quantification of rhamnolipids present in the biosurfactant of pseudomonas aeruginosa

Análisis comparativo de la producción de ramnolípidos en cepas de Pseudomonas aeruginosa

Pseudomonas aeruginosa is an environmental bacterium and an opportunistic pathogen of great concern due to its production of virulence-associated traits and its high antibiotic resistance1,2. A characteristic secondary metabolite produced by this bacterium is the biosurfactant RL, which is produced in a coordinated manner with several virulence-associated traits such as the phenazine pyocyanin, an antibiotic with redox activity, and the protease elastase3. The tensio-active and emulsification properties of RL have been exploited in different industrial applications and are currently commercialized4.
Most P. aeruginosa strains, belonging to phylogroups 1 and 2, produce two types of RL: mRL, which consists of one rhamnose moiety linked to a fatty acid dimer mainly of 10 carbons, and dRL, which contains an additional rhamnose moiety linked to the first rhamnose4 (see Figure 1). However, it has been reported that two minor P. aeruginosa phylogroups (groups 3 and 5) only produce mRL5,6. The two types of RL contain a mixture of fatty acid dimers, which, as mentioned, are mainly C10-C10, but smaller proportions of molecules containing C12-C10, C12-C12, and C10-C12:1 dimers are also produced. The characterization of the RL congeners produced by different strains using HPLC MS/MS has been reported7,8. The methods described in this work can only differentiate between mRL and dRL but cannot be used for the characterization of the RL congeners.
P. aeruginosa and some Burkholderia species are natural producers of RL9, but the former bacterium is the most efficient producer. However, commercially used RL is currently produced in Pseudomonas putida KT2440 derivatives expressing P. aeruginosa genes to avoid the use of this opportunistic pathogen10,11. The detection and quantification of RL produced by P. aeruginosa are of great importance for studying the molecular mechanisms involved in the expression of virulence-related traits12, in the characterization of strains belonging to clades 3 or 513, and for constructing P. aeruginosa derivatives that overproduce these biosurfactants while having reduced virulence14. The production of biosurfactants by different microorganisms has been detected based on some general characteristics of these compounds, such as the collapse drop method or emulsification index15, but these methods are neither accurate nor specific16.
Here, we describe the protocol to detect mRL and dRL using the liquid extraction of total RL from the culture supernatants of different P. aeruginosa-type strains and the separation of both types of RL using TLC. In this method, the RL extracted from the culture supernatant is separated by their differential solubility in the solvents used for TLC, causing differential migration on the silica gel plate. Thus, mRL have a more rapid migration than dRL, and they can be detected as separate spots when the plates are dried and stained with &#945;-naphthol.
The method described here for detecting mRL and dRL by TLC is based on a previously published article17, which is easy to perform and does not require expensive equipment. This method has been useful for detecting RL in various P. aeruginosa isolates13 using appropriate controls, such as a P. aeruginosa-derived mutant unable to produce RL. However, it is not the preferred method for characterizing novel biosurfactants produced by bacteria other than Pseudomonas aeruginosa due to its lack of specificity.
Additionally, a method for quantifying the rhamnose equivalents of total RL extracted from a P. aeruginosa culture supernatant is presented. This method quantifies these biosurfactants based on the reaction of orcinol with reductive sugars, resulting in a product that can be measured spectrophotometrically at 421 nm, as previously described18. Since the reaction with orcinol is not specific to rhamnose, it is important to perform this method with RL extracted from the culture supernatant that does not contain significant amounts of other sugar-containing molecules, such as lipopolysaccharides (LPS). An acidified chloroform/methanol mixture is used here for liquid extraction of RL18, but ethyl acetate can also be used, and solid-phase extraction (SPE) yields very good results19. The orcinol method described here does not require sophisticated equipment and can provide reliable results if performed with special care in preparing the analyzed samples, as discussed. To ensure proper sample preparation, it is important to include a Pseudomonas aeruginosa rhlA mutant unable to produce RL20 and to perform three biological and three technical replicates for each determination.
There has been significant controversy in the literature16,21regarding RL determination by the orcinol method, with some studies suggesting that RL production is overestimated and that the assay lacks specificity for rhamnose, potentially detecting other sugars. However, we demonstrate here that the methods described can be accurate and specific under the appropriate conditions. Furthermore, for comparison with the procedures outlined in this article, we utilize UPLC-MS/MS detection of a dRL standard and demonstrate that similar results are obtained with the orcinol method. The detailed protocol for quantifying RL using this method is included in Supplementary File 1.
These protocols are exemplified using the type strains PAO1 (phylogroup 1), PA14 (phylogroup 2), and PA7 (phylogroup 3). These strains were chosen because they are well-characterized and produce different RL profiles.

Autor destacado: Comprensión de la regulación de ramnolípidos en Pseudomonas aeruginosa

Detección y cuantificación de mono-ramnolípidos y di-ramnolípidos producidos por Pseudomonas aeruginosa

Cuantificación de ramnolípidos presentes en el biosurfactante de Pseudomonas aeruginosa

quantification-rhamnolipids-present-biosurfactant-pseudomonas

Research

JoVE Journal

Biochemistry

Quantification of Rhamnolipids Present in the Biosurfactant of Pseudomonas aeruginosa

75 vistas. Para empezar, pipetear 1,2 mililitros de un cultivo de 24 horas de Pseudomonas aeruginosa en un tubo de centrífuga de 1,5 mililitros. Centrifugar el tubo a 3000 g durante cuatro grados centígrados durante tres minutos. Recoja el sobrenadante en un tubo limpio.Pipetear 333 microlitros del sobrenadante en un tubo limpio. Luego agregue un mililitro de éter en el tubo. Mezcle la solución en un vórtice durante 30 segundos.Después de que todos l...

Video: Cuantificación de ramnolípidos presentes en el biosurfactante de Pseudomonas aeruginosa

Detection and Quantification of Mono-Rhamnolipids and Di-Rhamnolipids Produced by Pseudomonas aeruginosa

The environmental bacterium Pseudomonas aeruginosa is an opportunistic pathogen with high antibiotic resistance that represents a health hazard. This bacterium produces high levels of biosurfactants known as rhamnolipids (RL), which are molecules with significant biotechnological value but are also associated with virulence traits. In this respect, the detection and quantification of RL may be useful for both biotechnology applications and biomedical research projects. In this article, we demonstrate step-by-step the technique to detect the production of the two forms of RL produced by P. aeruginosa using thin-layer chromatography (TLC): mono-rhamnolipids (mRL), molecules constituted by a dimer of fatty acids (mainly C10-C10) linked to one rhamnose moiety, and di-rhamnolipids (dRL), molecules constituted by a similar fatty acid dimer linked to two rhamnose moieties. Additionally, we present a method to measure the total amount of RL based on the acid hydrolysis of these biosurfactants extracted from a P. aeruginosa culture supernatant and the subsequent detection of the concentration of rhamnose that reacts with orcinol. The combination of both techniques can be used to estimate the approximate concentration of mRL and dRL produced by a specific strain, as exemplified here with the type strains PAO1 (phylogroup 1), PA14 (phylogroup 2), and PA7 (phylogroup 3).

Pseudomonas aeruginosa&#160;produces the rhamnolipid biosurfactants. Thin-layer chromatography detects and determines the proportion of mono- and di-rhamnolipids produced by each strain. Quantification of total rhamnolipids involves assessing rhamnose equivalents present in these biosurfactants extracted from the culture supernatants using the orcinol method.

The environmental bacterium Pseudomonas aeruginosa is an opportunistic pathogen with high antibiotic resistance that represents a health hazard. This ...