(Re)-definition of the holo- and apo-Fur direct regulons of Helicobacter pylori.

Iron homeostasis is a critical process for living organisms because this metal is an essential co-factor for fundamental biochemical activities, like energy production and detoxification, albeit its excess quickly leads to cell intoxication. The protein Fur (ferric uptake regulator) controls iron homeostasis in bacteria by switching from its apo- to holo-form as a function of the cytoplasmic level of ferrous ions, thereby modulating gene expression. The Helicobacter pylori HpFur protein has the rare ability to operate as a transcriptional commutator; apo- and holo-HpFur function as two different repressors with distinct DNA binding recognition properties for specific sets of target genes. Although the regulation of apo- and holo-HpFur in this bacterium has been extensively investigated, we propose a genome-wide redefinition of holo-HpFur direct regulon in H. pylori by integration of RNA-seq and ChIP-seq data, and a large extension of the apo-HpFur direct regulon. We show that in response to iron availability, new coding sequences, non-coding RNAs, toxin-antitoxin systems, and transcripts within open reading frames are directly regulated by apo- or holo-HpFur. These new targets and the more thorough validation and deeper characterization of those already known provide a complete and updated picture of the direct regulons of this two-faced transcriptional regulator.

Molecular engineering of a spheroid-penetrating phage nanovector for photodynamic treatment of colon cancer cells.

Photodynamic therapy (PDT) represents an emerging strategy to treat various malignancies, including colorectal cancer (CC), the third most common cancer type. This work presents an engineered M13 phage retargeted towards CC cells through pentavalent display of a disulfide-constrained peptide nonamer. The M13CC nanovector was conjugated with the photosensitizer Rose Bengal (RB), and the photodynamic anticancer effects of the resulting M13CC-RB bioconjugate were investigated on CC cells. We show that upon irradiation M13CC-RB is able to impair CC cell viability, and that this effect depends on i) photosensitizer concentration and ii) targeting efficiency towards CC cell lines, proving the specificity of the vector compared to unmodified M13 phage. We also demonstrate that M13CC-RB enhances generation and intracellular accumulation of reactive oxygen species (ROS) triggering CC cell death. To further investigate the anticancer potential of M13CC-RB, we performed PDT experiments on 3D CC spheroids, proving, for the first time, the ability of engineered M13 phage conjugates to deeply penetrate multicellular spheroids. Moreover, significant photodynamic effects, including spheroid disruption and cytotoxicity, were readily triggered at picomolar concentrations of the phage vector. Taken together, our results promote engineered M13 phages as promising nanovector platform for targeted photosensitization, paving the way to novel adjuvant approaches to fight CC malignancies.

A modular phage vector platform for targeted photodynamic therapy of Gram-negative bacterial pathogens.

Growing antibiotic resistance has encouraged the revival of phage-inspired antimicrobial approaches. On the other hand, photodynamic therapy (PDT) is considered a very promising research domain for the protection against infectious diseases. Yet, very few efforts have been made to combine the advantages of both approaches in a modular, retargetable platform. Here, we foster the M13 bacteriophage as a multifunctional scaffold, enabling the selective photodynamic killing of bacteria. We took advantage of the well-defined molecular biology of M13 to functionalize its capsid with hundreds of photo-activable Rose Bengal sensitizers and contemporarily target this light-triggerable nanobot to specific bacterial species by phage display of peptide targeting moieties fused to the minor coat protein pIII of the phage. Upon light irradiation of the specimen, the targeted killing of diverse Gram(-) pathogens occurred at subnanomolar concentrations of the phage vector. Our findings contribute to the development of antimicrobials based on targeted and triggerable phage-based nanobiotherapeutics.

Putting a "C60 Ball" and Chain to Chlorin e6 Improves Its Cellular Uptake and Photodynamic Performances.

