Interactions of antimicrobial peptides with Leishmania and trypanosomes and their functional role in host parasitism

Antimicrobial peptides (AMPs) are multifunctional components of the innate systems of both insect and mammalian hosts of the pathogenic trypanosomatids Leishmania and Trypanosoma species. Structurally diverse AMPs from a wide range of organisms have in vitro activity against these parasites acting mainly to disrupt surface-membranes. In some cases AMPs also localize intracellularly to affect calcium levels, mitochondrial function and induce autophagy, necrosis and apoptosis. In this review we discuss the work done in the area of AMP interactions with trypanosomatid protozoa, propose potential targets of AMP activity at the cellular level and discuss how AMPs might influence parasite growth and differentiation in their hosts to determine the outcome of natural infection.     

Characterization of diverse antimicrobial peptides in skin secretions of Chungan torrent frog Amolops chunganensis

We have cloned, synthesized, and characterized 11 novel antimicrobial peptides from a skin derived cDNA library of the Chungan torrent frog, Amolops chunganensis. Seven of the 11 antimicrobial peptides were present in authentic A. chunganensis skin secretions. Sequence analysis indicated that the 11 peptides belonged to the temporin, esculentin-2, palustrin-2, brevinin-1, and brevinin-2 families. The peptides displayed potent antimicrobial activities against several strains of microorganisms. One peptide, brevinin-1CG5, demonstrated antimicrobial activity against all tested Gram-positive and Gram-negative bacteria and fungi, and showed high antimicrobial potency (MIC = 0.6 μM) against Gram-positive bacterium Rhodococcus rhodochrous. Some peptides also demonstrated weak hemolytic activity against human erythrocytes in vitro. Phylogenetic analysis based on the amino acid sequences of brevinin-1, brevinin-2, and esculentin-2 peptides from family Ranidae confirmed that the current taxonomic status of A. chunganensis is correct.     

Battle and balance at mucosal surfaces - The story of Shigella and antimicrobial peptides

Shigella is a major cause of morbidity and mortality for children in many developing countries. Emergence of antibiotic-resistance among Shigella demands the development of effective medicines. Antimicrobial peptides (AMPs) are expressed in phagocytes and at epithelial surfaces and are important effector molecules of innate immunity. We have found that pathogens are able to turn off the endogenous expression of AMPs, resulting in serious infections such as shigellosis. A therapeutic rationale to prevent microbial invasion would be to strengthen the epithelial line of defence through enhancing AMP expression. We have identified several inducers of AMP-production, including butyrate, phenylbutyrate and vitamin D, which have been investigated in animal models of shigellosis as well as in clinical trials. We believe that the conceptual framework presented here can be applied to additional clinical entities and that this novel approach can be an alternative or complement to traditional antibiotics in the future.     

Leishmania donovani: Thionins, plant antimicrobial peptides with leishmanicidal activity

The leishmanicidal activity of plant antibiotic peptides (PAPs) from the principal families, such wheat thionins, a barley lipid transfer protein and potato defensins and snakins were tested in vitro against Leishmania donovani. Only thionins and defensins were active against this human pathogen at a low micromolar range of concentrations. Thionins resulted as the most active peptides tested until now. They collapsed ionic and pH gradients across the parasite plasma membrane together with a rapid depletion of intracellular ATP without affecting mitochondrial potential. Hence the lethal effect of thionins was mostly associated to permeabilization of the plasma membrane leading to an immediate death of the parasite. The present work is the first evidence for leishmanicidal activity in plant peptides. Future prospects for their development as new antiparasite agents on human diseases are considered.     

