Franca Lisa Brancatisano

Use of antimicrobial peptides against microbial biofilms: advantages and limits.

The formation of surface-attached cellular agglomerates, the so-called biofilms, contributes significantly to bacterial resistance to antibiotics and innate host defenses. Bacterial biofilms are associated to various pathological conditions in humans such as cystic fibrosis, colonization of indwelling medical devices and dental plaque formation involved in caries and periodontitis. Over the last years, natural antimicrobial peptides (AMPs) have attracted considerable interest as a new class of antimicrobial drugs for a number of reasons. Among these, there are the broad activity spectrum, the relative selectivity towards their targets (microbial membranes), the rapid mechanism of action and, above all, the low frequency in selecting resistant strains. Since biofilm resistance to antibiotics is mainly due to the slow growth rate and low metabolic activity of bacteria in such community, the use of AMPs to inhibit biofilm formation could be potentially an attractive therapeutic approach. In fact, due to the prevalent mechanism of action of AMPs, which relies on their ability to permeabilize and/or to form pores within the cytoplasmic membranes, they have a high potential to act also on slow growing or even non-growing bacteria. This review will highlight the most important findings obtained testing AMPs in in vitro and in vivo models of bacterial biofilms, pointing out the possible advantages and limits of their use against microbial biofilm-related infections.

Direct binding of human NK cell natural cytotoxicity receptor NKp44 to the surfaces of mycobacteria and other bacteria.

ABSTRACT Our previous studies demonstrated that Mycobacterium bovis bacillus Calmette-Guérin (BCG) can directly interact with human NK cells and induce the proliferation, gamma interferon production, and cytotoxic activity of such cells without the need for accessory cells. Thus, the aim of the present study was to identify the putative receptor(s) responsible for the recognition of BCG by human NK cells and potentially involved in the activation of NK cells. To this end, we first investigated the surface expression of three NK cell-activating receptors belonging to the natural cytoxicity receptor (NCR) family on highly purified human NK cells upon in vitro direct stimulation with BCG. An induction of the surface expression of NKp44, but not of NKp30 or NKp46, was observed after 3 and 4 days of in vitro stimulation with live BCG. The NKp44 induction involved mainly a particular NK cell subset expressing the CD56 marker at high density, CD56bright. In order to establish whether NKp44 could directly bind to BCG, whole BCG cells were stained with soluble forms of the three NCRs chimeric for the human immunoglobulin G (IgG) Fc fragment (NKp30-Fc, NKp44-Fc, NKp46-Fc), followed by incubation with a phycoerythrin (PE)-conjugated goat anti-human IgG antibody. Analysis by flow cytometry of the complexes revealed a higher PE fluorescence intensity for BCG incubated with NKp44-Fc than for BCG incubated with NKp30-Fc, NKp46-Fc, or negative controls. The binding of NKp44-Fc to the BCG surface was confirmed with immunogold labeling using transmission electron microscopy, suggesting the presence of a putative ligand(s) for human NKp44 on the BCG cell wall. Similar binding assays performed on a number of gram-positive and gram-negative bacteria revealed a pattern of NKp44-Fc binding restricted to members of the genus Mycobacterium, to the mycobacterium-related species Nocardia farcinica, and to Pseudomonas aeruginosa. Altogether, the results obtained indicate, for the first time, that at least one member of the NCR family (NKp44) may be involved in the direct recognition of bacterial pathogens by human NK cells.

