Gabriela Seydlová

Review of surfactin chemical properties and the potential biomedical applications

Surfactin, a highly powerful biosurfactant produced by various strains of the genus Bacillus, exhibits antibacterial, antiviral, antitumor and hemolytic action. This anionic cyclic lipopeptide is constituted by a heptapeptide interlinked with a β-hydroxy fatty acid. Due to its amhipathic nature surfactin incorporates into the phospholipid bilayer and induces permeabilization and perturbation of target cells. The rising antibiotic resistance as well as a number of remarkable surfactin activities shows that it deserves special interest and is considered as a candidate compound for combating several health related issues. In this review, the current state of knowledge of surfactin properties, biomedical potential and limitations for its application is presented.

Surfactin production enhances the level of cardiolipin in the cytoplasmic membrane of Bacillus subtilis.

Surfactin is a cyclic lipopeptide antibiotic that disturbs the integrity of the cytoplasmic membrane. In this study, the role of membrane lipids in the adaptation and possible surfactin tolerance of the surfactin producer Bacillus subtilis ATCC 21332 was investigated. During a 1-day cultivation, the phospholipids of the cell membrane were analyzed at the selected time points, which covered both the early and late stationary phases of growth, when surfactin concentration in the medium gradually rose from 2 to 84μmol·l(-1). During this time period, the phospholipid composition of the surfactin producers membrane (Sf(+)) was compared to that of its non-producing mutant (Sf(-)). Substantial modifications of the polar head group region in response to the presence of surfactin were found, while the fatty acid content remained unaffected. Simultaneously with surfactin production, a progressive accumulation up to 22% of the stress phospholipid cardiolipin was determined in the Sf(+) membrane, whereas the proportion of phosphatidylethanolamine remained constant. At 24h, cardiolipin was found to be the second major phospholipid of the membrane. In parallel, the Laurdan generalized polarization reported an increasing rigidity of the lipid bilayer. We concluded that an enhanced level of cardiolipin is responsible for the membrane rigidification that hinders the fluidizing effect of surfactin. At the same time cardiolipin, due to its negative charge, may also prevent the surfactin-membrane interaction or surfactin pore formation activity.

Development of membrane lipids in the surfactin producer Bacillus subtilis

Processes occurring in the cytoplasmic membrane of the surfactin producer Bacillus subtilis were examined during a 3-d cultivation. The fatty acid composition was found to be almost stable within this interval, except for the early stationary phase when the nonbranched, mostly C16:0 and C18:0 (high melting fatty acids), prevailed transiently in the membrane. As for phospholipids, phosphatidylglycerol and phosphatidylethanolamine, representing 73 % of the total in the membranes of exponential cells were partly replaced by cardiolipin, which gradually rose from 5 to 28 % at the end of cultivation. In parallel, steady-state fluorescence anisotropy (rs) measurements with 1,6-diphenyl-1,3,5-hexatriene (DPH) indicated a remarkable increase of rs DPH during the long-term cultivation and implied a continuous rigidization of the membrane interior. By contrast, the almost constant values of rs 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene 4-toluenesulfonate (TMA-DPH) reflected stable microviscosity of the membrane surface region. Thus, the significant increase of high melting fatty acids and cardiolipin in the cytoplasmic membrane together with the progressive rigidization of the membrane interior reflected the cell adaptation to adverse conditions.

Surfactin - Novel Solutions for Global Issues

The constant demand for new, effective therapeutic agents has triggered intensive research in the field of diverse antimicrobials of natural origin. These compounds are synthesized by all forms of life and have important biomedical and biotechnological properties, and are thus widely considered a potential solution to the growing problem of resistance to conventional antibiotics, fungal infection and life-threatening diseases. Among these molecules, lipopeptides represent a unique class of bioactive secondary metabolites with increasing scientific, therapeutic and biotechnological interest. The principal representative of the anionic lipopeptide family is surfactin, which is produced by bacterium Bacillus subtilis. This most potent known biosurfactant (i.e. surface-active compound of microbial origin), was named surfactin due to its exceptional surface activity. Since its discovery (Arima et al., 1968) and the identification of its molecular structure as a macrolide lipopeptide (Kakinuma et al., 1969) it has been best recognized for its high amhiphilicity and strong tendency for self-aggregation (Ishigami et al., 1995). Due to these characteristics it shows remarkable surface-, interfaceand membrane-active properties, resulting in a number of promising biological activities, which are of great relevance in health care and biotechnology. These properties make surfactin a candidate drug for the resolution of a number of global issues in medicine (Banat et al., 2010; Cao et al., 2010), industry (Nitschke & Costa, 2007; Abdel-Mawgoud et al., 2008) and environmental protection (Mulligan, 2009).

