Computational Molecular Biology 2024, Vol.14, No.1, 36-44 http://bioscipublisher.com/index.php/cmb 38 2.2 Sample preparation and data analysis The process of sample collection and protein extraction is critical for proteomic studies. Proteins are typically extracted from bacteria grown under laboratory conditions or directly from the site of infection to understand the in vivo state of the pathogen (Cash, 2011). Following extraction, proteins are subjected to techniques such as MS or 2-DE for separation and identification. Data analysis in proteomics is complex and involves bioinformatics tools to interpret the large datasets generated, identifying protein biomarkers that correlate with virulence (Tjalsma et al., 2004). 2.3 Advantages over other biological approaches Proteomic approaches offer several advantages over genomic and other molecular biology techniques (Table 1). While genomics can predict potential virulence factors, proteomics can confirm their expression and modification in response to environmental cues or during infection. Proteomics provides a dynamic view of the pathogen's biology, revealing how proteins interact and change during the host-pathogen interaction, which is crucial for understanding bacterial virulence and developing targeted therapies (Pérez-Llarena and Bou, 2016). Additionally, proteomics can uncover the secretome and surface proteins, which are often key to pathogen-host interactions and potential vaccine targets (Dwivedi et al., 2016). 3 Proteomics in Identifying Virulence Factors 3.1 Identification of pathogenic proteins Proteomic techniques have been instrumental in identifying proteins that play a role in the virulence of bacterial pathogens. These techniques, including comparative genomics, transcriptomics, and proteomics, have been applied to a range of bacteria such as Neisseria meningitidis, Yersinia pestis, Mycobacterium tuberculosis, and Staphylococcus aureus (Figure 1) (Wu et al., 2008). Proteomics, in particular, has been successful in studying post-translationally modified proteins of bacterial pathogens, which are often critical to their virulence. The identification of these proteins provides a foundation for further investigation into their functions and mechanisms, which can be explored through phenotypic analyses such as mutagenesis and biochemical methods, as well as structural biology. The figure from Pivard et al. 2023, offers a detailed analysis of the proteomic variations in Staphylococcus aureus linked to genetic differences among strains. The use of principal component analysis (PCA) clearly differentiates the strains into distinct clusters based on two genetic markers: agr specificity groups and clonal complexes. Panel A showcases the distribution of various agr types, while Panel C does the same for clonal complexes, each represented by different colors, helping visualize the genetic diversity and its correlation with virulence profiles. The eigenvalue plot in Panel B quantitatively supports the PCA by showing the percentage of variance each principal component accounts for, with a significant drop after the first two dimensions. This study effectively highlights the complex relationship between genetic background and virulence in S. aureus, potentially guiding targeted therapeutic approaches. 3.2 Function of virulence proteins The functions of virulence proteins identified through proteomic analyses are diverse, but they often include toxins or enzymes that compromise plant health. The secretome, which encompasses proteins secreted by bacteria, plays a crucial role in bacterial survival and pathogenicity. For instance, the gram-positive bacterium Bacillus subtilis secretes approximately 90 extracellular proteins, many of which are involved in virulence. These proteins can include nonenzymatic toxins and enzymes that directly affect the host. Proteomics has revealed that the actual composition of the extracellular proteome can differ significantly from genome-based predictions, highlighting the importance of direct proteomic analysis in understanding bacterial virulence (Tjalsma et al., 2004; Bonar et al., 2015). 3.3 Comparative proteomics Comparative proteomics involves analyzing the protein profiles of pathogenic versus non-pathogenic strains or the changes in protein expression before and after infection onset. This approach has been used to identify protein biomarkers for virulent bacterial isolates and to correlate these biomarkers with the outcome of bacterial infections
RkJQdWJsaXNoZXIy MjQ4ODYzNA==