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.

BackgroundThe increase in antibiotic resistant bacteria has led to renewed interest in development of alternative antimicrobial compounds such as antimicrobial peptides (AMPs), either naturally-occurring or synthetically-derived. Knowledge of the mode of action (MOA) of synthetic compounds mimicking the function of AMPs is highly valuable both when developing new types of antimicrobials and when predicting resistance development. Despite many functional studies of AMPs, only a few of the synthetic peptides have been studied in detail.ResultsWe investigated the MOA of the lysine-peptoid hybrid, LP5, which previously has been shown to display antimicrobial activity against Staphylococcus aureus. At concentrations of LP5 above the minimal inhibitory concentration (MIC), the peptoid caused ATP leakage from bacterial cells. However, at concentrations close to the MIC, LP5 inhibited the growth of S. aureus without ATP leakage. Instead, LP5 bound DNA and inhibited macromolecular synthesis. The binding to DNA also led to inhibition of DNA gyrase and topoisomerase IV and caused induction of the SOS response.ConclusionsOur data demonstrate that LP5 may have a dual mode of action against S. aureus. At MIC concentrations, LP5 binds DNA and inhibits macromolecular synthesis and growth, whereas at concentrations above the MIC, LP5 targets the bacterial membrane leading to disruption of the membrane. These results add new information about the MOA of a new synthetic AMP and aid in the future design of synthetic peptides with increased therapeutic potential.

The rapid rise in antibiotic-resistant pathogens is causing increased health concerns, and consequently there is an urgent need for novel antimicrobial agents. Antimicrobial peptides (AMPs), which have been isolated from a wide range of organisms, represent a very promising class of novel antimicrobials. In the present study, the analogue FL9, based on the amphibian AMP fallaxin, was studied to elucidate its mode of action and antibacterial activity against the human pathogen Staphylococcus aureus. Our data showed that FL9 may have a dual mode of action against S. aureus. At concentrations around the MIC, FL9 bound DNA, inhibited DNA synthesis and induced the SOS DNA damage response, whereas at concentrations above the MIC the interaction between S. aureus and FL9 led to membrane disruption. The antibacterial activity of the peptide was maintained over a wide range of NaCl and MgCl 2 concentrations and at alkaline pH, while it was compromised by acidic pH and exposure to serum. Furthermore, at subinhibitory concentrations of FL9, S. aureus responded by increasing the expression of two major virulence factor genes, namely the regulatory rnaIII and hla, encoding a-haemolysin. In addition, the S. aureus-encoded natural tolerance mechanisms included peptide cleavage and the addition of positive charge to the cell surface, both of which minimized the antimicrobial activity of FL9. Our results add new information about FL9 and its effect on S. aureus, which may aid in the future development of analogues with improved therapeutic potential.

We determined mammalian cell invasion and virulence gene (inlA, inlB, and actA) sequences of Listeria monocytogenes strains belonging to a molecular subtype (RAPD 9) that often persists in Danish fish-processing plants. These strains invaded human placental trophoblasts less efficiently than other L. monocytogenes strains, including clinical strains, and they carry a premature stop codon in inlA. Eight of 15 strains, including the RAPD 9 and maternofetal strains, had a 105-nucleotide deletion in actA that did not affect cell-to-cell spread in mouse fibroblasts. The RAPD 9 strains may still be regarded as of low virulence with respect to human listeriosis.Listeria monocytogenes is a Gram-positive pathogenic bacterium that can cause foodborne listeriosis, which affects immunocompromised individuals, causing septicemia and meningitis, and pregnant women, causing preterm delivery, miscarriage, or stillbirth. It is a ubiquitous environmental bacterium, and it is therefore continuously introduced to food-processing plants, where some molecular subtypes are able to persist despite thorough cleaning and disinfection procedures (1,27,32,42). Such persistent strains are likely to contaminate the food products and may be the cause of foodborne infections (30).We have shown that specific molecular subtypes of L. monocytogenes can persist for years in the seafood-processing environment (45), and strains representing a particularly prevalent, persistent molecular subtype, RAPD type 9 ([RAPD 9] random amplified polymorphic DNA), had a lower virulence potential than clinical strains in simple eukaryotic models (12). However, in a more complex biological model using oral dosing of pregnant guinea pigs, the tested RAPD 9 strain (strain La111) surprisingly infected the placentas and fetuses just as efficiently as a clinical strain (13). We therefore hypothesized that this specific subtype may have an altered (enhanced) ability to invade placental cells (e.g., trophoblasts) or an enhanced ability to spread intracellularly. (29), the United States (25), and Japan (9), and these mutations lead to attenuation in the invasion of intestinal epithelial cells (25,28,33), but it is not known if invasion into trophoblasts is affected. ActA is important for cell-to-cell spread (5) and is involved in invasion of epithelial cells (39), and ActA-mediated cell-to-cell spread plays a major role in crossing the fetoplacental barrier in both a guinea pig and a mouse model (3,22).The purpose of this study was to determine if the high level of prevalence of a RAPD 9 strain in guinea pig fetuses after oral dosing could be explained by increased invasion into and spread between trophoblastic and fibroblastic cells, respectively. Subsequently, we sequenced selected virulence genes to determine if strain variations in cell invasion and spread could be explained by differences in sequences.Strains, culture conditions, and characterization. Fifteen L. monocytogenes strains representing different origins, RAPD types, serotypes, and lineages were used (Tabl...