Engineering of Escherichia coli for targeted delivery of transgenes to HER2/neu-positive tumor cells

Targeting of non‐phagocytic tumor cells and prompt release of gene cargos upon entry into tumors are two limiting steps in the bacterial gene delivery path. To tackle these problems, the non‐pathogenic Escherichia coli strain BL21(DE3) was engineered to display the anti‐HER2/neu affibody on the surface. After co‐incubation with tumor cells for 3 h, the anti‐HER2/neu affibody‐presenting E. coli strain was selectively internalized into HER2/neu‐positive SKBR‐3 cells. The invasion efficiency reached as high as 30%. Furthermore, the bacteria were equipped with the phage ϕX174 lysin gene E‐mediated autolysis system. Carrying the transgene (e.g., eukaryotic green fluorescent protein, GFP), the tumor‐targeting bacteria were subjected to the thermal shock to trigger the autolysis system upon entry into HER2/neu‐positive cells. Flow cytometric analysis revealed that 3% of infected cells expressed GFP 24 h post thermal induction. Overall, the results show a promise of the proposed approach for developing bacteria as a delivery carrier. Biotechnol. Bioeng. 2011; 108:1662–1672. © 2011 Wiley Periodicals, Inc.     

Bacteria in cancer therapy: a novel experimental strategy

Resistance to conventional anticancer therapies in patients with advanced solid tumors has prompted the need of alternative cancer therapies. Moreover, the success of novel cancer therapies depends on their selectivity for cancer cells with limited toxicity to normal tissues. Several decades after Coley's work a variety of natural and genetically modified non-pathogenic bacterial species are being explored as potential antitumor agents, either to provide direct tumoricidal effects or to deliver tumoricidal molecules. Live, attenuated or genetically modified non-pathogenic bacterial species are capable of multiplying selectively in tumors and inhibiting their growth. Due to their selectivity for tumor tissues, these bacteria and their spores also serve as ideal vectors for delivering therapeutic proteins to tumors. Bacterial toxins too have emerged as promising cancer treatment strategy. The most potential and promising strategy is bacteria based gene-directed enzyme prodrug therapy. Although it has shown successful results in vivo yet further investigation about the targeting mechanisms of the bacteria are required to make it a complete therapeutic approach in cancer treatment.     

靶向破坏铜绿假单胞菌生物被膜的定向蛋白投放技术

【目的】随着合成生物学的发展,通过在细菌体内设计合成复杂、多功能的基因线路进行靶向治疗已经取得巨大进展。虽然这种使用细菌作为治疗传递系统,选择性地在体内释放有效治疗成分的方式具有极大优势,但是如何使细菌在代谢负荷增加较低的情况下有效地分泌功能蛋白并发挥作用依旧是一个难题。【方法】针对这一难题,本研究提供了一种新的策略,即以细菌中广泛存在的蛋白类杀菌素和丝状噬菌体等相关编码基因作为生物模块,通过对铜绿假单胞菌的这些内源生物模块的重新编排和组装,构建了一种能在特定条件下裂解并投放功能蛋白的工程菌。为了评价工程菌中构建的生物模块能否工作,本研究选择胞外多糖水解酶PelA和PslG作为工程菌投放的功能蛋白,以此构建了工程菌PAO1102。通过对铜绿假单胞菌生物被膜的破坏实验、抑制形成实验以及抗生素耐药性实验,检验PAO1102对铜绿假单胞菌生物被膜的破坏和预防效果。【结果】与对照组相比,工程菌PAO1102的处理可以显著破坏已形成的生物被膜并抑制生物被膜的形成,同时还可显著增强生物被膜中的细菌对妥布霉素的敏感性,且这些功能主要通过外界Pf4丝状噬菌体侵染并使工程菌裂解而释放功能蛋白这一途径实现的。【结论】本研究所构建的工程菌可以作为一种微生物工具,用于靶向破坏铜绿假单胞菌生物被膜。在后续的研究中可根据不同的需求,在工程菌中表达不同的功能基因并实现功能蛋白的定向投放,从而执行不同的生物学功能。     

Construction and Application of Elastin Like Polypeptide Containing IL-4 Receptor Targeting Peptide

