Liposomal Rifabutin-A Promising Antibiotic Repurposing Strategy against Methicillin-Resistant Staphylococcus aureus Infections.

Methicillin-resistant Staphylococcus aureus (M RSA) infections, in particular biofilm-organized bacteria, remain a clinical challenge and a serious health problem. Rifabutin (RFB), an antibiotic of the rifamycins class, has shown in previous work excellent anti-staphylococcal activity. Here, we proposed to load RFB in liposomes aiming to promote the accumulation of RFB at infected sites and consequently enhance the therapeutic potency. Two clinical isolates of MRSA, MRSA-C1 and MRSA-C2, were used to test the developed formulations, as well as the positive control, vancomycin (VCM). RFB in free and liposomal forms displayed high antibacterial activity, with similar potency between tested formulations. In MRSA-C1, minimal inhibitory concentrations (MIC) for Free RFB and liposomal RFB were 0.009 and 0.013 µg/mL, respectively. Minimum biofilm inhibitory concentrations able to inhibit 50% biofilm growth (MBIC50) for Free RFB and liposomal RFB against MRSA-C1 were 0.012 and 0.008 µg/mL, respectively. Confocal microscopy studies demonstrated the rapid internalization of unloaded and RFB-loaded liposomes in the bacterial biofilm matrix. In murine models of systemic MRSA-C1 infection, Balb/c mice were treated with RFB formulations and VCM at 20 and 40 mg/kg of body weight, respectively. The in vivo results demonstrated a significant reduction in bacterial burden and growth index in major organs of mice treated with RFB formulations, as compared to Control and VCM (positive control) groups. Furthermore, the VCM therapeutic dose was two fold higher than the one used for RFB formulations, reinforcing the therapeutic potency of the proposed strategy. In addition, RFB formulations were the only formulations associated with 100% survival. Globally, this study emphasizes the potential of RFB nanoformulations as an effective and safe approach against MRSA infections.

Quantitative Imaging of the Action of vCPP2319, an Antimicrobial Peptide from a Viral Scaffold, against Staphylococcus aureus Biofilms of a Clinical Isolate.

The formation of biofilms is a common virulence factor that makes bacterial infections difficult to treat and a major human health problem. Biofilms are bacterial communities embedded in a self-produced matrix of extracellular polymeric substances (EPS). In this work, we show that vCPP2319, a polycationic peptide derived from the capsid protein of Torque teno douroucouli virus, is active against preformed Staphylococcus aureus biofilms produced by both a reference strain and a clinical strain isolated from a diabetic foot infection, mainly by the killing of biofilm-embedded bacteria. The direct effect of vCPP2319 on bacterial cells was imaged using atomic force and confocal laser scanning microscopy, showing that the peptide induces morphological changes in bacterial cells and membrane disruption. Importantly, vCPP2319 exhibits low toxicity toward human cells and high stability in human serum. Since vCPP2319 has a limited effect on the biofilm EPS matrix itself, we explored a combined effect with α-amylase (EC 3.2.1.1), an EPS matrix-degrading enzyme. In fact, α-amylase decreases the density of S. aureus biofilms by 2.5-fold. Nonetheless, quantitative analysis of bioimaging data shows that vCPP2319 partially restores biofilm compactness after digestion of the polysaccharides, probably due to electrostatic cross-bridging of the matrix nucleic acids, which explains why α-amylase fails to improve the antibacterial action of the peptide.

Extracellular Vesicles and Infection: From Hijacked Machinery to Therapeutic Tools.

Extracellular vesicles (EVs) comprise a broad range of secreted cell-derived membrane vesicles. Beyond their more well-characterized role in cell communication, in recent years, EVs have also been shown to play important roles during infection. Viruses can hijack the biogenesis of exosomes (which are small EVs) to promote viral spreading. Additionally, these exosomes are also important mediators in inflammation and immune responses during both bacterial and viral infections. This review summarizes these mechanisms while also describing the impact of bacterial EVs in regulating immune responses. Finally, the review also focuses on the potential and challenges of using EVs, in particular, to tackle infectious diseases.

Acetate modulates the inhibitory effect of Lactobacillus gasseri against the pathogenic yeasts Candida albicans and Candida glabrata.