Bacillus subtilis alters the proportion of major membrane phospholipids in response to surfactin exposure.

Surfactin, an anionic lipopeptide produced by Bacillus subtilis, is an antimicrobial that targets the cytoplasmic membrane. Nowadays it appears increasingly apparent that the mechanism of resistance against these types of antibiotics consists of target site modification. This prompted us to investigate whether the surfactin non-producing strain B. subtilis 168 changes its membrane composition in response to a sublethal surfactin concentration. Here we show that the exposure of B. subtilis to surfactin at concentrations of 350 and 650 μg/ml (designated as SF350 and SF650, respectively) leads to a concentration-dependent growth arrest followed by regrowth with an altered growth rate. Analysis of the membrane lipid composition revealed modifications both in the polar head group and the fatty acid region. The presence of either surfactin concentration resulted in a reduction in the content of the major membrane phospholipid phosphatidylglycerol (PG) and increase in phosphatidylethanolamine (PE), which was accompanied by elevated levels of phosphatidic acid (PA) in SF350 cultures. The fatty acid analysis of SF350 cells showed a marked increase in non-branched high-melting fatty acids, which lowered the fluidity of the membrane interior measured as the steady-state fluorescence anisotropy of DPH. The liposome leakage of carboxyfluorescein-loaded vesicles resembling the phospholipid composition of surfactin-adapted cells showed that the susceptibility to surfactin-induced leakage is strongly reduced when the PG/PE ratio decreases and/or PA is included in the target bilayer. We concluded that the modifications of the phospholipid content of B. subtilis cells might provide a self-tolerance of the membrane active surfactin.

The extent of the temperature-induced membrane remodeling in two closely related Bordetella species reflects their adaptation to diverse environmental niches

Changes in environmental temperature represent one of the major stresses faced by microorganisms as they affect the function of the cytoplasmic membrane. In this study, we have analyzed the thermal adaptation in two closely related respiratory pathogens Bordetella pertussis and Bordetella bronchiseptica. Although B. pertussis represents a pathogen strictly adapted to the human body temperature, B. bronchiseptica causes infection in a broad range of animals and survives also outside of the host. We applied GC-MS to determine the fatty acids of both Bordetella species grown at different temperatures and analyzed the membrane fluidity by fluorescence anisotropy measurement. In parallel, we also monitored the effect of growth temperature changes on the expression and production of several virulence factors. In response to low temperatures, B. pertussis adapted its fatty acid composition and membrane fluidity to a considerably lesser extent when compared with B. bronchiseptica. Remarkably, B. pertussis maintained the production of virulence factors at 24 °C, whereas B. bronchiseptica cells resumed the production only upon temperature upshift to 37 °C. This growth temperature-associated differential modulation of virulence factor production was linked to the phosphorylation state of transcriptional regulator BvgA. The observed differences in low-temperature adaptation between B. pertussis and B. bronchiseptica may result from selective adaptation of B. pertussis to the human host. We propose that the reduced plasticity of the B. pertussis membranes ensures sustained production of virulence factors at suboptimal temperatures and may play an important role in the transmission of the disease.

Rapid and effective method for the separation of Bacillus subtilis vegetative cells and spores.

A variety of techniques have been reported for separation of spores from vegetative cells with the aim to gain a pure spore fraction. These include differential sedimentation in water (Stewart and Halvorson 1953), separation of layers in centrifuged pellets (Long and Williams 1958) or flotation (Boyles and Lincoln 1958; Gaudin et al. 1960). Based on the higher density of spores over growing cells, isopycnic gradient cen-trifugation may also be applied (Sharpe et al. 1975; Tamir and Gilvarg 1966; Church and Halvorson 1959). Alternatively, bacterial spores can be concentrated in a two-phase aqueous system consisting of polyethylene glycol and potassium phosphate (Sacks and Alderton 1961). What all the above methods have in common is that they tend to be laborious, are often only partially successful, and are time demanding, or require extra equipment. Moreover, they originated in studies focused on spores, which are much more resistant to a number of chemical and physical factors applied in these techniques than their growing cell counterparts. Therefore it is highly probable that handling of culture during these procedures may cause them damage, bringing undesirable artifacts to the subpopu-lation of vegetative cells under the study. This paper describes a simple and rapid method to obtain a preparation of vegetative cells separated from the spore contamination based on a modified technique for the enzyme preparation of membrane vesicles (Bisschop and Konings 1976). Following this enzymatic treatment and centrifugation, intact spores remain in the sediment, whereas the cell-free supernatant contains the cytoplasmic and membrane fractions of vegetative cells. This technique was crucial for testing the phospholipid composition of Bacillus subtilis American Type Cultures Collection (ATCC) 21332 cytoplasmic membranes during surfactin production. Surfactin, a potent antibiotic that perturbs the phospholipid bilayer of the target cells, is produced by vegetative cells in the stationary phase of growth. In order to examine the possible adaptive modifications of membrane lipids of the cells generating surfactin induced by their own toxic product, it was necessary to separate these from the non-producing endospores. The B. subtilis ATCC 21332 strain was grown in a nutrient broth (pH 7.0; Oxoid) with aeration (120 rpm) at 30°C for 24 h. The pelleted culture (4 300×g, 15 min, 4°C) was resuspended in the 50 mmol/L potassium phosphate buffer (pH 8.0, 30°C) with phenylmethylsulfonyl fluoride (1 mmol/L) and digested with lysozyme (100,000 U/mg, Serva), DNase-I and RNase (all Sigma) at concentrations of 10 mg/mL and 25 and 25 …

