Hans-Henrik Kristensen

Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus

Animals and higher plants express endogenous peptide antibiotics called defensins. These small cysteine-rich peptides are active against bacteria, fungi and viruses. Here we describe plectasin—the first defensin to be isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella. Plectasin has primary, secondary and tertiary structures that closely resemble those of defensins found in spiders, scorpions, dragonflies and mussels. Recombinant plectasin was produced at a very high, and commercially viable, yield and purity. In vitro, the recombinant peptide was especially active against Streptococcus pneumoniae, including strains resistant to conventional antibiotics. Plectasin showed extremely low toxicity in mice, and cured them of experimental peritonitis and pneumonia caused by S. pneumoniae as efficaciously as vancomycin and penicillin. These findings identify fungi as a novel source of antimicrobial defensins, and show the therapeutic potential of plectasin. They also suggest that the defensins of insects, molluscs and fungi arose from a common ancestral gene.

The heme sensing response regulator HssR in Staphylococcus aureus

BackgroundHost defence peptides (HDPs), also known as antimicrobial peptides (AMPs), have emerged as potential new therapeutics and their antimicrobial spectrum covers a wide range of target organisms. However, the mode of action and the genetics behind the bacterial response to HDPs is incompletely understood and such knowledge is required to evaluate their potential as antimicrobial therapeutics. Plectasin is a recently discovered HDP active against Gram-positive bacteria with the human pathogen, Staphylococcus aureus (S. aureus) being highly susceptible and the food borne pathogen, Listeria monocytogenes (L. monocytogenes) being less sensitive. In the present study we aimed to use transposon mutagenesis to determine the genetic basis for S. aureus and L. monocytogenes susceptibility to plectasin.ResultsIn order to identify genes that provide susceptibility to plectasin we constructed bacterial transposon mutant libraries of S. aureus NCTC8325-4 and L. monocytogenes 4446 and screened for increased resistance to the peptide. No resistant mutants arose when L. monocytogenes was screened on plates containing 5 and 10 fold Minimal Inhibitory Concentration (MIC) of plectasin. However, in S. aureus, four mutants with insertion in the heme response regulator (hssR) were 2-4 fold more resistant to plectasin as compared to the wild type. The hssR mutation also enhanced resistance to the plectasin-like defensin eurocin, but not to other classes of HDPs or to other stressors tested. Addition of plectasin did not influence the expression of hssR or hrtA, a gene regulated by HssR. The genome of L. monocytogenes LO28 encodes a putative HssR homologue, RR23 (in L. monocytogenes EGD-e lmo2583) with 48% identity to the S. aureus HssR, but a mutation in the rr23 gene did not change the susceptibility of L. monocytogenes to plectasin.ConclusionsS. aureus HssR, but not the homologue RR23 from L. monocytogenes, provides susceptibility to the defensins plectasin and eurocin. Our data suggest that a functional difference between response regulators HssR and RR23 is responsible for the difference in plectasin susceptibility observed between S. aureus and L. monocytogenes.Background: Host defence peptides (HDPs), also known as antimicrobial peptides (AMPs), have emerged as potential new therapeutics and their antimicrobial spectrum covers a wide range of target organisms. However, the mode of action and the genetics behind the bacterial response to HDPs is incompletely understood and such knowledge is required to evaluate their potential as antimicrobial therapeutics. Plectasin is a recently discovered HDP active against Gram-positive bacteria with the human pathogen, Staphylococcus aureus (S. aureus) being highly susceptible and the food borne pathogen, Listeria monocytogenes (L. monocytogenes) being less sensitive. In the present study we aimed to use transposon mutagenesis to determine the genetic basis for S. aureus and L. monocytogenes susceptibility to plectasin. Results: In order to identify genes that provide susceptibility to plectasin we constructed bacterial transposon mutant libraries of S. aureus NCTC8325-4 and L. monocytogenes 4446 and screened for increased resistance to the peptide. No resistant mutants arose when L. monocytogenes was screened on plates containing 5 and 10 fold Minimal Inhibitory Concentration (MIC) of plectasin. However, in S. aureus, four mutants with insertion in the heme response regulator (hssR) were 2-4 fold more resistant to plectasin as compared to the wild type. The hssR mutation also enhanced resistance to the plectasin-like defensin eurocin, but not to other classes of HDPs or to other stressors tested. Addition of plectasin did not influence the expression of hssR or hrtA, a gene regulated by HssR. The genome of L. monocytogenes LO28 encodes a putative HssR homologue, RR23 (in L. monocytogenes EGD-e lmo2583) with 48% identity to the S. aureus HssR, but a mutation in the rr23 gene did not change the susceptibility of L. monocytogenes to plectasin. Conclusions: S. aureus HssR, but not the homologue RR23 from L. monocytogenes, provides susceptibility to the defensins plectasin and eurocin. Our data suggest that a functional difference between response regulators HssR and RR23 is responsible for the difference in plectasin susceptibility observed between S. aureus and L. monocytogenes. * Correspondence: leth@life.ku.dk Department of Veterinary Disease Biology, University of Copenhagen, DK1870 Frederiksberg C, Denmark Full list of author information is available at the end of the article Thomsen et al. BMC Microbiology 2010, 10:307 http://www.biomedcentral.com/1471-2180/10/307