Evaluation of the inhibitory effects of human serum components on bactericidal activity of human beta defensin 3

Naturally occurring cationic antimicrobial peptides (CAPs) are an essential component of the innate immune system of multicellular organisms. At concentrations generally higher than those found in vivo, most CAPs exhibit strong antibacterial properties in vitro, but their activity may be inhibited by body fluids, a fact that could limit their future use as antimicrobial and/or immunomodulatory agents. In the present study, we evaluated the effects of human serum components on bactericidal activity of the human beta-defensin 3 (hBD-3), a CAP considered particularly promising for future therapeutic employment. Human serum diluted to 20% strongly inhibited the bactericidal activity of the peptide against both the Gram-positive species Staphylococcus aureus and the Gram-negative species Acinetobacter baumannii. Such activity was not restored in serum devoid of salts (dialyzed), pre-treated with protease inhibitors, or subjected to both of these treatments. The addition of physiological concentrations of NaCl, CaCl2, and human albumin in the bactericidal assay abolished bactericidal activity of hBD-3 against S. aureus, while it only partially inhibited the activity of the peptide against A. baumannii. Although a proteolytic activity of serum on hBD-3 was demonstrated at the protein level by Western blot, addition of physiological concentrations of trypsin to the bactericidal assay only partially affected the antibacterial properties of the peptide. Altogether, these results demonstrate a major role of mono-divalent cations and serum proteins on inhibition of hBD-3 antibacterial properties and indicate a relative lack in sensitivity of the bactericidal activity of this peptide to trypsin and trypsin-like proteases.

Comparative Analysis of the Bactericidal Activities of Amphibian Peptide Analogues against Multidrug-Resistant Nosocomial Bacterial Strains

ABSTRACT Due to the widespread resistance of bacteria to the available drugs, the discovery of new classes of antibiotics is urgently needed, and naturally occurring antimicrobial peptides (AMPs) are considered promising candidates for future therapeutic use. Amphibian skin is one of the richest sources of such AMPs. In the present study we compared the in vitro bactericidal activities of five AMPs from three different species of anurans against multidrug-resistant clinical isolates belonging to species often involved in nosocomial infections (Staphylococcus aureus, Enterococcus faecium, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, and Acinetobacter baumannii). The peptides tested were temporins A, B, and G from Rana temporaria; the fragment from positions 1 to 18 of esculentin 1b [Esc(1-18)] from Rana esculenta; and bombinin H2 from Bombina variegata. When they were tested in buffer, all the peptides were bactericidal against all bacterial species tested (three strains of each species) at concentrations ranging from 0.5 to 48 μΜ, with only a few exceptions. The temporins were found to be more active against gram-positive bacteria, especially when they were assayed in human serum; Esc(1-18) showed fast and strong bactericidal activity, within 2 to 20 min, especially against the gram-negative species, which were killed by Esc(1-18) at concentrations ranging from 0.5 to 1 μΜ; bombinin H2 displayed similar bactericidal activity toward all isolates. Interestingly, while the activities of the temporins and bombinin H2 were almost completely inhibited in the presence of 20% human serum, the activity of Esc(1-18) against the gram-negative species was partially preserved in the presence of 40% serum. This property renders this peptide an attractive molecule for use in the development of new compounds for the treatment of infectious diseases.

Antimicrobial activity of human hepcidin 20 and 25 against clinically relevant bacterial strains: Effect of copper and acidic pH

Hepcidin 25 (hep-25) is a peptide primarily produced by human liver with a central role in iron homeostasis. Its isoform, hepcidin 20 (hep-20), has an unknown function and lacks the first five aminoacids of the amino-terminal portion. This sequence is crucial for iron regulation by hep-25 and contains a molecular motif able to bind metals. Aim of this study, was to evaluate the antibacterial properties of both peptides in vitro, against a wide range of bacterial clinical isolates and in different experimental conditions. Although both peptides were found to be bactericidal against a variety of clinical isolates with different antibiotic resistance profiles, hep-20 was active at lower concentrations than hep-25, in most of the cases. Killing kinetics, carried on in sodium-phosphate buffer at pH 7.4, demonstrated that bactericidal activity occurred not earlier than 30-90 min of incubation. Bactericidal activity of hep-25 was slightly enhanced in the presence of copper, while the same metal did not affect the activity of hep-20. Interestingly, bactericidal activity of both hepcidins was highly enhanced at acidic pH. Acidic pH (pH 5.0 and 6.6) not only reduced the microbicidal concentrations of hepcidins, but also shortened the killing times of both peptides, as compared to pH 7.4. Combining hep-20 and hep-25 at pH 5.0 a bactericidal effect could be obtained at very low concentrations of both peptides. These results render hepcidins interesting for the design of new drugs for the treatment of infections occurring in body districts with physiologic acidic pH.