Chlorin e6 (Ce6) and fullerene (C60) are among the most used photosensitizers (PSs) for photodynamic therapy (PDT). Through the combination of the chemical and photophysical properties of Ce6 and C60, in principle, we can obtain an "ideal" photosensitizer that is able to bypass the limitations of the two molecules alone, i.e., the low cellular uptake of Ce6 and the scarce solubility and absorption in the red region of the C60. Here, we synthesized and characterized a Ce6-C60 dyad. The UV-Vis spectrum of the dyad showed the typical absorption bands of both fullerene and Ce6, while a quenching of Ce6 fluorescence was observed. This behavior is typical in the formation of a fullerene-antenna system and is due to the intramolecular energy, or electron transfer from the antenna (Ce6) to the fullerene. Consequently, the Ce6-C60 dyad showed an enhancement in the generation of reactive oxygen species (ROS). Flow cytometry measurements demonstrated how the uptake of the Ce6 was strongly improved by the conjugation with C60. The Ce6-C60 dyad exhibited in A431 cancer cells low dark toxicity and a higher PDT efficacy than Ce6 alone, due to the enhancement of the uptake and the improvement of ROS generation.

Light-Enhanced Cytotoxicity of Doxorubicin by Photoactivation.

The combination of photodynamic therapy with chemotherapy (photochemotherapy, PCT) can lead to additive or synergistic antitumor effects. Usually, two different molecules, a photosensitizer (PS) and a chemotherapeutic drug are used in PCT. Doxorubicin is one of the most successful chemotherapy drugs. Despite its high efficacy, two factors limit its clinical use: severe side effects and the development of chemoresistance. Doxorubicin is a chromophore, able to absorb light in the visible range, making it a potential PS. Here, we exploited the intrinsic photosensitizing properties of doxorubicin to enhance its anticancer activity in leukemia, breast, and epidermoid carcinoma cells, upon irradiation. Light can selectively trigger the local generation of reactive oxygen species (ROS), following photophysical pathways. Doxorubicin showed a concentration-dependent ability to generate peroxides and singlet oxygen upon irradiation. The underlying mechanisms leading to the increase in its cytotoxic activity were intracellular ROS generation and the induction of necrotic cell death. The nuclear localization of doxorubicin represents an added value for its use as a PS. The use of doxorubicin in PCT, simultaneously acting as a chemotherapeutic agent and a PS, may allow (i) an increase in the anticancer effects of the drug, and (ii) a decrease in its dose, and thus, its dose-related adverse effects.

Local detection of pH-induced disaggregation of biocompatible micelles by fluorescence switch ON.

Fluorogenic nanoparticles (NPs) able to sense different physiological environments and respond with disaggregation and fluorescence switching OFF/ON are powerful tools in nanomedicine as they can combine diagnostics with therapeutic action. pH-responsive NPs are particularly interesting as they can differentiate cancer tissues from healthy ones, they can drive selective intracellular drug release and they can act as pH biosensors. Controlled polymerization techniques are the basis of such materials as they provide solid routes towards the synthesis of pH-responsive block copolymers that are able to assemble/disassemble following protonation/deprotonation. Ring opening metathesis polymerization (ROMP), in particular, has been recently exploited for the development of experimental nanomedicines owing to the efficient direct polymerization of both natural and synthetic functionalities. Here, we capitalize on these features and provide synthetic routes for the design of pH-responsive fluorogenic micelles via the assembly of ROMP block-copolymers. While detailed photophysical characterization validates the pH response, a proof of concept experiment in a model cancer cell line confirmed the activity of the biocompatible micelles in relevant biological environments, therefore pointing out the potential of this approach in the development of novel nano-theranostic agents.

pH Switchable Water Dispersed Photocatalytic Nanoparticles.

Photogeneration of Reactive Oxygen Species (ROS) finds applications in fields as different as nanomedicine, art preservation, air and water depollution and surface decontamination. Here we present photocatalytic nanoparticles (NP) that are active only at acidic pH while they do not show relevant ROS photo-generation at neutral pH. This dual responsivity (to light and pH) is achieved by stabilizing the surface of TiO2 NP with a specific organic shell during the synthesis and it is peculiar of the achieved core shell-structure, as demonstrated by comparison with commercial photocatalytic TiO2 NP. For the investigation of the photocatalytic activity, we developed two methods that allow real time detection of the process preventing any kind of artifact arising from post-treatments and delayed analysis. The reversibility of the pH response was also demonstrated as well as the selective photo-killing of cancer cells at acidic pH.

EGFR-Targeted Photodynamic Therapy.

