Hello,

I want to get some (positively charged) dyes into S.aureus cells.

Do you know about a shuttling transporter system of S.aureus one could use to get the dyes in life bacteria?
For example, coupling the dye to siderophores to get into the cell with the iron-uptake system, which would not work in my high-iron-environment!?

Thanks in advannce for your posts!

Best regards,

Gerhard

osiris-gdw:

Is there a particular dye that you are interested in using. If so, it would help to know what that is.

In the meantime, you can check out this new Invitrogen publication

Tools for Microbiology Detection and Research

Hello!

Thanks for the reply!

I just want to get some old dyes in the bacteria, something like TAMRA, methylene blue, FITC, Safranin, toluidine blue, something like these...

I would be glad if anyone could help!

Regards,

Gerhard

osiris-gdw:

You may want to investigate utilizing carboxyfluorescein diacetate (CFDA) or carboxyfluorescein diacetate succinimidyl ester (CFDA/SE).

Here is reference that may help you out:

Daniel Hoefel, Warwick L. Grooby, Paul T. Monis, Stuart Andrews and Christopher P. Saint. A comparative study of carboxyfluorescein diacetate and carboxyfluorescein diacetate succinimidyl ester as indicators of bacterial activity. Journal of Microbiological Methods
Volume 52, Issue 3, March 2003, Pages 379-388.


Below are some pertinent excerpts from this article...


"Carboxyfluorescein diacetate (CFDA) is an esterified fluorogenic substrate that has been widely used for assessing esterase activity in bacteria. CFDA is cell permeant and undergoes hydrolysis of the diacetate (DA) groups into fluorescent carboxyfluorescein (CF) by intracellular nonspecific
esterases (Fig. 1). As opposed to conventional fluorescein, CF contains extra negative charges, which leads to enhanced retention within the cell (Haugland, 1999)."

"CFDA has been used for for the enumeration of esterase-active bacteria in purified water used in pharmaceutical manufacturing
processes (Kawai et al., 1999) and to measure the viability of planktonic bacteria (Porter et al., 1995). Double staining with CFDA and propidium iodide is also commonly used to distinguish more easily between active and inactive bacteria. Determination
of Lactococcus lactis (Bunthof et al., 1999) and Escherichia coli O157:H7 (Tanaka et al., 2000) viability, as well as bacterial activity measurements in natural aquatic environments (Yamaguchi and Nasu, 1997), have been performed with this technique."

"Carboxyfluorescein diacetate succinimidyl ester (CFDA/SE) is another fluorogenic esterified substrate similar to CFDA but differing by the presence of a succinimidyl ester (SE) group that can bind strongly to free amines. CFDA/SE is also cell permeant
and the DA groups are hydrolysed intracellularly by nonspecific esterases, resulting in a highly fluorescent amine reactive fluorophore (CF/SE). This molecule can react with amine containing residues of intracellular proteins, forming highly stable dye protein adducts (Haugland, 1999) (Fig. 1). Conventionally, CFDA/SE has been used in conjunction with FCM for tracking cell division in mammalian cells (Lyons, 1999; Hodgkin and Lyons, 2000; Lyons et al., 2001) and bacteria (Ueckert et al., 1997). It also has wide applications in monitoring intracellular pH in bacteria (Breeuwer et al., 1996; Riondet et al.,
1997; Chitarra et al., 2000). In addition, CFDA/SE has been used for bacterial viability measurements (Fuller et al., 2000) and monitoring the transport of bacteria introduced into subsurface water over an extended period of time (DeFlaun et al., 2001; Fuller et al., 2001)."



Hope this helps!

Here is another reference that may be helpful which reviews various dyes and/or fluorescent markers commonly utilized during efflux pump evaluations.

M. Ines Borges-Walmsley, Kenneth S. McKeegan and Adrian R. Walmsley. Structure and function of efflux pumps that confer resistance to drugs. Biochem. J. (2003) 376 (313338).


And here are the pertinent excerpts from this open-source article...


"Fluorescent substrates
Once a suitable assay system for the transporter of choice has been determined, it allows a number of biochemical analyses to be undertaken that can measure transport function. Fluorescent probes are tools that are often used in such studies. The anti-tumour drug doxorubicin and the dyes R6G and EtBr are good examples of compounds that exhibit different levels of intracellular and extracellular fluorescence, which occurs due to their interaction with cellular components. In the case of R6G and doxorubicin, their fluorescence decreases in the intracellular environment, whereas the opposite is true of EtBr. This simple phenomenon can be used to directly monitor the efflux of these compounds out of treated cells. When de-energized E. coli strains that can efflux doxorubicin are loaded with the drug, active efflux upon energization can be monitored over time as an increase in fluorescence [176]. A similar phenomenon occurs with R6G [176]. In cells resistant to EtBr, efflux can be monitored as a decrease in fluorescence over time [177]. Fluorescence changes with these compounds that are associated with active efflux can be abolished when the preloaded cells are mutant strains lacking specific efflux pumps, such as the acrB mutant E. coli KAM3. The inability of KAM3 or other efflux mutants to expel doxorubicin, R6G or EtBr is shown by fluorescence changes that are associated with passive diffusion only [176,178].

