Current and Future Developments in Proteomics

Bacterial Biofilms are densely packed microbial communities formed by the microorganism to escape from any external threat. These Biofilms are composed of a polysaccharidic matrix, which formed a slimy layer outside the cell wall and protected the microbes from any damage (both physical and chemical). Biofilm can be developed by microorganisms on any surface, including medical devices, oral cavity and other biomaterials. These biofilms are difficult to treat and required almost 10,000 more concentrations of antibiotics compared to planktonic microorganisms. These biofilms are the main hindrance in the treatment of hospital-acquired infections. Majority of the treatment against microorganisms fails due to these biofilms in a clinical setting. Combination of different antibiotics, natural molecules and other strategies is in use to combat these biofilms. Microorganisms inside the Biofilm trigger some genes by quorum sensing and affect the expression of several protein factors. The current chapter will focus on the use of the proteomic approach for better understanding the nature and role of Biofilm in microbial pathogenesis and lower the emergence of drug resistance in these microorganisms.

Proteome deals with complete set of proteins, expressed by a genome of a cell present inside of an organism at a specific time period. It includes expression study, information of modifications in proteins, interactions with other protein biomolecules, etc. The goal of proteomics is to know new protein information, whether a protein is over expressed or under expressed in specific situation and overall gives information of its effect on an organism. Thousands of proteins can be examined at a time as compared to other methodologies. Many advanced technologies have been evolved to investigate proteome in depth and generate a huge amount of data. The most common high throughput techniques such as polyacrylamide & agarose gel electrophoresis and their advanced versions in combination with mass spectrometry are being used in modern proteomics. The study of microbial proteome data helps us to know how bacteria get resistant to particular drug and which biomolecules are involved in that process. Database also gives the opportunity to develop better drugs that target new places on bacterial surface or new drugs on the same target. The advancement of proteomics technique and their applications in microbial research has granted a new hope to explore disease biomarkers and the development of diagnostic assays. Microbial protein's benefits are extensively used in the agricultural sector. Proteomics profiling has a key role in disease identification in humans and animals. Thanks to protein information as new targets are identified and more safer and effective drugs are produced.

Almost 20 years after the publication of the first microbial sequences, today's world is growing in the direction of the post-genomic era with the understanding of transcriptomics and proteomics, which offer insight into cellular physiology. As the function of protein is associated with the phenotypic characters of an organism, the power to exceed the entire protein network of a microbial world has a huge impact on microbiology. Nowadays, mass spectroscopy (MS) has been extensively used in microbiology for the identification, characterization, and serotyping. In recent times, the large genome size, its complexity, and their study is a big task for the microbiologist. It is interesting to see how scientists examine microbial proteomics and analyze them. Proteomics has a key task to carry out in endeavors to develop far-reaching cell maps of biochemical procedures happening inside explicit microorganisms at given spatial and worldly focuses. Here we are going to discuss the methodologies for the identification of bacteria up to species level with great accuracy with the use of proteomes of bacterial pure culture. This chapter will also discuss the sample preparation and identification of a specific strain of bacteria for the liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the recent application of it.

Genomics and proteomics methods have witnessed a huge surge in the recent decade. Provided the pace of data accumulation from several advanced technologies such as next-generation sequencing, transcriptomics, ChipSeq and quantitative proteomics, the parallel growth in the functional annotation is relatively slow and needs continuous curation and analysis of existed data from repositories. Nevertheless, the standard procedures of functional annotations of proteins which were based on classical data sets, need to be revised in light of advanced and more comprehensive data. In fact, most omics technologies are now integrating and their merger can provide a much realistic and holistic picture of any biochemical and molecular scenario provided the data handling and curation is performed on cellular and molecular principles. A number of genomes and proteomes have been functionally annotated in the past; microbial genomes offer a better opportunity with less complex models. Nevertheless, microbes are among one of the most common causatives of pathogenic diseases and their diagnosis, treatment is limited by available information of proteins and their functions. Previously annotated hypothetical functions to proteins are likely to change in some cases therefore a number of research groups have attempted to re-annotate the microbial genomes with newer data-sets and new tools. Re-annotation of Mycobacterium tuberculosis was one such example. Besides pathogenic microbes, emerging trends in various useful microbes sequencing have shown a tremendous increase in information on human microflora and remain a highly prospective area in biology. Systems biology lies at the interphase of biology, mathematics and computational biology and involves a holistic approach to visualize a biological phenomenon. This chapter describes the basic principles involved in the functional annotation of hypothetical proteins in light of emerging datasets and tools. Besides the suggestions on improving standard pipelines, it also presents a summary of recently annotated microbial genomes and future prospects of involving systems biology in the functional annotation for improved quality and output.

