Smart Magnetotactic Bacteria Enable the Inhibition of Neuroblastoma under an Alternating Magnetic Field.

Magnetotactic bacteria are ubiquitous microorganisms in nature that synthesize intracellular magnetic nanoparticles called magnetosomes in a gene-controlled way and arrange them in chains. From in vitro to in vivo, we demonstrate that the intact body of Magnetospirillum magneticum AMB-1 has potential as a natural magnetic hyperthermia material for cancer therapy. Compared to chains of magnetosomes and individual magnetosomes, the entire AMB-1 cell exhibits superior heating capability under an alternating magnetic field. When incubating with tumor cells, the intact AMB-1 cells disperse better than the other two types of magnetosomes, decreasing cellular viability under the control of an alternating magnetic field. Furthermore, in vivo experiments in nude mice with neuroblastoma found that intact AMB-1 cells had the best antitumor activity with magnetic hyperthermia therapy compared to other treatment groups. These findings suggest that the intact body of magnetotactic bacteria has enormous promise as a natural material for tumor magnetic hyperthermia. In biomedical applications, intact and living magnetotactic bacteria play an increasingly essential function as a targeting robot due to their magnetotaxis.

Magnetosome mediated oral Insulin delivery and its possible use in diabetes management.

Our study investigates the effect of magnetosome mediated oral Insulin delivery on diabetic induced rat models. The study involves the development of Magnetosome-Insulin (MI) conjugates by direct and indirect (by means of PEG) coupling method and further characterized by microscopic and spectroscopic analysis. The in vivo oral delivery of magnetosome-Insulin conjugate against streptozotocin-induced rat models and its efficiency was investigated. The impact of MI showed a remarkable change in the reduction of FBG levels up to 65% than the standard (Insulin). Similarly, the serum parameters: triglycerides (43.81%), AST&ALT (39.4 and 57.2%), total cholesterol (43.8%) showed significant changes compared to the diabetic control. The histological results of MI treated rats were found similar to control rats. Thus, these significantly notable results on diabetic rats depicts that magnetosomes can be employed as a potential approach and a very promising alternative for the parenteral route of Insulin delivery.

Effects of Environmental Conditions on High-Yield Magnetosome Production by Magnetospirillum gryphiswaldense MSR-1

Background: Magnetotactic bacteria are a heterogeneous group of Gram-negative prokaryote cells that produce linear chains of magnetic particles called magnetosomes, intracellular organelles composed of magnetic iron particles. Many important applications have been defined for magnetic nanoparticles in biotechnology, such as cell separation applications, as well as acting as carriers of enzymes, antibodies, or anti-cancer drugs. Since the bacterial growth is difficult and the yield of magnetosome production is low, the application of magnetosome has not been developed on a commercial scale. Methods: Magnetospirillum gryphiswaldense strain MSR-1 was used in a modified current culture medium supplemented by different concentrations of oxygen, iron, carbon, and nitrogen, to increase the yield of magnetosomes. Results: Our improved MSR-1 culture medium increased magnetosome yield, magnetosome number per bacterial cell, magnetic response, and bacterial cell growth yield significantly. The yield of magnetosome increased approximately four times. The optimized culture medium containing 25 mM of Na-pyruvate, 40 mM of NaNO3, 200 µM of ferrous sulfate, and 5-10 ppm of dissolved oxygen (DO) resulted in 186.67 mg of magnetosome per liter of culture medium. The iron uptake increased significantly, and the magnetic response of the bacteria to the magnetic field was higher than threefold as compared to the previously reported procedures. Conclusion: This technique not only decreases the cultivation time but also reduces the production cost. In this modified method, the iron and DO are the major factors affecting the production of magnetosome by M. gryphiswaldense strain MSR-1. However, refining this technique will enable a further yield of magnetosome and cell density.

Influence of the bacterial growth phase on the magnetic properties of magnetosomes synthesized by Magnetospirillum gryphiswaldense.

BACKGROUND: The magnetosome biosynthesis is a genetically controlled process but the physical properties of the magnetosomes can be slightly tuned by modifying the bacterial growth conditions. METHODS: We designed two time-resolved experiments in which iron-starved bacteria at the mid-logarithmic phase are transferred to Fe-supplemented medium to induce the magnetosomes biogenesis along the exponential growth or at the stationary phase. We used flow cytometry to determine the cell concentration, transmission electron microscopy to image the magnetosomes, DC and AC magnetometry methods for the magnetic characterization, and X-ray absorption spectroscopy to analyze the magnetosome structure. RESULTS: When the magnetosomes synthesis occurs during the exponential growth phase, they reach larger sizes and higher monodispersity, displaying a stoichiometric magnetite structure, as fingerprinted by the well defined Verwey temperature. On the contrary, the magnetosomes synthesized at the stationary phase reach smaller sizes and display a smeared Verwey transition, that suggests that these magnetosomes may deviate slightly from the perfect stoichiometry. CONCLUSIONS: Magnetosomes magnetically closer to stoichiometric magnetite are obtained when bacteria start synthesizing them at the exponential growth phase rather than at the stationary phase. GENERAL SIGNIFICANCE: The growth conditions influence the final properties of the biosynthesized magnetosomes. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editors: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.