The epidermal growth factor receptor (EGFR) plays a pivotal role in the proliferation and metastatization of cancer cells. Aberrancies in the expression and activation of EGFR are hallmarks of many human malignancies. As such, EGFR-targeted therapies hold significant potential for the cure of cancers. In recent years, photodynamic therapy (PDT) has gained increased interest as a non-invasive cancer treatment. In PDT, a photosensitizer is excited by light to produce reactive oxygen species, resulting in local cytotoxicity. One of the critical aspects of PDT is to selectively transport enough photosensitizers to the tumors environment. Accordingly, an increasing number of strategies have been devised to foster EGFR-targeted PDT. Herein, we review the recent nanobiotechnological advancements that combine the promise of PDT with EGFR-targeted molecular cancer therapy. We recapitulate the chemistry of the sensitizers and their modes of action in PDT, and summarize the advantages and pitfalls of different targeting moieties, highlighting future perspectives for EGFR-targeted photodynamic treatment of cancer.

Carrying Temoporfin with Human Serum Albumin: A New Perspective for Photodynamic Application in Head and Neck Cancer.

Temoporfin (mTHPC) is approved in Europe for the photodynamic treatment of head and neck squamous cell carcinoma (HNSCC). Although it has a promising profile, its lipophilic character hampers the full exploitation of its potential due to high tendency of aggregation and a reduced ROS generation that compromise photodynamic therapy (PDT) efficacy. Moreover, for its clinical administration, mTHPC requires the presence of ethanol and propylene glycol as solvents, often causing adverse effects in the site of injection. In this paper we explored the efficiency of a new mTHPC formulation that uses human serum albumin (HSA) to disperse the photosensitizer in solution (mTHPC@HSA), investigating its anticancer potential in two HNSCC cell lines. Through a comprehensive characterization, we demonstrated that mTHPC@HSA is stable in physiological environment, does not aggregate, and is extremely efficient in PDT performance, due to its high singlet oxygen generation and the high dispersion as monomolecular form in HSA. This is supported by the computational identification of the specific binding pocket of mTHPC in HSA. Moreover, mTHPC@HSA-PDT induces cytotoxicity in both HNSCC cell lines, increasing intracellular ROS generation and the number of γ-H2AX foci, a cellular event involved in the global response to cellular stress. Taken together these results highlight the promising phototoxic profile of the complex, prompting further studies to assess its clinical potential.

Advanced photodynamic therapy with an engineered M13 phage targeting EGFR: Mitochondrial localization and autophagy induction in ovarian cancer cell lines.

Photodynamic therapy (PDT) is a potential synergistic approach to chemotherapy for treating ovarian cancer, the most lethal gynecologic malignancy. Here we used M13 bacteriophage as a targeted vector for the efficient photodynamic killing of SKOV3 and COV362 cells. The M13 phage was refactored (M13r) to display an EGFR binding peptide in its tip that is frequently overexpressed in ovarian cancer. The refactored phage was conjugated with chlorin e6 (Ce6), one of the most widely used photosensitizers (M13r-Ce6). The new platform, upon irradiation, generated ROS by type I mechanism and showed activity in killing SKOV3 and COV362 cells even at concentrations in which Ce6 alone was ineffective. A microscopy analysis demonstrated an enhanced cellular uptake of M13r-Ce6 compared to free Ce6 and its mitochondrial localization. Western blot analysis revealed significant downregulation in the expression of EGFR in cells exposed to M13r-Ce6 after PDT. Following PDT treatment, autophagy induction was supported by an increased expression of LC3II, along with a raised autophagic fluorescent signal, as observed by fluorescence microscopy analysis for autophagosome visualization. As a conclusion we have herein proposed a bacteriophage-based receptor targeted photodynamic therapy for EGFR-positive ovarian cancer.

Orthogonal nanoarchitectonics of M13 phage for receptor targeted anticancer photodynamic therapy.

Photodynamic therapy (PDT) represents a promising therapeutic modality for cancer. Here we used an orthogonal nanoarchitectonics approach (genetic/chemical) to engineer M13 bacteriophages as targeted vectors for efficient photodynamic killing of cancer cells. M13 was genetically refactored to display on the phage tip a peptide (SYPIPDT) able to bind the epidermal growth factor receptor (EGFR). The refactored M13EGFR phages demonstrated EGFR-targeted tropism and were internalized by A431 cancer cells, that overexpress EGFR. Using an orthogonal approach to the genetic display, M13EGFR phages were then chemically modified, conjugating hundreds of Rose Bengal (RB) photosensitizing molecules on the capsid surface, without affecting the selective recognition of the SYPIPDT peptides. Upon internalization, the M13EGFR-RB derivatives generated intracellularly reactive oxygen species, activated by an ultralow intensity white light irradiation. The killing activity of cancer cells is observed at picomolar concentrations of the M13EGFR phage.

