Retinal Pigment Epithelial Cells Express Antimicrobial Peptide Lysozyme - A Novel Mechanism of Innate Immune Defense of the Blood-Retina Barrier.

Purpose: For this study we aimed to understand if retinal pigment epithelial (RPE) cells express antimicrobial peptide lysozyme as a mechanism to protect the neuroretina from blood-borne pathogens. Methods: The expression of lysozyme in human and mouse RPE cells was examined by RT-PCR or immune (cyto)histochemistry in cell cultures or retinal sections. RPE cultures were treated with different concentrations of Pam3CSK4, lipopolysaccharides (LPS), staphylococcus aureus-derived peptidoglycan (PGN-SA), Poly(I:C), and Poly(dA:dT). The mRNA expression of lysozyme was examined by qPCR and protein expression by ELISA. Poly(I:C) was injected into the subretinal space of C57BL/6J mice and eyes were collected 24 hours later and processed for the evaluation of lysozyme expression by confocal microscopy. Bactericidal activity was measured in ARPE19 cells following LYZ gene deletion using Crispr/Cas9 technology. Results: The mRNA and protein of lysozyme were detected in mouse and human RPE cells under normal conditions, although the expression levels were lower than mouse microglia BV2 or human monocytes THP-1 cells, respectively. Immunohistochemistry showed punctate lysozyme expression inside RPE cells. Lysozyme was detected by ELISA in normal RPE lysates, and in live bacteria-treated RPE supernatants. Treatment of RPE cells with Pam3CSK4, LPS, PGN-SA, and Poly(I:C) enhanced lysozyme expression. CRISPR/Cas9 deletion of lysozyme impaired bactericidal activity of ARPE19 cells and reduced their response to LPS and Poly(I:C) stimulation. Conclusions: RPE cells constitutively express antimicrobial peptide lysozyme and the expression is modulated by pathogenic challenges. RPE cells may protect the neuroretina from blood-borne pathogens by producing antimicrobial peptides, such as lysozyme.

The Microbial Lipopeptide Paenibacterin Disrupts Desiccation Resistance in Salmonella enterica Serovars Tennessee and Eimsbuettel.

Salmonella enterica is increasingly linked to disease outbreaks associated with consumption of low-water-activity (low-aw) foods. Persistence of the pathogen in these foods was attributed to its ability to implement desiccation resistance mechanisms. Published knowledge about methods that disrupt desiccation resistance in S. enterica is lacking. We hypothesize that strong membrane-active compounds disrupt the desiccation resistance that S. enterica may acquire in low-aw foods or environments. The newly discovered antimicrobial lipopeptide paenibacterin was the membrane-active agent investigated in this study. Strains of S. enterica serovars Tennessee and Eimsbuettel, with a history of association with low-moisture foods, were investigated. The viability of these strains did not decrease significantly during dehydration and subsequent storage in the dehydrated state. Considering that the paenibacterin MIC against S. enterica strains was 8 µg/ml, concentrations of 4 to 16 µg/ml paenibacterin were tested. Within this range, desiccation-adapted S. Eimsbuettel was much more tolerant to the antimicrobial agent than the desiccation-adapted S. Tennessee. Pretreatment with 8 µg/ml paenibacterin increased inactivation of S. enterica during desiccation. The use of paenibacterin at 16 µg/ml or higher concentrations resulted in leakage of intracellular potassium ions from desiccation-adapted cells. Paenibacterin significantly decreased the biosynthesis of the intracellular osmoprotectant solute, trehalose, in a concentration-dependent manner. Treatment with 64 µg/ml paenibacterin increased the permeability of the cytoplasmic membranes of desiccation-adapted cells. Transcription of the desiccation-related genes proV, STM1494, kdpA, and otsB in response to paenibacterin treatment was investigated using reverse transcription-quantitative PCR. Transcription of some of these genes was downregulated in a concentration- and strain-dependent manner.IMPORTANCESalmonella enterica adapts effectively and persists for a long time in low-aw foods or environments through resistance mechanisms to desiccation stress. Desiccation-resistant cells compromise food safety and constitute a serious health hazard. Strategies to combat desiccation resistance in S. enterica are needed to sensitize the pathogen to lethal processes used in food preservation. The study proved that the membrane-active lipopeptide paenibacterin disrupts the resistance in desiccation-adapted S. enterica, as measured by phenotypic, biochemical, and genetic analyses. This study highlighted the role of the lipopeptide paenibacterin in disrupting mechanisms employed by S. enterica to resist desiccation. This knowledge may lead to the design of novel control measures to improve the safety of low-aw foods.