Various human solid tumors highly express IL-4 receptors which amplify the expression of some of anti-apoptotic proteins, preventing drug-induced cancer cell death. Thus, IL-4 receptor targeted drug delivery can possibly increase the therapeutic efficacy in cancer treatment. Macromolecular carriers with multivalent targeting moieties offered great advantages in cancer therapy as they not only increase the plasma half-life of the drug but also allow delivery of therapeutic drugs to the cancer cells with higher specificity, minimizing the deleterious effects of the drug on normal cells. In this study we designed a library of elastin like polypeptide (ELP) polymers containing tumor targeting AP1 peptide using recursive directional ligation method. AP1 was previously discovered as an atherosclerotic plaque and breast tumor tissue homing peptide using phage display screening method, and it can selectively bind to the interleukin 4 receptor (IL-4R). The fluorescently labeled [AP1-V₁₂]₆, an ELP polymer containing six AP1 enhanced tumor-specific targeting ability and uptake efficiency in H226 and MDA-MB-231 cancer cell lines in vitro. Surface plasmon resonance analysis showed that multivalent presentation of the targeting ligand in the ELP polymer increased the binding affinity towards IL-4 receptor compared to free peptide. The binding of [AP1-V₁₂]₆ to cancer cells was remarkably reduced when IL-4 receptors were blocked by antibody against IL-4 receptor further confirmed its binding. Importantly, the Cy5.5-labeled [AP1-V₁₂]₆ demonstrated excellent homing and longer retention in tumor tissues in MDA-MB-231 xenograft mouse model. Immunohistological studies of tumor tissues further validated the targeting efficiency of [AP1-V₁₂]₆ to tumor tissue. These results indicate that designed [AP1-V₁₂]₆ can serve as a novel carrier for selective delivery of therapeutic drugs to tumors.     

Impediments to Enhancement of CPT-11 Anticancer Activity by E. coli Directed Beta-Glucuronidase Therapy

CPT-11 is a camptothecin analog used for the clinical treatment of colorectal adenocarcinoma. CPT-11 is converted into the therapeutic anti-cancer agent SN-38 by liver enzymes and can be further metabolized to a non-toxic glucuronide SN-38G, resulting in low SN-38 but high SN-38G concentrations in the circulation. We previously demonstrated that adenoviral expression of membrane-anchored beta-glucuronidase could promote conversion of SN-38G to SN-38 in tumors and increase the anticancer activity of CPT-11. Here, we identified impediments to effective tumor therapy with E. coli that were engineered to constitutively express highly active E. coli beta-glucuronidase intracellularly to enhance the anticancer activity of CPT-11. The engineered bacteria, E. coli (lux/βG), could hydrolyze SN-38G to SN-38, increased the sensitivity of cultured tumor cells to SN-38G by about 100 fold and selectively accumulated in tumors. However, E. coli (lux/βG) did not more effectively increase CPT-11 anticancer activity in human tumor xenografts as compared to non-engineered E. coli. SN-38G conversion to SN-38 by E. coli (lux/βG) appeared to be limited by slow uptake into bacteria as well as by segregation of E. coli in necrotic regions of tumors that may be relatively inaccessible to systemically-administered drug molecules. Studies using a fluorescent glucuronide probe showed that significantly greater glucuronide hydrolysis could be achieved in mice pretreated with E. coli (lux/βG) by direct intratumoral injection of the glucuronide probe or by intratumoral lysis of bacteria to release intracellular beta-glucuronidase. Our study suggests that the distribution of beta-glucuronidase, and possibly other therapeutic proteins, in the tumor microenvironment might be an important barrier for effective bacterial-based tumor therapy. Expression of secreted therapeutic proteins or induction of therapeutic protein release from bacteria might therefore be a promising strategy to enhance anti-tumor activity.     

Survival, Physiology, and Lysis of Lactococcus lactis in the Digestive Tract

The survival and the physiology of lactococcal cells in the different compartments of the digestive tracts of rats were studied in order to know better the fate of ingested lactic acid bacteria after oral administration. For this purpose, we used strains marked with reporter genes, the luxA-luxB gene of Vibrio harveyi and the gfp gene of Aequora victoria, that allowed us to differentiate the inoculated bacteria from food and the other intestinal bacteria. Luciferase was chosen to measure the metabolic activity of Lactococcus lactis in the digestive tract because it requires NADH, which is available only in metabolically active cells. The green fluorescent protein was used to assess the bacterial lysis independently of death. We report not only that specific factors affect the cell viability and integrity in some digestive tract compartments but also that the way bacteria are administrated has a dramatic impact. Lactococci which transit with the diet are quite resistant to gastric acidity (90 to 98% survival). In contrast, only 10 to 30% of bacteria survive in the duodenum. Viable cells are metabolically active in each compartment of the digestive tract, whereas most dead cells appear to be subject to rapid lysis. This property suggests that lactococci could be used as a vector to deliver specifically into the duodenum the proteins produced in the cytoplasm. This type of delivery vector would be particularly appropriate for targeting digestive enzymes such as lipase to treat pancreatic deficiencies.     