"The dye Hoechst 33342, a substrate of a number of multidrug resistance pumps, is another fluorescent probe that is particularly useful in membrane transport studies [172,177,179182]. Hoechst 33342 is strongly fluorescent in the membrane environment, but has low fluorescence in aqueous solutions. This ability has been has been used to characterize a number of MDR transporters, including P-gp, LmrA, LmrP, NorA, YyvC, MdfA and HorA [82,165,172,174,175,180,183,184]. Transport of Hoechst 33342 in everted vesicles containing the Staphylococcus aureus MDR transporter NorA was saturable. Additionally, Hoechst 33342 transport was shown to be inhibited competitively by the presence of the known MDR inhibitor verapamil [172]. Using similar methods, LmrP-mediated transport of Hoechst 33342 was inhibited competitively by quinine and verapamil, non-competitively with nicardipin and vinblastine, and uncompetitively with TPP+ [180]. This finding strongly suggests that LmrP can interact with substrates via a number of different sites [180]. Many of the drugs that LmrP has been shown to transport are highly hydrophobic and will readily partition into the lipid bilayer.

"Fluorescent substrates can also be used to indicate the site of interaction between efflux pumps and their substrates. Using the non-fluorescent compound BCECF-AM [2,7-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester], which partitions into the membrane bilayer, LmrP-overexpressing cells have been shown to efflux BCECF-AM from the cell before it is hydrolysed by cytoplasmic esterases into the fluorescent compound BCECF [185]. This provides evidence that LmrP transports drugs from the membrane and not from the cytoplasm [185]. The point of interaction between LmrP and transported substrate was localized to the inner-membrane leaflet of the bilayer using the fluorescent probe TMA-DPH [1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene iodide] [185]. This probe shares similar characteristics with Hoescht 33342, i.e. it is fluorescent in the membrane but not in aqueous environments. TMA-DPH partitioning into the membrane of Lactococcus lactis was shown to be a biphasic process. This behaviour was shown to be a reflection of the fast entry of TMA-DPH into the outer leaflet, followed by a slower transbilayer movement to the inner leaflet of the membrane [185]. The initial rate of TMA-DPH extrusion correlates with the amount of probe associated with the inner leaflet, providing direct evidence that the ability of amphiphilic substrates to partition into the inner leaflet of the membrane is a prerequisite for recognition by multidrug transporters [185].

Fluorescent probes
Fluorescent probes do not need to be transported substrates. Site-directed labelling with fluorescent probes directly on to the efflux protein can provide alternative strategies. These probes are particularly useful tools when used to monitor conformational changes. Extensive studies by Liu and Sharom [186] have used the thiol-specific probe MIANS [1-(4-maleimidylanilino)napthalene-6-sulphonic acid] to label P-gp via the two cysteine residues within the Walker A motifs of the NBDs. The MIANS group displays saturable quenching in the presence of ATP, ADP and non-hydrolysable analogues such as p[NH]ppA [187]. Quenching of the MIANs label is most probably due to local conformational changes caused by nucleotide binding. MIANS-dependent fluorescence changes also occur in the presence of P-gp substrates, suggesting cross-talk between the drug-binding domains and the NBDs [82]. Further evidence of cross-talk between the domains of P-gp was discussed in an earlier section [121]. MIANS-labelled P-gp also exhibits saturable quenching in the presence of P-gp substrates, and provides a useful tool that allows the determination of P-gp substrate affinities [82].

Intrinsic fluorescence
Proteins contain natural fluorophores, most notably the aromatic amino acid residues tryptophan and tyrosine. This phenomenon is known as intrinsic fluorescence (see [188] for a practical review of this topic). Tryptophan residues are specifically excited maximally by light with a wavelength of 295 nm, and emit light maximally at 340 nm. The excitation and emission maxima of tyrosine are 275 nm and 310 nm respectively. In most cases tryptophan fluorescence predominates. Intrinsic fluorescence can be used to monitor proteinligand interactions when the fluorescence of the proteinligand complex differs from that of the free protein or ligand. The binding of ligand to the protein often results in a conformational change within the protein, and fluorescence changes occur due to movements of the aromatic residues. Changes in the intrinsic fluorescence of proteins in the presence of ligands, if saturable, can provide a useful means by which the dissociation constants of particular ligands can be calculated. In this respect, intrinsic fluorescence has been used successfully to monitor the interactions between P-gp, transported substrates and nucleotides such as ADP and ATP [80,82,189]. A major advantage of intrinsic fluorescence is that the native protein can be used; for example, MIANS-labelled P-gp, which has seemingly unaltered affinity for substrates and cofactors, is catalytically inactive [82]. Using intrinsic fluorescence and MIANS labelling techniques, Sharom and co-workers [82] have determined the Kd values for over 80 P-gp substrates, finding a range of values from 25 nM to 250 mM. The ability of P-gp to bind such a wide range of substrates with differential binding activities over four orders of magnitude is a stark demonstration of it substrate promiscuity. Intrinsic fluorescence has also been used to measure the affinities of glycerol 2-phosphate, glycerol 3-phosphate and Pi towards the E. coli MF transporter GlpT [190]. As yet, the use of intrinsic fluorescence has not been applied to bacterial efflux transporters; this probably reflects difficulties in overexpression and purification, the unsuitability of the substrates for these assays, or a lack of suitable intrinsic fluorophores."



