Effects of Escherichia coli LPS Structure on Antibacterial and Anti-Endotoxin Activities of Host Defense Peptides.

The binding of Host Defense Peptides (HDPs) to the endotoxin of Gram-negative bacteria has important unsolved aspects. For most HDPs, it is unclear if binding is part of the antibacterial mechanism or whether LPS actually provides a protective layer against HDP killing. In addition, HDP binding to LPS can block the subsequent TLR4-mediated activation of the immune system. This dual activity is important, considering that HDPs are thought of as an alternative to conventional antibiotics, which do not provide this dual activity. In this study, we systematically determine, for the first time, the influence of the O-antigen and Lipid A composition on both the antibacterial and anti-endotoxin activity of four HDPs (CATH-2, PR-39, PMAP-23, and PMAP36). The presence of the O-antigen did not affect the antibacterial activity of any of the tested HDPs. Similarly, modification of the lipid A phosphate (MCR-1 phenotype) also did not affect the activity of the HDPs. Furthermore, assessment of inner and outer membrane damage revealed that CATH-2 and PMAP-36 are profoundly membrane-active and disrupt the inner and outer membrane of Escherichia coli simultaneously, suggesting that crossing the outer membrane is the rate-limiting step in the bactericidal activity of these HDPs but is independent of the presence of an O-antigen. In contrast to killing, larger differences were observed for the anti-endotoxin properties of HDPs. CATH-2 and PMAP-36 were much stronger at suppressing LPS-induced activation of macrophages compared to PR-39 and PMAP-23. In addition, the presence of only one phosphate group in the lipid A moiety reduced the immunomodulating activity of these HDPs. Overall, the data strongly suggest that LPS composition has little effect on bacterial killing but that Lipid A modification can affect the immunomodulatory role of HDPs. This dual activity should be considered when HDPs are considered for application purposes in the treatment of infectious diseases.

Novel insights in antimicrobial and immunomodulatory mechanisms of action of PepBiotics CR-163 and CR-172.

OBJECTIVES: Our group recently developed a new group of antimicrobial peptides termed PepBiotics, of which peptides CR-163 and CR-172 showed optimized antibacterial activity against Pseudomonas aeruginosa and Staphylococcus aureus without inducing antimicrobial resistance. In this study, the antibacterial mechanism of action and the immunomodulatory activity of these two PepBiotics was explored. METHODS: RAW264.7 cells were used to determine the ability of PepBiotics to neutralize Lipopolysaccharide (LPS)-and Lipoteichoic acid (LTA)-induced activation of macrophages. Isothermal titration calorimetry and competition assays with dansyl-labeled polymyxin B determined binding characteristics to LPS and LTA. Combined bacterial killing with subsequent macrophage activation assays was performed to determine so-called 'silent killing'. Finally, flow cytometry of peptide-treated genetically engineered Escherichia coli expressing Green Fluorescent Protein (GFP) and mCherry in the cytoplasm and periplasm, respectively, further established the antimicrobial mechanism of PepBiotics. RESULTS: Both CR-163 and CR-172 were shown to have broad-spectrum activity against ESKAPE pathogens and E. coli using a membranolytic mechanism of action. PepBiotics could exothermically bind LPS/LTA and were able to replace polymyxin B. Finally, it was demonstrated that bacteria killed by PepBiotics were less prone to stimulate immune cells, contrary to gentamicin and heat-killed bacteria that still elicited a strong immune response. CONCLUSIONS: These studies highlight the multifunctional nature of the two peptide antibiotics as both broad-spectrum antimicrobial and immunomodulator. Their ability to kill bacteria and reduce unwanted subsequent immune activation is a major advantage and highlights their potential for future therapeutic use.

Modulation of outer membrane vesicle-based immune responses by cathelicidins.