The exploration of the interference prompted by commensal bacteria over fungal pathogens is an interesting alternative to develop new therapies. In this work we scrutinized how the presence of the poorly studied vaginal species Lactobacillus gasseri affects relevant pathophysiological traits of Candida albicans and Candida glabrata. L. gasseri was found to form mixed biofilms with C. albicans and C. glabrata resulting in pronounced death of the yeast cells, while bacterial viability was not affected. Reduced viability of the two yeasts was also observed upon co-cultivation with L. gasseri under planktonic conditions. Either in planktonic cultures or in biofilms, the anti-Candida effect of L. gasseri was augmented by acetate in a concentration-dependent manner. During planktonic co-cultivation the two Candida species counteracted the acidification prompted by L. gasseri thus impacting the balance between dissociated and undissociated organic acids. This feature couldn't be phenocopied in single-cultures of L. gasseri resulting in a broth enriched in acetic acid, while in the co-culture the non-toxic acetate prevailed. Altogether the results herein described advance the design of new anti-Candida therapies based on probiotics, in particular, those based on vaginal lactobacilli species, helping to reduce the significant burden that infections caused by Candida have today in human health.

A Glimpse into Dendrimers Integration in Cancer Imaging and Theranostics.

Cancer is a result of abnormal cell proliferation. This pathology is a serious health problem since it is a leading cause of death worldwide. Current anti-cancer therapies rely on surgery, radiation, and chemotherapy. However, these treatments still present major associated problems, namely the absence of specificity. Thus, it is urgent to develop novel therapeutic strategies. Nanoparticles, particularly dendrimers, have been paving their way to the front line of cancer treatment, mostly for drug and gene delivery, diagnosis, and disease monitoring. This is mainly derived from their high versatility, which results from their ability to undergo distinct surface functionalization, leading to improved performance. In recent years, the anticancer and antimetastatic capacities of dendrimers have been discovered, opening new frontiers to dendrimer-based chemotherapeutics. In the present review, we summarize the intrinsic anticancer activity of different dendrimers as well as their use as nanocarriers in cancer diagnostics and treatment.

The role of RNA regulators, quorum sensing and c-di-GMP in bacterial biofilm formation.

Biofilms provide an ecological advantage against many environmental stressors, such as pH and temperature, making it the most common life-cycle stage for many bacteria. These protective characteristics make eradication of bacterial biofilms challenging. This is especially true in the health sector where biofilm formation on hospital or patient equipment, such as respirators, or catheters, can quickly become a source of anti-microbial resistant strains. Biofilms are complex structures encased in a self-produced polymeric matrix containing numerous components such as polysaccharides, proteins, signalling molecules, extracellular DNA and extracellular RNA. Biofilm formation is tightly controlled by several regulators, including quorum sensing (QS), cyclic diguanylate (c-di-GMP) and small non-coding RNAs (sRNAs). These three regulators in particular are fundamental in all stages of biofilm formation; in addition, their pathways overlap, and the significance of their role is strain-dependent. Currently, ribonucleases are also of interest for their potential role as biofilm regulators, and their relationships with QS, c-di-GMP and sRNAs have been investigated. This review article will focus on these four biofilm regulators (ribonucleases, QS, c-di-GMP and sRNAs) and the relationships between them.

Cost-Effective Mechanical Aggregation of Cardiac Progenitors and Encapsulation in Matrigel Support Self-Organization in a Dynamic Culture Environment.

Human iPSC-derived self-organized cardiac tissues can be valuable for the development of platforms for disease modeling and drug screening, enhancing test accuracy and reducing pharmaceutical industry financial burden. However, current differentiation systems still rely on static culture conditions and specialized commercial microwells for aggregation, which hinders the full potential of hiPSC-derived cardiac tissues. Herein, we integrate cost-effective and reproducible manual aggregation of hiPSC-derived cardiac progenitors with Matrigel encapsulation and a dynamic culture to support hiPSC cardiac differentiation and self-organization. Manual aggregation at day 7 of cardiac differentiation resulted in 97% of beating aggregates with 78% of cTnT-positive cells. Matrigel encapsulation conjugated with a dynamic culture promoted cell migration and the creation of organized structures, with observed cell polarization and the creation of lumens. In addition, encapsulation increased buoyancy and decreased coalescence of the hiPSC-derived cardiac aggregates. Moreover, VEGF supplementation increased over two-fold the percentage of CD31-positive cells resulting in the emergence of microvessel-like structures. Thus, this study shows that the explored culture parameters support the self-organization of hiPSC-derived cardiac microtissues containing multiple cardiac cell types. Additional stimuli (e.g., BMP) in long-term scalable and fully automatized cultures can further potentiate highly structured and mature hiPSC-derived cardiac models, contributing to the development of reliable platforms for high-throughput drug screening and disease modeling.