Toxoplasma gondii-derived heat shock protein 70 stimulates the maturation of human monocyte-derived dendritic cells

We investigated the role of Toxoplasma gondii-derived heat shock protein 70 (TgHSP70) as a dendritic cell (DC) maturation-inducing molecule. TgHSP70 induced the maturation of human monocyte-derived dendritic cells as determined by increased levels of surface markers, namely, CD40, CD80, CD86, and HLA-DR. Moreover, TgHSP70 also reduced phagocytic activity and increased the allostimulatory capacity of DCs, suggesting the functional maturation of DCs by TgHSP70. Maturation of DCs by TgHSP70 also elicited a significant increase in IL-12 production in a polymyxin B-insensitive manner. TgHSP70 also stimulated extracellular signal-regulated kinase and p38 kinase pathways in DCs, and TgHSP70-induced IL-12 production was inhibited by SB203580 but not by PD98059, thus indicating the role of p38 kinase in the maturation of DCs by TgHSP70. This study demonstrates the role of TgHSP70 in the functional maturation of DCs and suggests TgHSP70 as a useful molecule for the development of a vaccine against T. gondii.     

Anti-Legionella activity of staphylococcal hemolytic peptides

A collection of various Staphylococci was screened for their anti-Legionella activity. Nine of the tested strains were found to secrete anti-Legionella compounds. The culture supernatants of the strains, described in the literature to produce hemolytic peptides, were successfully submitted to a two step purification process. All the purified compounds, except one, corresponded to previously described hemolytic peptides and were not known for their anti-Legionella activity. By comparison of the minimal inhibitory concentrations, minimal permeabilization concentrations, decrease in the number of cultivable bacteria, hemolytic activity and selectivity, the purified peptides could be separated in two groups. First group, with warnericin RK as a leader, corresponds to the more hemolytic and bactericidal peptides. The peptides of the second group, represented by the PSMα from Staphylococcus epidermidis, appeared bacteriostatic and poorly hemolytic.     

Leishmanicidal cycloartane-type triterpene glycosides from Astragalus oleifolius

Two new cycloartane-type glycosides oleifoliosides A (1) and B (2) were isolated from the lower stem parts of Astragalus oleifolius. Their structures were identified as 3-O-[beta-xylopyranosyl-(1 --> 2)-alpha-arabinopyranosyl]-6-O-beta-xylopyranosyl-3beta,6alpha,16beta,24(S),25-pentahydroxycycloartane and 3-O-[beta-xylopyranosyl-(1 --> 2)-alpha-arabinopyranosyl]-6-O-beta-glucopyranosyl-3beta,6alpha,16beta,24(S),25-pentahydroxycycloartane, respectively, by means of spectroscopic methods (IR, 1D and 2D NMR, ESI-MS). Three known cycloartane glycosides cyclocanthoside E (3), astragaloside II (4) and astragaloside IV (5) were also isolated and characterized. All five compounds were evaluated for in vitro trypanocidal, leishmanicidal and antiplasmodial activities as well as their cytotoxic potential on primary mammalian (L6) cells. Except for the compound 5, all compounds showed notable growth inhibitory activity against Leishmania donovani with IC50 values ranging from 13.2 to 21.3 microg/ml. Only weak activity against Trypanosoma brucei rhodesiense was observed with the known compounds astragaloside II (4, IC50 66.6 microg/ml) and cyclocanthoside E (3, IC50 85.2 microg/ml), while all compounds were inactive against Trypanosoma cruzi and Plasmodium falciparum. None of the compounds were toxic to mammalian cells (IC50's > 90 microg/ml). This is the first report of leishmanicidal and trypanocidal activity of cycloartane-type triterpene glycosides.     

Toxoplasma gondii: Impaired maturation and pro-inflammatory response of dendritic cells in MIF-deficient mice favors susceptibility to infection

Macrophage migration inhibitory factor (MIF) has been found to be involved in host resistance to several parasitic infections. To determine the mechanisms of MIF-dependent responses to Toxoplasma gondii, we investigated host resistance in MIF−/− mice (BALB/c background) during natural oral infection. We focused on the potential involvement of MIF in Dendritic Cell (DC) maturation and IL-12 production. Following oral T. gondii infection, wild type mice developed a strong IL-12 response with an adequate maturation of their draining mesenteric lymph node DC (MLNDC) population and were resistant to challenge with either 40 or 100 cysts (ME49 strain). In contrast, similarly infected MIF−/− mice mounted a weak IL-12 response, displayed immature MLNDCs in the early phases of infection and rapidly succumbed to both type of challenges. Lack of maturation and IL-12 production of DCs in response to T. gondii antigens was confirmed by in vitro studies, and these effects were reversed following treatment with recombinant MIF. These findings demonstrate that MIF-induced early DC maturation and IL-12 production mediate resistance to T. gondii infection.     