Interaction of Mycobacterium tuberculosis Cell Wall Components with the Human Natural Killer Cell Receptors NKp44 and Toll‐Like Receptor 2

We have previously demonstrated that a soluble form of the human NK cell natural cytotoxicity receptor NKp44, binds to the surface of Mycobacterium tuberculosis (MTB). Herein, we investigated the interaction of MTB cell wall components (CWC) with NKp44 or with Toll‐like receptor 2 (TLR2) and the role of NKp44 and TLR2 in the direct activation of NK cells upon stimulation with MTB CWC. By using several purified bacterial CWC in an ELISA, we demonstrated that NKp44 was able to bind to the MTB cell wall core mycolyl‐arabinogalactan‐peptidoglycan (mAGP) as well as to mycolic acids (MA) and arabinogalactan (AG), while soluble TLR2 bound to MTB peptidoglycan (PG), but not to MA or AG. The mAGP complex induced NK cell expression of CD25, CD69, NKp44 and IFN‐γ production at levels comparable to M. bovis Bacillus Calmette–Guérin‐stimulated (BCG) cells. While AG and MA used alone failed to induce NK cell activation, mycobacterial PG‐exhibited NK cell stimulatory capacity. Activation of resting NK cells by mAGP and IFN‐γ production were inhibited by anti‐TLR2 MAb, but not by anti‐NKp44 MAb. Differently, anti‐NKp44 MAb partially inhibited CD69 expression on NK cells pre‐activated with IL‐2 and then stimulated with mAGP or whole BCG. Overall, these results provide evidence that components abundant in mycobacterial cell wall are able to interact with NKp44 (AG, MA) and TLR‐2 (PG), respectively. While interaction of TLR2 with mycobacterial cell wall promotes activation of resting NK cells and IFN‐γ production, NKp44 interaction with its putative ligands could play a secondary role in maintaining cell activation.

Gingipains produced by Porphyromonas gingivalis ATCC49417 degrade human-β-defensin 3 and affect peptide's antibacterial activity in vitro

Porphyromonas gingivalis, one of the major pathogen associated with periodontitis, is a highly proteolytic bacterial species. Production of proteases is a common microbial virulence factor that enables the destruction of host tissues and evasion from host defense mechanisms. Antimicrobial peptides are important effector molecules of the innate immune system with a broad range of antimicrobial and immunoregulatory activities. We and others have previously demonstrated that P. gingivalis is relatively resistant to the bactericidal activity of the human β-defensin 3 (hBD3). In this study, ability of proteases released by the pathogenic strain of P. gingivalis ATCC 49417 to degrade hBD3 and to affect the antibacterial properties of the peptide was assessed. P. gingivalis culture supernatants (CS) were found to degrade hBD3 in a concentration- and time-dependent manner. Such degradation was mainly due to the activity of Arg and Lys-gingipains, as pretreatment of CS with inhibitors selective for this class of proteases abolished CS ability to degrade hBD3. Importantly, preincubation of hBD3 with CS reduced peptides antibacterial activity against a susceptible strain of Staphylococcus aureus, while the presence of gingipain inhibitors in the bactericidal assay increased P. gingivalis susceptibility to hBD3. Altogether these results suggest that gingipains may have a role in the resistance of P. gingivalis ATCC 49417 to hBD3.

