Decreased susceptibility to viscosin in Streptococcus pneumoniae.

Growing numbers of infections caused by antibiotic-resistant Streptococcus pneumoniae strains are a major concern for healthcare systems that will require new antibiotics for treatment as well as preventative measures that reduce the number of infections. Lipopeptides are antimicrobial molecules, of which some are used as antibiotics, including the last resort antibiotics daptomycin and polymyxins. Here we have studied the antimicrobial effect of the cyclic lipopeptide viscosin on S. pneumoniae growth and morphology. Most lipopeptides function as surfactants that create pores in membrane layers, which is regarded as their main antimicrobial activity. We show that viscosin can inhibit growth of S. pneumoniae without disintegration of the cytoplasmic membrane. Instead, the cells developed abnormal shapes and misplaced new division sites. The cell wall of these bacteria appeared less dense in electron microscopy images, suggesting that viscosin interfered with normal cell wall synthesis. Corroborating this observation, a luciferase reporter assay was used to show that the two-component systems LiaFSR and CiaRH, which are known to be activated upon cell wall stress, were strongly induced by viscosin. Furthermore, a mutant displaying 1.8-fold decreased susceptibility to viscosin was generated by sequential exposure to increasing concentrations of the lipopeptide. The mutant suffered from significant fitness loss and had mutations in genes involved in fatty acid synthesis, teichoic acid synthesis, and cell wall synthesis as well as transcription and translation. How these mutations might be linked to decreased viscosin susceptibility is discussed.IMPORTANCEStreptococcus pneumoniae is a leading cause of bacterial pneumonia, sepsis, and meningitis in children, and the incidence of infections caused by antibiotic-resistant strains is increasing. Development of new antibiotics is therefore necessary to treat these types of infections in the future. Here, we have studied the activity of the antimicrobial lipopeptide viscosin on S. pneumoniae and show that in addition to having the typical membrane destabilizing activity of lipopeptides, viscosin inhibits pneumococcal growth by obstructing normal cell wall synthesis. This suggests a more specific mode of action than just the surfactant activity. Furthermore, we show that S. pneumoniae does not easily acquire resistance to viscosin, which makes it a promising molecule to explore further, for example, by synthesizing less toxic derivates that can be tested for therapeutic potential.

Characterization and Production of a Biosurfactant Viscosin from Pseudomonas sp. HN11 and its Application on Enhanced oil Recovery During oily Sludge Cleaning.

Biosurfactants are renewable resources with versatile applications on environmental bioremediation and industrial processes. Pseudomonas species are one of the promising biosurfactant producers. However, besides rhamnolipids, little is known about Pseudomonas-derived biosurfactants on solubilization of polycyclic aromatic hydrocarbons (PAHs) and oily sludge treatment. In this study, Pseudomonas sp. HN11-derived biosurfactant was purified by chromatographic methods and was characterized as viscosin via bioinformatic analysis, spectrometric and spectroscopic analyses, Marfey's method and (C-H)α NMR fingerprint matching approach. Viscosin is a potent biosurfactant with critical micelle concentration of 5.79 mg/L and is stable under various stresses. Moreover, viscosin was produced at 0.42 g/L at 48 h of liquid fermentation. Further data have shown that emulsifying agent viscosin is capable of promoting the solubilization of PAHs and displays enhanced oil recovery during oily sludge treatment. More specifically, viscosin has shown significantly enhanced solubilization on fluoranthene compared with control (0.04 mg/L), 2.21 mg/L and 1.27 mg/L fluoranthene was recovered from 100 mg/L and 200 mg/L viscosin treatment, respectively. However, only 200 mg/L viscosin has significantly enhanced the solubilization of phenanthrene (0.75 mg/L) and benzo[a]pyrene (0.51 mg/L) compared to each control (0.23 mg/L for phenanthrene and 0.09 mg/L for benzo[a]pyrene). Viscosin treatment of oily sludge (recovering of 0.58 g oil) has shown a significant oil recovery compared to that of control (recovering of 0.42 g oil). This study shows the great potential of viscosin-type biosurfactant on oily sludge treatment.

Biosurfactants Produced by Phyllosphere-Colonizing Pseudomonads Impact Diesel Degradation but Not Colonization of Leaves of Gnotobiotic Arabidopsis thaliana.