Transcriptome analysis reveals physiological characteristics required for magnetosome formation in Magnetospirillum gryphiswaldense MSR-1.

Magnetosome synthesis ability of Magnetospirillum gryphiswaldense MSR-1 in an autofermentor can be precisely controlled through strict control of dissolved oxygen concentration. In this study, using transcriptome data we discovered gene transcriptional differences and compared physiological characteristics of MSR-1 cells cultured under aerobic (high-oxygen) and micro-aerobic (low-oxygen) conditions. The results showed that 77 genes were up-regulated and 95 genes were down-regulated significantly under micro-aerobic situation. These genes were involved primarily in the categories of cell metabolism, transport, regulation and unknown-function proteins. The nutrient transport and physiological metabolism were slowed down under micro-aerobic condition, whereas dissimilatory denitrification pathways were activated and it may supplemental energy was made available for magnetosome synthesis. The result suggested that the genes of magnetosome membrane proteins (Mam and Mms) are not directly regulated by oxygen level, or are constitutively expressed. A proposed regulatory network of differentially expressed genes reflects the complexity of physiological metabolism in MSR-1, and suggests that some yet-unknown functional proteins play important roles such as ferric iron uptake and transport during magnetosome synthesis. The transcriptome data provides a holistic view of the responses of MSR-1 cells to differing oxygen levels. This approach will give new insights into general principles of magnetosome formation.

Iron response regulator protein IrrB in Magnetospirillum gryphiswaldense MSR-1 helps control the iron/oxygen balance, oxidative stress tolerance, and magnetosome formation.

Magnetotactic bacteria are capable of forming nanosized, membrane-enclosed magnetosomes under iron-rich and oxygen-limited conditions. The complete genomic sequence of Magnetospirillum gryphiswaldense strain MSR-1 has been analyzed and found to contain five fur homologue genes whose protein products are predicted to be involved in iron homeostasis and the response to oxidative stress. Of these, only the MGMSRv2_3149 gene (irrB) was significantly downregulated under high-iron and low-oxygen conditions, during the transition of cell growth from the logarithmic to the stationary phase. The encoded protein, IrrB, containing the conserved HHH motif, was identified as an iron response regulator (Irr) protein belonging to the Fur superfamily. To investigate the function of IrrB, we constructed an irrB deletion mutant (ΔirrB). The levels of cell growth and magnetosome formation were lower in the ΔirrB strain than in the wild type (WT) under both high-iron and low-iron conditions. The ΔirrB strain also showed lower levels of iron uptake and H2O2 tolerance than the WT. Quantitative real-time reverse transcription-PCR analysis indicated that the irrB mutation reduced the expression of numerous genes involved in iron transport, iron storage, heme biosynthesis, and Fe-S cluster assembly. Transcription studies of the other fur homologue genes in the ΔirrB strain indicated complementary functions of the Fur proteins in MSR-1. IrrB appears to be directly responsible for iron metabolism and homeostasis and to be indirectly involved in magnetosome formation. We propose two IrrB-regulated networks (under high- and low-iron conditions) in MSR-1 cells that control the balance of iron and oxygen metabolism and account for the coexistence of five Fur homologues.

Magnetotactic bacteria for cancer therapy.

Cancer is characterized by anomalous cell growth. Conventional therapies face many challenges and hence alternative treatment methods are in great demand. In addition, nature offers the best inspiration and recently many therapies of natural origin have proved multi-targeted, multi-staged, and a multi-component mode of action against cancer. Magnetotactic bacteria and magnetosomes-based treatment methods are among them. Present paper reviews various routes by which magnetotactic bacteria and magnetosomes contribute to cancer therapy.

Magnetic nanoparticles from Magnetospirillum gryphiswaldense increase the efficacy of thermotherapy in a model of colon carcinoma.

Magnetic nanoparticles (MNPs) are capable of generate heating power under the influence of alternating magnetic fields (AMF); this behaviour recently opened new scenarios for advanced biomedical applications, mainly as new promising tumor therapies. In this paper we have tested magnetic nanoparticles called magnetosomes (MNs): a class of MNPs naturally produced by magnetotactic bacteria. We extracted MNs from Magnetospirillum gryphiswaldense strain MSR-1 and tested the interaction with cellular elements and anti-neoplastic activity both in vitro and in vivo, with the aim of developing new therapeutic approaches for neoplastic diseases. In vitro experiments performed on Human Colon Carcinoma HT-29 cell cultures demonstrated a strong uptake of MNs with no evident signs of cytotoxicity and revealed three phases in the interaction: adherence, transport and accumulation in Golgi vesicles. In vivo studies were performed on subcutaneous tumors in mice; in this model MNs are administered by direct injection in the tumor volume, then a protocol consisting of three exposures to an AMF rated at 187 kHz and 23kA/m is carried out on alternate days, over a week. Tumors were monitored by Magnetic Resonance Imaging (MRI) to obtain information about MNs distribution and possible tissue modifications induced by hyperthermia. Histological analysis showed fibrous and necrotic areas close to MNs injection sites in mice subjected to a complete thermotherapy protocol. These results, although concerning a specific tumor model, could be useful to further investigate the feasibility and efficacy of protocols based on MFH. Magnetic nanoparticles naturally produced and extracted from bacteria seem to be promising candidates for theranostic applications in cancer therapy.