Spiky Gold Nanoparticles for the Photothermal Eradication of Colon Cancer Cells.

Colorectal cancer (CRC) is a widespread and lethal disease. Relapses of the disease and metastasis are very common in instances of CRC, so adjuvant therapies have a crucial role in its treatment. Systemic toxic effects and the development of resistance during therapy limit the long-term efficacy of existing adjuvant therapeutic approaches. Consequently, the search for alternative strategies is necessary. Photothermal therapy (PTT) represents an innovative treatment for cancer with great potential. Here, we synthesize branched gold nanoparticles (BGNPs) as attractive agents for the photothermal eradication of colon cancer cells. By controlling the NP growth process, large absorption in the first NIR biological window was obtained. The FBS dispersed BGNPs are stable in physiological-like environments and show an extremely efficient light-to-heat conversion capability when irradiated with an 808-nm laser. Sequential cycles of heating and cooling do not affect the BGNP stability. The uptake of BGNPs in colon cancer cells was confirmed using flow cytometry and confocal microscopy, exploiting their intrinsic optical properties. In dark conditions, BGNPs are fully biocompatible and do not compromise cell viability, while an almost complete eradication of colon cancer cells was observed upon incubation with BGNPs and irradiation with an 808-nm laser source. The PTT treatment is characterized by an extremely rapid onset of action that leads to cell membrane rupture by induced hyperthermia, which is the trigger that promotes cancer cell death.

A role for circular code properties in translation.

Circular codes represent a form of coding allowing detection/correction of frame-shift errors. Building on recent theoretical advances on circular codes, we provide evidence that protein coding sequences exhibit in-frame circular code marks, that are absent in introns and are intimately linked to the keto-amino transformation of codon bases. These properties strongly correlate with translation speed, codon influence and protein synthesis levels. Strikingly, circular code marks are absent at the beginning of coding sequences, but stably occur 40 codons after the initiator codon, hinting at the translation elongation process. Finally, we use the lens of circular codes to show that codon influence on translation correlates with the strong-weak dichotomy of the first two bases of the codon. The results can lead to defining new universal tools for sequence indicators and sequence optimization for bioinformatics and biotechnological applications, and can shed light on the molecular mechanisms behind the decoding process.

Concanavalin A-Rose Bengal bioconjugate for targeted Gram-negative antimicrobial photodynamic therapy.

Photodynamic therapy (PDT) is considered a very promising therapeutic modality for antimicrobial therapy. Although several studies have demonstrated that Gram-positive bacteria are very sensitive to PDT, Gram-negative bacteria are more resistant to photodynamic action. This difference is due to a different cell wall structure. Gram-negative bacteria have an outer cell membrane containing lipopolysaccharides (LPS) that hinder the binding of photosensitizer molecules, protecting the bacterial cells from chemical attacks. Combination of the lipopolysaccharides-binding activity of Concanavalin A (ConA) with the photodynamic properties of Rose Bengal (RB) holds the potential of an innovative protein platform for targeted photodynamic therapy against Gram-negative bacteria. A ConA-RB bioconjugate was synthesized and characterized. Approximately 2.4 RB molecules were conjugated per ConA monomer. The conjugation of RB to ConA determines a decrease of the singlet oxygen generation and an increase of superoxide and peroxide production. The photokilling efficacy of the ConA-RB bioconjugate was demonstrated in a planktonic culture of E. coli. Irradiation with white light from a LED lamp produced a dose-dependent photokilling of bacteria. ConA-RB conjugates exhibited a consistent improvement over RB (up to 117-fold). The improved uptake of the photosensitizer explains the enhanced PDT effect accompanying increased membrane damages induced by the ConA-RB conjugate. The approach can be readily generalized (i) using different photo/sonosensitizers, (ii) to target other pathogens characterized by cell membranes containing lipopolysaccharides (LPS).

