Mariagrazia Di Luca

Nanoscale protein diffusion by STED-based pair correlation analysis.

We describe for the first time the combination between cross-pair correlation function analysis (pair correlation analysis or pCF) and stimulated emission depletion (STED) to obtain diffusion maps at spatial resolution below the optical diffraction limit (super-resolution). Our approach was tested in systems characterized by high and low signal to noise ratio, i.e. Capsid Like Particles (CLPs) bearing several (>100) active fluorescent proteins and monomeric fluorescent proteins transiently expressed in living Chinese Hamster Ovary cells, respectively. The latter system represents the usual condition encountered in living cell studies on fluorescent protein chimeras. Spatial resolution of STED-pCF was found to be about 110 nm, with a more than twofold improvement over conventional confocal acquisition. We successfully applied our method to highlight how the proximity to nuclear envelope affects the mobility features of proteins actively imported into the nucleus in living cells. Remarkably, STED-pCF unveiled the existence of local barriers to diffusion as well as the presence of a slow component at distances up to 500–700 nm from either sides of nuclear envelope. The mobility of this component is similar to that previously described for transport complexes. Remarkably, all these features were invisible in conventional confocal mode.

Treatment of microbial biofilms in the post-antibiotic era: prophylactic and therapeutic use of antimicrobial peptides and their design by bioinformatics tools

The treatment for biofilm infections is particularly challenging because bacteria in these conditions become refractory to antibiotic drugs. The reduced effectiveness of current therapies spurs research for the identification of novel molecules endowed with antimicrobial activities and new mechanisms of antibiofilm action. Antimicrobial peptides (AMPs) have been receiving increasing attention as potential therapeutic agents, because they represent a novel class of antibiotics with a wide spectrum of activity and a low rate in inducing bacterial resistance. Over the past decades, a large number of naturally occurring AMPs have been identified or predicted from various organisms as effector molecules of the innate immune system playing a crucial role in the first line of defense. Recent studies have shown the ability of some AMPs to act against microbial biofilms, in particular during early phases of biofilm development. Here, we provide a review of the antimicrobial peptides tested on biofilms, highlighting their advantages and disadvantages for prophylactic and therapeutic applications. In addition, we describe the strategies and methods for de novo design of potentially active AMPs and discuss how informatics and computational tools may be exploited to improve antibiofilm effectiveness.

BaAMPs: the database of biofilm-active antimicrobial peptides

Antimicrobial peptides (AMPs) are increasingly being considered as novel agents against biofilms. The development of AMP-based anti-biofilm strategies strongly relies on the design of sequences optimized to target specific features of sessile bacterial/fungal communities. Although several AMP databases have been created and successfully exploited for AMP design, all of these use data collected on peptides tested against planktonic microorganisms. Here, an open-access, manually curated database of AMPs specifically assayed against microbial biofilms (BaAMPs) is presented for the first time. In collecting relevant data from the literature an effort was made to define a minimal standard set of essential information including, for each AMP, the microbial species and biofilm conditions against which it was tested, and the specific assay and peptide concentration used. The availability of these data in an organized framework will benefit anti-biofilm research and support the design of novel molecules active against biofilm. BaAMPs is accessible at http://www.baamps.it

Antimicrobial Peptides Design by Evolutionary Multiobjective Optimization

Antimicrobial peptides (AMPs) are an abundant and wide class of molecules produced by many tissues and cell types in a variety of mammals, plant and animal species. Linear alpha-helical antimicrobial peptides are among the most widespread membrane-disruptive AMPs in nature, representing a particularly successful structural arrangement in innate defense. Recently, AMPs have received increasing attention as potential therapeutic agents, owing to their broad activity spectrum and their reduced tendency to induce resistance. The introduction of non-natural amino acids will be a key requisite in order to contrast host resistance and increase compounds life. In this work, the possibility to design novel AMP sequences with non-natural amino acids was achieved through a flexible computational approach, based on chemophysical profiles of peptide sequences. Quantitative structure-activity relationship (QSAR) descriptors were employed to code each peptide and train two statistical models in order to account for structural and functional properties of alpha-helical amphipathic AMPs. These models were then used as fitness functions for a multi-objective evolutional algorithm, together with a set of constraints for the design of a series of candidate AMPs. Two ab-initio natural peptides were synthesized and experimentally validated for antimicrobial activity, together with a series of control peptides. Furthermore, a well-known Cecropin-Mellitin alpha helical antimicrobial hybrid (CM18) was optimized by shortening its amino acid sequence while maintaining its activity and a peptide with non-natural amino acids was designed and tested, demonstrating the higher activity achievable with artificial residues.

