Transducing Particles Research Articles

Phage-host co-evolution has led to distinct generalized transduction strategies.

Generalized transduction is pivotal in bacterial evolution but lacks comprehensive understanding regarding the facilitating features and variations among phages. We addressed this gap by sequencing and comparing the transducing particle content of three different Salmonella Typhimurium phages (i.e. Det7, ES18 and P22) that share a headful packaging mechanism that is typically initiated from a cognate pac site within the phage chromosome. This revealed substantial disparities in both the extent and content of transducing particles among these phages. While Det7 outperformed ES18 in terms of relative number of transducing particles, both phages contrasted with P22 in terms of content. In fact, we found evidence for the presence of conserved P22 pac-like sequences in the host chromosome that direct tremendously increased packaging and transduction frequencies of downstream regions by P22. More specifically, a ca. 561 kb host region between oppositely oriented pac-like sequences in the purF and minE loci was identified as highly packaged and transduced during both P22 prophage induction and lytic infection. Our findings underscore the evolution of phage transducing capacity towards attenuation, promiscuityor directionality, and suggest that pac-like sequences in the host chromosome could become selected as sites directing high frequency of transduction.

Structure of the Portal Complex from Staphylococcus aureus Pathogenicity Island 1 Transducing Particles In Situ and In Isolation

Staphylococcus aureus is an important human pathogen, and the prevalence of antibiotic resistance is a major public health concern. The evolution of pathogenicity and resistance in S. aureus often involves acquisition of mobile genetic elements (MGEs). Bacteriophages play an especially important role, since transduction represents the main mechanism for horizontal gene transfer. S. aureus pathogenicity islands (SaPIs), including SaPI1, are MGEs that carry genes encoding virulence factors, and are mobilized at high frequency through interactions with specific “helper” bacteriophages, such as 80α, leading to packaging of the SaPI genomes into virions made from structural proteins supplied by the helper. Among these structural proteins is the portal protein, which forms a ring-like portal at a fivefold vertex of the capsid, through which the DNA is packaged during virion assembly and ejected upon infection of the host. We have used high-resolution cryo-electron microscopy to determine structures of the S. aureus bacteriophage 80α portal itself, produced by overexpression, and in situ in the empty and full SaPI1 virions, and show how the portal interacts with the capsid. These structures provide a basis for understanding portal and capsid assembly and the conformational changes that occur upon DNA packaging and ejection.

Peptide ligands targeting the vesicular stomatitis virus G (VSV-G) protein for the affinity purification of lentivirus particles.

The recent uptick in the approval of ex vivo cell therapies highlights the relevance of lentivirus (LV) as an enabling viral vector of modern medicine. As labile biologics, however, LVs pose critical challenges to industrial biomanufacturing. In particular, LV purification-currently reliant on filtration and anion-exchange or size-exclusion chromatography-suffers from long process times and low yield of transducing particles, which translate into high waiting time and cost to patients. Seeking to improve LV downstream processing, this study introduces peptides targeting the enveloped protein Vesicular stomatitis virus G (VSV-G) to serve as affinity ligands for the chromatographic purification of LV particles. An ensemble of candidate ligands was initially discovered by implementing a dual-fluorescence screening technology and a targeted in silico approach designed to identify sequences with high selectivity and tunable affinity. The selected peptides were conjugated on Poros resin and their LV binding-and-release performance was optimized by adjusting the flow rate, composition, and pH of the chromatographic buffers. Ligands GKEAAFAA and SRAFVGDADRD were selected for their high product yield (50%-60% of viral genomes; 40%-50% of HT1080 cell-transducing particles) upon elution in PIPES buffer with 0.65 M NaCl at pH 7.4. The peptide-based adsorbents also presented remarkable values of binding capacity (up to 3·109 TU per mL of resin, or 5·1011 vp per mL of resin, at the residence time of 1 min) and clearance of host cell proteins (up to a 220-fold reduction of HEK293 HCPs). Additionally, GKEAAFAA demonstrated high resistance to caustic cleaning-in-place (0.5 M NaOH, 30 min) with no observable loss in product yield and quality.