The Helicobacter pylori Heat-Shock Repressor HspR: Definition of Its Direct Regulon and Characterization of the Cooperative DNA-Binding Mechanism on Its Own Promoter.

The ability of pathogens to perceive environmental conditions and modulate gene expression accordingly is a crucial feature for bacterial survival. In this respect, the heat-shock response, a universal cellular response, allows cells to adapt to hostile environmental conditions and to survive during stress. In the major human pathogen Helicobacter pylori the expression of chaperone-encoding operons is under control of two auto-regulated transcriptional repressors, HrcA and HspR, with the latter acting as the master regulator of the regulatory circuit. To further characterize the HspR regulon in H. pylori, we used global transcriptome analysis (RNA-sequencing) in combination with Chromatin Immunoprecipitation coupled with deep sequencing (ChIP-sequencing) of HspR genomic binding sites. Intriguingly, these analyses showed that HspR is involved in the regulation of different crucial cellular functions through a limited number of genomic binding sites. Moreover, we further characterized HspR-DNA interactions through hydroxyl-radical footprinting assays. This analysis in combination with a nucleotide sequence alignment of HspR binding sites, revealed a peculiar pattern of DNA protection and highlighted sequence conservation with the HAIR motif (an HspR-associated inverted repeat of Streptomyces spp.). Site-directed mutagenesis demonstrated that the HAIR motif is fundamental for HspR binding and that additional nucleotide determinants flanking the HAIR motif are required for complete binding of HspR to its operator sequence spanning over 70 bp of DNA. This finding is compatible with a model in which possibly a dimer of HspR recognizes the HAIR motif overlapping its promoter for binding and in turn cooperatively recruits two additional dimers on both sides of the HAIR motif.

Targeting Helicobacter pylori urease activity and maturation: In-cell high-throughput approach for drug discovery.

BACKGROUND: Helicobacter pylori is a bacterium strongly associated with gastric cancer. It thrives in the acidic environment of the gastric niche of large portions of the human population using a unique adaptive mechanism that involves the catalytic activity of the nickel-dependent enzyme urease. Targeting urease represents a key strategy for drug design and H. pylori eradication. METHOD: Here, we describe a novel method to screen, directly in the cellular environment, urease inhibitors. A ureolytic Escherichia coli strain was engineered by cloning the entire urease operon in an expression plasmid and used to test in-cell urease inhibition with a high-throughput colorimetric assay. A two-plasmid system was further developed to evaluate the ability of small peptides to block the protein interactions that lead to urease maturation. RESULTS: The developed assay is a robust cellular model to test, directly in the cell environment, urease inhibitors. The efficacy of a co-expressed peptide to affect the interaction between UreF and UreD, two accessory proteins necessary for urease activation, was observed. This event involves a process that occurs through folding upon binding, pointing to the importance of intrinsically disordered hot spots in protein interfaces. CONCLUSIONS: The developed system allows the concomitant screening of a large number of drug candidates that interfere with the urease activity both at the level of the enzyme catalysis and maturation. GENERAL SIGNIFICANCE: As inhibition of urease has the potential of being a global antibacterial strategy for a large number of infections, this work paves the way for the development of new candidates for antibacterial drugs.

Comprehensive mapping of the Helicobacter pylori NikR regulon provides new insights in bacterial nickel responses.

Nickel homeostasis is important for pathogenic and ureolytic bacteria, which use this metal ion as enzymatic cofactor. For example, in the human pathogen Helicobacter pylori an optimal balance between nickel uptake and incorporation in metallo-enzymes is fundamental for colonization of the host. Nickel is also used as cofactor to modulate DNA binding of the NikR regulator, which controls transcription of genes involved in nickel trafficking or infection in many bacteria. Accordingly, there is much interest in a systematic characterization of NikR regulation. Herein we use H. pylori as a model to integrate RNA-seq and ChIP-seq data demonstrating that NikR not only regulates metal-ion transporters but also virulence factors, non-coding RNAs, as well as toxin-antitoxin systems in response to nickel stimulation. Altogether, results provide new insights into the pathobiology of H. pylori and contribute to understand the responses to nickel in other bacteria.

Insight into the essential role of the Helicobacter pylori HP1043 orphan response regulator: genome-wide identification and characterization of the DNA-binding sites.

