Team:Freiburg/Shampoo

Future application: The probiotic shampoo
Since we see great potential in our approach of specific binding to a structure of interest, we came across another way of using this system, namely to target structures of epithelia with our nanobody-expressing spores.

The skin is the human’s largest organ and the habitat of millions of microorganisms. Normally those organisms do no harm and can even be beneficial for the human skin. However this condition can change into imbalance due to an increased expression of cells, leading to undesirable effects.

The fungus Malassezia furfur, a natural part of the skin flora, is mostly located on the human scalp. However, overpopulation of M.furfur can lead to irritation and production of additional skin cells. The excessive cells will die off, which results in (so called) dandruff.1

The common treatments for dandruff are mainly based on the use of anti-fungal shampoos, which inhibit the division of fungal cells. These shampoos include a high amount of detergents to prune of the dead cells, often containing strong chemical compounds like zinc pyrithione2,3. Such components can provoke irritation of the skin, since they affect the whole skin flora and not only M. furfur.

Ideally, our shampoo only targets M. furfur through specific binding, instead of affecting each cell equally. Resulting in an improvement of the efficiency of anti-fungal shampoos and decreasing the needed amount of detergents or chemical compounds.

We believe that applying our Nanocillus for the specific binding to M. furfur could be a viable solution. We hereby want to reduce the population of the fungus without harming the overall skin flora.

As nanobodies, single-chain antibodies, are able to specifically bind to surfaces, they could be used as a binding component in a “targeting-shampoo”. Their advantages include enormous specificity, simplicity, stability and their producibility in bacteria4. In general leading to a low cost in production and to receive multivalency they can be variously expressed on our modified B.subtilis. In doing so, a higher efficiency is obtained.

For this approach specific marker proteins are required for targeting to reduce dandruff. We would target the surface protein Malf11, which is expressed on M. furfur cells.

To establish this method in the lab, we used a surface coated with GFP as a model for the surface of M. furfur with the targetable proteins and an anti-GFP nanobody as the nanobody, which is able to bind Malf1. The GFP was coated on a 96-well plate and washed with either the pure detergent solution, nanobodies in the solution or a combination of a detergent solution and the nanobody-expressing spores of Bacillus subtilis. The plates were treated with the different solutions like described here in the methods.

With the resulting data we were able to analyze the effect the spores have on the process of washing. As a positive control we used 2% of SDS and the negative control was PBS together with one construct at a time. To determine the washing efficiency of our nanobody-spores we measured the fluorescence-intensity of coated GFP plates followed by washing them with spores and shampoo. Thereafter the difference between the GFP-fluorescence before and after the washing procedure was calculated. Afterwards the difference was divided by the fluorescence of the coated GFP to determine the washing efficiency.



Results
For the application of the Nanocillus as an anti-dandruff agent, we tested a 16% (v/v) solution of a commercially available shampoo with two B. subtilis spore constructs in which the nanobody was fused to the surface protein CotZ over a G4S-linker (BBa_K2114001) and an α-helical-linker (BBa_K2114002) which were expressed in the wild type and in a ΔCotZ strain. As a further check we used the detergent together with only the nanobody.

The shampoo treated wells show an increase in efficiency with the use of the spores rather than the use of only the nanobody. Although the negative controls show that the addition of nanobody to PBS increases the efficiency more than the addition of wild type spores(Figure 1). This means that the improvement of the nanobody-expressing spores is higher than the improvement by the wild type spores.

Figure 1: Efficiency of washing. Calculated column chart of the wash efficiency of shampoo in combination with anti-GFP nanobody [Nb] in solution, BBa_K2114002 aGFPnano_HA_G4S_cotZ ΔcotZ spore [SP2], BBa_K2114002 aGFPnano_HA_G4S_cotZ spore [SP1], BBa_K2114001_aGFPnano_HA_aHelix_cotZ ΔcotZ spore [SP3]. none washed [NC1], WT spore [NC3], PBS [NC2], anti-GFP nanobody in PBS [NC5], 2% SDS [PC].

The efficiency of only the shampoo was around 54%. By using the anti-GFP nanobody spores the wash efficiency increased to almost 70%. The addition of the nanobody to the shampoo doesn’t increase the washing efficiency rather shows how the use of the spore makes a better improvement to the efficiency. The negative controls show how the wild type spores provide lesser improvement to the efficiency than the nanobodies.

The best result was achieved with the detergent with the ΔCotZ spore expressing BBa_K2114002.

Discussion


We could determine an increased washing efficiency using our nanobody-displaying spores in comparison to the shampoo only and the shampoo supplemented with purified nanobody, respectively. Additionally, we see an increase of the washing efficiency in our nanobody-displaying spores compared to washing with the nanobody only. We hypothesize that the modified spores bound to the coated GFP and resulted in an aggregation therefore facilitating the removal.

For further validation of this trend, additional repeats are necessary to confirm statistical significance. Expanding the approach of such a probiotic shampoo could include the utilization of antifungals drugs, which would only target the abundant fungus, such as M. furfur in case dandruff1. A further major benefit of this probiotic shampoo could be represented by the B. subtilis spores themselves, which could replace plastic microbeads5 in many commercial shampoos. By reducing the amount of micro plastics, the positive impact on the environment would be enormous! Since the spores of B. subtilis are ecologically compatible and even beneficial for the flora!6

There are no limits to the various possibilities using our spores to improve and change for the better.

‘cause truly… spore is more! :)

References

1. Dolk, E. et al. Isolation of llama antibody fragments for prevention of dandruff by phage display in shampoo. Applied and environmental microbiology 71, 442–450 (2005).
2. Chandler, C. J. & Segel, I. H. Mechanism of the Antimicrobial Action of Pyrithione. Effects on Membrane Transport, ATP Levels, and Protein Synthesis. Antimicrobial Agents and Chemotherapy 14, 60–68 (1978).
3. Lamore, S. D., Cabello, C. M. & Wondrak, G. T. The topical antimicrobial zinc pyrithione is a heat shock response inducer that causes DNA damage and PARP-dependent energy crisis in human skin cells. Cell stress & chaperones 15, 309–322 (2010).
4. Muyldermans, S. et al. Camelid immunoglobulins and nanobody technology. Veterinary immunology and immunopathology 128, 178–183 (2009).
5. Mazurais, D. et al. Evaluation of the impact of polyethylene microbeads ingestion in European sea bass (Dicentrarchus labrax) larvae. Marine environmental research 112, 78–85 (2015).
6. Hirooka, K. Transcriptional response machineries of Bacillus subtilis conducive to plant growth promotion. Bioscience, biotechnology, and biochemistry 78, 1471–1484 (2014).

Posted by: iGEM Freiburg

Nanocillus - 'cause spore is more!

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