Insights into the Mechanism of Action of Bactericidal Lipophosphonoxins.

The advantages offered by established antibiotics in the treatment of infectious diseases are endangered due to the increase in the number of antibiotic-resistant bacterial strains. This leads to a need for new antibacterial compounds. Recently, we discovered a series of compounds termed lipophosphonoxins (LPPOs) that exhibit selective cytotoxicity towards Gram-positive bacteria that include pathogens and resistant strains. For further development of these compounds, it was necessary to identify the mechanism of their action and characterize their interaction with eukaryotic cells/organisms in more detail. Here, we show that at their bactericidal concentrations LPPOs localize to the plasmatic membrane in bacteria but not in eukaryotes. In an in vitro system we demonstrate that LPPOs create pores in the membrane. This provides an explanation of their action in vivo where they cause serious damage of the cellular membrane, efflux of the cytosol, and cell disintegration. Further, we show that (i) LPPOs are not genotoxic as determined by the Ames test, (ii) do not cross a monolayer of Caco-2 cells, suggesting they are unable of transepithelial transport, (iii) are well tolerated by living mice when administered orally but not peritoneally, and (iv) are stable at low pH, indicating they could survive the acidic environment in the stomach. Finally, using one of the most potent LPPOs, we attempted and failed to select resistant strains against this compound while we were able to readily select resistant strains against a known antibiotic, rifampicin. In summary, LPPOs represent a new class of compounds with a potential for development as antibacterial agents for topical applications and perhaps also for treatment of gastrointestinal infections.

Lipophosphonoxins II: Design, Synthesis, and Properties of Novel Broad Spectrum Antibacterial Agents

The increase in the number of bacterial strains resistant to known antibiotics is alarming. In this study we report the synthesis of novel compounds termed Lipophosphonoxins II (LPPO II). We show that LPPO II display excellent activities against Gram-positive and -negative bacteria, including pathogens and multiresistant strains. We describe their mechanism of action-plasmatic membrane pore-forming activity selective for bacteria. Importantly, LPPO II neither damage nor cross the eukaryotic plasmatic membrane at their bactericidal concentrations. Further, we demonstrate LPPO II have low propensity for resistance development, likely due to their rapid membrane-targeting mode of action. Finally, we reveal that LPPO II are not toxic to either eukaryotic cells or model animals when administered orally or topically. Collectively, these results suggest that LPPO II are highly promising compounds for development into pharmaceuticals.

A Single Tim Translocase in the Mitosomes of Giardia intestinalis Illustrates Convergence of Protein Import Machines in Anaerobic Eukaryotes

Abstract Mitochondria have evolved diverse forms across eukaryotic diversity in adaptation to anoxia. Mitosomes are the simplest and the least well-studied type of anaerobic mitochondria. Transport of proteins via TIM complexes, composed of three proteins of the Tim17 protein family (Tim17/22/23), is one of the key unifying aspects of mitochondria and mitochondria-derived organelles. However, multiple experimental and bioinformatic attempts have so far failed to identify the nature of TIM in mitosomes of the anaerobic metamonad protist, Giardia intestinalis, one of the few experimental models for mitosome biology. Here, we present the identification of a single G. intestinalis Tim17 protein (GiTim17), made possible only by the implementation of a metamonad-specific hidden Markov model. While very divergent in primary sequence and in predicted membrane topology, experimental data suggest that GiTim17 is an inner membrane mitosomal protein, forming a disulphide-linked dimer. We suggest that the peculiar GiTim17 sequence reflects adaptation to the unusual, detergent resistant, inner mitosomal membrane. Specific pull-down experiments indicate interaction of GiTim17 with mitosomal Tim44, the tethering component of the import motor complex. Analysis of TIM complexes across eukaryote diversity suggests that a “single Tim” translocase is a convergent adaptation of mitosomes in anaerobic protists, with Tim22 and Tim17 (but not Tim23), providing the protein backbone.