Monique L. van Hoek

Natural and synthetic cathelicidin peptides with anti-microbial and anti-biofilm activity against Staphylococcus aureus.

BackgroundChronic, infected wounds typically contain multiple genera of bacteria, including Staphylococcus aureus, many of which are strong biofilm formers. Bacterial biofilms are thought to be a direct impediment to wound healing. New therapies that focus on a biofilm approach may improve the recovery and healing rate for infected wounds. In this study, cathelicidins and related short, synthetic peptides were tested for their anti-microbial effectiveness as well as their ability to inhibit the ability of S. aureus to form biofilms.ResultsThe helical human cathelicidin LL-37 was tested against S. aureus, and was found to exhibit effective anti-microbial, anti-attachment as well as anti-biofilm activity at concentrations in the low μg/ml range. The effect of peptide chirality and associated protease-resistance was explored through the use of an all-D amino acid peptide, D-LL-37, and in turn compared to scrambled LL-37. Helical cathelicidins have been identified in other animals such as the Chinese cobra, Naja atra (NA-CATH). We previously identified an 11-residue imperfectly repeated pattern (ATRA motif) within the sequence of NA-CATH. A series of short peptides (ATRA-1, -2, -1A), as well as a synthetic peptide, NA-CATH:ATRA1-ATRA1, were designed to explore the significance of the conserved residues within the ATRA motif for anti-microbial activity. The CD spectrum of NA-CATH and NA-CATH:ATRA1-ATRA1 revealed the structural properties of these peptides and suggested that helicity may factor into their anti-microbial and anti-biofilm activities.ConclusionsThe NA-CATH:ATRA1-ATRA1 peptide inhibits the production of biofilm by S. aureus in the presence of salt, exhibiting anti-biofilm activity at lower peptide concentrations than NA-CATH, LL-37 and D-LL-37; and demonstrates low cytoxicity against host cells but does not affect bacterial attachment. The peptides utilized in this anti-biofilm approach may provide templates for a new group of anti-microbials and potential future topical therapeutics for treating chronic wound infections.

Susceptibility of Pseudomonas aeruginosa Biofilm to Alpha-Helical Peptides: D-enantiomer of LL-37

Pseudomonas aeruginosa is a highly versatile opportunistic pathogen and its ability to produce biofilms is a direct impediment to the healing of wounds and recovery from infection. Interest in anti-microbial peptides (AMPs) has grown due to their potential therapeutic applications and their possible use against antibiotic resistant bacteria. LL-37 is the only cathelicidin expressed by humans. In this study, we tested LL-37 and the effect of a protease-resistant LL-37 peptide mimetic, the peptide enantiomer D-LL-37, for anti-microbial and anti-biofilm activity against P. aeruginosa. Both forms of the peptide were equally effective as AMPs with similar killing kinetics. Circular dichroism spectra were obtained to demonstrate the chirality of D- and L-LL-37, and the trypsin resistance of D-LL-37 was confirmed. The helical cathelicidin from the cobra Naja atra (NA-CATH), and synthetic peptide variations (ATRA-1, ATRA-2, NA-CATH:ATRA1-ATRA1) were also tested. Although the cobra cathelicidin and related peptides had strong anti-microbial activity, those tested did not inhibit Pseudomonas biofilm formation, neither did control peptides. Both D- and L-LL-37 inhibited the attachment of Pseudomonas to a 96-well plate and decreased the amount of pre-formed (established) biofilm. D-LL-37 is able to promote Pseudomonas motility and decrease biofilm formation by altering the rate of twitching as well as by downregulating the expression of the biofilm-related genes, rhlA and rhlB, similar to L-LL-37. Both L- and D-LL-37 protected Galleria mellonella in vivo against Pseudomonas infection, while NA-CATH:ATRA1-ATRA1 peptide did not. This study demonstrates the ability and equivalence of D-LL-37 compared to L-LL-37 to promote bacterial twitching motility and inhibit biofilm formation, and protect against in vivo infection, and suggests that this peptide could be a critical advancement in the development of new treatments for P. aeruginosa infection.