Bacteria are simple organisms, optimized with basic but robust cell regulation mechanisms for efficient growth. Nonetheless, fitting in the description, they possess remarkable adaptive capacity with diverse survival strategies. Post-translational modifications (PTMs) provide a competitive edge to adapt bacteria with limiting, fluctuating nutrients and extremes of environment variables. PTM is one of the cellular processes in bacteria and helps them to adapt to a new environment for their survival. Several post-translation modifications regulate bacterial functions and provide strength to bacteria for surviving in adverse conditions. On-going investigations revealed many reversible or irreversible novel bacterial PTMs like the addition of simple group (acetylation, phosphorylation, methylation and hydroxylation) or composite molecules (AMPylation, ADP-ribosylation, glycosylation and isoprenylation), or small protein ubiquitin and modifying the side chain residues like (elimination and deamidation). PTMs are recognized as important players in directing cellular dynamics like cell metabolism, stress response, pathogenesis and virulence factors. Bacteria with several PTMs are capable of modulating the signaling pathways by destabilising the host cell defense machinery, protein-protein interactions, ultimately promoting their replication. Currently, many studies focus on the relationship between PTMs and antibiotic resistance, increasing bacterial tolerance to various antimicrobials. A paradoxical behaviour is that a single protein may be modified at one or variable positions in interspecies, but changes also exist in the same species. So, to characterize the multifaceted interactions of PTMs, it is still a challenge in metabolic engineering, synthetic biology, and medical sciences. Therefore, understanding of bacterial PTMs and PTMs directed host proteins modification will provide better insights into hostpathogen interaction. This chapter focuses on the roles of PTMs in nutrition sequestration and cellular response. Furthermore, we discuss the prospects and advances of proteomics tools in enhancing knowledge related to PTMs of human gut microbiota.

Posttranslational modification of proteins is a prevalent method for the regulation of cells according to changes in the surrounding environment and diversifications. Pupylation, architecturally similar but not homologous to eukaryotic proteasomal degradation machinery, exists in a certain order of bacteria, especially actinobacteria. Pupylation supports the bacteria to survive under challenging environmental conditions like stress (physical or chemical) and nutrient starvation. Pupylation is also involved in iron homeostasis, which is necessary for cellular metabolism and the normal growth of bacteria. Pupylation is a posttranslational modification through which intrinsically disordered proteins are tagged for proteasomal degradation. Although this process is functionally reminiscent of ubiquitination in eukaryotes; it is carried out by a different set of enzymes in evolutionarily connected bacterial carboxylate-amine ligases. In this chapter, we will discuss the recent advances in the understanding of how proteins are tagged for proteasomal degradation in actinobacteria and its role in the survival of mycobacterium during pathogenesis in the host. Furthermore, we will examine the role of accessory factors associated with the proteasomal system in bacteria that function independently of proteolysis.

Sample preparation is the most crucial step in the proteome research of microbes. 2D gel electrophoresis (2-DGE) is a high throughput approach by which all proteins imprint on the gel in the form of spots or dots. Various protocols for the extraction of proteins exist in the literatures that are compatible with 2-DGE based proteome analysis from different microbes. Analysis of low abundance proteins, the solubility of proteins, and their resolution on the gel are some major issues observed with sample preparation and 2-DGE. To combat these issues, researchers have developed improved versions of the existing protocols using detergent/chaotropes for the enrichment of proteins during extraction along with the compatible chemical precipitation for better resolution of 2D gel pattern. In this chapter, we will discuss the overview, challenges encountered during the protein sample preparation, and their possible solutions in order to get a better 2-D gel of microbial proteins.

Pathogens like viruses, bacteria, protozoa, fungi and helminths have caused and are causing diseases as well as the death of mankind since time immemorial. A proper understanding of the infection mechanisms will aid in designing efficient vaccines, diagnostics and drugs for efficient disease management. When compared to genes, proteins are involved in effector functions of the cell (of the pathogen as well as the host) and play a pivotal role in pathogen entry into host cells, its transmission and disease pathogenesis. Hence, the study of proteins will decipher the intricate communication networks involved in disease pathogenesis and will also elucidate their use as biomarkers in diagnostic and therapeutic scenarios. Thus, this chapter will highlight the proteomic and mass spectrometry-based approaches in comprehending the pathogen interactions with the host at the molecular level as well as biomarker development for use in diagnostics, prognostics and disease management.

