Physiological consequences of inactivation of lgmB and lpxL1, two genes involved in lipid A synthesis in Bordetella bronchiseptica.

To develop a Bordetella bronchiseptica vaccine with reduced endotoxicity, we previously inactivated lpxL1, the gene encoding the enzyme that incorporates a secondary 2-hydroxy-laurate in lipid A. The mutant showed a myriad of phenotypes. Structural analysis showed the expected loss of the acyl chain but also of glucosamine (GlcN) substituents, which decorate the phosphates in lipid A. To determine which structural change causes the various phenotypes, we inactivated here lgmB, which encodes the GlcN transferase, and lpxL1 in an isogenic background and compared the phenotypes. Like the lpxL1 mutation, the lgmB mutation resulted in reduced potency to activate human TLR4 and to infect macrophages and in increased susceptibility to polymyxin B. These phenotypes are therefore related to the loss of GlcN decorations. The lpxL1 mutation had a stronger effect on hTLR4 activation and additionally resulted in reduced murine TLR4 activation, surface hydrophobicity, and biofilm formation, and in a fortified outer membrane as evidenced by increased resistance to several antimicrobials. These phenotypes, therefore, appear to be related to the loss of the acyl chain. Moreover, we determined the virulence of the mutants in the Galleria mellonella infection model and observed reduced virulence of the lpxL1 mutant but not of the lgmB mutant.

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.

The cathelicidin CATH-2 efficiently neutralizes LPS- and E. coli-induced activation of porcine bone marrow derived macrophages.

Infectious diseases in pigs cause monetary loss to farmers and pose a zoonotic risk. Therefore, it is important to obtain more porcine specific immunological knowledge as a measure to protect against infectious diseases, for example by exploring immunomodulators that are usable as vaccine adjuvants. Cathelicidins are a class of host defence peptides (HDPs) able to directly kill microbes as well as exert a diverse range of effects on the immune system. The peptides have shown promise as immunomodulatory peptides in many applications, including vaccines. However, it is currently unknown what the precise effect of these peptides is on porcine immune cells and whether peptides of other species might also have a strong immunomodulatory effect on porcine macrophages. Mononuclear bone marrow cells of pigs, aged 5-6 months, were cultured into M1 or M2 macrophages and stimulated with LPS or whole bacteria in the presence of host defence peptides (HDPs). CATH-2 and LL-37 strongly inhibited LPS-induced activation of M1 macrophages, the inhibition of LPS-induced activation of M2 macrophages by HDPs was milder, showing that the peptides have selective effects on different cell types. Upon stimulation with whole bacteria, only CATH-2 could effectively inhibit macrophage activation, showing the potent anti-inflammatory potential of this peptide. These results show that porcine peptides are not necessarily the most active in a porcine system, and that CATH-2 is effective in a porcine system as an anti-inflammatory immune modulator, which can be used, for example, in inactivated pathogen vaccines.

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.

Reduction of endotoxicity in Bordetella bronchiseptica by lipid A engineering: Characterization of lpxL1 and pagP mutants.

Whole-cell vaccines against Gram-negative bacteria commonly display high reactogenicity caused by the endotoxic activity of lipopolysaccharide (LPS), one of the major components of the bacterial outer membrane. Underacylation of the lipid A moiety of LPS has been related with reduced endotoxicity in several Gram-negative species. Here, we evaluated whether the inactivation of two genes encoding lipid A acylases of Bordetella bronchiseptica, i.e. pagP and lpxL1, could be used for the development of less reactogenic vaccines against this pathogen for livestock and companion animals. Inactivation of pagP resulted in the loss of the secondary palmitate chain at position 3' of lipid A, but hardly affected the potency of the LPS to activate the Toll-like receptor 4 (TLR4). Inactivation of lpxL1 resulted in the loss of the secondary 2-hydroxy laurate group present at position 2 of lipid A and, unexpectedly, in the additional loss of the glucosamines that decorate the phosphate groups at positions 1 and 4' and in an increase in LPS molecules carrying O-antigen. The resulting LPS showed greatly reduced potency to activate TLR4 in HEK-Blue reporter cells expressing human or mouse TLR4 as well as in porcine macrophages. Characterization of the lpxL1 mutant revealed many pleiotropic phenotypes, including increased resistance to SDS and rifampicin, increased susceptibility to cationic antimicrobial peptides, decreased auto-aggregation and biofilm formation, and a tendency to decreased infectivity of macrophages, which are all related to the altered LPS structure. We suggest that the lpxL1 mutant will be useful for the generation of safer vaccines.