Proteomic Characterization and Functional Analysis of Outer Membrane Vesicles of Francisella novicida Suggests Possible Role in Virulence and Use as a Vaccine

We have isolated and characterized outer membrane vesicles (OMVs) from Francisella. Transport of effector molecules through secretion systems is a major mechanism by which Francisella tularensis alters the extracellular proteome and interacts with the host during infection. Outer membrane vesicles produced by Francisella were examined using TEM and AFM and found to be 43-125 nm in size, representing another potential mechanism for altering the extracellular environment. A proteomic analysis (LC-MS/MS) of OMVs from F. novicida and F. philomiragia identified 416 (F. novicida) and 238 (F. philomiragia) different proteins, demonstrating that OMVs are an important contributor to the extracellular proteome. Many of the identified OMV proteins have a demonstrated role in Francisella pathogenesis. Biochemical assays demonstrated that Francisella OMVs possess acid phosphatase and hemolytic activities that may affect host cells during infection, and are cytotoxic toward murine macrophages in cell culture. OMVs have been previously used as a human vaccine against Neisseria meningitidis . We hypothesized that Francisella OMVs could be useful as a novel Francisella vaccine. Vaccinated BALB/C mice challenged with up to 50 LD50 of Francisella showed statistically significant protection when compared to control mice. In the context of these new findings, we discuss the relevance of OMVs in Francisella pathogenesis as well as their potential use as a vaccine.

The Human Cathelicidin Antimicrobial Peptide LL-37 as a Potential Treatment for Polymicrobial Infected Wounds

Diabetic patients often have ulcers on their lower-limbs that are infected by multiple biofilm-forming genera of bacteria, and the elimination of the biofilm has proven highly successful in resolving such wounds in patients. To that end, antimicrobial peptides have shown potential as a new anti-biofilm approach. The single human cathelicidin peptide LL-37 has been shown to have antimicrobial and anti-biofilm activity against multiple Gram-positive and Gram-negative human pathogens, and have wound-healing effects on the host. The combination of the anti-biofilm effect and wound-healing properties of LL-37 may make it highly effective in resolving polymicrobially infected wounds when topically applied. Such a peptide or its derivatives could be a platform from which to develop new therapeutic strategies to treat biofilm-mediated infections of wounds. This review summarizes known mechanisms that regulate the endogenous levels of LL-37 and discusses the anti-biofilm, antibacterial, and immunological effects of deficient vs. excessive concentrations of LL-37 within the wound environment. Here, we review recent advances in understanding the therapeutic potential of this peptide and other clinically advanced peptides as a potential topical treatment for polymicrobial infected wounds.

Antimicrobial and antibiofilm activity of cathelicidins and short, synthetic peptides against Francisella.

Francisella infects the lungs causing pneumonic tularemia. Focusing on the lungs host defense, we have examined antimicrobial peptides as part of the innate immune response to Francisella infection. Interest in antimicrobial peptides, such as the cathelicidins, has grown due their potential therapeutic applications and the increasing problem of bacterial resistance to commonly used antibiotics. Only one human cathelicidin, LL-37, has been characterized. Helical cathelicidins have also been discovered in snakes including the Chinese King Cobra, Naja atra (NA-CATH). Four synthetic 11-residue peptides (ATRA-1, -2, -1A and -1P) containing variations of a repeated motif within NA-CATH were designed. We hypothesized that these smaller synthetic peptides could have excellent antimicrobial effectiveness with shorter length (and less cost), making them strong potential candidates for development into broad-spectrum antimicrobial compounds. We tested the susceptibility of F. novicida to four ATRA peptides, LL-37, and NA-CATH. Two of the ATRA peptides had high antimicrobial activity (microM), while the two proline-containing ATRA peptides had low activity. The ATRA peptides did not show significant hemolytic activity even at high peptide concentration, indicating low cytotoxicity against host cells. NA-CATH killed Francisella bacteria more quickly than LL-37. However, LL-37 was the most effective peptide against F. novicida (EC50=50 nM). LL-37 mRNA was induced in A549 cells by Francisella infection. We recently demonstrated that F. novicida forms in vitro biofilms. LL-37 inhibited F. novicida biofilm formation at sub-antimicrobial concentrations. Understanding the properties of these peptides, and their endogenous expression in the lung could lead to potential future therapeutic interventions for this lung infection.