Efficacy of NZ2114, a Novel Plectasin-Derived Cationic Antimicrobial Peptide Antibiotic, in Experimental Endocarditis Due to Methicillin-Resistant Staphylococcus aureus

ABSTRACT Cationic antimicrobial peptides (CAPs) play important roles in host immune defenses. Plectasin is a defensin-like CAP isolated from the saprophytic fungus Pseudoplectania nigrella. NZ2114 is a novel variant of plectasin with potent activity against Gram-positive bacteria. In this study, we investigated (i) the in vivo pharmacokinetic and pharmacodynamic (PK/PD) characteristics of NZ2114 and (ii) the in vivo efficacy of NZ2114 in comparison with those of two conventional antibiotics, vancomycin or daptomycin, in an experimental rabbit infective endocarditis (IE) model due to a methicillin-resistant Staphylococcus aureus (MRSA) strain (ATCC 33591). All NZ2114 regimens (5, 10, and 20 mg/kg of body weight, intravenously [i.v.], twice daily for 3 days) significantly decreased MRSA densities in cardiac vegetations, kidneys, and spleen versus those in untreated controls, except in one scenario (5 mg/kg, splenic MRSA counts). The efficacy of NZ2114 was clearly dose dependent in all target tissues. At 20 mg/kg, NZ2114 showed a significantly greater efficacy than vancomycin (P < 0.001) and an efficacy similar to that of daptomycin. Of importance, only NZ2114 (in 10- and 20-mg/kg regimens) prevented posttherapy relapse in cardiac vegetations, kidneys, and spleen, while bacterial counts in these target tissues continued to increase in vancomycin- and daptomycin-treated animals. These in vivo efficacies were equivalent and significantly correlated with three PK indices investigated: fCmax/MIC (the maximum concentration of the free, unbound fraction of a drug in serum divided by the MIC), fAUC/MIC (where AUC is the area under the concentration-time curve), and f%T>MIC (%T>MIC is the cumulative percentage of a 24-h period that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions), as analyzed by a sigmoid maximum-effect (Emax) model (R2 > 0.69). The superior efficacy of NZ2114 in this MRSA IE model suggests the potential for further development of this compound for treating serious MRSA infections.

Eurocin, a New Fungal Defensin: STRUCTURE, LIPID BINDING, AND ITS MODE OF ACTION*

Background: Antimicrobial peptides are new antibiotics avoiding resistance problems. Results: Eurocin is a new antimicrobial peptide featuring a cysteine-stabilized αβ-fold. Eurocin binds the cell wall precursor lipid II but does not disrupt cell membranes. Conclusion: Eurocin acts by inhibiting cell wall synthesis. Its structure is typical for invertebrate defensins. Significance: Knowing the mode of action and structure is a prerequisite for pharmaceutical application of an antibiotic. Antimicrobial peptides are a new class of antibiotics that are promising for pharmaceutical applications because they have retained efficacy throughout evolution. One class of antimicrobial peptides are the defensins, which have been found in different species. Here we describe a new fungal defensin, eurocin. Eurocin acts against a range of Gram-positive human pathogens but not against Gram-negative bacteria. Eurocin consists of 42 amino acids, forming a cysteine-stabilized α/β-fold. The thermal denaturation data point shows the disulfide bridges being responsible for the stability of the fold. Eurocin does not form pores in cell membranes at physiologically relevant concentrations; it does, however, lead to limited leakage of a fluorophore from small unilamellar vesicles. Eurocin interacts with detergent micelles, and it inhibits the synthesis of cell walls by binding equimolarly to the cell wall precursor lipid II.