Immunomodulatory effect of resistin in human dendritic cells stimulated with lipoteichoic acid from Staphylococcus aureus

Resistin is an adipokine whose physiologic role in obesity, type II diabetes mellitus, and inflammatory diseases has been a subject of debate because while it is expressed in adipocytes and adipose tissue in mouse, it is expressed in leukocytes, such as macrophages, in human. In the present study, we attempt to define the effect of resistin on human dendritic cells (DCs) derived from CD14⁺ monocytes. When DCs were stimulated with lipoteichoic acid (LTA) and treated with various concentrations of resistin, antigen-uptake process and the endocytic capacity of DCs were decreased. It is intriguing that resistin attenuated cytokine production in LTA-primed DCs. Consequently, T cell activity was reduced when lymphocytes were mixed with Staphylococcus aureus-primed autologous DCs treated with resistin compared to S. aureus-primed DCs without resistin. Our results suggest that resistin interferes with the efficacy of immune responses activated by Gram-positive bacterial infection in human DCs.     

Identification and characterization of antimicrobial peptides from skin of Amolops ricketti (Anura: Ranidae)

As one of large amphibian group, there are a total of 45 species of Amolops in the world. However, the antimicrobial peptides (AMPs) existing in this genus has not been extensively studied. In this study, cDNAs encoding five novel AMP precursors were cloned by screening the skin-derived cDNA library of Amolops ricketti, a frog species that exists in southern and western parts of China. Protein sequence analysis led to the identification of five deduced peptides, three belonging to the brevinin-1 family and two belonging to the brevinin-2 family of amphibian AMPs. Thus, they were named as brevinin-1RTa (FLPLLAGVVANFLPQIICKIARKC), brevinin-1RTb (FLGSLLGLVGKVVPTLFCKISKKC), brevinin-1RTc (FLGSLLGLVGKIVPTLICKISKKC), brevinin-2RTa (GLMSTLKDFGKTAAKEIAQSLLSTASCKLAKTC), and brevinin-2RTb (GILDTLKEFGKTAAKGIAQSLLSTASCKLAKTC), respectively. The purification of brevinin-1RTa, brevinin-1RTb, and brevinin-2RTb was carried out by RP-HPLC, and confirmed by the LC-MS/MS-based proteomics approach. All of the peptides displayed different antimicrobial potency against a variety of microorganisms. In addition, brevinin-2RTa and brevinin-2RTb were found to have relatively low hemolytic activity (>400μg/ml) against mammalian red blood cells in vitro, which could potentially be as candidates for developing novel anti-infection agents.     

Expression systems for heterologous production of antimicrobial peptides

Antimicrobial peptides (AMPs) consist of molecules that act on the defense systems of numerous organisms toward multiple pathogens such as bacteria, fungi, parasites and viruses. These compounds have become extremely significant due to the increasing resistance of microorganisms to common antibiotics. However, the low quantity of peptides obtained from direct purification is, to date, still a remarkable bottleneck for scientific and industrial research development. Therefore, this review describes the main heterologous systems currently used for AMP production, including bacteria, fungi and plants, and also the related strategies for reaching greater functional peptide production. The main difficulties of each system are also described in order to provide some directions for AMP production. In summary, data revised here indicate that large-scale production of AMPs can be obtained using biotechnological tools, and the products may be applied in the pharmaceutical industry as well as in agribusiness.     

S-thanatin in vitro prevents colistin resistance and improves its efficacy in an animal model of Pseudomonas aeruginosa sepsis

An experimental study was performed to evaluate the interaction between s-thanatin and colistin both in vitro and in vivo, using two Pseudomonas aeruginosa strains with different patterns of susceptibilities. We evaluated whether selecting for colistin-resistant P. aeruginosa could be prevented in vitro by combining colistin with s-thanatin. The strains were serially exposed in broth to twofold stepwise increasing concentrations of colistin alone or in combination with a fixed concentration [0.25× minimum inhibitory concentration (MIC)] of s-thanatin. We also performed an in vitro synergy study. For in vivo studies, a mouse model of Pseudomonas sepsis has been used. Main outcome measures were lethality and quantitative blood cultures. Exposure to colistin alone gradually selected for Pseudomonas strains with an increased MIC. In vitro studies, s-thanatin showed a positive interaction with colistin, and was able to prevent its resistance. In vivo studies, s-thanatin combined with colistin exhibited the highest efficacy on all main outcome measurements. These results highlight the potential usefulness of this combination and provide a future therapeutic alternative in severe Pseudomonas infections.     