Are Sphingolipids and Serine Dipeptide Lipids Underestimated Virulence Factors of Porphyromonas gingivalis?

The keystone periodontal pathogen Porphyromonas gingivalis produces phosphorylated dihydroceramide lipids (sphingolipids) such as phosphoethanolamine dihydroceramide (PE DHC) and phosphoglycerol dihydroceramide (PG DHC) lipids. Phosphorylated DHCs (PDHCs) from P. gingivalis can affect a number of mammalian cellular functions, such as potentiation of prostaglandin secretion from gingival fibroblasts, promotion of RANKL-induced osteoclastogenesis, promotion of apoptosis, and enhancement of autoimmunity. In P. gingivalis, these lipids affect anchoring of surface polysaccharides, resistance to oxidative stress, and presentation of surface polysaccharides (anionic polysaccharides and K-antigen capsule). In addition to phosphorylated dihydroceramide lipids, serine dipeptide lipids of P. gingivalis are implicated in alveolar bone loss in chronic periodontitis through interference with osteoblast differentiation and function and promotion of osteoclast activity. As a prerequisite for designation as bacterial virulence factors, bacterial sphingolipids and serine dipeptide lipids are recovered in gingival/periodontal tissues, tooth calculus, human blood, vascular tissues, and brain. In addition to P. gingivalis, other bacteria of the genera Bacteroides, Parabacteroides, Porphyromonas, Tannerella, and Prevotella produce sphingolipids and serine dipeptide lipids. The contribution of PDHCs and serine dipeptide lipids to the pathogenesis of periodontal and extraoral diseases may be an underappreciated area in microbe-host interaction and should be more intensively investigated.

Insights into the molecular basis of biocontrol of Brassica pathogens by Bacillus amyloliquefaciens UCMB5113 lipopeptides.

Background and Aims: Certain micro-organisms can improve plant protection against pathogens. The protective effect may be direct, e.g. due to antibiotic compounds, or indirect, by priming of plant defence as induced systemic resistance (ISR). The plant growth-promoting rhizobacterium Bacillus amyloliquefaciens UCMB5113 shows potential for disease management of oilseed rape. To investigate the mode of action of this protection, especially in relation to jasmonic acid-dependent ISR, Bacillus UCMB5113 was tested with Arabidopsis thaliana mutants and several important fungal pathogens of Brassica species. Methods: Secreted lipopeptide fractions from Bacillus UCMB5113, together with synthetic peptide mimics, were evaluated for their effects on fungal phytopathogens and A. thaliana . The structures of secreted lipopeptides were analysed using mass spectrometry. Plant mutants and reporter lines were used to identify signalling steps involved in disease suppression by lipopeptides. Key Results: In plate tests Bacillus UCMB5113 and lipopeptide extracts suppressed growth of several fungal pathogens infecting Brassica plants. Separation of secreted lipopeptides using reversed-phase high-performance liquid chromatography revealed several fractions that inhibited fungal growth. Analysis by mass spectrometry identified the most potent compounds as novel linear forms of antifungal fengycins, with synthetic peptide mimics confirming the biological activity. Application of the lipopeptide extracts on Arabidopsis roots provided systemic protection against Alternaria brassicicola on leaves. Arabidopsis signalling mutants and PDF1.2 and VSP2 promoter-driven GUS lines indicated that the lipopeptide fraction involved jasmonic-acid-dependent host responses for suppression of fungal growth indicative of ISR. Conclusions: The ability of Bacillus UCMB5113 to counteract pathogens using both antagonistic lipopeptides and through ISR provides a promising tool for sustainable crop production.