Many bacterial regulatory genes appear to be dispensable, as they can be deleted from the genome without loss of bacterial functionalities. In Helicobacter pylori, the hp1043 gene, also known as hsrA, is one of the transcriptional regulator that is essential for cell viability. This gene could not be deleted, nor the amount of protein modulated, supporting the hypothesis that HP1043 could be involved in the regulation of crucial cellular processes. Even though detailed structural data are available for the HP1043 protein, its targets are still ill-defined. Using Chromatin Immunoprecipitation-sequencing (ChIP-seq), one of the most powerful approaches to characterize protein-DNA interactions in vivo, we were able to identify genome-wide several new HP1043 binding sites. Moreover, in vitro DNA binding assays enabled precise mapping of the HP1043 binding sites on the new targets, revealing the presence of a conserved nucleotide sequence motif. Intriguingly, a significant fraction of the newly identified binding sites overlaps promoter regions controlling the expression of genes involved in translation. Accordingly, when protein translation was blocked, a significant induction of almost all HP1043 target genes was detected. These observations prompted us to propose HP1043 as a key regulator in H. pylori, likely involved in sensing and in coordinating the response to environmental conditions that provoke an arrest of protein synthesis. The essential role of HP1043 in coordinating central cellular processes is discussed.

Metal-responsive promoter DNA compaction by the ferric uptake regulator.

Short-range DNA looping has been proposed to affect promoter activity in many bacterial species and operator configurations, but only few examples have been experimentally investigated in molecular detail. Here we present evidence for a metal-responsive DNA condensation mechanism controlled by the Helicobacter pylori ferric uptake regulator (Fur), an orthologue of the widespread Fur family of prokaryotic metal-dependent regulators. H. pylori Fur represses the transcription of the essential arsRS acid acclimation operon through iron-responsive oligomerization and DNA compaction, encasing the arsR transcriptional start site in a repressive macromolecular complex. A second metal-dependent regulator NikR functions as nickel-dependent anti-repressor at this promoter, antagonizing the binding of Fur to the operator elements responsible for the DNA condensation. The results allow unifying H. pylori metal ion homeostasis and acid acclimation in a mechanistically coherent model, and demonstrate, for the first time, the existence of a selective metal-responsive DNA compaction mechanism controlling bacterial transcriptional regulation.

The cag-pathogenicity island encoded CncR1 sRNA oppositely modulates Helicobacter pylori motility and adhesion to host cells.

Small regulatory RNAs (sRNAs) are emerging as key post-transcriptional regulators in many bacteria. In the human pathobiont Helicobacter pylori a plethora of trans- and cis-encoded sRNAs have been pinpointed by a global transcriptome study. However, only two have been studied in depth at the functional level. Here we report the characterization of CncR1, an abundant and conserved sRNA encoded by the virulence-associated cag pathogenicity island (cag-PAI) of H. pylori. Growth-phase dependent transcription of CncR1 is directed by the PcagP promoter, which resulted to be a target of the essential transcriptional regulator HsrA (HP1043). We demonstrate that the 213 nt transcript arising from this promoter ends at an intrinsic terminator, few bases upstream of the annotated cagP open reading frame, establishing CncR1 as the predominant gene product encoded by the cagP (cag15) locus. Interestingly, the deletion of the locus resulted in the deregulation en masse of σ(54)-dependent genes, linking CncR1 to flagellar functions. Accordingly, the enhanced motility recorded for cncR1 deletion mutants was complemented by ectopic reintroduction of the allele in trans. In silico prediction identified fliK, encoding a flagellar checkpoint protein, as likely regulatory target of CncR1. The interaction of CncR1 with the fliK mRNA was thus further investigated in vitro, demonstrating the formation of strand-specific interactions between the two RNA molecules. Accordingly, the full-length translational fusions of fliK with a lux reporter gene were induced in a cncR1 deletion mutant in vivo. These data suggest the involvement of CncR1 in the post-transcriptional modulation of H. pylori motility functions through down-regulation of a critical flagellar checkpoint factor. Concurrently, the cncR1 mutant revealed a decrease of transcript levels for several H. pylori adhesins, resulting in a phenotypically significant impairment of bacterial adhesion to a host gastric cell line. The data presented support a model in which the cag-PAI encoded CncR1 sRNA is able to oppositely modulate bacterial motility and adhesion to host cells.