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Laboratoire : SPECTROMETRIE DE MASSE BIOLOGIQUE ET PROTEOMIQUE
Institution d’accueil : Paris Sciences et Lettres - PSL
Ecole doctorale : Chimie physique et chimie analytique de Paris Centre - ED 388
Description :
Clouds are key components in Earth’s functioning. In addition of acting as obstacles to light radiations and chemical reactors, they are atmospheric oases for airborne microorganisms. The presence of microbes in the atmosphere and their transport over long distances through the Earth’s surface has been reported by many groups. They can disperse a contaminant locally over a few kilometers or on a continent-wide basis and can also play a role in climate change and ecological processes. They can even be beneficial to human health and the environment. It will also be a valuable tool for the study of the dispersion of allergen species by air displacement. Microorganisms have been detected and partially characterized by DNA and RNA studies.
The present project aims at the development of a strategy for the study of bacterial spreading at the proteomic level. Because proteins, and subsequent functional mature proteoforms, are the final products of the genes, they could be very valuable descriptors of the aeromicrobiology of the clouds. Their characterization should allow a better understanding of the metabolic activity of living bacteria in bioaerosols and their identification according to atmospheric fluxes and precipitations. To reach this goal, the analytical challenges of low concentration (requiring high sensitivity), complex mixture (metaproteomic) and non-sequenced species (proteogenomic) will be addressed.
At the interface of environmental science, biology and chemistry, clouds proteomics addresses some issues concerning the effect of the climate on cloud microbiome
metabolism, and reversely the impact of microbial activity on the « bioprecipitation » and the climate by accelerating cloud formation.
Context and Motivation :
Water scarcity is becoming a major problem throughout the world due to increased demand for population growth and urbanization. As part of a sustainable economy, rainwater harvesting as an alternative source of water is of considerable concern.
However, the use of rainwater as drinking water is limited by water quality problems in terms of chemical and microbiological contamination and its potential health risks. Many studies on the chemical contamination of rainwater have been carried out, but microbial contamination remains relatively unknown due to the analytical challenges involved in this type of study for the characterization of pathogen and allergenic species (Kaushik R et al, 2013).
Clouds represent one of the most important bioaerosols. Within the atmospheric system, clouds are atmospheric interface with the ground : they physically connect high altitude with the surface by being to a large extent at the origin of wet deposition of aerosols, including microorganisms. Cloud water is a complex mixture of soluble gas and particles dissolved into millions of micron-sized water droplets, and forming very reactive and dynamic systems (Vaïtilingom et al., 2013, Vaïtilingom et al., 2010). As non-soluble biological particles, some microorganisms can physically impact clouds by acting as embryos for the formation of water droplets and ice crystals, with subsequent impacts on hydrological cycles (Besaury et al., 2017). Observations of microbiological features in fog and clouds raised the possibility that these also represent habitats for microorganisms, where they would actively take part in the chemical reactivity through metabolic activity and nutrient utilization (Amato et al., 2017).
Scientific Objectives :
Biologicals particles including bacteria, pollen and fungi can all be found in airborne particles, or bioaerosols. Bioaerosols can spread disease to human, plants, animals on the scale to meters to continent, due to the potential for long-distance transport in the atmosphere (DeLeon-Rodriguez et al 2013). Bioaerosols may also play a role in climate change and ecological processes and may even be beneficial for human health and the environment (Barr et al 2013). A large variety of bioorganisms can be spread by bioaerosols, as they could be generated from water containing microbes but also from soils (Joung et al., 2017).
Clouds droplets host living cells. A study on microbial communities in cloud water by high throughput sequencing from DNA and RNA extracts, allowed to identify active species among community members. Communities consisted of 103−104 bacteria and archaea/mL and 102−103 eukaryote cells/mL, with a high diversity : more than 28000 distinct bacteria detected and 2 600 in eukaryotes. Proteobacteria and Bacteroidetes were the major bacteria, while eukaryotes were essentially distributed among Fungi, Stramenopiles and Alveolata (Amato et al 2017).
The present project aims at the development of a strategy for the study of microorganisms in clouds at the proteomic level. In the last years, proteomic has proved its complementary to genomic and transcriptomic, leading to a new field of sciences called proteogenomic.
On the present project, proteomic analysis should allow a better annotation of genomic data leading to a better comprehension of metabolism state of living bacteria and ecosystems present in the clouds. This metabolism is likely to be strongly impacted by the presence of specific gas such as CO2, Nox or O3.
The bacterial metabolism signature will be correlated with the sampling geographical area, the season, the pollution level (CO2, Nox) and the climate. This will be to our knowledge the first analysis of the aeromicrobiome without a priori.
Methodology and Planning :
Methodology :
The first objective of this project is to develop a strategy for the study of bacteria at the proteomic level. The samples will be prepared in sterile condition to avoid contamination. Different places of sampling at altitude will be considered, notably the station of Puy de Dome, which has a unique "cloud vacuum cleaner". Then, high-resolution, high-accuracy mass spectrometry coupled to nanoseparation techniques will allow the analysis of complex mixtures with high sensitivity. This is an essential benefit for this type of sample, containing a large number of organisms whose genome is not known.
After analysis, protein identification is usually performed by comparison with protein or genomic databases. Here we will need a proteogenomics approach. Different strategies will be used. After using the classic database filtering software, the de Novo sequencing will be performed. This will result in a home database containing new sequences. Overall, all of our data will be intercut with genomic data for better exploitation of proteomic data and a genomic annotation project.
The second objective will be to carry out the analysis at different times, from the same collection site. Due to the multiple origin of the bacteria in the cloud, we expect a great variability between these samples. The analysis of protein co-expression will consist of an initial description of a cloud ecosystem. Organisms but also protéoformes can give an indication of the particular chemistry present in the cloud.
Analytical challenges related to low concentration (requiring high sensitivity), complex mixture (proteomics) and unsequenced species (proteogenomic) will be addressed. Proteogenomics and proteomics are emerging sciences, and new tools to carry out these analyses are regularly proposed and improved. But the peculiarity of the sample type very diluted in sterile conditions is also to be considered and validated.
Planning :
Sample preparation protocol 3 months
NanoLC MS/MS analysis protocol : 2 months
Construction of a database according to literature : 1 month
10 campaigns of sampling/proteogenomics/library searching and updating (nanoLC MS/MS and RT PCR) : 18 months
Statistical treatment and analysis of the data : 4 months
Biochemical validation of some proteoforms as biomarkers : 2 months
Publication/communication : 6 months
Compétences requises :
Master M2 with a dominant in analytical chemistry, or analytical instrumentation. A (theoretical) knowledge in mass spectrometry and miniaturized separation techniques is expected. The candidates should be interested in analytical chemistry applied to biology since this doctoral project is at the interface of the two domains. He/she should have validated a full M2 master degree, or engineering degree (if possible with a final transcript >14/20).