Good luck - hope this helps!

Here are a few more references that may help...

Rhena Schumann, Ulrich Schiewer, Ulf Karsten, Thorsten Rieling. Viability of bacteria from different aquatic habitats. II. Cellular fluorescent markers for membrane integrity and metabolic activity. Aquatic Microbioal Ecology. Vol. 32: 137150, 2003

Abstract:
We applied different types of fluorescent markers to natural bacterioplankton from different aquatic systems to investigate microscopically the percentage of viable bacteria. To characterise viable bacteria, cell-specific respiration was measured by cyanoditolyltetrazolium chloride (CTC) reduction.
Membrane integrity was investigated with 3 dead cell stains (SYTOX Green, propidium iodide and ethidium homodimer-2). Cellular enzyme activity was detected by artificial substrate analogs with a high cell retention (CellTracker Green CMFDA for cellular esterase and 7-amino-4-chloromethylcoumarin
L-leucine amide, hydrochloride [CMAC-Leu] for cellular peptidase). The percentage of impermeable, i.e. morphologically intact, cells accounted for 22 to 81% of the total cell number at all locations. Although up to 48% of all bacteria were respiring, they averaged between 10 and 14% in freshwater, estuarine
waters and in the Baltic Sea. The portion of esterase-positive cells correlated significantly with the concentrations of dissolved (DOC) and particulate organic carbon (POC) as well as with chlorophyll a (chl a) content. Cellular esterase was shown by this labelling technique in only 9% of freshwater, 12% of estuarine and 5% of Baltic Sea bacteria, . The percentages of bacteria with cellular peptidase were even lower with 6, 5 and 3%, respectively. The different amounts of intact and respiring bacteria as well as those with cellular hydrolytic enzyme activities require not only correct operational definitions of
active and viable bacteria, but also the appropriate choice of fluorescent markers regarding the goals of investigation. Fluorescent labels for cellular hydrolytic enzymes will also provide a new tool to localise active cells in aggregates or on sediment particles, where, besides the respiration of organic carbon, hydrolysis of organic substances is an important conversion process.



JK Sugden. Photochemistry of dyes and fluorochromes used in biology and medicine: some physicochemical background and current applications. Biotechnic and Histochemistry, Volume 79, Issue 2 April 2004 , pages 71 - 90.

Abstract:
An overview of the basic principles of photochemistry is presented to facilitate discussion of fluorescence, quenching and quantum yields. These topics in turn provide the foundation for an account of fluorescence spectroscopy and its application to microscopy. A brief overview of light microscopy and the application of fluorescence microscopy is given. The influences of molecular features, such as aromatic character and substitution patterns, on color and fluorescence are described. The concept of color fading is considered with particular reference to its effect on microscopic preparations. A survey of representative fluorescent probes is provided, and their sensitivity, application, and limitations are described. The phototoxicity of fluorescent molecules is discussed using biomembranes and DNA as examples of targets of toxicity. Photodynamic therapy, a relatively new clinical application of phototoxicity, is described. Both anticancer and antimicrobial applications are noted, and an assessment is given of the current ideas on the ideal physicochemical properties of the sensitizing agents for such applications.

Hello Tony,

these are real birthday presents... (my B-day is August, 7th :-)!
I am just reading all the literature, but YES, some of these proposal really could solve my problem...

As soon as I am through the literature, Ill be back with my conclusions! Please be patient :-))

Best regards,

Gerhard

Gerhard:

I'm glad these were able to point you in the right direction!

Good luck!

Im back...

Tony:
I read all the literature, and YES, I think this is the direction I should go with my next experiments...
I will do some of these assays today to adress my rather special problem, but I am rather sure, it will work out!

I will post the outcome of the experiment in the next few days...

Regards,

Gerhard

Great to hear you're heading in the right direction.

Can't wait to hear the outcome of your preliminary experiments.