Reduced Human Beta Defensin 3 in Individuals with Periodontal Disease

The human beta defensin 3 (hBD3) is widely expressed in the oral cavity and exerts strong antibacterial and immunomodulatory activities. Hence, we hypothesized that hBD3 could play a protective role in the maintenance of periodontal homeostasis, and that it could be found in gingival crevicular fluid (GCF) of healthy individuals and those with periodontitis at levels correlating with the degree of periodontal health. By using an ELISA assay to quantify hBD3 in GCF, we demonstrated that the peptide is present at levels easily detectable in the majority of healthy individuals, but it is drastically reduced in GCF from those with periodontitis. Furthermore, hBD3 levels inversely correlate with the severity of the disease and the degree of colonization by combinations of bacterial species with elevated periodontopathogenic potential. Both genetic factors and host/bacterial proteases released in diseased sites may be responsible for the observed low/null hBD3 levels in GCF from individuals with periodontitis.

pH-dependent disruption of Escherichia coli ATCC 25922 and model membranes by the human antimicrobial peptides hepcidin 20 and 25

The human hepcidin 25 (hep‐25) and its isoform hepcidin 20 (hep‐20) are histidine‐containing, cystein rich, β‐sheet structured peptides endowed with antimicrobial activity. We previously reported that, similar to other histidine‐containing peptides, the microbicidal effects of hep‐25 and hep‐20 are highly enhanced at acidic pH. In the present study, we investigated whether pH influences the mode of action of hep‐25 and hep‐20 on Escherichia coli American Type Culture Collection 25922 and model membranes. A striking release of β‐galactosidase by hepcidin‐treated E. coli was observed at pH 5.0, whereas no inner membrane permeabilization capacity was seen at pH 7.4, even at bactericidal concentrations. Similar results were obtained by flow cytometry when assessing the internalization of propidium iodide by hepcidin‐treated E. coli. Scanning electron microscope imaging revealed that both peptides induced the formation of numerous blebs on the surface of bacterial cells at acidic pH but not at neutral pH. Moreover, a phospholipid/polydiacetylene colourimetric vesicle assay revealed a more evident membrane damaging effect at pH 5.0 than at pH 7.4. The leakage of entrapped dextrans of increasing molecular size from liposomes was also assessed at pH 7.4. Consistent with the lack of β‐galactosidase release from whole E. coli observed at such a pH value, evident leakage of only the smallest 4‐kDa dextran (and not of dextrans of 20 or 70 kDa) was observed, indicating a poor ability of hepcidin peptides to permeabilize liposome vesicles at pH 7.4. Altogether, the data obtained in the present study using different approaches strongly suggest that the ability of hepcidins to perturb bacterial membranes is markedly pH‐dependent.

Inhibitory effect of the human liver-derived antimicrobial peptide hepcidin 20 on biofilms of polysaccharide intercellular adhesin (PIA)-positive and PIA-negative strains of Staphylococcus epidermidis

Staphylococcus epidermidis plays a major role in biofilm-related medical device infections. Herein the anti-biofilm activity of the human liver-derived antimicrobial peptide hepcidin 20 (hep20) was evaluated against polysaccharide intercellular adhesin (PIA)-positive and PIA-negative clinical isolates of S. epidermidis. Hep20 markedly inhibited biofilm formation and bacterial cell metabolism of PIA-positive and PIA-negative strains, but the decrease in biofilm biomass only partially correlated with a decrease in viable bacteria. Confocal microscope images revealed that, in the presence of hep20, both PIA-positive and PIA-negative strains formed biofilms with altered architectures and reduced amounts of extracellular matrix. Co-incubation of hep20 with vancomycin produced no synergistic effect, evaluated as number of viable cells, both in preventing biofilm formation and in treating preformed biofilms. In contrast, biofilms obtained in the presence of hep20, and then exposed to vancomycin, displayed an increased susceptibility to vancomycin. These results suggest that hep20 may inhibit the production/accumulation of biofilm extracellular matrix.