Francisella novicida forms in vitro biofilms mediated by an orphan response regulator.

Francisella tularensis is associated with water and waterways and infects many species of animals, insects, and protists. The mechanism Francisella utilizes to persist in the environment and in tick vectors is currently unknown. We have demonstrated for the first time that Francisella novicida, a model organism of F. tularensis, forms a biofilm in vitro. Selected F. novicida transposon mutants were tested for their ability to form biofilm compared to the wildtype F. novicida strain. Mutation of the putative qseB gene led to an impairment in the ability to form biofilm with no impairment in bacterial growth. A qseC mutant had impaired growth but demonstrated a marked impairment in biofilm production. Mutation in capC affected both bacterial growth and biofilm formation, but no biofilm production impairment was seen with capB or pilE mutants. A deletion mutant in the orphan response regulator FTN_1465, which we propose is the putative QseB, formed significantly less biofilm than the wildtype. When FTN_1465 was complemented back into the deletion mutant, biofilm formation was restored. Thus, the orphan response regulator FTN_1465 is an important factor in biofilm production in vitro in F. novicida. These results demonstrate that Francisella species are able to form biofilms in vitro, suggesting that biofilm formation may be important for the lifecycle of this organism.

Extracellular vesicles from infected cells: potential for direct pathogenesis.

Infections that result in natural or manmade spread of lethal biological agents are a concern and require national and focused preparedness. In this manuscript, as part of an early diagnostics and pathogen treatment strategy, we have focused on extracellular vesicles (EVs) that arise following infections. Although the field of biodefense does not currently have a rich resource in EVs literature, none the less, similar pathogens belonging to the more classical emerging and non-emerging diseases have been studied in their EV/exosomal contents and function. These exosomes are formed in late endosomes and released from the cell membrane in almost every cell type in vivo. These vesicles contain proteins, RNA, and lipids from the cells they originate from and function in development, signal transduction, cell survival, and transfer of infectious material. The current review focuses on how different forms of infection exploit the exosomal pathway and how exosomes can be exploited artificially to treat infection and disease and potentially also be used as a source of vaccine. Virally-infected cells can secrete viral as well as cellular proteins and RNA in exosomes, allowing viruses to cause latent infection and spread of miRNA to nearby cells prior to a subsequent infection. In addition to virally-infected host cells, bacteria, protozoa, and fungi can all release small vesicles that contain pathogen-associated molecular patterns, regulating the neighboring uninfected cells. Examples of exosomes from both virally and bacterially infected cells point toward a re-programming network of pathways in the recipient cells. Finally, many of these exosomes contain cytokines and miRNAs that in turn can effect gene expression in the recipient cells through the classical toll-like receptor and NFκB pathway. Therefore, although exosomes do not replicate as an independent entity, they however facilitate movement of infectious material through tissues and may be the cause of many pathologies seen in infected hosts.

Antimicrobial activity of the Naja atra cathelicidin and related small peptides

We have identified an 11-residue pattern (KR(F/A)KKFFKK(L/P)K), which we have named the ATRA motif, within the sequence of the Chinese cobra (Naja atra) cathelicidin. A series of 11-residue peptides (ATRA-1, -2, -1A and -1P) were designed to probe the significance of the conserved residues within the ATRA motif, and their contributions to antimicrobial performance. The antimicrobial activities of the peptides were assessed against Escherichia coli K12 strain and Aggregatibacter actinomycetemcomitans Y4. ATRA-1 and -1A, demonstrated potencies comparable to that of N. atra cathelicidin. Structural examination by circular dichroism of the four short peptides suggested the significance of specific amino acid positions within the motif by their contribution to helicity. The results of these studies indicate that short peptides derived from the repeated ATRA motif from the N. atra cathelicidin can demonstrate both low toxicity against host cells and high antimicrobial activity against the gram-negative bacteria used in this study. They constitute novel, effective antimicrobial peptides that are much shorter (and thus less expensive to produce) than the natural cathelicidins, and they may represent new templates for therapeutic drug development.