Microbial metalloproteomics involves a detailed analysis of the proteins that have metals as an important part or known to bind to metals in biological samples. Recent updates showed that metalloproteome helps in understanding the role of different environments/conditions in the survival of microbes and is involved in microbial pathogenesis. Microbial metalloproteomics could also be used in understanding the resistance mechanisms of microbes. We have explored different metalloproteomic approaches such as inductively coupled plasma-Mass spectroscopy (ICP)-MS, X-ray absorption/fluorescence, radionuclide, and bioinformatics. We have also discussed the role of metalloproteins such as metallo-beta-lactamases in microbial drug resistance, the alternation of the microbial proteome in response to the metal, and their role in host-pathogen interactions. We have also surveyed different therapeutics targeting the microbial metalloproteins. Current advancements in the metalloproteome would help in understanding the mechanism better and the adequate role of metalloenzymes and proteins in conferring the drug resistance in microbes.

Tuberculosis (TB) is a universally prevalent disease caused by an aerobic, gram-positive bacterium Mycobacterium tuberculosis (M. tuberculosis). It has continued to pose a significant threat to human health. The emergence of multi-drug resistance (MDR) strains of Mycobacterium tuberculosis (M. tuberculosis) has further worsened the situation worldwide. Its genome has been primarily focusing on the past exploration of the molecular basis of disease. The genetic information or genes are transcribed into mRNA that is then processed, spliced, and translated into a single or multitude of proteins. Proteomics is the large-scale study of proteins, focusing on their structure and functions. For understanding the biology of any living organisms including humans, we need to decode the information encoded by proteins or its associated genes, as it reflects the true status of the cell. Although proteins play a major role in the whole life of the organism, its profile may vary from cell to cell due to spontaneous changes or biochemical interaction of microbial genome with environments. Even an immense amount of DNA/gene sequences data has been deposited by the scientific community in the databases, which is very useful in determining the virulencity, pathogenicity of the organisms. It simply contains a complete sequence of genomes that lacks its usefulness to illuminate biological function. The regulation process of a single cell involves complex mechanisms, including a multitude of metabolic and regulatory pathways for its survival. To date, no strict linear relationship has been documented between genes and proteome of the cell. The available technologies, i.e. microarray, identify a large number of differentially expressed genes very quickly. However, these have failed to identify the functional significance of the associated genes. In this chapter, we highlight prospects of the advances in the proteomics study that could be beneficial to mycobacterial research. Also, it will provide information to explore the possibility of protein-based biomarkers in the development of new diagnostic therapeutics for TB.

Mycobacterium tuberculosis (MTB) has been an exceptionally successful human pathogen over the centuries, infecting almost one-third of the global population. An exponential increase in tuberculosis (TB) cases, mainly by the drug-resistant (DR) strains of MTB, has created an urgent need for identifying and developing new antituberculosis drugs acting via novel mechanisms. The multi drug-resistant (MDR) TB and extensively drug-resistant (XDR) TB strains, accelerating through drug specific resistance amplification, are resistant to a majority of antibiotics used in the treatment and are challenging to remove from the host’s system. Since proteins are the functional beings of the biological arrangement, they make promising drug targets for immunodiagnostics or therapeutics. To identify and characterize such novel proteins, which directly or indirectly regulatedrug resistance in mycobacteria, proteomics approaches could be successfully employed. Serological techniques like immunoassay have higher chances of rendering false positive or false negative results and hence could be rectified by using more sophisticated techniques like mass spectrometry. In the past two to three decades, proteomics-based approach has seen a pivotal rise. The application of proteomics-based approaches has helped to gain insights into MTB and its relevance to clinical science. They have aided in the identification and characterization of novel proteins. To have a better understanding of pathophysiology of MTB, proteome-based science could help simultaneously in the identification of proteins, which can be potential targets. Recent progress in the area of proteomics has opened up the doors to address many previously unanswered questions, with studies on DR-TB being no exception. The Beijing family of MTB forms an interesting candidate for proteomic analysis as it constitutes 13% of the global isolates and has higher chances of acquiring drug resistance. Proteomics can play an important role in the discovery of biomarkers for TB and other diseases. Also, it can aid in the development of effective vaccines as well as simple, rapid, and cost-effective tests for the diagnosis of TB, which are crucial for the management and control of the disease.