Synergistic antibiotic activity against planktonic and biofilm-embedded Streptococcus agalactiae, Streptococcus pyogenes and Streptococcus oralis

Objectives To determine the antimicrobial activity against streptococcal biofilm in species mostly isolated from implant-associated infections and examine the effect of enzyme treatment of biofilm on the antimicrobial activity of different antibiotics. Methods The activities of fosfomycin, rifampicin, benzylpenicillin, daptomycin, gentamicin, levofloxacin, proteinase K and their combinations on planktonic and/or biofilm-embedded standard laboratory strains of Streptococcus agalactiae, Streptococcus pyogenes and Streptococcus oralis were investigated in vitro by standard methods and isothermal microcalorimetry. Results MIC values obtained for the tested antimicrobials against planktonic bacteria ranged from 0.016 to 128 mg/L for the three species tested. Higher antibiotic concentrations were usually required to reduce biofilm in comparison with planktonic bacteria, with the exception of gentamicin, for which similar concentrations (4-16 mg/L) exerted an effect on both planktonic and biofilm cells. A synergistic effect against the streptococcal biofilm of the three species was observed when gentamicin was combined with benzylpenicillin or with rifampicin. Moreover, antibiotic concentrations comparable to the MIC observed against planktonic cells induced a strong reduction of viable bacteria in proteinase K pre-treated biofilm. Conclusions This study shows that the combination of gentamicin with either benzylpenicillin or rifampicin exerts a synergistic effect against biofilms produced by the tested streptococci strains in vitro. Our results also suggest that coupling a dispersal agent with conventional antibiotics may facilitate their access to the bacteria within the biofilm. In vivo and clinical studies are needed in order to confirm whether such a strategy may be effective in the treatment of implant-associated infections caused by streptococci.

In Silico Design of Antimicrobial Peptides

The rapid spread of drug-resistant pathogenic microbial strains has created an urgent need for the development of new anti-infective molecules, having different mechanism of action in comparison to existing drugs. Natural antimicrobial peptides (AMPs) represent a novel class of molecules with a broad spectrum of activity and a low rate in inducing bacterial resistance. In particular, linear alpha-helical cationic antimicrobial peptides are among the most widespread membrane-disruptive AMPs in nature, representing a particularly successful structural arrangement of the innate defense against microbes. However, until now, many AMPs have failed in clinical trials because of several drawbacks that strongly limit their applicability such as degradation, cytotoxicity, and high production cost. Thus, to overcome the limitations of native peptides, a rational in silico approach to AMPs design becomes a promising strategy that drastically reduce production costs and the time required for evaluation of activity and toxicity. This chapter focuses on the strategies and methods for de novo design of potentially active AMPs. In particular, statistical-based design strategies and MD methods for modelling AMPs are elucidated.

In vitro anti-biofilm activity of a biphasic gentamicin-loaded calcium sulfate/hydroxyapatite bone graft substitute

Bone and implant-associated infections caused by microorganisms that grow in biofilms are difficult to treat because of persistence and recurrence of infection. Along with surgical debridement, the combination of systemic and local administration of antimicrobials represents the background for an efficient treatment strategy. Gentamicin is one of most used antibiotics for the local treatment of bone-related infections, alone or in combination, due to its bactericidal and broad-range activity. Gentamicin-loaded beads (GLBs), composed of calcium sulfate/hydroxyapatite, were assessed for their in vitro antimicrobial activity against planktonic and biofilm S. agalactiae, S. aureus, S. epidermidis, E. faecalis and E. coli, using standard methods and ultra-sensitive isothermal microcalorimetry. Gentamicin released from GLBs to clinically relevant concentrations (200-2500μg/mL) within 1h was able to kill planktonic S. agalactiae, S. epidermidis and E. coli at lower concentrations (MIC: ≤4μg/mL). Moreover, 12 and 23μg/mL of released gentamicin were able to prevent bacterial adhesion and suppress a 24h-old biofilm of E. coli, respectively. Conversely, higher amounts of antibiotic, ranging from 171 to 1260μg/mL, were needed to prevent and eradicate biofilms of gram-positive bacteria. Likewise, the emergence of resistance to GLBs in vitro and the bacterial attachment on the bone graft substitute, when the amount of gentamicin in the material is reduced, were also reported. This study provides further information regarding the in vitro anti-biofilm activity of the biphasic gentamicin-loaded bone graft substitute, suggesting the validity of this antibiotic-loaded material for the prophylaxis and treatment of bone and implant-associated infections.