Induced systemic resistance responses in perennial ryegrass against Magnaporthe oryzae elicited by semi-purified surfactin lipopeptides and live cells of Bacillus amyloliquefaciens.

The suppressive ability of several strains of cyclic lipopeptide-producing Bacillus rhizobacteria to grey leaf spot disease caused by Magnaporthe oryzae has been documented previously; however, the underlying mechanism(s) involved in the induced systemic resistance (ISR) activity in perennial ryegrass (Lolium perenne L.) remains unknown. Root-drench application of solid-phase extraction (SPE)-enriched surfactin and live cells of mutant Bacillus amyloliquefaciens strain FZB42-AK3 (produces surfactin, but not bacillomycin D and fengycin) significantly reduced disease incidence and severity on perennial ryegrass. The application of the treatments revealed a pronounced multilayered ISR defence response activation via timely and enhanced accumulation of hydrogen peroxide (H2O2), elevated cell wall/apoplastic peroxidase activity, and deposition of callose and phenolic/polyphenolic compounds underneath the fungal appressoria in naïve leaves, which was significantly more intense in treated plants than in mock-treated controls. Moreover, a hypersensitive response (HR)-type reaction and enhanced expression of LpPrx (Prx, peroxidase), LpOXO4 (OXO, oxalate oxidase), LpPAL (PAL, phenylalanine ammonia lyase), LpLOXa (LOX, lipoxygenase), LpTHb (putative defensin) and LpDEFa (DEFa, putative defensin) in perennial ryegrass were associated with SPE-enriched surfactin and live AK3 cell treatments, acting as a second layer of defence when pre-invasive defence responses failed. The results indicate that ISR activity following surfactin perception may sensitize H2O2 -mediated defence responses, thereby providing perennial ryegrass with enhanced protection against M. oryzae.

Macrophage-activating lipopeptide-2 requires Mal and PI3K for efficient induction of heme oxygenase-1.

AIMS: This study is to investigate the mechanisms by which macrophage-activating lipopeptide-2 (MALP-2) induces heme oxygenase (HO)-1, a cytoprotective enzyme that catalyzes the degradation of heme, in human monocytes. METHODS: Human monocytic THP-1 cells were cultured for transient transfection with plasmids and stimulation with MALP-2 for indicative time intervals. After incubation with MALP-2, cells were collected and disrupted, before being tested for promoter activity using luciferase assay. For analysis of proteins, immunoreactive bands were detected using an enhanced chemiluminescence Western blotting system, and the band intensity was measured by densitometryic analysis. For the detection of co-immunoprecipitation, SDS-PAGE was performed and the membranes were probed using respective antibodies. To investigate the cellular localization of NF-E2-related factor 2 (Nrf2), cells underwent immunofluorescence staining and confocal microscopy, and were analyzed using electrophoretic mobility shift assay. RESULTS: MALP-2-induced HO-1 expression and promoter activity were abrogated by transfection with dominant negative (DN) plasmids of TLR2 and TLR6, or their neutralizing antibodies. However, inhibition of MyD88 or transfection with the DN-MyD88 was insufficient to attenuate HO-1 expression. In contrast, mutation or silencing of MyD88 adapter-like (Mal) by DN-Mal or siRNA almost completely blocked HO-1 induction. Btk, c-Src and PI3K were also involved in MALP-2-induced HO-1 expression, as revealed by specific inhibitors LFM-A13, PP1 and LY294002, or by transfection with siRNA of c-Src. MALP-2-induced activation of PI3K was attenuated by transfection with DN mutant of Mal, and by pretreatment with LFM-A13 or PP1. Furthermore, MALP-2 stimulated the translocation of Nrf2 from the cytosol to the nucleus and Nrf2 binding to the ARE site in the HO-1 promoter, which could also be inhibited by pretreatment with a PI3K inhibitor, LY294002. CONCLUSIONS: These results indicated that MALP-2 required TLR2/6, Btk, Mal and c-Src to activate PI3K, which in turn initiated the activation of Nrf2 for efficient HO-1 induction.