Azithromycin effectiveness against intracellular infections of Francisella

BackgroundMacrolide antibiotics are commonly administered for bacterial respiratory illnesses. Azithromycin (Az) is especially noted for extremely high intracellular concentrations achieved within macrophages which is far greater than the serum concentration. Clinical strains of Type B Francisella (F.) tularensis have been reported to be resistant to Az, however our laboratory Francisella strains were found to be sensitive. We hypothesized that different strains/species of Francisella (including Type A) may have different susceptibilities to Az, a widely used and well-tolerated antibiotic.ResultsIn vitro susceptibility testing of Az confirmed that F. tularensis subsp. holarctica Live Vaccine Strain (LVS) (Type B) was not sensitive while F. philomiragia, F. novicida, and Type A F. tularensis (NIH B38 and Schu S4 strain) were susceptible. In J774A.1 mouse macrophage cells infected with F. philomiragia, F. novicida, and F. tularensis LVS, 5 μg/ml Az applied extracellularly eliminated intracellular Francisella infections. A concentration of 25 μg/ml Az was required for Francisella- infected A549 human lung epithelial cells, suggesting that macrophages are more effective at concentrating Az than epithelial cells. Mutants of RND efflux components (tolC and ftlC) in F. novicida demonstrated less sensitivity to Az by MIC than the parental strain, but the tolC disc-inhibition assay demonstrated increased sensitivity, indicating a complex role for the outer-membrane transporter. Mutants of acrA and acrB mutants were less sensitive to Az than the parental strain, suggesting that AcrAB is not critical for the efflux of Az in F. novicida. In contrast, F. tularensis Schu S4 mutants ΔacrB and ΔacrA were more sensitive than the parental strain, indicating that the AcrAB may be important for Az efflux in F. tularensis Schu S4. F. novicida LPS O-antigen mutants (wbtN, wbtE, wbtQ and wbtA) were found to be less sensitive in vitro to Az compared to the wild-type. Az treatment prolonged the survival of Galleria (G.) mellonella infected with Francisella.ConclusionThese studies demonstrate that Type A Francisella strains, as well as F. novicida and F. philomiragia, are sensitive to Az in vitro. Francisella LPS and the RND efflux pump may play a role in Az sensitivity. Az also has antimicrobial activity against intracellular Francisella, suggesting that the intracellular concentration of Az is high enough to be effective against multiple strains/species of Francisella, especially in macrophages. Az treatment prolonged survival an in vivo model of Francisella- infection.

Antimicrobial peptides in reptiles.

Reptiles are among the oldest known amniotes and are highly diverse in their morphology and ecological niches. These animals have an evolutionarily ancient innate-immune system that is of great interest to scientists trying to identify new and useful antimicrobial peptides. Significant work in the last decade in the fields of biochemistry, proteomics and genomics has begun to reveal the complexity of reptilian antimicrobial peptides. Here, the current knowledge about antimicrobial peptides in reptiles is reviewed, with specific examples in each of the four orders: Testudines (turtles and tortosises), Sphenodontia (tuataras), Squamata (snakes and lizards), and Crocodilia (crocodilans). Examples are presented of the major classes of antimicrobial peptides expressed by reptiles including defensins, cathelicidins, liver-expressed peptides (hepcidin and LEAP-2), lysozyme, crotamine, and others. Some of these peptides have been identified and tested for their antibacterial or antiviral activity; others are only predicted as possible genes from genomic sequencing. Bioinformatic analysis of the reptile genomes is presented, revealing many predicted candidate antimicrobial peptides genes across this diverse class. The study of how these ancient creatures use antimicrobial peptides within their innate immune systems may reveal new understandings of our mammalian innate immune system and may also provide new and powerful antimicrobial peptides as scaffolds for potential therapeutic development.