appearance.

The second step was performed using IDT Technologies' proprietary tool, which aimed to eliminate repeated sequences and low complexity regions that could complicate the synthesis process. Using the same algorithm, a total of four forbidden restriction sites, present in the two sequences, were also deleted, to ensure compatibility with the Biobrick standard.

In the end to assemble the final cassettes all the aforementioned elements were put together in the right order, with some minor adjustments. For both the constructs the standard prefix and suffix were added at the 5’ and 3’ ends respectively. The suffix is then followed by the pL-lacO-1 sequence accompanied by the RBS. To the cassette expressing the Gp3_cut gene was added a start codon (ATG), coding for methionine and a double stop codon (TAA). Whereas to the cassette expressing the Gp3_Pf3 was added just a double stop codon (TAA). Both the coding sequences are then followed by the T1 terminator and the suffix. Thus, the two cassettes were assembled and registered as part BBa_K4727006 (G3 deleted gene) and BBa_K4727007 (Pf3 gene).

Once the cassettes were cloned into the helper plasmid pSB3KM13, the construct was transformed into the E. coli MG1655 Z1 cell line, which has the characteristic of constitutively expressing the regulatory genes LacI, AraC, and TetR at high levels. Consequently, this allows the utilization of the regulated cassettes under suitable promoters in presence of the correct inducer (in this case IPTG). Finally, to produce phage particles with altered tropism, it is sufficient to co-transform the helper plasmid here assembled with an M13 phagemid, such as pTZ19R. The phagemid, due to the presence of the encapsidation sequence, can be inserted into the assembled phage particles. Consequently, it becomes possible to generate phages carrying a desired genome with modified tropism, thereby targeting specific cellular receptors. Thus, the described modifications propose a versatile platform for facile and consistent production of phage particles carrying a desired genome, with the potential to easily alter tropism through standardized genetic components. This method allows the tropism-determining genes of the phages to become standardized parts, readily exchangeable among them.

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