Antimicrobial Peptides for the Control of Biofilm Formation

Antimicrobial peptides (AMPs) are an abundant and varied group of molecules recognized as the most ancient components of the innate immune system. They are found in a wide group of organisms including bacteria, plants and animals as a defense mechanism against different kinds of infectious pathogens. Over the past two decades, a fast-growing number of AMPs have been identified/designed and their wide-spectrum antimicrobial activity has been deeply investigated. In recent years, there has been an increasing interest in the use of AMPs as alternative anti-biofilm molecules for the control of biofilm-related infections. Biofilms are sessile communities of microbial cells embedded in a self-produced matrix and characterized by a low metabolic activity. Due to their peculiar physiological properties, bacteria/fungi in biofilms result more resistant to conventional antibiotic therapies compared with their planktonic counterparts. AMPs may be a promising strategy to combat biofilm-related infections, as many of them target the microbial membrane, thus being potentially effective also on metabolically inactive cells. Investigations conducted so far evidenced that these peptides may be active in either eradicating established biofilms or preventing their formation, depending on the specific molecule. Here we present a detailed review of the literature describing the latest results of both in vitro and in vivo experiments aimed at evaluating AMP potential usage in biofilm control. In addition, we provide the reader with an overview on AMP local delivery systems, and we discuss their potential application in the coating of medical indwelling devices.

Detection of Biofilms in Biopsies from Chronic Rhinosinusitis Patients: In Vitro Biofilm Forming Ability and Antimicrobial Susceptibility Testing in Biofilm Mode of Growth of Isolated Bacteria

Chronic rhinosinusitis (CRS) is the most common illness among chronic disorders that remains poorly understood from a pathogenic standpoint and has a significant impact on patient quality of life, as well as healthcare costs. Despite being widespread, little is known about the etiology of the CRS. Recent evidence, showing the presence of biofilms within the paranasal sinuses, suggests a role for biofilm in the pathogenesis. To elucidate the role of biofilm in the pathogenesis of CRS, we assessed the presence of biofilm at the infection site and the ability of the aerobic flora isolated from CRS patients to form biofilm in vitro. For selected bacterial strains the susceptibility profiles to antibiotics in biofilm condition was also evaluated.Staphylococci represented the majority of the isolates obtained from the infection site, with S. epidermidis being the most frequently isolated species. Other isolates were represented by Enterobacteriaceae or by species present in the oral flora. Confocal laser scanning microscopy (CLSM) of the mucosal biopsies taken from patients with CRS revealed the presence of biofilm in the majority of the samples. Strains isolated from the specific infection site of the CRS patients were able to form biofilm in vitro at moderate or high levels, when tested in optimized conditions. No biofilm was observed by CLSM in the biopsies from control patients, although the same biopsies were positive for staphylococci in microbiological culture analysis. Drug-susceptibility tests demonstrated that the susceptibility profile of planktonic bacteria differs from that of sessile bacteria in biofilms.

Diffusion Tensor Analysis by Two-Dimensional Pair Correlation of Fluorescence Fluctuations in Cells.

In a living cell, the movement of biomolecules is highly regulated by the cellular organization into subcompartments that impose barriers to diffusion, can locally break the spatial isotropy, and ultimately guide these molecules to their targets. Despite the pivotal role of these processes, experimental tools to fully probe the complex connectivity (and accessibility) of the cell interior with adequate spatiotemporal resolution are still lacking. Here, we show how the heterogeneity of molecular dynamics and the location of barriers to molecular motion can be mapped in live cells by exploiting a two-dimensional (2D) extension of the pair correlation function (pCF) analysis. Starting from a time series of images collected for the same field of view, the resulting 2D pCF is calculated in the proximity of each point for each time delay and allows us to probe the spatial distribution of the molecules that started from a given pixel. This 2D pCF yields an accurate description of the preferential diffusive routes. Furthermore, we combine this analysis with the image-derived mean-square displacement approach and gain information on the average nanoscopic molecular displacements in different directions. Through these quantities, we build a fluorescence-fluctuation-based diffusion tensor that contains information on speed and directionality of the local dynamical processes. Contrary to classical fluorescence correlation spectroscopy and related methods, this combined approach can distinguish between isotropic and anisotropic local diffusion. We argue that the measurement of this iMSD tensor will contribute to advance our understanding of the role played by the intracellular environment in the regulation of molecular diffusion at the nanoscale.