Biosurfactant production is a common trait in leaf surface-colonizing bacteria that has been associated with increased survival and movement on leaves. At the same time, the ability to degrade aliphatics is common in biosurfactant-producing leaf colonizers. Pseudomonads are common leaf colonizers and have been recognized for their ability to produce biosurfactants and degrade aliphatic compounds. In this study, we investigated the role of biosurfactants in four non-plant-pathogenic Pseudomonas strains by performing a series of experiments to characterize their surfactant properties and their role during leaf colonization and diesel degradation. The biosurfactants produced were identified using mass spectrometry. Two strains produced viscosin-like biosurfactants, and the other two produced massetolide A-like biosurfactants, which aligned with the phylogenetic relatedness between the strains. To further investigate the role of surfactant production, random Tn5 transposon mutagenesis was performed to generate knockout mutants. The knockout mutants were compared to their respective wild types with regard to their ability to colonize gnotobiotic Arabidopsis thaliana and to degrade diesel or dodecane. It was not possible to detect negative effects during plant colonization in direct competition or individual colonization experiments. When grown on diesel, knockout mutants grew significantly slower than their respective wild types. When grown on dodecane, knockout mutants were less impacted than during growth on diesel. By adding isolated wild-type biosurfactants, it was possible to complement the growth of the knockout mutants.IMPORTANCE Many leaf-colonizing bacteria produce surfactants and are able to degrade aliphatic compounds; however, whether surfactant production provides a competitive advantage during leaf colonization is unclear. Furthermore, it is unclear if leaf colonizers take advantage of the aliphatic compounds that constitute the leaf cuticle and cuticular waxes. Here, we tested the effect of surfactant production on leaf colonization, and we demonstrate that the lack of surfactant production decreases the ability to degrade aliphatic compounds. This indicates that leaf surface-dwelling, surfactant-producing bacteria contribute to degradation of environmental hydrocarbons and may be able to utilize leaf surface waxes. This has implications for plant-microbe interactions and future studies.

Biosynthesis and Antimicrobial Activity of Pseudodesmin and Viscosinamide Cyclic Lipopeptides Produced by Pseudomonads Associated with the Cocoyam Rhizosphere.

Pseudomonas cyclic lipopeptides (CLPs) are encoded non-ribosomally by biosynthetic gene clusters (BGCs) and possess diverse biological activities. In this study, we conducted chemical structure and BGC analyses with antimicrobial activity assays for two CLPs produced by Pseudomonas strains isolated from the cocoyam rhizosphere in Cameroon and Nigeria. LC-MS and NMR analyses showed that the Pseudomonas sp. COR52 and A2W4.9 produce pseudodesmin and viscosinamide, respectively. These CLPs belong to the Viscosin group characterized by a nonapeptidic moiety with a 7-membered macrocycle. Similar to other Viscosin-group CLPs, the initiatory non-ribosomal peptide synthetase (NRPS) gene of the viscosinamide BGC is situated remotely from the other two NRPS genes. In contrast, the pseudodesmin genes are all clustered in a single genomic locus. Nano- to micromolar levels of pseudodesmin and viscosinamide led to the hyphal distortion and/or disintegration of Rhizoctonia solani AG2-2 and Pythium myriotylum CMR1, whereas similar levels of White Line-Inducing Principle (WLIP), another member of the Viscosin group, resulted in complete lysis of both soil-borne phytopathogens. In addition to the identification of the biosynthetic genes of these two CLPs and the demonstration of their interaction with soil-borne pathogens, this study provides further insights regarding evolutionary divergence within the Viscosin group.

Identification of the Molecular Determinants Involved in Antimicrobial Activity of Pseudodesmin A, a Cyclic Lipopeptide From the Viscosin Group.

Cyclic lipo(depsi)peptides (CLiPs) from Pseudomonas constitute a class of natural products involved in a broad range of biological functions for their producers. They also display interesting antimicrobial potential including activity against Gram-positive bacteria. Literature has indicated that these compounds can induce membrane permeabilization, possibly through pore-formation, leading to the general view that the cellular membrane constitutes the primary target in their mode of action. In support of this view, we previously demonstrated that the enantiomer of pseudodesmin A, a member of the viscosin group of CLiPs, shows identical activity against a test panel of six Gram-positive bacterial strains. Here, a previously developed total organic synthesis route is used and partly adapted to generate 20 novel pseudodesmin A analogs in an effort to derive links between molecular constitution, structure and activity. From these, the importance of a macrocycle closed by an ester bond as well as a critical length of ß-OH fatty acid chain capping the N-terminus is conclusively demonstrated, providing further evidence for the importance of peptide-membrane interactions in the mode of action. Moreover, an alanine scan is used to unearth the contribution of specific amino acid residues to biological activity. Subsequent interpretation in terms of a structural model describing the location and orientation of pseudodesmin A in a membrane environment, allows first insight in the peptide-membrane interactions involved. The biological screening also identified residue positions that appear less sensitive to conservative modifications, allowing the introduction of a non-perturbing tryptophan residue which will pave the way toward biophysical studies using fluorescence spectroscopy.

Natural C. elegans Microbiota Protects against Infection via Production of a Cyclic Lipopeptide of the Viscosin Group.