Prediction of bacterial proteins carrying a nuclear localization signal and nuclear targeting of HsdM from Klebsiella pneumoniae

Nuclear targeting of bacterial proteins is an emerging pathogenic mechanism whereby bacterial proteins can interact with nuclear molecules and alter the physiology of host cells. The fully sequenced bacterial genome can predict proteins that target the nuclei of host cells based on the presence of nuclear localization signal (NLS). In the present study, we predicted bacterial proteins with the NLS sequences from Klebsiella pneumoniae by bioinformatic analysis, and 13 proteins were identified as carrying putative NLS sequences. Among them, HsdM, a subunit of KpnAl that is a type I restriction-modification system found in K. pneumoniae, was selected for the experimental proof of nuclear targeting in host cells. HsdM carried the NLS sequences, ₇KKAKAKK₁₃, in the N-terminus. A transient expression of HsdM-EGFP in COS-1 cells exhibited exclusively a nuclear localization of the fusion proteins, whereas the fusion proteins of HsdM with substitutions in residues lysine to alanine in the NLS sequences, ₇AAAKAAA₁₃, were localized in the cytoplasm. HsdM was co-localized with importin o in the nuclei of host cells. Recombinant HsdM alone methylated the eukaryotic DNA in vitro assay. Although HsdM tested in this study has not been considered to be a virulence factor, the prediction of NLS motifs from the full sequenced genome of bacteria extends our knowledge of functional genomics to understand subcellular targeting of bacterial proteins.     

A Novel Balanced-Lethal Host-Vector System Based on glmS

During the last decade, an increasing number of papers have described the use of various genera of bacteria, including E. coli and S. typhimurium, in the treatment of cancer. This is primarily due to the facts that not only are these bacteria capable of accumulating in the tumor mass, but they can also be engineered to deliver specific therapeutic proteins directly to the tumor site. However, a major obstacle exists in that bacteria because the plasmid carrying the therapeutic gene is not needed for bacterial survival, these plasmids are often lost from the bacteria. Here, we report the development of a balanced-lethal host-vector system based on deletion of the glmS gene in E. coli and S. typhimurium. This system takes advantage of the phenotype of the GlmS⁻ mutant, which undergoes lysis in animal systems that lack the nutrients required for proliferation of the mutant bacteria, D-glucosamine (GlcN) or N-acetyl-D-glucosamine (GlcNAc), components necessary for peptidoglycan synthesis. We demonstrate that plasmids carrying a glmS gene (GlmS⁺p) complemented the phenotype of the GlmS⁻ mutant, and that GlmS⁺p was maintained faithfully both in vitro and in an animal system in the absence of selection pressure. This was further verified by bioluminescent signals from GlmS ⁺pLux carried in bacteria that accumulated in grafted tumor tissue in a mouse model. The signal was up to several hundred-fold stronger than that from the control plasmid, pLux, due to faithful maintenance of the plasmid. We believe this system will allow to package a therapeutic gene onto an expression plasmid for bacterial delivery to the tumor site without subsequent loss of plasmid expression as well as to quantify bioluminescent bacteria using in vivo imaging by providing a direct correlation between photon flux and bacterial number.     