Effects of the Newly Isolated T4-like Phage on Transmission of Plasmid-Borne Antibiotic Resistance Genes via Generalized Transduction.

Bacteriophages are the most abundant biological entities on earth and may play an important role in the transmission of antibiotic resistance genes (ARG) from host bacteria. Although the specialized transduction mediated by the temperate phage targeting a specific insertion site is widely explored, the carrying characteristics of “transducing particles” for different ARG subtypes in the process of generalized transduction remains largely unclear. Here, we isolated a new T4-like lytic phage targeting transconjugant Escherichia coli C600 that contained plasmid pHNAH67 (KX246266) and encoded 11 different ARG subtypes. We found that phage AH67C600_Q9 can misload plasmid-borne ARGs and package host DNA randomly. Moreover, for any specific ARG subtype, the carrying frequency was negatively correlated with the multiplicity of infection (MOI). Further, whole genome sequencing (WGS) identified that only 0.338% (4/1183) of the contigs of an entire purified phage population contained ARG sequences; these were floR, sul2, aph(4)-Ia, and fosA. The low coverage indicated that long-read sequencing methods are needed to explore the mechanism of ARG transmission during generalized transduction.

Engineering Bacteria to Produce Pure Phage-like Particles for Gene Delivery.

Natural and engineered phages have been used in many applications, but their use to deliver user-defined genetic cargoes has been hampered by contamination with replicative phage, restricting use of the technology beyond the laboratory. Here we present a method to produce transducing particles without contamination. In addition, we demonstrate the use of a helper phage-free transducing particle preparation as an antimicrobial agent. This will pave the way for the development of new phage-based technologies with greater scope than lytic phage therapy.

Overexpression of ER Protein Processing and Apoptosis Regulator Genes in Human Embryonic Kidney 293 Cells Improves Gene Therapy Vectors Production.

Gammaretroviral and lentiviral vectors (γ-RV and LV) are among the most used vectors in gene therapy. Currently, human embryonic kidney (HEK) 293 cells, the manufacture platform of choice for these vectors, provide low transducing particle yields, challenging their therapeutic applications and commercialization. This work studies metabolic pathways, focusing on endoplasmic reticulum (ER) protein processing and anti-apoptotic mechanisms, influencing vector productivity in HEK 293 cell substrates. To that end, four candidate genes-protein disulfide isomerase family A member 2 gene, heat shock protein family A (Hsp70) member 5 gene, X-box binding protein 1 gene (ER protein processing), and B-cell lymphoma 2 protein gene (anti-apoptotic)-are individually stably expressed in the cells. How their overexpression level influence vector yields is analyzed by establishing cell populations with incremental genomic copies of each. γ-RV volumetric productivity increases up to 97% when overexpressing ER protein processing genes. LV volumetric production increases 53% when overexpressing the anti-apoptotic gene. Improvements are associated with higher cell specific productivities and dependent on gene overexpression level, highlighting the importance of fine-tuning gene expression. Overall, this work discloses gene engineering targets enabling efficient gene therapy product manufacture showing that ER protein processing and anti-apoptotic pathways are pivotal to producer cell performance.

Bacteriophages benefit from generalized transduction.

Temperate phages are bacterial viruses that as part of their life cycle reside in the bacterial genome as prophages. They are found in many species including most clinical strains of the human pathogens, Staphylococcus aureus and Salmonella enterica serovar Typhimurium. Previously, temperate phages were considered as only bacterial predators, but mounting evidence point to both antagonistic and mutualistic interactions with for example some temperate phages contributing to virulence by encoding virulence factors. Here we show that generalized transduction, one type of bacterial DNA transfer by phages, can create conditions where not only the recipient host but also the transducing phage benefit. With antibiotic resistance as a model trait we used individual-based models and experimental approaches to show that antibiotic susceptible cells become resistant to both antibiotics and phage by i) integrating the generalized transducing temperate phages and ii) acquiring transducing phage particles carrying antibiotic resistance genes obtained from resistant cells in the environment. This is not observed for non-generalized transducing temperate phages, which are unable to package bacterial DNA, nor for generalized transducing virulent phages that do not form lysogens. Once established, the lysogenic host and the prophage benefit from the existence of transducing particles that can shuffle bacterial genes between lysogens and for example disseminate resistance to antibiotics, a trait not encoded by the phage. This facilitates bacterial survival and leads to phage population growth. We propose that generalized transduction can function as a mutualistic trait where temperate phages cooperate with their hosts to survive in rapidly-changing environments. This implies that generalized transduction is not just an error in DNA packaging but is selected for by phages to ensure their survival.