The immunomodulatory effect of cathelicidin-B1 on chicken macrophages.

Cathelicidins (CATHs) play an important role in the innate immune response against microbial infections. Among the four chicken cathelicidins, CATH-B1 is studied the least. In this study, the effect of CATH-B1 on the macrophage response towards avian pathogenic E. coli (APEC) and bacterial ligands was investigated. Our results show that APEC induced CATH-B1 gene expression in both a chicken macrophage cell line (HD11 cells) and primary macrophages, while expression of the other three CATHs was virtually unaffected. While the antimicrobial activity of CATH-B1 is very low under cell culture conditions, it enhanced bacterial phagocytosis by macrophages. Interestingly, CATH-B1 downregulated APEC-induced gene expression of pro-inflammatory cytokines (IFN-ß, IL-1ß, IL-6 and IL-8) in primary macrophages. In addition, CATH-B1 pre-incubated macrophages showed a significantly higher gene expression of IL-10 after APEC challenge, indicating an overall anti-inflammatory profile for CATH-B1. Using isothermal titration calorimetry (ITC), CATH-B1 was shown to bind LPS. This suggests that CATH-B1 reduces toll like receptor (TLR) 4 dependent activation by APEC which may partly explain the decreased production of pro-inflammatory cytokines by macrophages. On the contrary, direct binding of CATH-B1 to ODN-2006 enhanced the TLR21 dependent activation of macrophages as measured by nitric oxide production. In conclusion, our results show for the first time that CATH-B1 has several immunomodulatory activities and thereby could be an important factor in the chicken immune response.

Cathelicidins Modulate TLR-Activation and Inflammation.

Cathelicidins are short cationic peptides that are part of the innate immune system. At first, these peptides were studied mostly for their direct antimicrobial killing capacity, but nowadays they are more and more appreciated for their immunomodulatory functions. In this review, we will provide a comprehensive overview of the various effects cathelicidins have on the detection of damage- and microbe-associated molecular patterns, with a special focus on their effects on Toll-like receptor (TLR) activation. We review the available literature based on TLR ligand types, which can roughly be divided into lipidic ligands, such as LPS and lipoproteins, and nucleic-acid ligands, such as RNA and DNA. For both ligand types, we describe how direct cathelicidin-ligand interactions influence TLR activation, by for instance altering ligand stability, cellular uptake and receptor interaction. In addition, we will review the more indirect mechanisms by which cathelicidins affect downstream TLR-signaling. To place all this information in a broader context, we discuss how these cathelicidin-mediated effects can have an impact on how the host responds to infectious organisms as well as how these effects play a role in the exacerbation of inflammation in auto-immune diseases. Finally, we discuss how these immunomodulatory activities can be exploited in vaccine development and cancer therapies.

Chicken CATH-2 Increases Antigen Presentation Markers on Chicken Monocytes and Macrophages.

BACKGROUND: Cathelicidins are a family of Host Defense Peptides (HDPs), that play an important role in the innate immune response. They exert both broad-spectrum antimicrobial activity against pathogens, and strong immunomodulatory functions that affect the response of innate and adaptive immune cells. OBJECTIVE: The aim of this study was to investigate immunomodulation by the chicken cathelicidin CATH-2 and compare its activities to those of the human cathelicidin LL-37. METHODS: Chicken macrophages and chicken monocytes were incubated with cathelicidins. Activation of immune cells was determined by measuring surface markers Mannose Receptor Ctype 1 (MRC1) and MHC-II. Cytokine production was measured by qPCR and nitric oxide production was determined using the Griess assay. Finally, the effect of cathelicidins on phagocytosis was measured using carboxylate-modified polystyrene latex beads. RESULTS: CATH-2 and its all-D enantiomer D-CATH-2 increased MRC1 and MHC-II expression, markers for antigen presentation, on primary chicken monocytes, whereas LL-37 did not. D-CATH- 2 also increased the MRC1 and MHC-II expression if a chicken macrophage cell line (HD11 cells) was used. In addition, LPS-induced NO production by HD11 cells was inhibited by CATH-2 and D-CATH-2. CONCLUSION: These results are a clear indication that CATH-2 (and D-CATH-2) affect the activation state of monocytes and macrophages, which leads to optimization of the innate immune response and enhancement of the adaptive immune response.