The return of the phage

Phages have been used to treat infectious diseases since their discovery nearly a century ago. Modern sequencing and genetic engineering technologies now enable researchers to vastly expand the use of phages as general drug delivery vehicles....it is only in the past five years that the regulatory guidelines for the approval of phage products—both in therapy and food safety—have been createdOver the past decade, bacteriophages have occasionally stirred public and media interest because of their potential as biological weapons against bacterial infections. Such reports have tended to come from Russian or Georgian laboratories, whereas Western research institutes and companies have usually found that phages do not live up to their promise. More than a decade later, however, the view of bacteriophages is set to change. Spurred on by advances in sequencing and other molecular techniques, research into phages has yielded its first applications. Not only are phages proving effective as therapeutic agents, but they are also playing a role in food safety and as delivery vehicles for drugs against a wide range of diseases.Interest in phages as therapeutic agents emerged almost immediately after their discovery nearly a century ago (Twort, 1915; d''Hérelle, 1917). This interest evaporated quickly in the West after the discovery of penicillin, but phage research was kept alive in the old Soviet Union and continued after its collapse in the 1990s. Ongoing studies there, although not always conforming to the most rigorous standards, provided the only evidence of the therapeutic potential of phages.Eventually, especially in the light of the increasing threat from drug-resistant bacteria, Western researchers turned to exploring phages again. However, it is only in the past five years that the regulatory guidelines for the approval of phage products—in both therapy and food safety—have been created. Previously, the US Food and Drug Administration (FDA) had lacked the appropriate regulatory measures; it took them four years to approve the first phage product for use in food safety in 2006. ListShield^TM is a cocktail of several phages that target Listeria monocytogenes, contaminants in meat and poultry products. Approvals for other food safety products have followed with greater speed (Sulakvelidze, 2011). Moreover, in 2008, the FDA approved the first phase 1 clinical trial of phages. This again involved a cocktail of eight phages to target various bacteria including Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli, in venous leg ulcers. This trial eventually established the safety of the phage preparation and cleared the way for more phage therapy trials (www.clinicaltrials.gov).The recent acceptance in the West of phages as anti-pathogenic agents was preceded by their use for diagnostic purposes to identify bacteria...The recent acceptance in the West of phages as anti-pathogenic agents was preceded by their use for diagnostic purposes to identify bacteria, according to Martin Loessner from the Institute of Food, Nutrition and Health in Zürich, Switzerland. “It then became possible to [...] harness the specificity of phage for applications such as recognition of the host cell, and also for reporter phage, which is a genetically modified phage with a gene so [you] can easily see the phage''s impact on the target cell,” he explained. “Later on we figured why not go and revisit the idea of using phages against pathogens.”This approach turned out to be highly successful against key food pathogens, Loessner said, because of the way phages work: “[T]he phage has been very finely tuned through zillions of generations in the evolutionary arms race, and is highly specific.” This specificity is important for targeting the few bacteria that cause food poisoning while sparing the bacteria in fermented food—such as soft cheeses—that are harmless and contribute flavour. “The phage is also immune to development of resistance by the host bacteria, because if not it would have become extinct a long time ago,” Loessner said.It is bacterial toxins that cause food poisoning rather than bacteria themselves, so phages are used as a preventive measure to stop the growth of bacteria such as Listeria in the first place. As such, it is important to bombard food products with a large number of phages to ensure that virtually all target bacteria are eradicated. “I always have this magic number of 10⁸, or 100 million per gram of food,” Loessner said. “In 1 g of food there are often only 500 target bacteria, so there is not enough to amplify the phage and you need really high numbers to kill the bacteria in one round of infection.” He added that, in his view, phages would soon become the main treatment for preventing bacterial contamination. “Phage in the near future will be the number one [treatment against] Listeria and Salmonella. It''s becoming number one already, especially in the US.”In Europe, the use of phages in food safety therapy is being held back by the requirement that foods treated with them are labelled as containing viruses, which means they are likely to meet consumer resistance, as happened with foods containing or made from genetically modified organisms. Loessner commented that education is required to raise awareness that the properly controlled use of phages involves minimal risk and could greatly enhance food safety. However, he also emphasized that the use of phages should represent an extra level of protection, not replace existing quality control measures....because phage lysins are often specific to a single bacterial genus, they would allow the specific targeting of pathogenic bacteriaThe ability of phages to target specific bacteria while leaving others alone also has great potential for treating bacterial infections, particularly in the light of increasing antibiotic resistance. Such treatments would not necessarily involve the phage themselves, but rather the use of their lysins—the enzymes that weaken the bacterial cell wall to allow newly formed viruses to exit the host cell. Lysins can be administered as antibiotics, at least for gram-positive bacteria that lack a separate outer membrane around the cell wall. Moreover, because phage lysins are often specific to a single bacterial genus, they would allow the specific targeting of pathogenic bacteria. “The fact that phage lysins leave the commensal microflora undisturbed is particularly significant,” commented Olivia McAuliffe, Senior Research Officer at the Teagasc Food Research Centre in Cork, Ireland. “Most of the antibiotics used clinically have broad-activity spectra and treatment with these antibiotics can have devastating effects on the normal flora, in particular for those taking long-term antibiotic courses.”Phages also have another great advantage over most conventional antibiotics in being potent against both dividing and non-dividing cells. “Because most antibiotics target pathways such as protein synthesis, DNA replication, and cell wall biosynthesis, they can only act when the cells are actively growing,” McAuliffe added. “Because lysins are enzymes, they will chew away the peptidoglycan in both viable and non-viable cells, dividing and non-dividing cells. This would be particularly important in the case of slow-growing organisms that cause infection, an example being Mycobacterium species.”This specificity of phages and their lysins is particularly important for treating chronic conditions resulting from persistent bacterial infection, particularly in the respiratory system or digestive tract. Broad-spectrum antibiotics also attack harmless and beneficial commensal bacteria, and can even worsen the condition by encouraging the growth of resistant bacteria. This is the case with Clostridium difficile, a cause of secondary infections and a major nosocomial (hospital-acquired) antibiotic-resistant pathogen, according to McAuliffe. It is a Gram-positive, rod-shaped, spore-forming bacterium that is the most serious and common cause of diarrhoea and other intestinal disease when competing bacteria in the