Molecular Mechanisms That Contribute to Horizontal Transfer of Plasmids by the Bacteriophage SPP1.

Natural transformation and viral-mediated transduction are the main avenues of horizontal gene transfer in Firmicutes. Bacillus subtilis SPP1 is a generalized transducing bacteriophage. Using this lytic phage as a model, we have analyzed how viral replication and recombination systems contribute to the transfer of plasmid-borne antibiotic resistances. Phage SPP1 DNA replication relies on essential phage-encoded replisome organizer (G38P), helicase loader (G39P), hexameric replicative helicase (G40P), recombinase (G35P) and in less extent on the partially dispensable 5′→3′ exonuclease (G34.1P), the single-stranded DNA binding protein (G36P) and the Holliday junction resolvase (G44P). Correspondingly, the accumulation of linear concatemeric plasmid DNA, and the formation of transducing particles were blocked in the absence of G35P, G38P, G39P, and G40P, greatly reduced in the G34.1P, G36P mutants, and slightly reduced in G44P mutants. In contrast, establishment of injected linear plasmid DNA in the recipient host was independent of viral-encoded functions. DNA homology between SPP1 and the plasmid, rather than a viral packaging signal, enhanced the accumulation of packagable plasmid DNA. The transfer efficiency was also dependent on plasmid copy number, and rolling-circle plasmids were encapsidated at higher frequencies than theta-type replicating plasmids.

Derepression of SaPIbov1 Is Independent of φNM1 Type 2 dUTPase Activity and Is Inhibited by dUTP and dUMP

Staphylococcus aureus is an opportunistic human pathogen able to transfer virulence genes to other cells through the mobilization of S. aureus pathogenicity islands (SaPIs). SaPIs are derepressed and packaged into phage-like transducing particles by helper phages like 80α or φNM1. Phages 80α and φNM1 encode structurally distinct dUTPases, Dut80α (type 1) and DutNM1 (type 2). Both dUTPases can interact with the SaPIbov1 Stl master repressor, leading to derepression and mobilization. That two structurally distinct dUTPases bind the same repressor led us to speculate that dUTPase activity may be important to the derepression process. In type 1 dUTPases, Stl binding is inhibited by dUTP. The purpose of this study was to assess the involvement of dUTP binding and dUTPase activity in derepression by DutNM1. DutNM1 activity mutants were created and tested for dUTPase activity using a novel NMR-based assay. We found that all DutNM1 null activity mutants interacted with the SaPIbov1 Stl C-terminal domain, formed DutNM1–Stl heterodimers, and caused the release of the Pstr promoter. However, promoter release was inhibited in the presence of dUTP or dUMP. We tested two φNM1 mutant phages that had null enzyme activity and found that they could still mobilize SaPIbov1. These results show that only the apo form of DutNM1 is active in Stl derepression and that dUTPase activity is not necessary for the mobilization of SaPIbov1 by DutNM1.

Resistance gene transfer: induction of transducing phage by sub-inhibitory concentrations of antimicrobials is not correlated to induction of lytic phage.