Inhibition of autoimmune diabetes by TLR2 tolerance.

We have reported that apoptotic ß cells undergoing secondary necrosis, called "late apoptotic (LA) ß cells," stimulated APCs and induced diabetogenic T cell priming through TLR2, which might be one of the initial events in autoimmune diabetes. Indeed, diabetogenic T cell priming and the development of autoimmune diabetes were significantly inhibited in TLR2-null NOD mice, suggesting the possibility that TLR2 blockade could be used to inhibit autoimmune diabetes. Because prolonged TLR stimulation can induce TLR tolerance, we investigated whether repeated TLR2 administration affects responses to LA ß cells and inhibits autoimmune diabetes in NOD mice by inducing TLR2 tolerance. Treatment of primary peritoneal macrophages with a TLR2 agonist, Pam3CSK(4), suppressed cytokine release in response to LA insulinoma cells or further TLR2 stimulation. The expression of signal transducer IRAK-1 and -4 proteins was decreased by repeated TLR2 stimulation, whereas expression of IRAK-M, an inhibitory signal transducer, was enhanced. Chronic Pam3CSK(4) administration inhibited the development of diabetes in NOD mice. Diabetogenic T cell priming by dendritic cells and upregulation of costimulatory molecules on dendritic cells by in vitro stimulation were attenuated by Pam3CSK(4) administration in vivo. Pam3CSK(4) inhibited diabetes after adoptive transfer of diabetogenic T cells or recurrence of diabetes after islet transplantation by pre-existing sensitized T cells. These results showed that TLR2 tolerance can be achieved by prolonged treatment with TLR2 agonists, which could inhibit priming of naive T cells, as well as the activity of sensitized T cells. TLR2 modulation could be used as a novel therapeutic modality against autoimmune diabetes.

TLR2 ligand induces protection against cerebral ischemia/reperfusion injury via activation of phosphoinositide 3-kinase/Akt signaling.

This study examined the effect of TLR2 activation by its specific ligand, Pam3CSK4, on cerebral ischemia/reperfusion (I/R) injury. Mice (n = 8/group) were treated with Pam3CSK4 1 h before cerebral ischemia (60 min), followed by reperfusion (24 h). Pam3CSK4 was also given to the mice (n = 8) 30 min after ischemia. Infarct size was determined by triphenyltetrazolium chloride staining. The morphology of neurons in brain sections was examined by Nissl staining. Pam3CSK4 administration significantly reduced infarct size by 55.9% (p < 0.01) compared with untreated I/R mice. Therapeutic treatment with Pam3CSK4 also significantly reduced infarct size by 55.8%. Morphologic examination showed that there was less neuronal damage in the hippocampus of Pam3CSK4-treated mice compared with untreated cerebral I/R mice. Pam3CSK4 treatment increased the levels of Hsp27, Hsp70, and Bcl2, and decreased Bax levels and NF-κB-binding activity in the brain tissues. Administration of Pam3CSK4 significantly increased the levels of phospho-Akt/Akt and phospho-GSK-3ß/GSK-3ß compared with untreated I/R mice. More significantly, either TLR2 deficiency or PI3K inhibition with LY29004 abolished the protection by Pam3CSK4. These data demonstrate that activation of TLR2 by its ligand prevents focal cerebral ischemic damage through a TLR2/PI3K/Akt-dependent mechanism. Of greater significance, these data indicate that therapy with a TLR2-specific agonist during cerebral ischemia is effective in reducing injury.

Peripheral and central cyclooxygenase (COX) products may contribute to the manifestation of brain-controlled sickness responses during localized inflammation induced by macrophage-activating lipopeptide-2 (MALP-2).