Caenorhabditis elegans is associated in nature with a species-rich, distinct microbiota, which was characterized only recently [1]. Thus, our understanding of the relevance of the microbiota for nematode fitness is still at its infancy. One major benefit that the intestinal microbiota can provide to its host is protection against pathogen infection [2]. However, the specific strains conferring the protection and the underlying mechanisms of microbiota-mediated protection are often unclear [3]. Here, we identify natural C. elegans microbiota isolates that increase C. elegans resistance to pathogen infection. We show that isolates of the Pseudomonas fluorescens subgroup provide paramount protection from infection with the natural pathogen Bacillus thuringiensis through distinct mechanisms. We found that the P. lurida isolates MYb11 and MYb12 (members of the P. fluorescens subgroup) protect C. elegans against B. thuringiensis infection by directly inhibiting growth of the pathogen both in vitro and in vivo. Using genomic and biochemical analyses, we further demonstrate that MYb11 and MYb12 produce massetolide E, a cyclic lipopeptide biosurfactant of the viscosin group [4, 5], which is active against pathogenic B. thuringiensis. In contrast to MYb11 and MYb12, P. fluorescens MYb115-mediated protection involves increased resistance without inhibition of pathogen growth and most likely depends on indirect, host-mediated mechanisms. This work provides new insight into the functional significance of the C. elegans natural microbiota and expands our knowledge of bacteria-derived compounds that can influence pathogen colonization in the intestine of an animal.

Membrane Interactions of Natural Cyclic Lipodepsipeptides of the Viscosin Group.

Many Pseudomonas spp. produce cyclic lipodepsipeptides (CLPs), which, besides their role in biological functions such as motility, biofilm formation and interspecies interactions, are antimicrobial. It has been established that interaction with the cellular membrane is central to the mode of action of CLPs. In this work, we focus on the CLPs of the so-called viscosin group, aiming to assess the impact of the main structural variations observed within this group on both the antimicrobial activity and the interaction with model membranes. The antimicrobial activity of viscosin, viscosinamide A, WLIP and pseudodesmin A were all tested on a broad panel of mainly Gram-positive bacteria. Their capacity to permeabilize or fuse PG/PE/cardiolipin model membrane vesicles is assessed using fluorescent probes. We find that the Glu2/Gln2 structural variation within the viscosin group is the main factor that influences both the membrane permeabilization properties and the minimum inhibitory concentration of bacterial growth, while the configuration of the Leu5 residue has no apparent effect. The CLP-membrane interactions were further evaluated using CD and FT-IR spectroscopy on model membranes consisting of PG/PE/cardiolipin or POPC with or without cholesterol. In contrast to previous studies, we observe no conformational change upon membrane insertion. The CLPs interact both with the polar heads and aliphatic tails of model membrane systems, altering bilayer fluidity, while cholesterol reduces CLP insertion depth.

Role of secondary metabolites in the interaction between Pseudomonas fluorescens and soil microorganisms under iron-limited conditions.

Microorganisms can be versatile in their interactions with each other, being variously beneficial, neutral or antagonistic in their effect. Although this versatility has been observed among many microorganisms and in many environments, little is known regarding the mechanisms leading to these changes in behavior. In the present work, we analyzed the mechanism by which the soil bacterium Pseudomonas fluorescens BBc6R8 shifts from stimulating the growth of the ectomycorrhizal fungus Laccaria bicolor S238N to killing the fungus. We show that among the three secondary metabolites produced by the bacterial strain-the siderophores enantio-pyochelin and pyoverdine, and the biosurfactant viscosin-the siderophores are mainly responsible for the antagonistic activity of the bacterium under iron-limited conditions. While the bacterial strain continues to produce beneficial factors, their effects are overridden by the action of their siderophores. This antagonistic activity of the strain P. fluorescens BBC6R8 in iron-depleted environments is not restricted to its influence on L. bicolor, since it was also seen to inhibit the growth of the actinomycete Streptomyces ambofaciens ATCC23877. We show that the strain P. fluorescens BBc6R8 uses different strategies to acquire iron, depending on certain biotic and abiotic factors.

Characterization of Pseudomonas chlororaphis from Theobroma cacao L. rhizosphere with antagonistic activity against Phytophthora palmivora (Butler).

AIM: To isolate and characterize rhizobacteria from Theobroma cacao with antagonistic activity against Phytophthora palmivora, the causal agent of the black pod rot, which is one of the most important diseases of T. cacao. METHODS AND RESULTS: Among 127 rhizobacteria isolated from cacao rhizosphere, three isolates (CP07, CP24 and CP30) identified as Pseudomonas chlororaphis, showed in vitro antagonistic activity against P. palmivora. Direct antagonism tested in cacao detached leaves revealed that the isolated rhizobacteria were able to reduce symptom severity upon infection with P. palmivora Mab1, with Ps. chlororaphis CP07 standing out as a potential biocontrol agent. Besides, reduced symptom severity on leaves was also observed in planta where cacao root system was pretreated with the isolated rhizobacteria followed by leaf infection with P. palmivora Mab1. The production of lytic enzymes, siderophores, biosurfactants and HCN, as well as the detection of genes encoding antibiotics, the formation of biofilm, and bacterial motility were also assessed for all three rhizobacterial strains. By using a mutant impaired in viscosin production, derived from CP07, it was found that this particular biosurfactant turned out to be crucial for both motility and biofilm formation, but not for the in vitro antagonism against Phytophthora, although it may contribute to the bioprotection of T. cacao. CONCLUSIONS: In the rhizosphere of T. cacao, there are rhizobacteria, such as Ps. chlororaphis, able to protect plants against P. palmivora. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides a theoretical basis for the potential use of Ps. chlororaphis CP07 as a biocontrol agent for the protection of cacao plants from P. palmivora infection.