Two bifunctional enzymes with ferric reduction ability play complementary roles during magnetosome synthesis in Magnetospirillum gryphiswaldense MSR-1.

The bacterial strain Magnetospirillum gryphiswaldense MSR-1 does not produce siderophores, but it absorbs a large amount of ferric iron and synthesizes magnetosomes. We demonstrated previously the presence of six types of ferric reductase isozymes (termed FeR1 through FeR6) in MSR-1. Of these isozymes, FeR5 was the most abundant and FeR6 showed the highest ferric reductase activity. In the present study, we cloned the fer5 and fer6 genes from MSR-1 and expressed them separately in Escherichia coli. FeR5 and FeR6 were shown to be bifunctional enzymes through analysis of amino acid sequence homologies, structural predictions (using data from GenBank), and detection of enzyme activities. FeR5 is a thioredoxin reductase and FeR6 is a flavin reductase, in addition to being ferric reductases. To elucidate the functions of the enzymes, we constructed two single-gene-deletion mutant strains (Δfer5 and Δfer6 mutants) and a double-gene-deletion mutant strain (Δfer5 Δfer6 [Δfer5+6] mutant) along with its complemented strains (C5 and C6). An evaluation of phenotypic and physiological properties did not reveal significant differences between the wild-type and single-gene-deletion strains, whereas the double-gene-deletion strain showed reduced iron absorption and no magnetosome synthesis. Complementation of the double-gene-deletion strain using either fer5 or fer6 resulted in the partial recovery of magnetosome synthesis. Quantitative real-time PCR analysis of fer5 and fer6 transcriptional levels in the wild-type and complemented strains demonstrated consistent transcription of the two genes and confirmed that FeR5 and FeR6 are bifunctional enzymes that play complementary roles during the process of magnetosome synthesis in MSR-1.

Magnetosomes eliminate intracellular reactive oxygen species in Magnetospirillum gryphiswaldense MSR-1.

Magnetotactic bacteria synthesize magnetic particles called magnetosomes that cause them to orient to their external magnetic fields. However, the physiological significance and other possible functions of these magnetosomes have not been explored in detail. In this study, we have investigated the biological functions of magnetosomes with respect to their ability to scavenge reactive oxygen species (ROS) in Magnetospirillum gryphiswaldense MSR-1. To assess the changes in ROS levels under different conditions, cells were cultured under aerobic or micro-aerobic conditions in medium containing high and low amounts of iron. To ensure that the observed results were not due to nonspecific interactions, reactions were carried out using a mutant deficient in synthesizing magnetite (mamO-deficient mutant), its complementary strain or the wild-type MSR-1. We observed that the levels of intercellular ROS under micro-aerobic conditions with high-iron medium were much higher when the non-synthetic Fe(3) O(4) crystals mutant Mu21-415 was employed for the assay, compared with the wild-type or complementary strain, or when conditions were aerobic with low-iron medium. These results indicated that magnetosomes function in the scavenging of intracellular ROS. Furthermore, we have demonstrated that the magnetosomes exhibit peroxidase-like properties, by using the earlier reported in vitro horseradish peroxidase assay for artificial magnetic nanoparticles. In addition to possessing peroxidase-like activity, the magnetosomes also exhibited a more enzymatic kinetic response, suggesting that proteins on the membranes of the magnetosomes likely contribute to the enzymatic activity. This is the first study to demonstrate that magnetosomes play an important role in decreasing or eliminating ROS.

Desulfovibrio magneticus RS-1 contains an iron- and phosphorus-rich organelle distinct from its bullet-shaped magnetosomes.

Intracellular magnetite crystal formation by magnetotactic bacteria has emerged as a powerful model for investigating the cellular and molecular mechanisms of biomineralization, a process common to all branches of life. Although magnetotactic bacteria are phylogenetically diverse and their crystals morphologically diverse, studies to date have focused on a few, closely related species with similar crystal habits. Here, we investigate the process of magnetite biomineralization in Desulfovibrio magneticus sp. RS-1, the only reported species of cultured magnetotactic bacteria that is outside of the alpha-Proteobacteria and that forms bullet-shaped crystals. Using a variety of high-resolution imaging and analytical tools, we show that RS-1 cells form amorphous, noncrystalline granules containing iron and phosphorus before forming magnetite crystals. Using NanoSIMS (dynamic secondary ion mass spectroscopy), we show that the iron-phosphorus granules and the magnetite crystals are likely formed through separate cellular processes. Analysis of the cellular ultrastructure of RS-1 using cryo-ultramicrotomy, cryo-electron tomography, and tomography of ultrathin sections reveals that the magnetite crystals are not surrounded by membranes but that the iron-phosphorus granules are surrounded by membranous compartments. The varied cellular paths for the formation of these two minerals lead us to suggest that the iron-phosphorus granules constitute a distinct bacterial organelle.