Cathelicidins: Immunomodulatory Antimicrobials.

Cathelicidins are host defense peptides with antimicrobial and immunomodulatory functions. These effector molecules of the innate immune system of many vertebrates are diverse in their amino acid sequence but share physicochemical characteristics like positive charge and amphipathicity. Besides being antimicrobial, cathelicidins have a wide variety in immunomodulatory functions, both boosting and inhibiting inflammation, directing chemotaxis, and effecting cell differentiation, primarily towards type 1 immune responses. In this review, we will examine the biology and various functions of cathelicidins, focusing on putting in vitro results in the context of in vivo situations. The pro-inflammatory and anti-inflammatory functions are highlighted, as well both direct and indirect effects on chemotaxis and cell differentiation. Additionally, we will discuss the potential and limitations of using cathelicidins as immunomodulatory or antimicrobial drugs.

Multidrug-Resistant Enterococcal Infections: New Compounds, Novel Antimicrobial Therapies?

Over the past two decades infections due to antibiotic-resistant bacteria have escalated world-wide, affecting patient morbidity, mortality, and health care costs. Among these bacteria, Enterococcus faecium and Enterococcus faecalis represent opportunistic nosocomial pathogens that cause difficult-to-treat infections because of intrinsic and acquired resistance to a plethora of antibiotics. In recent years, a number of novel antimicrobial compound classes have been discovered and developed that target Gram-positive bacteria, including E. faecium and E. faecalis. These new antibacterial agents include teixobactin (targeting lipid II and lipid III), lipopeptides derived from nisin (targeting lipid II), dimeric vancomycin analogues (targeting lipid II), sortase transpeptidase inhibitors (targeting the sortase enzyme), alanine racemase inhibitors, lipoteichoic acid synthesis inhibitors (targeting LtaS), various oxazolidinones (targeting the bacterial ribosome), and tarocins (interfering with teichoic acid biosynthesis). The targets of these novel compounds and mode of action make them very promising for further antimicrobial drug development and future treatment of Gram-positive bacterial infections. Here we review current knowledge of the most favorable anti-enterococcal compounds along with their implicated modes of action and efficacy in animal models to project their possible future use in the clinical setting.

De novo identification of lipid II binding lipopeptides with antibacterial activity against vancomycin-resistant bacteria.

Creative strategies for identifying new antibiotics are essential to addressing the looming threat of a post-antibiotic era. We here report the use of a targeted peptide phage display screen as a means of generating novel antimicrobial lipopeptides. Specifically, a library of phage displayed bicyclic peptides was screened against a biomolecular target based on the bacterial cell wall precursor lipid II. In doing so we identified unique lipid II binding peptides that upon lipidation were found to be active against a range of Gram-positive bacteria including clinically relevant strains of vancomycin resistant bacteria. Optimization of the peptide sequence led to variants with enhanced antibacterial activity and reduced hemolytic activity. Biochemical experiments further confirm a lipid II mediated mode of action for these new-to-nature antibacterial lipopeptides.

Total Synthesis of Laspartomycin C and Characterization of Its Antibacterial Mechanism of Action.

Laspartomycin C is a lipopeptide antibiotic with activity against a range of Gram-positive bacteria including drug-resistant pathogens. We report the first total synthesis of laspartomycin C as well as a series of structural variants. Laspartomycin C was found to specifically bind undecaprenyl phosphate (C55-P) and inhibit formation of the bacterial cell wall precursor lipid II. While several clinically used antibiotics target the lipid II pathway, there are no approved drugs that act on its C55-P precursor.