Objectives: Horizontal gene transfer of antimicrobial resistance (AMR) genes between clinical isolates via transduction is poorly understood. MRSA are opportunistic pathogens resistant to all classes of antimicrobial agents but currently no strains are fully drug resistant. AMR gene transfer between Staphylococcus aureus isolates is predominantly due to generalized transduction via endogenous bacteriophage, and recent studies have suggested transfer is elevated during host colonization. The aim was to investigate whether exposure to sub-MIC concentrations of antimicrobials triggers bacteriophage induction and/or increased efficiency of AMR gene transfer. Methods: Isolates from MRSA carriers were exposed to nine antimicrobials and supernatants were compared for lytic phage particles and ability to transfer an AMR gene. A new technology, droplet digital PCR, was used to measure the concentration of genes in phage particles. Results: All antibiotics tested induced lytic phage and AMR gene transduction, although the ratio of transducing particles to lytic particles differed substantially for each antibiotic. Mupirocin induced the highest ratio of transducing versus lytic particles. Gentamicin and novobiocin reduced UV-induced AMR transduction. The genes carried in phage particles correlated with AMR transfer or lytic particle activity, suggesting antimicrobials influence which DNA sequences are packaged into phage particles. Conclusions: Sub-inhibitory antibiotics induce AMR gene transfer between clinical MRSA, while combination therapy with an inhibiting antibiotic could potentially alter AMR gene packaging into phage particles, reducing AMR transfer. In a continually evolving environment, pathogens have an advantage if they can transfer DNA while lowering the risk of lytic death.

Bacterial viruses enable their host to acquire antibiotic resistance genes from neighbouring cells.

Prophages are quiescent viruses located in the chromosomes of bacteria. In the human pathogen, Staphylococcus aureus, prophages are omnipresent and are believed to be responsible for the spread of some antibiotic resistance genes. Here we demonstrate that release of phages from a subpopulation of S. aureus cells enables the intact, prophage-containing population to acquire beneficial genes from competing, phage-susceptible strains present in the same environment. Phage infection kills competitor cells and bits of their DNA are occasionally captured in viral transducing particles. Return of such particles to the prophage-containing population can drive the transfer of genes encoding potentially useful traits such as antibiotic resistance. This process, which can be viewed as ‘auto-transduction', allows S. aureus to efficiently acquire antibiotic resistance both in vitro and in an in vivo virulence model (wax moth larvae) and enables it to proliferate under strong antibiotic selection pressure. Our results may help to explain the rapid exchange of antibiotic resistance genes observed in S. aureus.

Mycobacterial recombineering.

The precise knockout or modification of Mycobacterium tuberculosis genes has been critical for the identification of functions important for the growth and pathogenicity of this important bacterium. Schemes have been previously described, using both non-replicating vectors and transducing particles, for the introduction of gene knockout substrates into M. tuberculosis, where the endogenous recombination systems of the host (both homologous and illegitimate) compete for transfer of the modified allele to the chromosome. Recombineering technologies, first introduced in laboratory and pathogenic strains of Escherichia coli over the last 16 years, have been developed for use in M. tuberculosis. Described in this chapter is the use of the mycobacterial Che9c phage RecET recombination system, which has been used to make gene knockouts, reporter fusions, promoter replacements, and single base pair modifications within the M. tuberculosis and M. smegmatis chromosomes at very high frequency. Higher success rates, in a shorter period of time, are routinely observed when recombineering is compared to previously described M. tuberculosis gene knockout protocols.

Unravelling bacteriophage ϕ11 requirements for packaging and transfer of mobile genetic elements in Staphylococcus aureus

Bacteriophages play a major role in spreading mobile genetic elements (MGEs)-encoded genes among bacterial populations. In spite of this, the molecular requirements for building phage transducing particles have not been completely deciphered. Here, we systematically inactivated each ORF from the packaging and lysis modules of the staphylococcal phage ϕ11, used as a model for the Siphoviridae phages infecting Gram-positive bacteria, and determined their functional role in transferring different MGEs including plasmids, staphylococcal pathogenicity islands (SaPIs) and the phage itself. In a previous report, we identified seven of these ORFs as being required for the production of functional phage or SaPI particles. In this report, we have completed the mutational analysis and have identified and characterized 15 additional phage-encoded proteins required for the production of mature phage, SaPI, or transducing particles. Apart from these, we have not yet ascertained any specific function for the six remaining ϕ11 genes, though they are highly conserved among the staphylococcal bacteriophages. To the best of our knowledge, this study represents the first systematic deletion analysis of all the ORFs comprising the morphogenetic and lysis modules of a phage, clearly defining the molecular requirements involved in phage-mediated MGEs transfer.