We investigated whether an inflammation-dependent activation of the brain occurs in response to systemic intraperitoneal (i.p.) or local injections of macrophage-activating lipopeptide-2 (MALP-2) into a subcutaneous (s.c.) air pouch, and whether local (peripheral) or central cyclooxygenase (COX)-2-dependent formations of prostaglandin E(2) (PGE(2)) are involved in MALP-2-induced illness responses. Body temperature, activity, food and water intake were measured telemetrically. Local (s.c.) and circulating levels of PGE(2) were measured by an ELISA. Inflammatory activation of the brain in response to MALP-2 was determined by immunohistochemical detection of the transcription factors NFkappaB and STAT3 in cell nuclei as well as the appearance of COX-2 at the same sites. S.c. treatment with the preferential COX-2 inhibitor meloxicam attenuated, but not abolished fever induced by local injections of MALP-2 into the pouch. Local MALP-2-induced formation of PGE(2) was blunted by treatment with meloxicam. In the brain, i.p. stimulation with MALP-2-induced nuclear STAT3- and NFkappaB-translocation in the vasculature and the sensory circumventricular organs, which was accompanied by an increase in COX-2 immunoreactivity (IR) in endothelial cells. Local MALP-2-treatment induced a moderate STAT3 activation and a small but significant increase in COX-2 IR while no NFkappaB-activation could be observed in the brains of these animals. We demonstrated that the activation of the brain STAT3 (NFkappaB)-COX-2 singling cascade seems to be involved in the manifestation of brain-controlled illness symptoms induced by systemic and local inflammatory stimulation with MALP-2. The present data further suggest a contribution of peripherally produced PGE(2) to MALP-2-induced activation of brain sites implicated in fever.

Effect of the hydrophobicity to net positive charge ratio on antibacterial and anti-endotoxin activities of structurally similar antimicrobial peptides.

The interaction between host-defense antimicrobial peptides (AMPs) and the bacterial lipopolysaccharide (LPS) governs both the susceptibility of the bacteria to the peptide and the ability of the peptide to inhibit LPS activation of immune cells. Both functions depend on the biophysical properties of the peptides. However, the sequence and structural diversity of AMPs makes it difficult to determine common denominators required for antimicrobial and LPS neutralizing activities. Toward this end, we synthesized and investigated a series of nine 12-amino acid peptides and their fatty acid-conjugated analogues composed of both D- and L-isomers of Leu and Lys at various ratios. The positions of the D-amino acids were preserved. These peptides differ in their net positive charge and hydrophobicity. However, their overall structure in the membrane is similar, as determined by Fourier transform infrared spectroscopy. The peptides and their analogues were functionally tested for their antibacterial and hemolytic activity, their ability to permeate LPS vesicles, their ability to neutralize LPS activation of macrophages, and their effect on LPS morphology, determined by negative staining electron microscopy. The data revealed that increasing the ratio between hydrophobicity and the net positive charge increases both antimicrobial and LPS neutralization activities, but with different modes of contributions. Whereas antimicrobial activity increases linearly with the increase in the peptides' hydrophobicity, peptides with different hydrophobicities are endowed with similar LPS neutralizing activities. Besides adding important information regarding AMP parameters involved in antimicrobial and anti-LPS activities, this study suggests the use of such diastereomers as potential templates for the development of simple molecules that conduct both types of functions.

CD47 and TLR-2 cross-talk regulates neutrophil transmigration.