gut flora have been wiped out by antibiotics. The bacterium and its spores, which form in aerobic conditions outside the body, are widespread in the environment and are present in the guts of 3% of healthy individuals and 66% of infants, according to the UK''s Health Protection Agency. Clostridium spreads readily on the hands of healthcare staff and visitors in hospitals. The ability of the bacteria to form spores resistant to heat, drying and disinfectants, which then adhere to surfaces, enables them to persist in the hospital environment.Because Clostridium is resistant to most conventional antibiotics, it has for some years usually been treated with metronidazole, which exploits the fact that Clostridium is anaerobic during infection. Metronidazole has proven particularly appealing as it has relatively little impact on human cells or commensal aerobic bacteria in the gut as it does not work in the presence of oxygen. But metronidazole does not always work, and physicians have therefore been using vancomycin, a stronger but more toxic antibiotic, as a last resort. Moreover, even in cases where antibiotics seem to eliminate Clostridium and cure the associated diarrhoea, infection recurs in as many as 20% of hospital patients (Kelly & LaMont, 2008). About one-fifth of these 20%, or 4% of the total number of patients succumbing to Clostridium, end up with a long-term infection that at present is difficult to eradicate.This is where phages step in, because they are well tolerated by patients and their specificity means that they will not target other gut bacteria. Clostridium phages have already been demonstrated to work selectively and there is the possibility of extracting lysins against Clostridium from the phage itself; an avenue being pursued by Aidan Coffey''s group at the Department of Biological Sciences at the Cork Institute of Technology in Bishoptown, Ireland.There is also growing interest in using phages to tackle various other infections that are resistant to existing drugs—for example, in wounds that fail to heal, which are a major risk for diabetics. The application of phages in such cases is not new—before penicillin it was often the only option—but the difference now is that modern molecular techniques for isolating bacterial strains from biopsies and matching them to phages greatly increases efficiency. One clinical trial, organized by the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences, is currently recruiting patients to evaluate the use of phage preparations against a range of drug-resistant bacteria, including MRSA (methicillin-resistant Staphylococcus aureus), Enterococcus, Escherichia, Citrobacter, Enterobacter, Klebsiella, Shigella and Salmonella. The intention is to isolate bacterial strains from each patient and to identify matching phages from the Institute''s bacteriophage collection in Wrocław.Although the potential of phages or their lysins to combat bacterial pathogens, whether in food or those causing infectious diseases, has long been recognized, more recent work has identified new applications as delivery vehicles for vaccines or cytotoxic drugs to treat cancer. These applications do not exploit the phage''s natural targeting of bacteria, but make use of their ability to carry surface ligands that attract them to specific host cells.Even though phages do not attack human cells, they elicit an immune response and can be used as vectors to carry an engineered antigen on their surface to vaccinate against viral or bacterial disease. This approach has been tested in rabbits with a DNA vaccine against hepatitis B (Clark et al, 2011). The study compared the phage DNA vaccine with Engerix B—a commercially available vaccine based on a homologous recombinant protein—and found that the phage vaccine produced a significantly higher antibody response more quickly, as well as being potentially cheaper to produce and stable at a wider range of temperatures. This hepatitis B vaccine is now being developed by the UK biotech firm BigDNA in Edinburgh, Scotland, which has been granted a European patent, pending future clinical trials in humans.Modified phages could also serve as nanoparticles to deliver cytotoxic drugs straight to tumour cells, bypassing healthy cellsModified phages could also serve as nanoparticles to deliver cytotoxic drugs straight to tumour cells, bypassing healthy cells. Phages are a promising candidate vehicle because they can be readily engineered both to display appropriate ligands for targeting tumour cells specifically, and to carry a cytotoxic payload that is only released inside the target. One Israeli group has developed a technology for manufacturing phage nanoparticles that in principle can be used to target drugs to either tumour cells or pathogens (Bar et al, 2008). The group chose one particular phage family, known as filamentous phages, because of their small size and the relative ease of engineering them. Filamentous phages comprise just 10 genes with a sheath of several thousand identical α-helical coat proteins in a helical array assembled around a single-stranded circular DNA molecule. The Israeli scientists combine genetic modification and chemical engineering to create a phage that is able to attach to its target cell and release cytotoxic molecules. “Genetic engineering makes it possible to convert the phage to a targeted particle by displaying a target-specifying molecule on the phage coat,” explained Itai Benhar from Tel-Aviv University, the lead author of the paper. “Genetic engineering also makes it possible to design a drug-release mechanism. Finally chemical engineering makes it possible to load the particle with a large payload of cargo.”The group has used the same approach to target two bacteria species, Staphylococcus aureus and Escherichia coli, with the antibiotic chloramphenicol, which was first developed in 1949 but has raised concerns over its toxicity. According to the Israeli group, the phage nanoparticle loaded with the drug was 20,000 times more potent against both bacteria than the drug administered on its own. Just as importantly, the phage particles do not affect other cells. The overall advantage of the phage-based delivery approach is that it can deliver highly effective and toxic drugs in a safe way. The other point is that this and other methods in which phages are engineered to reach specific targets have nothing directly to do with the natural ability of phage viruses to attack bacteria. “The phage''s natural ability to infect bacteria is totally irrelevant to their application for targeting non-bacterial cells,” said Benhar. “In fact, they are not relevant for targeting bacteria either in this case, since the chemical modification we subject the phages to renders them non-infective.”However, the phage nanoparticles retain their immunogenic effect, which is a problem if the objective is merely to deliver a drug to the target while minimizing all other impacts. “Phages are immunogenic, and although we found a way to reduce their immunogenicity we did not totally eliminate it,” Benhar said. The other challenge is that, as the particles carry the payload drug on their surface, the physical and chemical properties change every time a new drug is loaded. Although the payload itself is inert until it reaches the target, the varying characteristics could alter the host response and therefore affect regulatory approval for each new phage construct, as safety would have to be demonstarted in each case.The use of phages is no longer confined to directly attacking infectious bacteria, but has vastly expanded in terms of methods, applications and the diseases that can be tackledNevertheless, this approach holds great promise as a novel way of delivering not just new drugs but also existing ones that are effective but too toxic for healthy cells. This is exactly the most exciting aspect of recent therapeutic phage research. The use of phages is no longer confined to directly attacking infectious bacteria, but has vastly expanded in terms of methods, applications and the diseases that can be tackled.     