Cestode antigens induce a tolerogenic-like phenotype and inhibit LPS inflammatory responses in human dendritic cells

Pathogens have developed strategies to modify Dendritic Cells (DCs) phenotypes and impair their functions in order to create a safer environment for their survival. DCs responses to helminths and their derivatives vary among different studies. Here we show that excretory/secretory products of the cestode Taenia crassiceps (TcES) do not induce the maturation of human DCs judged by a lack of increment in the expression of CD83, HLA-DR, CD80 and CD86 molecules but enhanced the production of IL-10 and positively modulated the expression of the C-type lectin receptor MGL and negatively modulated the expression of DC-SIGN. Additionally, these antigens were capable of down-modulating the inflammatory response induced by LPS in these cells by reducing the expression of the maturation markers and the production of the inflammatory cytokines IL-1β, TNF, IL-12 and IL-6. The effects of TcES upon the DCs responses to LPS were stronger if cells were exposed during their differentiation to the helminth antigens. All together, these findings suggest the ability of TcES to induce the differentiation of human DCs into a tolerogenic-like phenotype and to inhibit the effects of inflammatory stimuli.     

EST-based in silico identification and in vitro test of antimicrobial peptides in Brassica napus

Background

Brassica napus is the third leading source of vegetable oil in the world after soybean and oil palm. The accumulation of gene sequences, especially expressed sequence tags (ESTs) from plant cDNA libraries, has provided a rich resource for genes discovery including potential antimicrobial peptides (AMPs). In this study, we used ESTs including those generated from B. napus cDNA libraries of seeds, pathogen-challenged leaves and deposited in the public databases, as a model, to perform in silico identification and consequently in vitro confirmation of putative AMP activities through a highly efficient system of recombinant AMP prokaryotic expression.

Results

In total, 35,788 were generated from cDNA libraries of pathogen-challenged leaves and 187,272 ESTs from seeds of B. napus, and the 644,998 ESTs of B. napus were downloaded from the EST database of PlantGDB. They formed 201,200 unigenes. First, all the known AMPs from the AMP databank (APD2 database) were individually queried against all the unigenes using the BLASTX program. A total of 972 unigenes that matched the 27 known AMP sequences in APD2 database were extracted and annotated using Blast2GO program. Among these unigenes, 237 unigenes from B. napus pathogen-challenged leaves had the highest ratio (1.15 %) in this unigene dataset, which is 13 times that of the unigene datasets of B. napus seeds (0.09 %) and 2.3 times that of the public EST dataset. About 87 % of each EST library was lipid-transfer protein (LTP) (32 % of total unigenes), defensin, histone, endochitinase, and gibberellin-regulated proteins. The most abundant unigenes in the leaf library were endochitinase and defensin, and LTP and histone in the pub EST library. After masking of the repeat sequence, 606 peptides that were orthologous matched to different AMP families were found. The phylogeny and conserved structural motifs of seven AMPs families were also analysed. To investigate the antimicrobial activities of the predicted peptides, 31 potential AMP genes belonging to different AMP families were selected to test their antimicrobial activities after bioinformatics identification. The AMP genes were all optimized according to Escherichia coli codon usage and synthetized through one-step polymerase chain reaction method. The results showed that 28 recombinant AMPs displayed expected antimicrobial activities against E. coli and Micrococcus luteus and Sclerotinia sclerotiorum strains.

Conclusion

The study not only significantly expanded the number of known/predicted peptides, but also contributed to long-term plant genetic improvement for increased resistance to diverse pathogens of B.napus. These results proved that the high-throughput method developed that combined an in silico procedure with a recombinant AMP prokaryotic expression system is considerably efficient for identification of new AMPs from genome or EST sequence databases.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1849-x) contains supplementary material, which is available to authorized users.     