Neutrophil (PMN) infiltration into tissues is a hallmark of acute inflammation and is crucial for the rapid removal of microbial pathogens. Previous studies have shown that PMN transmigration is regulated by the cell surface protein CD47. However this phenomenon in the context of microbial invasion and subsequent TLR signaling is poorly understood. In this study, we assessed the role of TLR2 and CD47 costimulation in regulating PMN transmigration. Human PMN transmigration across acellular collagen-coated filters toward the bacterial chemoattractant fMLP was more significantly inhibited by MALP-2 (TLR2/6 agonist) than Pam(3)CSK(4) (TLR2/1 agonist). Subsequent experiments demonstrated that treatment with MALP-2 or anti-human CD47 mAbs delayed human PMN transfilter migration, while combined treatment led to further delayed inhibition. Interestingly, stimulation of PMNs with MALP-2 resulted in an increase in surface expression of CD11b, but not CD47. In experiments addressing the role of TLR agonists in regulating CD47-mediated PMN transmigration, incubation with MALP-2 or with anti-mouse CD47 mAbs did not inhibit transfilter migration of TLR2(-/-) or MyD88(-/-)-deficient murine bone marrow-derived PMNs. Similarly, inhibition of MyD88 homodimerization reversed the attenuation of human PMN transmigration induced by MALP-2 or anti-human CD47 mAbs. Separate experiments demonstrated that CD47(-/-) murine bone marrow-derived PMNs exhibited 4-fold decreased sensitivity toward MALP-2. Collectively, these findings suggest that activation of CD47 signaling enhances PMN sensitivity toward TLR2 activation which, in turn, signals their arrival at a site of invasion and may facilitate antimicrobial function.

Microbial interactions: bacteria talk to (some of) their neighbors.

A recent study reports that Bacillus subtilis biofilm formation depends upon paracrine signaling where the signal-producing and target-responsive cells are different.

A novel cell wall lipopeptide is important for biofilm formation and pathogenicity of Mycobacterium avium subspecies paratuberculosis.

Biofilm formation by pathogenic bacteria plays a key role in their pathogenesis. Previously, the pstA gene was shown to be involved in the virulence of Mycobacterium avium subspecies paratuberculosis (M. ap), the causative agent of Johne's disease in cattle and a potential risk factor for Crohn's disease. Scanning electron microscopy and colonization levels of the M. ap mutant indicated that the pstA gene significantly contributes to the ability of M. ap to form biofilms. Digital measurements taken during electron microscopy identified a unique morphology for the DeltapstA mutant, which consisted of significantly shorter bacilli than the wild type. Analysis of the lipid profiles of the mycobacterial strains identified a novel lipopeptide that was present in the cell wall extracts of wild-type M. ap, but missing from the DeltapstA mutant. Interestingly, the calf infection model suggested that pstA contributes to intestinal invasion of M. ap. Furthermore, immunoblot analysis of peptides encoded by pstA identified a specific and significant level of immunogenicity. Taken together, our analysis revealed a novel cell wall component that could contribute to biofilm formation and to the virulence and immunogenicity of M. ap. Molecular tools to better control M. ap infections could be developed utilizing the presented findings.

Genome analysis of Bacillus amyloliquefaciens FZB42 reveals its potential for biocontrol of plant pathogens.

The genome of plant-associated Bacillus amyloliquefaciens FZB42 harbors an array of giant gene clusters involved in synthesis of lipopeptides and polyketides with antifungal, antibacterial and nematocidal activity. Five gene clusters, srf, bmy, fen, nrs, dhb, covering altogether 137 kb, were shown to direct synthesis of the cyclic lipopeptides surfactin, bacillomycin, fengycin, an unknown peptide, and the iron-siderophore bacillibactin. In addition, one gene cluster encoding enzymes involved in synthesis and export of the antibacterial dipeptide bacilysin is also functional in FZB42. Three gene clusters, mln, bae, and dfn, with a total size of 199 kb were shown to direct synthesis of the antibacterial acting polyketides macrolactin, bacillaene, and difficidin. In total, FZB42 dedicates about 340 kb, corresponding to 8.5% of its total genetic capacity, to synthesis of secondary metabolites. On the contrary, genes involved in ribosome-dependent synthesis of lantibiotics and other peptides are scarce. Apart from two incomplete gene clusters directing immunity against mersacidin and subtilin, only one peptide-like compound has been detected in the culture fluid that inhibits the growth of B. subtilis lacking the alternative sigma factor W.