Antibiotic resistance is increasing and one strategy to prevent resistance development is the use of bacterial vaccines. For Gram-negative bacteria, natural outer membrane vesicles (OMVs) could be used for vaccine development. These vesicular structures are naturally produced by all Gram-negative bacteria and contain several antigens in their native environment. However, despite that the presence of lipopolysaccharide (LPS) may aid as intrinsic adjuvant, there is a risk that it may also cause undesired immune responses. Therefore, molecules to dampen LPS-induced toll-like receptor (TLR) 4 activation may be needed. Here host defense peptides (HDPs), like cathelicidins, can play an important role. They have been shown to interact with LPS and thereby neutralize LPS-induced TLR4 activation. However, there is currently no knowledge about neutralization in an OMV-based setting. Therefore, in this paper the immune modulating capacity of HDPs was investigated after macrophage stimulation with either spontaneous or heat-induced B. bronchiseptica OMVs. This revealed that the cathelicidins LL-37, CATH-2, PMAP-36 and K9CATH were able to modulate immune responses. Interestingly, immune modulation by these cathelicidins was different for spontaneous compared to heat-induced OMVs. Interaction studies revealed that the mode of binding of cathelicidins to OMVs slightly differed between OMV classes. Furthermore, TLR screening revealed that TLR2, 4, 5 and 9 were involved in stimulation of macrophages by OMVs, with TLR4-mediated activation being the most important pathway. Uptake of OMVs did not play a major role in macrophage activation. Taken together, this study shows how OMVs can activate macrophages and how cathelicidins may modulate these immune responses.

Pal depletion results in hypervesiculation and affects cell morphology and outer-membrane lipid asymmetry in bordetellae.

Current vaccines against Bordetella pertussis do not prevent colonization and transmission of the bacteria, and vaccine-induced immunity wanes rapidly. Besides, efficacy of vaccines for Bordetella bronchiseptica remains unclear. Novel vaccines could be based on outer-membrane vesicles (OMVs), but vesiculation of bordetellae needs to be increased for cost-effective vaccine production. Here, we focused on increasing OMV production by reducing the anchoring of the outer membrane to the peptidoglycan layer. Inactivation of rmpM, tolR, and pal failed, presumably because their products are essential in bordetellae. Conditional pal mutants were constructed, which were hypervesiculating under Pal-depletion conditions. SDS-PAGE and Western blot analyses showed that the protein composition of OMVs produced under Pal-depletion conditions resembled that of the outer membrane but differed from that of OMVs released by the wild type. Pal depletion affected the cell morphology and appeared to increase the amounts of cell-surface-exposed phospholipids, possibly reflecting a role for the Tol-Pal system in retrograde phospholipid transport. We also identified additional lipoproteins in bordetellae with a putative peptidoglycan-anchoring domain. However, their inactivation did not influence OMV production. We conclude that the conditional pal mutants could be valuable for the development of OMV-based vaccines.

d-enantiomers of CATH-2 enhance the response of macrophages against Streptococcus suis serotype 2.

Introduction: Due to the increase of antibiotic resistant bacterial strains, there is an urgent need for development of alternatives to antibiotics. Cathelicidins can be such an alternative to antibiotics having both a direct antimicrobial capacity as well as an immunomodulatory function. Previously, the full d-enantiomer of chicken cathelicidin-2 (d-CATH-2) has shown to prophylactically protect chickens against infection 7 days post hatch when administered in ovo three days before hatch. Objectives: To further evaluate d-CATH-2 in mammals as a candidate for an alternative to antibiotics.In this study, the prophylactic capacity of d-CATH-2 and two truncated derivatives, d-C(1-21) and d-C(4-21), was determined in mammalian cells. Methods: Antibacterial assays; immune cell differentiation and modulation; cytotoxicity, isothermal titration calorimetry; in vivo prophylactic capacity of peptides in an S. suis infection model. Results: d-CATH-2 and its derivatives were shown to have a strong direct antibacterial capacity against four different S. suis serotype 2 strains (P1/7, S735, D282, and OV625) in bacterial medium and even stronger in cell culture medium. In addition, d-CATH-2 and its derivatives ameliorated the efficiency of mouse bone marrow-derived macrophages (BMDM) and skewed mouse bone marrow-derived dendritic cells (BMDC) towards cells with a more macrophage-like phenotype. The peptides directly bind lipoteichoic acid (LTA) and inhibit LTA-induced activation of macrophages. In addition, S. suis killed by the peptide was unable to further activate mouse macrophages, which indicates that S. suis was eliminated by the previously reported silent killing mechanism. Administration of d-C(1-21) at 24 h or 7 days before infection resulted in a small prophylactic protection with reduced disease severity and reduced mortality of the treated mice. Conclusion: d-enantiomers of CATH-2 show promise as anti-infectives against pathogenic S. suis for application in mammals.

Heat shock enhances outer-membrane vesicle release in Bordetella spp.