Xeno-Free Integrated Platform for Robust Production of Cardiomyocyte Sheets from hiPSCs.

Human induced pluripotent stem cells (hiPSCs) can be efficiently differentiated into cardiomyocytes (CMs), which can be used for cardiac disease modeling, for drug screening, and to regenerate damaged myocardium. Implementation of xeno-free culture systems is essential to fully explore the potential of these cells. However, differentiation using xeno-free adhesion matrices often results in low CM yields and lack of functional CM sheets, capable of enduring additional maturation stages. Here, we established a xeno-free CM differentiation platform using TeSR/Synthemax, including a replating step and integrated with two versatile purification/enrichment metabolic approaches. Results showed that the replating step was essential to reestablish a fully integrated, closely-knit CM sheet. In addition, replating contributed to increase the cTnT expression from 65% to 75% and the output from 2.2 to 3.1 CM per hiPSC, comparable with the efficiency observed when using TeSR/Matrigel. In addition, supplementation with PluriSin1 and Glu-Lac+ medium allowed increasing the CM content over 80% without compromising CM sheet integrity or functionality. Thus, this xeno-free differentiation platform is a reliable and robust method to produce hiPSC-derived CMs, increasing the possibility of using these cells safely for a wide range of applications.

Optically traceable PLGA-silica nanoparticles for cell-triggered doxorubicin delivery.

Fluorescent silica nanoparticles with a polymer shell of poly (D, L-lactide-co-glycolide) (PLGA) can provide traceable cell-triggered delivery of the anticancer drug doxorubicin (DOX), protecting the cargo while in transit and releasing it only intracellularly. PLGA with 50:50 lactide:glycolide ratio was grown by surface-initiated ring-opening polymerization (ROP) from silica nanoparticles of ca. 50 nm diameter, doped with a perylenediimide (PDI) fluorescent dye anchored to the silica structure. After loading DOX, release from the core-shell particles was evaluated in solution at physiological pH (7.4), and in human breast cancer cells (MCF-7) after internalization. The hybrid silica-PLGA nanoparticles can accommodate a large cargo of DOX, and the release in solution (PBS) due to PLGA hydrolysis is negligible for at least 72 h. However, once internalized in MCF-7 cells, the nanoparticles release the DOX cargo by degradation of the PLGA. Accumulation of DOX in the nucleus causes cell apoptosis, with the drug-loaded nanoparticles found to be as potent as free DOX. Our fluorescently traceable hybrid silica-PLGA nanoparticles with cell-triggered cargo release offer excellent prospects for the controlled delivery of anticancer drugs, protecting the cargo while in transit and efficiently releasing the drug once inside the cell.

A designed cyclic analogue of gomesin has potent activity against Staphylococcus aureus biofilms.

BACKGROUND: Infections caused by bacterial biofilms are very difficult to treat. The use of currently approved antibiotics even at high dosages often fails, making the treatment of these infections very challenging. Novel antimicrobial agents that use distinct mechanisms of action are urgently needed. OBJECTIVES: To explore the use of [G1K,K8R]cGm, a designed cyclic analogue of the antimicrobial peptide gomesin, as an alternative approach to treat biofilm infections. METHODS: We studied the activity of [G1K,K8R]cGm against biofilms of Staphylococcus aureus, a pathogen associated with several biofilm-related infections. A combination of atomic force and real-time confocal laser scanning microscopies was used to study the mechanism of action of the peptide. RESULTS: The peptide demonstrated potent activity against 24 h-preformed biofilms through a concentration-dependent ability to kill biofilm-embedded cells. Mechanistic studies showed that [G1K,K8R]cGm causes morphological changes on bacterial cells and permeabilizes their membranes across the biofilm with a half-time of 65 min. We also tested an analogue of [G1K,K8R]cGm without disulphide bonds, and a linear unfolded analogue, and found both to be inactive. CONCLUSIONS: The results suggest that the 3D structure of [G1K,K8R]cGm and its stabilization by disulphide bonds are essential for its antibacterial and antibiofilm activities. Moreover, our findings support the potential application of this stable cyclic antimicrobial peptide to fight bacterial biofilms.

