Proteomic response of bacteria during the interaction with a host cell in a model of Mycoplasma gallisepticum

Abstract

Parasitism, which is a way of the coexistence of bacteria with a host, is a very interesting phenomenon. Although the pathogenicity of many microorganisms has been studied in detail, many aspects of this phenomenon remain poorly understood for most bacteria. Among interesting objects for studying pathogen–host interactions are the members of the Mycoplasma genus. Despite a very broad geographical range of habitat of these bacteria, their pathogenicity has been poorly studied. Almost all living beings are the hosts of mycoplasmas, and the reduced genome of these microorganisms makes them a convenient object for model studies. Here, we have described for the first time an experimental label-free method for the analysis of the protein profile of a bacterium immediately during its interaction with a host cell, which made it possible to understand what changes the pathogen undergoes at this stage of infection. The method involves the sampling of eukaryotic cells at different times of cultivation with the pathogen, the sedimentation of infected cells, the preparation of a cell lysate, the hydrolytic cleavage of cell proteins, and their identification by mass spectrometry (HPLC–MS). Changes in the protein abundance were analyzed by the method of multiple reaction monitoring (MRM). In the present work, a model of the interaction of the bacterium Mycoplasma gallisepticum with chicken erythroblasts (HD3 cells) at the stage of acute infection was studied. A panel of 100 proteins was taken for the analysis, which were chosen based on their functions and literature data. To obtain the greatest number of identified proteins, several methods of their hydrolytic cleavage with subsequent preparation of the peptide extract were used: digestion in solution using the RapiGest SF surfactant, digestion using sodium deoxycholate and urea, and the digestion of proteins in gel. As a result, proteins were identified that, probably, are involved in the interaction of M. gallisepticum with the host cell at early infection stages; most of them were the proteins of adhesion, glycolysis, and ribosomal proteins.