Phage formation in Staphylococcus muscae cultures. X. The relationship between virus synthesis,the release of bacterial ribonucleic acid,virus liberation,and cellular lysis

1. Under a variety of conditions in which cells are infected with one or a few virus particles and the host cells are killed, but no infective particles or virus material is formed as indicated by plaque count, one-step growth curve, or protein or desoxyribonucleic determinations, the cells neither lyse nor release ribonucleic acid into the medium. 2. The "killing" effect of S. muscae phage is separate from its lytic property. 3. The release of ribonucleic acid into the medium is not simply due to the killing of the cell by the virus, and ribonucleic acid is never found in the medium unless virus material is synthesized. 4. Infected cells of S. muscae synthesizing virus release ribonucleic acid into the medium before cellular lysis begins and before any virus is liberated. 5. The higher the phage yield the more ribonucleic acid is released into the medium before any virus is released. 6. Phage may be released from one strain of Staphylococcus muscae without cellular lysis, although bacterial lysis begins shortly after the virus is released. In another strain, infected under similar conditions, virus liberation occurs simultaneously with cellular lysis. 7. The viruses liberated from both bacterial strains appear to be the same in so far as they cannot be distinguished by serological tests, have the same plaque type and plaque size, and need the same amino acids added to the medium in order to grow. Furthermore, the virus liberated from one strain can infect and multiply in the other strain and vice versa. 8. It is suggested that virus synthesis, in S. muscae cells infected with one or a few phage particles, leads to a disturbance of the normal cellular metabolism, resulting in lysis of the host cell.     