Core-shell polycationic polyurea pharmadendrimers: new-generation of sustainable broad-spectrum antibiotics and antifungals.

The efficacy of conventional antimicrobials is falling to critical levels and raising alarming concerns around the globe. In this scenery, engineered nanoparticles emerged as a solid strategy to fight growing deadly infections. Here, we show the in vitro and in vivo performance of pharmadendrimers, a novel class of engineered polyurea dendrimers that are synthetic mimics of antibacterial peptides, against a collection of both Gram-positive and Gram-negative bacteria and fungi. These nanobiomaterials are stable solids prepared by low-cost and green processes, display a dense positively charged core-shell, and are biocompatible and hemocompatible drugs. Mechanistic data, corroborated by coarse-grained molecular dynamics simulations, points towards a fast-killing mechanism via membrane disruption, triggered by electrostatic interactions. Altogether this study provides strong evidence and support for the future use of polyurea pharmadendrimers in antibacterial and antifungal nanotherapeutics.

Multivalent NHS-activated acrylates for orthogonal site-selective functionalisation of peptides at cysteine residues.

The site-selective chemical appendage of multiple functionalities on a native peptide backbone is a highly demanding and complex tool of modern chemical biology. Here, novel NHS-activated acrylates were designed to hold various payloads in a single bioconjugation handle that is able to site-selectively and orthogonally target the N-terminal cysteine of peptides.

Fusions of a carbohydrate binding module with the small cationic hexapeptide RWRWRW confer antimicrobial properties to cellulose-based materials.

The emergence of antibiotic-resistant bacteria is a critical worldwide healthcare problem. In the specific case of wound care, new and effective alternatives to currently available solutions are urgently needed. Cellulose-based dressings, for example, could be made more attractive if rendered antimicrobial. This work proposes a new strategy to modify cellulose-based materials with the short antimicrobial hexapeptide MP196 (RWRWRW-NH2) that relies on a biomolecular recognition approach based on carbohydrate binding modules (CBMs). Specifically, we focused on the modification of hydrogels, paper, and microfibrillated cellulose (MFC) with fusions of the CBM3 from Clostridium thermocellum (C. thermocellum) with derivatives of MP196. The fusions are prepared by promoting the formation of a disulfide bond between Cys-terminated derivatives of MP196 and a CBM3 that is pre-anchored in the materials. The CBM3-MP196-modified materials displayed antibacterial activity against Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) that was significantly higher when compared with the activity of materials prepared by physical adsorption of MP196. The biomolecular strategy provides a more favorable orientation, exposure, and distancing of the peptide from the matrix. This versatile concept provides a toolbox for the functionalization of cellulose materials of different origins and architectures with a broad choice in peptides. Functionalization under mild biological conditions avoids further purification steps, allowing for translational research and multiple applications as drug delivery systems, scaffolds for tissue engineering and biomaterials. STATEMENT OF SIGNIFICANCE: The emergence of antibiotic-resistant bacteria is a critical worldwide healthcare problem. In the specific case of wound care, new and effective alternatives to currently available solutions are urgently needed. This work proposes a new strategy to modify cellulose-based materials with a short antimicrobial hexapeptide that relies on a biomolecular recognition approach based on carbohydrate binding modules. The modified materials displayed antibacterial activity against both Gram-negative and Gram-positive bacteria. The biomolecular strategy provides a favorable orientation, exposure, and distancing of the peptide from the matrix. This versatile concept offers a toolbox for the functionalization of different cellulose materials with a broad choice in peptides. Functionalization under mild biological conditions avoids further purification steps, allowing for translational research and multiple applications.

RNase R, a New Virulence Determinant of Streptococcus pneumoniae.