High efficiency generalized transduction in Escherichia coli O157:H7

Genetic manipulation in enterohemorrhagic E. coli O157:H7 is currently restricted to recombineering, a method that utilizes the recombination system of bacteriophage lambda, to introduce gene replacements and base changes inter alia into the genome. Bacteriophage 933W is a prophage in E. coli O157:H7 strain EDL933, which encodes the genes ( stx2AB) for the production of Shiga toxin which is the basis for the potentially fatal Hemolytic Uremic Syndrome in infected humans. We replaced the stx2AB genes with a kanamycin cassette using recombineering. After induction of the prophage by ultra-violet light, we found that bacteriophage lysates were capable of transducing to wildtype, point mutations in the lactose, arabinose and maltose genes. The lysates could also transduce tetracycline resistant cassettes. Bacteriophage 933W is also efficient at transducing markers in E. coli K-12. Co-transduction experiments indicated that the maximal amount of transferred DNA was likely the size of the bacteriophage genome, 61 kB. All tested transductants, in both E. coli K-12 and O157:H7, were kanamycin-sensitive indicating that the transducing particles contained host DNA.

Efficient transfer of antibiotic resistance plasmids by transduction within methicillin-resistant Staphylococcus aureus USA300 clone

The epidemic community-associated methicillin-resistant clone Staphylococcus aureus USA300 is a major source of skin and soft tissue infections and involves strains with a diverse set of resistance genes. In this study, we report efficient transduction of penicillinase and tetracycline resistance plasmids by bacteriophages φ80α and φJB between clinical isolates belonging to the USA300 clone. High transduction frequencies (10(-5) - 10(-6) CFU/PFU) were observed using phages propagated on donor strains as well as prophages induced from donors by ultraviolet light. Quantitative real-time PCR was employed to detect penicillinase plasmids in transducing phage particles and determine the ratio of transducing particles in phage lysates to infectious phage particles (determined as approximately 1 : 1700). Successful transfer of plasmids between strains in USA300 clone proves transduction is an effective mechanism for spreading plasmids within the clone. Such events contribute to its evolution and to emergence of new multiple drug-resistant strains of this successful clone.

Mobilization of pathogenicity islands by Staphylococcus aureus strain Newman bacteriophages

Staphylococcus aureus pathogenicity islands (SaPIs) are mobile genetic elements that encode virulence factors and depend on helper phages for their mobilization. Such mobilization is specific and depends on the ability of a phage protein to inactivate the SaPI repressor Stl. Phage 80α can mobilize several SaPIs, including SaPI1 and SaPIbov1, via its Sri and Dut proteins, respectively. In many cases, the capsids formed in the presence of the SaPI are smaller than those normally produced by the phage. Two SaPI-encoded proteins, CpmA and CpmB, are involved in this size determination process. S. aureus strain Newman contains four prophages, named φNM1 through φNM4. Phages φNM1 and φNM2 are very similar to phage 80α in the structural genes, and encode almost identical Sri proteins, while their Dut proteins are highly divergent. We show that φNM1 and φNM2 are able to mobilize both SaPI1 and SaPIbov1 and yield infectious transducing particles. The majority of the capsids formed in all cases are small, showing that both SaPIs can redirect the capsid size of both φNM1 and φNM2.

The Staphylococcus aureus Pathogenicity Island 1 Protein gp6 Functions as an Internal Scaffold during Capsid Size Determination

Staphylococcus aureus pathogenicity island 1 (SaPI1) is a mobile genetic element that carries genes for several superantigen toxins. SaPI1 is normally stably integrated into the host genome but can become mobilized by “helper” bacteriophage 80α, leading to the packaging of SaPI1 genomes into phage-like transducing particles that are composed of structural proteins supplied by the helper phage but having smaller capsids. We show that the SaPI1-encoded protein gp6 is necessary for efficient formation of small capsids. The NMR structure of gp6 reveals a dimeric protein with a helix–loop–helix motif similar to that of bacteriophage scaffolding proteins. The gp6 dimer matches internal densities that bridge capsid subunits in cryo-electron microscopy reconstructions of SaPI1 procapsids, suggesting that gp6 acts as an internal scaffolding protein in capsid size determination.

