Article pp.97-100 du Vol.23 n°1 (2003)

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SCIENCES DES ALIMENTS, 23(2003) 97-100

FOCUS : JSMTV

The proteomic approach for better understanding the physiology of bacteria adaptation

to food plant environments

M. Hébraud¹

The proteome is a new word which indicates the PROTEins expressed by the genOME of a living organism. Proteomic analysis consists in studying the global protein expression of an organism at the level of the different cell types, tissues, physiological liquids, or even in the subcellular compartments (nucleus, mito- chondria, chloroplasts …). The release of complete DNA sequences of several prokaryotes and eukaryotes organisms gives to the scientists the dictionary of all the genes which constitute the genetic inheritance of each of these organisms. In the cells, genes are first expressed as transcripts (mRNA) which are then transla- ted into proteins. The products from these two steps, mRNA and proteins, can be qualitatively and quantitatively different in the cells according to their nature, their localization, their physiological state and their surrounding environment.

Thus, the dynamic sights of transcriptomic and proteomic evolutions come to complete the static information given by the genomic sequences. The transcriptomic and proteomic approaches concern molecular entities which are linked in the cellular functions but whose qualitative and quantitative production are not always correlated. They may provide complementary informations on how work and are regulated some metabolic pathways, some physiological responses or other cellular mechanisms. So, genomic and post genomic represent what is cal- led today the integrative biology.

The proteomic analysis constitutes a tool of research suitable to bring a lot of very rich and accurate informations. It gives the possibility to obtain a global and dynamic sight of the organism functioning and of the events which occur during various cellular processes (differenciation, ageing, pathological modifications, effect of pharmacological treatments, environmental variations or stresses…). The protein analysis is the global image of a physiological state at a given time and it appeared that an organism or a cell can have many proteomes since the cellular regulations on the whole, and thus the qualitative and quantitative variations of the protein expression, are taken into account. Thus, the post-translational modifications (in particular phosphorylations and glycosyla- tions), which play a significant role, and even an essential role in the regulation of protein activity, can be visualised. This gives to the proteomic approach a

1. Station de Recherches sur la Viande – Microbiologie Plate-forme protéomique, INRA de Theix, 63122 Saint-Genès Champanelle

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98 Sci. Aliments 23(1), 2003 M. Hébraud

crucial interest for the study of cellular mechanisms through the proteins, these molecular entities which are finally the true acting agents in the cell.

The proteomic analysis comprises three main steps which each one con- cerns different technologies (fig. 1). The initial step is the two-dimensional elec- trophoresis (2-DE) in polyacrylamide gel which is the only method currently available capable for the simultaneously separation of several hundreds of proteins with a very high resolution. It utilizes two successive electrophoretic separations according to independent criteria, the isoelectric point (pI), then the molecular mass (Mr) of the proteins. The second step includes the setting in image, the digitalisation, the image treatment and comparison of the protein patterns constituted by the 2-DE gels. The last step, essential, is the identification of protein spots of interest which is, to date, generally realised by the mass spectrometry technology. The evolutions of methods and technologies during these last years have significantly improved the reproducibility, the resolution and analytical possibilities of proteomic. Thus, we have passed from the con- templative era, which consisted to compare de visu 2 or 3 physiological states with sometimes the expensive identification of some proteins, to the analytic era with the qualitative and quantitative comparison between proteomes, thanks to softwares, and the identification of several tens protein spots.

However, the proteomic approach doesn’t allow an exhaustive study of the whole proteins present in an organism or a cell and it is necessary to keep in mind that the analysis spectrum is limited by the first steps of solubilisation and electrophoretic separations. Indeed, many proteins, in particular membrane one, are insoluble or slightly soluble, which makes their extraction very difficult and even impossible. In fact, 2-DE classically allows to separate proteins of pI ranging between 3 and 8 and of Mr ranging between 10 and 100 kDa. Conse- quently, it is necessary to take into account that these limitations result in a par- tial analysis of proteomes. In spite of that, 2-DE remains one of the most efficient technique for proteomic analysis because it combines two fundamental characteristics which are the ability to separate several hundreds of proteins and the possibility to follow quantitatively the kinetic of protein expression during a biological phenomenon.

For these reasons, the proteomic approach become essential in various fields for the comprehension of the organisation and the functioning of cellular factory. In the field of food industry, the microbiological safety of foodstuff and food plants is a major public health concern of our society. Moreover, the eco- nomic consequences may be sometimes dramatic. In this context, it is important to better understand the physiology of foodborne micro-organisms. The proteomic analysis allows to investigate the mechanisms of adaptation to environmental variations and stresses they have to undergo such as refrigeration, salt addition, pH modifications, nutritional depletion, desiccation or cleaning disinfection treatments. Generally, when bacteria are subjected to these stresses, they are in a sessile mode of growth and form a biofilm or aggregates which adhere on the surfaces. In this condition, bacteria are much more resis- tant than in a planktonic mode of growth, i.e. in suspension in a liquid medium, and can survive several weeks. Thus, they constitute an important risk of (re)contamination of foodstuffs. So, the study of the physiology of sessile cells adhering on biotic or abiotic surfaces is particularly crucial.

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The proteomic approach for better understanding the physiology… 99

Figure 1

Different steps and technical settings involved in proteomic approach

The stress adaptation and the physiology of growth in biofilms are two topics we study by a proteomic approach. Two bacteria are mainly concern by our studies, the foodborne pathogen Listeria monocytogenes and the food spoi- lage Pseudomonas fragi. For example, we have shown that the cold shock adaptation of P. fragi resulted in the over-expression of four small proteins (1).

These four proteins share 80% sequence identity and function by pairs with two different kinetic of expression and probably different mechanisms of regulation.

In another study, we have reported that the expression of 20% of the L. monocytogenes proteome was affected by the biofilm mode of growth in bio- film by comparison to the planktonic mode of growth (2). Some of the proteins over-expressed when the bacteria adhere onto a surface and begin to form a biofilm are involved either in the metabolism of carbohydrates or in the metabolism of amino acid, or in the RNA or protein synthesis. The identification of other proteins by Maldi-Tof mass spectrometry is in progress.

Protein extraction

Isoelectrofocalisation

3 pI 10

SDS-PAGE

Digitalisation

Database query Spectrum treatment

SM Maldi-tof Spot extraction/

hydrolysis

Staining or autoradiography

Identification of protein spots Analysis

Image treatment

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100 Sci. Aliments 23(1), 2003 M. Hébraud

REFERENCES

1. MICHEL V., LEHOUX I., DEPRET G., ANGLADE P., LABADIE J. and HÉBRAUD M., 1997. J. Bacteriol., 179: 7331-7342.

2. CHAVANT P., 2001. Étude physiologique et moléculaire de Listeria monocytogenes en biofilms. Doctorat de l’Université Blaise Pascal de Clermont-Ferrand.