Plectasin Shows Intracellular Activity against Staphylococcus aureus in Human THP-1 Monocytes and in a Mouse Peritonitis Model

ABSTRACT Antimicrobial therapy of infections with Staphylococcus aureus can pose a challenge due to slow response to therapy and recurrence of infection. These treatment difficulties can partly be explained by intracellular survival of staphylococci, which is why the intracellular activity of antistaphylococcal compounds has received increased attention within recent years. The intracellular activity of plectasin, an antimicrobial peptide, against S. aureus was determined both in vitro and in vivo. In vitro studies using THP-1 monocytes showed that some intracellular antibacterial activity of plectasin was maintained (maximal relative efficacy [Emax], 1.0- to 1.3-log reduction in CFU) even though efficacy was inferior to that of extracellular killing (Emax, >4.5-log CFU reduction). Animal studies included a novel use of the mouse peritonitis model, exploiting extra- and intracellular differentiation assays, and assessment of the correlations between activity and pharmacokinetic (PK) parameters. The intracellular activity of plectasin was in accordance with the in vitro studies, with an Emax of a 1.1-log CFU reduction. The parameter most important for activity was fCpeak/MIC, where fCpeak is the free peak concentration. These findings stress the importance of performing studies of extra- and intracellular activity since these features cannot be predicted from traditional MIC and killing kinetic studies. Application of both the THP-1 and the mouse peritonitis models showed that the in vitro results were similar to findings in the in vivo model with respect to demonstration of intracellular activity. Therefore the in vitro model was a good screening model for intracellular activity. However, animal models should be applied if further information on activity, PK/pharmacodynamic parameters, and optimal dosing regimens is required.

High Cerebrospinal Fluid (CSF) Penetration and Potent Bactericidal Activity in CSF of NZ2114, a Novel Plectasin Variant, during Experimental Pneumococcal Meningitis

ABSTRACT Plectasin is the first defensin-type antimicrobial peptide isolated from a fungus and has potent activity against gram-positive bacteria. By using an experimental meningitis model, the penetration of plectasin into the cerebrospinal fluid (CSF) of infected and uninfected rabbits and the bactericidal activities in CSF of the plectasin variant NZ2114 and ceftriaxone against a penicillin-resistant Streptococcus pneumoniae strain (NZ2114 and ceftriaxone MICs, 0.25 and 0.5 μg/ml, respectively) were studied. Pharmacokinetic analysis showed that there was a significantly higher level of CSF penetration of NZ2114 through inflamed than through noninflamed meninges (area under the concentration-time curve for CSF/area under the concentration-time curve for serum, 33% and 1.1%, respectively; P = 0.03). The peak concentrations of NZ2114 in purulent CSF were observed ∼3 h after the infusion of an intravenous bolus of either 20 or 40 mg/kg of body weight and exceeded the MIC >10-fold for a 6-h study period. Treatment with NZ2114 (40 and 20 mg/kg at 0 and 5 h, respectively; n = 11) caused a significantly higher reduction in CSF bacterial concentrations than therapy with ceftriaxone (125 mg/kg at 0 h; n = 7) at 3 h (median changes, 3.7 log10 CFU/ml [interquartile range, 2.5 to 4.6 log10 CFU/ml] and 2.1 log10 CFU/ml [interquartile range, 1.7 to 2.6 log10 CFU/ml], respectively; P = 0.001), 5 h (median changes, 5.2 log10 CFU/ml [interquartile range, 3.6 to 6.1 log10 CFU/ml] and 3.1 log10 CFU/ml [interquartile range, 2.6 to 3.7 log10 CFU/ml], respectively; P = 0.01), and 10 h (median changes, 5.6 log10 CFU/ml [interquartile range, 5.2 to 5.9 log10 CFU/ml] and 4.2 log10 CFU/ml [interquartile range, 3.6 to 5.0 log10 CFU/ml], respectively; P = 0.03) after the start of therapy as well compared to the CSF bacterial concentrations in untreated rabbits with meningitis (n = 7, P < 0.05). Also, significantly more rabbits had sterile CSF at 5 and 10 h when they were treated with NZ2114 than when they were treated with ceftriaxone (67% [six of nine rabbits] and 0% [zero of seven rabbits], respectively, at 5 h and 75% [six of eight rabbits] and 14% [one of seven rabbits], respectively, at 10 h; P < 0.05). Due to its excellent CSF penetration and potent bactericidal activity in CSF, the plectasin variant NZ2114 could be a promising new option for the treatment of CNS infections caused by gram-positive bacteria, including penicillin-resistant pneumococcal meningitis.

Antimicrobial peptides effectively kill a broad spectrum of Listeria monocytogenes and Staphylococcus aureus strains independently of origin, sub-type, or virulence factor expression.