Intracellular localization of adeno‐associated viral proteins expressed in insect cells

Production of vectors derived from adeno-associated virus (AAVv) in insect cells represents a feasible option for large-scale applications. However, transducing particles yields obtained in this system are low compared with total capsid yields, suggesting the presence of genome encapsidation bottlenecks. Three components are required for AAVv production: viral capsid proteins (VP), the recombinant AAV genome, and Rep proteins for AAV genome replication and encapsidation. Little is known about the interaction between the three components in insect cells, which have intracellular conditions different to those in mammalian cells. In this work, the localization of AAV proteins in insect cells was assessed for the first time with the purpose of finding potential limiting factors. Unassembled VP were located either in the cytoplasm or in the nucleus. Their transport into the nucleus was dependent on protein concentration. Empty capsids were located in defined subnuclear compartments. Rep proteins expressed individually were efficiently translocated into the nucleus. Their intranuclear distribution was not uniform and differed from VP distribution. While Rep52 distribution and expression levels were not affected by AAV genomes or VP, Rep78 distribution and stability changed during coexpression. Expression of all AAV components modified capsid intranuclear distribution, and assembled VP were found in vesicles located in the nuclear periphery. Such vesicles were related to baculovirus infection, highlighting its role in AAVv production in insect cells. The results obtained in this work suggest that the intracellular distribution of AAV proteins allows their interaction and does not limit vector production in insect cells.

A Conformational Switch Involved in Maturation of Staphylococcus aureus Bacteriophage 80α Capsids

Bacteriophages are involved in many aspects of the spread and establishment of virulence factors in Staphylococcus aureus, including the mobilization of genetic elements known as S. aureus pathogenicity islands (SaPIs), which carry genes for superantigen toxins and other virulence factors. SaPIs are packaged into phage-like transducing particles using proteins supplied by the helper phage. We have used cryo-electron microscopy and icosahedral reconstruction to determine the structures of the procapsid and the mature capsid of 80α, a bacteriophage that can mobilize several different SaPIs. The 80α capsid has T=7 icosahedral symmetry with the capsid protein organized into pentameric and hexameric clusters that interact via prominent trimeric densities. The 80α capsid protein was modeled based on the capsid protein fold of bacteriophage HK97 and fitted into the 80α reconstructions. The models show that the trivalent interactions are mediated primarily by a 22-residue β hairpin structure called the P loop that is not found in HK97. Capsid expansion is associated with a conformational switch in the spine helix that is propagated throughout the subunit, unlike the domain rotation mechanism in phage HK97 or P22.

Plasmid transduction using bacteriophage Phi(adh) for expression of CC chemokines by Lactobacillus gasseri ADH.

Vaginal mucosal microfloras are typically dominated by Gram-positive Lactobacillus species, and colonization of vaginal mucosa by exogenous microbicide-secreting Lactobacillus strains has been proposed as a means of enhancing this natural mucosal barrier against human immunodeficiency virus (HIV) infection. We asked whether an alternative strategy could be utilized whereby anti-HIV molecules are expressed within the cervicovaginal milieu by endogenous vaginal Lactobacillus populations which have been engineered in situ via transduction. In this study, we therefore investigated the feasibility of utilizing transduction for the expression of two HIV coreceptor antagonists, the CC chemokines CCL5 and CCL3, in a predominant vaginal Lactobacillus species, Lactobacillus gasseri. Modifying a previously established transduction model, which utilizes L. gasseri ADH and its prophage Phiadh, we show that mitomycin C induction of L. gasseri ADH transformants containing pGK12-based plasmids with CCL5 and CCL3 expression and secretion cassettes (under the control of promoters P6 and P59, respectively) and a 232-bp Phiadh cos site fragment results in the production of transducing particles which contain 8 to 9 copies of concatemeric plasmid DNA. High-frequency transduction for these particles (almost 6 orders of magnitude greater than that for pGK12 alone) was observed, and transductants were found to contain recircularized expression plasmids upon subsequent culture. Importantly, transductants produced CC chemokines at levels comparable to those produced by electroporation-derived transformants. Our findings therefore lend support to the potential use of transduction in vaginal Lactobacillus species as a novel strategy for the prevention of HIV infection across mucosal membranes.