Characterization of two crustin antimicrobial peptides from the freshwater crayfish Pacifastacus leniusculus

The two bacteria-induced crustin genes, Plcrustin1 and Plcrustin2, previously found in the hemocyte cDNA library of Pacifastacus leniusculus, contain the open reading frames of 357 bp encoding a putative protein of 118 amino acid residues and 330 bp encoding a putative protein of 109 amino acid residues, respectively. The carboxyl-terminal part of the two crustins possesses, respectively, 7 and 8 conserved cysteine residues representation of a WAP domain that is found in carcinins and crustins in other several crustaceans. The amino acid sequences of Plcrustin1 and Plcrustin2 show that they belong to type I crustins. In order to characterize their properties and biological activities, the two recombinant crustin proteins were produced in the Escherichia coli expression system. Antimicrobial assays showed that the growth of only one Gram-positive bacterium, Micrococcus luteus M1 11, was inhibited by the recombinant Plcrustin1 and Plcrustin2 with MIC of about 0.07-0.27 μM and 3.5-8 μM, respectively. In addition, the study of inhibition mechanism revealed that the antimicrobial activity of the two recombinant crustin proteins was a result of bactericidal effect. However, the two crustins did not exhibit the inhibitory activities against trypsin, chymotrypsin, elastase and subtilisin A.     

Variation in Streptococcus pneumoniae susceptibility to human antimicrobial peptides may mediate intraspecific competition

Streptococcus pneumoniae is a facultative pathogen inhabiting the nasopharynx of humans where it is exposed to a range of antimicrobial peptides (AMPs) of the innate immune response. It is possible therefore that the susceptibility of strains to AMPs plays a role in determining their ability to colonize, and furthermore, that AMPs could mediate competitive interactions between co-colonizing genotypes. However, little is known about patterns of natural variation in AMP susceptibility of S. pneumoniae, and it is unclear whether the susceptibilities of an isolate to multiple human AMPs are correlated. We tested this by characterizing the susceptibility of 31 S. pneumoniae natural isolates to human neutrophil peptide (HNP-1) (α-defensin) and LL-37 (cathelicidin). We observed significant variation in susceptibility between isolates to both AMPs, and in the majority of isolates, susceptibilities to HNP-1 and LL-37 were uncorrelated. Clinical isolates were more susceptible to AMPs than were carriage isolates. The polysaccharide capsule of S. pneumoniae is thought to protect cells against AMPs. However, serotype alone could not explain the observed variation in susceptibility suggesting that genetic background plays an equally important role. We tested directly whether AMPs could mediate competition between isolates using competition experiments in the presence and absence of AMPs. These experiments demonstrated that AMPs could indeed reverse the outcome of competition between selected isolates. AMP-mediated competition could therefore contribute to the maintenance of intraspecific genetic diversity in S. pneumoniae.     

Two novel antimicrobial peptides purified from the symbiotic bacteria Xenorhabdus budapestensis NMC-10

Symbiotic bacteria, which are carried in the intestinal vesicle of the infective stage of juvenile entomopathogenic nematodes, produce broad-spectrum antibiotics. In this study, we aimed to isolate the antimicrobial peptides from the culture of the entomopathogenic bacterium Xenorhabdus budapestensis NMC-10. By screening chromatography columns and optimizing flow rate, pH, salinity and other purification conditions, we identified the final purification procedures which consisted of Q ion-exchange chromatography, gel filtration chromatography and two-step reverse-phase chromatography. Two novel antimicrobial peptides were identified via Q-TOF-TOF and de novo sequencing, and designated as GP-19 and EP-20. Both natural and synthetic peptides demonstrated broad-spectrum antimicrobial activities. The synthetic GP-19 peptide was active against Verticillium dahlia with EC(50) values of 17.54 μg/ml and highly inhibited the growth of a variety of bacteria, while the synthetic EP-20 peptide was highly active against Phytophthora capsici with EC(50) values of 3.14 μg/ml.