THE ACCELERATING EFFECT OF MANGANOUS IONS ON PHAGE ACTION

Dilute solutions of MnCl₂ or MnSO₄ accelerate the lytic effect of phage upon susceptible staphylococci. Under the conditions of our experiments the manganese-containing mixtures lysed regularly 0.5 hour sooner than the controls. The effect is shown to be due to a lowering of the lytic threshold, i.e. the quantity of phage/bacterium requisite for lysis; Mn⁺⁺ reduces the ratio from 54 to about 12. In the presence of Mn⁺⁺ phage distribution is altered and in growing phage-bacteria mixtures the extracellular phage concentration is increased by manganese to approximately 4 times that occurring in the absence of manganese. There appears to be no enhancement of phage formation nor any affect on the rate of bacterial growth. As would be anticipated, for any given initial phage concentration the end titre after completion of lysis is less in the presence of manganese than in its absence. This is due to the reduced lytic threshold produced by Mn⁺⁺, there consequently being less phage needed to bring about lytic destruction of the bacteria.     

Intracellular events during infection by Haemophilus influenzae phage and transfection by its DNA

Intracellular events following infection of competent Haemophilus influenzae by HPlcl phage, or transfection by DNA from the phage, were examined. Physical separation of a large fraction of the intracellular phage DNA from the bulk of the host DNA was achieved by lysis of infected or transfected cells with digitonin, followed by low-speed centrifugation. The small amount of bacterial DNA remaining with the phage DNA in the supernatants could be distinguished from phage DNA by its ability to yield transformants. After infection by whole phage, three forms of intracellular phage DNA were observable by sedimentation velocity analysis: form III, the slowest-sedimenting one; form II, which sedimented 1.1 times faster than III, and form I, which sedimented 1.6 times faster than III. It was shown by electron microscopy, velocity sedimentation in alkali, and equilibrium sedimentation with ethidium bromide, that forms I, II and III are twisted circles, open circles, and linear duplexes, respectively.After the entry of phage DNA into wild-type cells in transfection, the DNA is degraded at early times, but later some of the fragments are reassembled, resulting in molecules that sediment faster than the monomer length of phage DNA. Some of the fast-sedimenting molecules are presumably concatemers and are generated by recombination. In strain rec₁^? the fast-sedimenting molecules do not appear and degradation of phage DNA is even more pronounced than in wild-type cells. In strain rec₂^? there is little degradation of phage DNA, and the proportion of fast-sedimenting molecules is much smaller than in wild-type cells. Since rec₁^? and rec₂^? are transfected with much lower efficiency than wild type, our hypothesis is that both fragmentation and generation of fast-sedimenting phage DNA by recombination are required for more efficient transfection.     

A Liposomal Drug Platform Overrides Peptide Ligand Targeting to a Cancer Biomarker,Irrespective of Ligand Affinity or Density

One method for improving cancer treatment is the use of nanoparticle drugs functionalized with targeting ligands that recognize receptors expressed selectively by tumor cells. In theory such targeting ligands should specifically deliver the nanoparticle drug to the tumor, increasing drug concentration in the tumor and delivering the drug to its site of action within the tumor tissue. However, the leaky vasculature of tumors combined with a poor lymphatic system allows the passive accumulation, and subsequent retention, of nanosized materials in tumors. Furthermore, a large nanoparticle size may impede tumor penetration. As such, the role of active targeting in nanoparticle delivery is controversial, and it is difficult to predict how a targeted nanoparticle drug will behave in vivo. Here we report in vivo studies for α_vβ₆-specific H2009.1 peptide targeted liposomal doxorubicin, which increased liposomal delivery and toxicity to lung cancer cells in vitro. We systematically varied ligand affinity, ligand density, ligand stability, liposome dosage, and tumor models to assess the role of active targeting of liposomes to α_vβ₆. In direct contrast to the in vitro results, we demonstrate no difference in in vivo targeting or efficacy for H2009.1 tetrameric peptide liposomal doxorubicin, compared to control peptide and no peptide liposomes. Examining liposome accumulation and distribution within the tumor demonstrates that the liposome, and not the H2009.1 peptide, drives tumor accumulation, and that both targeted H2009.1 and untargeted liposomes remain in perivascular regions, with little tumor penetration. Thus H2009.1 targeted liposomes fail to improve drug efficacy because the liposome drug platform prevents the H2009.1 peptide from both actively targeting the tumor and binding to tumor cells throughout the tumor tissue. Therefore, using a high affinity and high specificity ligand targeting an over-expressed tumor biomarker does not guarantee enhanced efficacy of a liposomal drug. These results highlight the complexity of in vivo targeting.     