Pertussis, also known as whooping cough, is caused by the Gram-negative bacterium Bordetella pertussis, an obligate human pathogen. Despite high vaccination rates in high-income countries, resurgence of pertussis cases is an occurring problem that urges the necessity of developing an improved vaccine. Likewise, the efficacy of vaccines for Bordetella bronchiseptica, which causes similar disease in pigs and companion animals, is debatable. A promising approach for novel vaccines is the use of outer membrane vesicles (OMVs). However, spontaneous OMV (sOMV) release by Bordetella spp. is too low for cost-effective vaccine production. Therefore, we investigated the influence of growth in various media commonly used for culturing Bordetella in the Bvg+, i.e. virulent, phase and of a heat shock applied to inactivate the cells on OMV production. Inactivation of the bacterial cells at 56 °C before OMV isolation greatly enhanced OMV release in both Bordetella spp. without causing significant cell lysis. The growth medium used barely affected the efficiency of OMV release but did affect the protein pattern of the OMVs. Differences were found to be related, at least in part, to different availability of the nutrient metals iron and zinc in the media and include expression of potentially relevant vaccine antigens, such as the receptors FauA and ZnuD. The protein content of OMVs released by heat shock was comparable to that of sOMVs as determined by SDS-PAGE and Western blot analysis, and their heat-modifiable electrophoretic mobility suggests that also protein conformation is unaffected. However, significant differences were noticed between the protein content of OMVs and that of a purified outer membrane fraction, with two major outer membrane proteins, porin OmpP and the peptidoglycan-associated RmpM, being underrepresented in the OMVs. Altogether, these results indicate that the application of a heat shock is potentially an important step in the development of cost-effective, OMV-based vaccines for both Bordetella spp.

PMAP-36 reduces the innate immune response induced by Bordetella bronchiseptica-derived outer membrane vesicles.

Host defense peptides (HDPs), such as cathelicidins, are small, cationic, amphipathic peptides and represent an important part of the innate immune system. Most cathelicidins, including the porcine PMAP-36, are membrane active and disrupt the bacterial membrane. For example, a chicken cathelicidin, CATH-2, has been previously shown to disrupt both Escherichia coli membranes and to release, at sub-lethal concentrations, outer membrane vesicles (OMVs). Since OMVs are considered promising vaccine candidates, we sought to investigate the effect of sub-bactericidal concentrations of PMAP-36 on both OMV release by a porcine strain of Bordetella bronchiseptica and on the modulation of immune responses to OMVs. PMAP-36 treatment of bacteria resulted in a slight increase in OMV release. The characteristics of PMAP-36-induced OMVs were compared with those of spontaneously released OMVs and OMVs induced by heat treatment. The stability of both PMAP-36- and heat-induced OMVs was decreased compared to spontaneous OMVs, as shown by dynamic light scattering. Furthermore, treatment of bacteria with PMAP-36 or heat resulted in an increase in negatively charged phospholipids in the resulting OMVs. A large increase in lysophospholipid content was observed in heat-induced OMVs, which was at least partially due to the activity of the outer-membrane phospholipase A (OMPLA). Although PMAP-36 was detected in OMVs isolated from PMAP-36-treated bacteria, the immune response of porcine bone-marrow-derived macrophages to these OMVs was similar as those against spontaneous or heat-induced OMVs. Therefore, the effect of PMAP-36 addition after OMV isolation was investigated. This did decrease cytokine expression of OMV-stimulated macrophages. These results indicate that PMAP-36 is a promising molecule to attenuate undesirable immune responses, for instance in vaccines.

Outer Membrane Vesicles Protect Gram-Negative Bacteria against Host Defense Peptides.

Host defense peptides (HDPs) are part of the innate immune system and constitute a first line of defense against invading pathogens. They possess antimicrobial activity against a broad spectrum of pathogens. However, pathogens have been known to adapt to hostile environments. Therefore, the bacterial response to treatment with HDPs was investigated. Previous observations suggested that sublethal concentrations of HDPs increase the release of outer membrane vesicles (OMVs) in Escherichia coli. First, the effects of sublethal treatment with HDPs CATH-2, PMAP-36, and LL-37 on OMV release of several Gram-negative bacteria were analyzed. Treatment with PMAP-36 and CATH-2 induced release of OMVs, but treatment with LL-37 did not. The OMVs were further characterized with respect to morphological properties. The HDP-induced OMVs often had disc-like shapes. The beneficial effect of bacterial OMV release was studied by determining the susceptibility of E. coli toward HDPs in the presence of OMVs. The minimal bactericidal concentration was increased in the presence of OMVs. It is concluded that OMV release is a means of bacteria to dispose of HDP-affected membrane. Furthermore, OMVs act as a decoy for HDPs and thereby protect the bacterium. IMPORTANCE Antibiotic resistance is a pressing problem and estimated to be a leading cause of mortality by 2050. Antimicrobial peptides, also known as host defense peptides (HDPs), and HDP-derived antimicrobials have potent antimicrobial activity and high potential as alternatives to antibiotics due to low resistance development. Some resistance mechanisms have developed in bacteria, and complete understanding of bacterial defense against HDPs will aid their use in the clinic. This study provides insight into outer membrane vesicles (OMVs) as potential defense mechanisms against HDPs, which will allow anticipation of unforeseen resistance to HDPs in clinical use and possibly prevention of bacterial resistance by the means of OMVs.