BackgroundHost defense peptides (HDPs), or antimicrobial peptides (AMPs), are important components of the innate immune system that bacterial pathogens must overcome to establish an infection and HDPs have been suggested as novel antimicrobial therapeutics in treatment of infectious diseases. Hence it is important to determine the natural variation in susceptibility to HDPs to ensure a successful use in clinical treatment regimes.ResultsStrains of two human bacterial pathogens, Listeria monocytogenes and Staphylococcus aureus, were selected to cover a wide range of origin, sub-type, and phenotypic behavior. Strains within each species were equally sensitive to HDPs and oxidative stress representing important components of the innate immune defense system. Four non-human peptides (protamine, plectasin, novicidin, and novispirin G10) were similar in activity profile (MIC value spectrum) to the human β-defensin 3 (HBD-3). All strains were inhibited by concentrations of hydrogen peroxide between 0.1% – 1.0%. Sub-selections of both species differed in expression of several virulence-related factors and in their ability to survive in human whole blood and kill the nematode virulence model Caenorhabditis elegans. For L. monocytogenes, proliferation in whole blood was paralleled by high invasion in Caco-2 cells and fast killing of C. elegans, however, no such pattern in phenotypic behavior was observed for S. aureus and none of the phenotypic differences were correlated to sensitivity to HDPs.ConclusionStrains of L. monocytogenes and S. aureus were within each species equally sensitive to a range of HDPs despite variations in subtype, origin, and phenotypic behavior. Our results suggest that therapeutic use of HDPs will not be hampered by occurrence of naturally tolerant strains of the two species investigated in the present study.

Design of Novispirin Antimicrobial Peptides by Quantitative Structure–Activity Relationship

Novispirin G10 is an α‐helical antimicrobial peptide designed in an effort to develop alternative treatments against multidrug‐resistant micro‐organisms. To further optimize the antimicrobial activity, 58 novispirin analogs were constructed and used to establish a quantitative structure–activity relationship model. A statistically significant model (r2 = 0.73, q2 = 0.61) was obtained using a set of 69 selected molecular descriptors. Among these, VolSurf and charged partial surface area descriptors played a dominant role. Analysis of the model indicated that hydrophobicity, amphipathicity and charge were the most important features influencing activity for this set of peptides. Furthermore, the ability of the quantitative structure–activity relationship model to predict bioactivity was evaluated by analyzing a set of 400 novispirin analogs designed by molecular modeling. Out of these 400, 16 new novispirins with a higher predicted antimicrobial activity were tested in the suicide expression system, and about three out of four appeared more potent than the parent novispirin G10. Combination of VolSurf and charged partial surface area descriptors seems relevant to depict the interaction between novispirin and its target(s), presumably the microbial cell membrane. The presented findings show that modeling and quantitative structure–activity relationship methods can be useful in the construction of and/or optimization of the bioactivity of antimicrobial peptides for further development as effective antibiotic therapeutics.

Intracellular activity of the peptide antibiotic NZ2114: studies with Staphylococcus aureus and human THP-1 monocytes, and comparison with daptomycin and vancomycin.

OBJECTIVES Staphylococcus aureus survives inside eukaryotic cells. Our objective was to assess the activity of NZ2114, a novel peptidic antibiotic, against intracellular S. aureus in comparison with established antistaphylococcal agents acting on the bacterial envelope with a distinct mechanism. METHODS The extracellular (broth) and intracellular (THP-1 monocytes) activities of NZ2114 were compared with those of vancomycin and daptomycin against methicillin-susceptible S. aureus (MSSA), methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA). RESULTS All three compounds showed an extracellular bactericidal effect (>3 log(10) kill) against MSSA and MRSA. Daptomycin and NZ2114 also exhibited bactericidal activity against VRSA. The extracellular killing was concentration dependent for all three compounds within the range of drug concentrations tested. The intracellular experiments demonstrated a maximal intracellular effect of NZ2114 after 24 h as a 5 log(10) cfu reduction against MSSA (ATCC 25923), while the activity was a 0.9 log(10) cfu reduction against MRSA and a 0.2 log(10) cfu reduction against VRSA. For comparison, the intracellular activity of daptomycin was a 1.0 log(10) cfu reduction against MSSA, a 0.8 log(10) cfu reduction against MRSA and a 0.3 log(10) cfu reduction against VRSA. Vancomycin showed activity against both MSSA and MRSA (0.6 log(10) cfu reduction), whereas VRSA was resistant to vancomycin. CONCLUSIONS NZ2114 displayed similar extracellular and intracellular activities as daptomycin, and was more effective than vancomycin against the intracellular forms of susceptible bacteria. However, the study also showed that the intracellular activities of NZ2114 and daptomycin are weaker than their extracellular activities.

Using antimicrobial host defense peptides as anti-infective and immunomodulatory agents

Virtually all life forms express short antimicrobial cationic peptides as an important component of their innate immune defenses. They serve as endogenous antibiotics that are able to rapidly kill an unusually broad range of bacteria, fungi and viruses. Consequently, considerable efforts have been expended to exploit the therapeutic potential of these antimicrobial peptides. Within the last couple of years, it has become increasingly clear that many of these peptides, in addition to their direct antimicrobial activity, also have a wide range of functions in modulating both innate and adaptive immunity. For one class of antimicrobial peptides, such as the human defensins, their primary role may even be as immunomodulators. These properties potentially provide entirely new therapeutic approaches to anti-infective therapy.