Pneumococcal infections have increasingly high mortality rates despite the availability of vaccines and antibiotics. Therefore, the identification of new virulence determinants and the understanding of the molecular mechanisms behind pathogenesis have become of paramount importance in the search of new targets for drug development. The exoribonuclease RNase R has been involved in virulence in a growing number of pathogens. In this work, we used Galleria mellonella as an infection model to demonstrate that the presence of RNase R increases the pneumococcus virulence. Larvae infected with the RNase R mutant show an increased expression level of antimicrobial peptides. Furthermore, they have a lower bacterial load in the hemolymph in the later stages of infection, leading to a higher survival rate of the larvae. Interestingly, pneumococci expressing RNase R show a sudden drop in bacterial numbers immediately after infection, resembling the eclipse phase observed after intravenous inoculation in mice. Concomitantly, we observed a lower number of mutant bacteria inside larval hemocytes and a higher susceptibility to oxidative stress when compared to the wild type. Together, our results indicate that RNase R is involved in the ability of pneumococci to evade the host immune response, probably by interfering with internalization and/or replication inside the larval hemocytes.

Lipid Droplets in Cancer: From Composition and Role to Imaging and Therapeutics.

Cancer is the second most common cause of death worldwide, having its origin in the abnormal growth of cells. Available chemotherapeutics still present major drawbacks, usually associated with high toxicity and poor distribution, with only a small fraction of drugs reaching the tumour sites. Thus, it is urgent to develop novel therapeutic strategies. Cancer cells can reprogram their lipid metabolism to sustain uncontrolled proliferation, and, therefore, accumulate a higher amount of lipid droplets (LDs). LDs are cytoplasmic organelles that store neutral lipids and are hypothesized to sequester anti-cancer drugs, leading to reduced efficacy. Thus, the increased biogenesis of LDs in neoplastic conditions makes them suitable targets for anticancer therapy and for the development of new dyes for cancer cells imaging. In recent years, cancer nanotherapeutics offered some exciting possibilities, including improvement tumour detection and eradication. In this review we summarize LDs biogenesis, structure and composition, and highlight their role in cancer theranostics.

The BASHY Platform Enables the Assembly of a Fluorescent Bortezomib-GV1001 Conjugate.

In this study, we show that fluorescent boronic-acid derived salicylidenehydrazone complexes (BASHY) can function as fluorescent linkers for bioconjugates that were used to monitor the delivery of the proteasome inhibitor bortezomib (Btz) to HT-29 cancer cells. BASHY complexes were structurally optimized to improve the stability of the complex in buffered conditions (ammonium acetate, pH 7 up to t 1/2 = 40 h), photophysically characterized regarding their fluorescence properties and used in confocal microscopy colocalization studies that revealed their intracellular sequestration by lipid droplets. The accumulation in these hydrophobic organelles limited the hydrolysis of the complex and consequently the drug release, a problem that was circumvented by the conjugation of the BASHY-Btz complex with a cell-penetrating peptide GV1001-C. The conjugate exhibited an improved cytoplasmic availability as confirmed by confocal fluorescence microscopy studies and an improved potency against HT-29 cancer cells (IC50 = 100 nM) as compared to the nontargeted complex (IC50 = 450 nM).

The Two Weapons against Bacterial Biofilms: Detection and Treatment.

Bacterial biofilms are defined as complex aggregates of bacteria that grow attached to surfaces or are associated with interfaces. Bacteria within biofilms are embedded in a self-produced extracellular matrix made of polysaccharides, nucleic acids, and proteins. It is recognized that bacterial biofilms are responsible for the majority of microbial infections that occur in the human body, and that biofilm-related infections are extremely difficult to treat. This is related with the fact that microbial cells in biofilms exhibit increased resistance levels to antibiotics in comparison with planktonic (free-floating) cells. In the last years, the introduction into the market of novel compounds that can overcome the resistance to antimicrobial agents associated with biofilm infection has slowed down. If this situation is not altered, millions of lives are at risk, and this will also strongly affect the world economy. As such, research into the identification and eradication of biofilms is important for the future of human health. In this sense, this article provides an overview of techniques developed to detect and imaging biofilms as well as recent strategies that can be applied to treat biofilms during the several biofilm formation steps.

Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P2 Confinement.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an essential plasma membrane component involved in several cellular functions, including membrane trafficking and cytoskeleton organization. This function multiplicity is partially achieved through a dynamic spatiotemporal organization of PI(4,5)P2 within the membrane. Here, we use a Förster resonance energy transfer (FRET) approach to quantitatively assess the extent of PI(4,5)P2 confinement within the plasma membrane. This methodology relies on the rigorous evaluation of the dependence of absolute FRET efficiencies between pleckstrin homology domains (PHPLCδ) fused with fluorescent proteins and their average fluorescence intensity at the membrane. PI(4,5)P2 is found to be significantly compartmentalized at the plasma membrane of HeLa cells, and these clusters are not cholesterol-dependent, suggesting that membrane rafts are not involved in the formation of these nanodomains. On the other hand, upon inhibition of actin polymerization, compartmentalization of PI(4,5)P2 is almost entirely eliminated, showing that the cytoskeleton network is the critical component responsible for the formation of nanoscale PI(4,5)P2 domains in HeLa cells.

Liposomes as a Nanoplatform to Improve the Delivery of Antibiotics into Staphylococcus aureus Biofilms.

Staphylococcus aureus biofilm-associated infections are a major public health concern. Current therapies are hampered by reduced penetration of antibiotics through biofilm and low accumulation levels at infected sites, requiring prolonged usage. To overcome these, repurposing antibiotics in combination with nanotechnological platforms is one of the most appealing fast-track and cost-effective approaches. In the present work, we assessed the potential therapeutic benefit of three antibiotics, vancomycin, levofloxacin and rifabutin (RFB), through their incorporation in liposomes. Free RFB displayed the utmost antibacterial effect with MIC and MBIC50 below 0.006 µg/mL towards a methicillin susceptible S. aureus (MSSA). RFB was selected for further in vitro studies and the influence of different lipid compositions on bacterial biofilm interactions was evaluated. Although positively charged RFB liposomes displayed the highest interaction with MSSA biofilms, RFB incorporated in negatively charged liposomes displayed lower MBIC50 values in comparison to the antibiotic in the free form. Preliminary safety assessment on all RFB formulations towards osteoblast and fibroblast cell lines demonstrated that a reduction on cell viability was only observed for the positively charged liposomes. Overall, negatively charged RFB liposomes are a promising approach against biofilm S. aureus infections and further in vivo studies should be performed.

Carbapenem-Resistant Klebsiella pneumoniae Clinical Isolates: In Vivo Virulence Assessment in Galleria mellonella and Potential Therapeutics by Polycationic Oligoethyleneimine.

Klebsiella pneumoniae, one of the most common pathogens found in hospital-acquired infections, is often resistant to multiple antibiotics. In fact, multidrug-resistant (MDR) K. pneumoniae producing KPC or OXA-48-like carbapenemases are recognized as a serious global health threat. In this sense, we evaluated the virulence of K. pneumoniae KPC(+) or OXA-48(+) aiming at potential antimicrobial therapeutics. K. pneumoniae carbapenemase (KPC) and the expanded-spectrum oxacillinase OXA-48 isolates were obtained from patients treated in medical care units in Lisbon, Portugal. The virulence potential of the K. pneumonia clinical isolates was tested using the Galleria mellonella model. For that, G. mellonella larvae were inoculated using patients KPC(+) and OXA-48(+) isolates. Using this in vivo model, the KPC(+) K. pneumoniae isolates showed to be, on average, more virulent than OXA-48(+). Virulence was found attenuated when a low bacterial inoculum (one magnitude lower) was tested. In addition, we also report the use of a synthetic polycationic oligomer (L-OEI-h) as a potential antimicrobial agent to fight infectious diseases caused by MDR bacteria. L-OEI-h has a broad-spectrum antibacterial activity and exerts a significantly bactericidal activity within the first 5-30 min treatment, causing lysis of the cytoplasmic membrane. Importantly, the polycationic oligomer showed low toxicity against in vitro models and no visible cytotoxicity (measured by survival and health index) was noted on the in vivo model (G. mellonella), thus L-OEI-h is foreseen as a promising polymer therapeutic for the treatment of MDR K. pneumoniae infections.