The presence of professional phagocytes dictates the number of host cells targeted for Yop translocation during infection

Type III secretion systems deliver effector proteins from Gram‐negative bacterial pathogens into host cells, where they disarm host defences, allowing the pathogens to establish infection. Although Yersinia pseudotuberculosis delivers its effector proteins, called Yops, into numerous cell types grown in culture, we show that during infection Y. pseudotuberculosis selectively targets Yops to professional phagocytes in Peyer's patches, mesenteric lymph nodes and spleen, although it colocalizes with B and T cells as well as professional phagocytes. Strikingly, in the absence of neutrophils, the number of cells with translocated Yops was significantly reduced although the bacterial loads were similar, indicating that Y. pseudotuberculosis did not arbitrarily deliver Yops to the available cells. Using isolated splenocytes, selective binding and selective targeting to professional phagocytes when bacteria were limiting was also observed, indicating that tissue architecture was not required for the tropism for professional phagocytes. In isolated splenocytes, YadA and Invasin increased the number of all cells types with translocated Yops, but professional phagocytes were still preferentially translocated with Yops in the absence of these adhesins. Together these results indicate that Y. pseudotuberculosis discriminates among cells it encounters during infection and selectively delivers Yops to phagocytes while refraining from translocation to other cell types.     

Agrobacterium tumefaciens Conn IV. Bacteriophage PB21 and Its Inhibitory Effect on Tumor Induction

Strain B2 of Agrobacterium tumefaciens Conn produces plaques when seeded against strain B6-806 of the same organism. From such a plaque, a highly virulent bacteriophage was obtained by use of D'Herelle's technique of selecting for virulent phage. On nutrient agar, this phage, PB2₁, produced large clear plaques which did not overgrow. Plaques produced on a glutamate medium and on White's plant tissue culture medium were even larger and in White's medium had a three-dimensional appearance. PB2₁ does not appear to be an oncogenic virus. To the contrary, the addition of phage under circumstances which insure mass lysis completely inhibited tumor initiation. Fewer than 10 phage particles present at the beginning of a 21-hr induction period were able, at times, to inhibit completely tumor induction by highly virulent bacteria (strain B6). The data lend further support to the concept that anything which interferes with the metabolic activity associated with the growth of the bacteria interferes with the tumor-inducing process. Attempts to use the phage to rid crown gall tissue of bacteria were unsuccessful.     

Encapsulation of Bacteriophage in Liposome Accentuates Its Entry in to Macrophage and Shields It from Neutralizing Antibodies

Phage therapy has been a centre of attraction for biomedical scientists to treat infections caused by drug resistant strains. However, ability of phage to act only on extracellular bacteria and probability of interference by anti-phage antibodies in vivo is considered as a important limitation of bacteriophage therapy. To overcome these hurdles, liposome were used as delivery vehicle for phage in this study. Anti-phage antibodies were raised in mice and pooled serum was evaluated for its ability to neutralize free and liposome entrapped phage. Further, ability of phage and liposome-entrapped phage to enter mouse peritoneal macrophages and kill intracellular Klebsiella pneumoniae was compared. Also, an attempt to compare the efficacy of free phage and liposome entrapped phage, alone or in conjunction with amikacin in eradicating mature biofilm was made. The entrapment of phage in liposome provided 100% protection to phage from neutralizing antibody. On the contrary un-entrapped phage got neutralized within 3 h of its interaction with antibody. Compared to the inability of free phage to enter macrophages, the liposome were able to deliver entrapped phage inside macrophages and cause 94.6% killing of intracellular K. pneumoniae. Liposome entrapped phage showed synergistic activity along with amikacin to eradicate mature biofilm of K. pneumoniae. Our study reinforces the growing interest in using phage therapy as a means of targeting multidrug resistant bacterial infections as liposome entrapment of phage makes them highly effective in vitro as well as in vivo by overcoming the majority of the hurdles related to clinical use of phage.     

Direct injection of functional single-domain antibodies from E. coli into human cells

Intracellular proteins have a great potential as targets for therapeutic antibodies (Abs) but the plasma membrane prevents access to these antigens. Ab fragments and IgGs are selected and engineered in E. coli and this microorganism may be also an ideal vector for their intracellular delivery. In this work we demonstrate that single-domain Ab (sdAbs) can be engineered to be injected into human cells by E. coli bacteria carrying molecular syringes assembled by a type III protein secretion system (T3SS). The injected sdAbs accumulate in the cytoplasm of HeLa cells at levels ca. 10⁵–10⁶ molecules per cell and their functionality is shown by the isolation of sdAb-antigen complexes. Injection of sdAbs does not require bacterial invasion or the transfer of genetic material. These results are proof-of-principle for the capacity of E. coli bacteria to directly deliver intracellular sdAbs (intrabodies) into human cells for analytical and therapeutic purposes.