Outer Membrane Vesicle Induction and Isolation for Vaccine Development.

Gram-negative bacteria release vesicular structures from their outer membrane, so called outer membrane vesicles (OMVs). OMVs have a variety of functions such as waste disposal, communication, and antigen or toxin delivery. These vesicles are the promising structures for vaccine development since OMVs carry many surface antigens that are identical to the bacterial surface. However, isolation is often difficult and results in low yields. Several methods to enhance OMV yield exist, but these do affect the resulting OMVs. In this review, our current knowledge about OMVs will be presented. Different methods to induce OMVs will be reviewed and their advantages and disadvantages will be discussed. The effects of the induction and isolation methods used in several immunological studies on OMVs will be compared. Finally, the challenges for OMV-based vaccine development will be examined and one example of a successful OMV-based vaccine will be presented.

Antiviral Activity of Chicken Cathelicidin B1 Against Influenza A Virus.

Cathelicidins (CATHs) are host defense peptides (HDPs) that play an important role in the innate immune response against infections. Although multiple functions of cathelicidins have been described, including direct antimicrobial activity and several immunomodulatory effects on the host, relatively little is known about their antiviral activity. Therefore, in vitro antiviral activity of chicken cathelicidins and the underlying mechanism was investigated in this study against different influenza A virus (IAV) strains. Our results show that chicken CATH-B1 has broad anti-IAV activity compared to other cathelicidins (CATH-1, -2, -3, LL-37, PMAP-23, and K9CATH) with an inhibition of viral infection up to 80% against three tested IAV strains (H1N1, H3N1, and H5N1). In agreement herewith, CATH-B1 affected virus-induced inflammatory cytokines expression (IFN-ß, IL-1ß, IL-6, and IL-8). Incubation of cells with CATH-B1 prior to or after their inoculation with virus did not reduce viral infection indicating that direct interaction of virus with the peptide was required for CATH-B1's antiviral activity. Experiments using combined size exclusion and affinity-based separation of virus and peptide also indicated that CATH-B1 bound to viral particles. In addition, using electron microscopy, no morphological change of the virus itself was seen upon incubation with CATH-B1 but large aggregates of CATH-B1 and viral particles were observed, indicating that aggregation might be the mechanism of action reducing IAV infectivity. Neuraminidase (NA) activity assays using monovalent or multivalent substrates, indicated that CATH-B1 did not affect NA activity per se, but negatively affected the ability of virus particles to interact with multivalent receptors, presumably by interfering with hemagglutinin activity. In conclusion, our results show CATH-B1 has good antiviral activity against IAV by binding to the viral particle and thereby blocking viral entry.

Studies on citrullinated LL-37: detection in human airways, antibacterial effects and biophysical properties.

Arginine residues of the antimicrobial peptide LL-37 can be citrullinated by peptidyl arginine deiminases, which reduce the positive charge of the peptide. Notably, citrullinated LL-37 has not yet been detected in human samples. In addition, functional and biophysical properties of citrullinated LL-37 are not fully explored. The aim of this study was to detect citrullinated LL-37 in human bronchoalveolar lavage (BAL) fluid and to determine antibacterial and biophysical properties of citrullinated LL-37. BAL fluid was obtained from healthy human volunteers after intra-bronchial exposure to lipopolysaccharide. Synthetic peptides were used for bacterial killing assays, transmission electron microscopy, isothermal titration calorimetry, mass-spectrometry and circular dichroism. Using targeted proteomics, we were able to detect both native and citrullinated LL-37 in BAL fluid. The citrullinated peptide did not kill Escherichia coli nor lysed human red blood cells. Both peptides had similar α-helical secondary structures but citrullinated LL-37 was more stable at higher temperatures, as shown by circular dichroism. In conclusion, citrullinated LL-37 is present in the human airways and citrullination impaired bacterial killing, indicating that a net positive charge is important for antibacterial and membrane lysing effects. It is possible that citrullination serves as a homeostatic regulator of AMP-function by alteration of key functions.