**5. Proteomics**

repressed. Additionally, genes encoding tricarboxylic acid, malic enzyme, PEP carboxykinase, gluconate-glyceraldehyde-3-phosphate dehydrogenase and ATP synthase cycle enzymes

The microarray technique provided an advance in the research with important organisms, such as the members of the genus *Corynebacterium*. Nevertheless, this technique has some limitations, such as high noise interference, inability to detect transcripts with a low number of copies per cell, low coverage of transcripts, and dependence on prior knowledge about the genome for the preparation of the probes, consequently generating little information about

As a result of these limitations and the advent of NGS platforms, a promising alternative technique was developed, RNA-seq. Through this technique, it was possible to obtain more accurate, fast and reliable analyzes from cDNA sequencing. The advantages of this method are: low occurrence or absence of interference, detection of small transcripts that would not be detected by other methods, low cost and reduction of time and work to prepare the samples. RNA-Seq is considered an ideal tool for the analysis of complete transcriptomes and is applied in the exploration of expression profile, and characterization of differentially expressed genes. Thus, it represents an important tool to uncover the mechanisms of virulence and pathogenic-

Relating to this, two studies with *C. pseudotuberculosis* simulating the stress conditions faced by the bacterium during infection in host were performed. The first study was with strain *C. pseudotuberculosis* 1002, biovar *ovis*, which underwent three stress conditions: thermal, acidic and osmotic. Most of the identified targets were related to oxidation and reduction, cell division and cell cycle, and the *stimulon* of the three stresses presented induced genes that participate in the mechanisms of virulence, defense against oxidative stress, adhesion and regulation, revealing that they have important role in the infection process [53]. The other study, with strain 258, biovar *equi*, was performed using the thermal stress condition, similar to the conditions performed on strain 1002. Herein, 113 genes were considered induced, in which *hspR, grpE, dnaK* and *clpB* were highlighted due to its expression rates and participation

Recently, the first analysis of RNA-Seq with *C. diphtheriae* was developed, in which it was sought to investigate the alteration of the transcription profile between a wild strain and a Δ*dtxR* mutant, also to detect the operon structures from the transcriptome data of the wild type strain. The authors revealed that approximately 15% of the genome was differentially transcribed and that DtxR may also play a role in other regulatory functions, in addition to regulating the metabolism of iron and diphtheria toxin. Finally, they identified 471 operons

One of the representatives of the genus that had the gene expression regulation most studied is *C. glutamicum*. The RNA-Seq approach elucidated the regulatory mechanisms of several industrially relevant compounds, such as the dissolved oxygen concentration (DO), which is important in industrial microbial processes, providing new information on the relationship

in the mechanism of adaptation of the pathogen to high temperatures [54].

subdivided into 167 sub-operon structures [55].

between oxygen supply and bacterial metabolism [56].

were induced [50].

the transcript sequence [47].

66 Basic Biology and Applications of Actinobacteria

ity in microorganisms [51, 52].

The central base of molecular biology involves understanding how cells work and interact among each other. These cellular processes occur through the activity of biomolecules that act together throughout specialized mechanisms. This whole process involves storing the genetic information in the DNA molecule and the unidirectional flow of this information to the RNA and proteins. Proteins make up a large part of the cell molecular machinery, and the overall analysis of them provides the information needed to understand how cells work. This analysis is referred to as proteomics [61].

In 1995, the term "proteome" was taken as the set of proteins produced by a cell or tissue at a given time and condition [62]. As early as 1996, the term "proteomics" appeared to define the large characterization of all protein contents of a cell line, tissue or organism [63]. The study of the proteome currently refers not only to the knowledge of the protein content of a given organism in a given condition, but also includes the quantification, location, modifications, interactions and function of these proteins [64, 65].

This area has three strands: expression, structural and functional. Expression proteomics generally involves studies to investigate the pattern of protein expression in abnormal cells. This classification encompasses studies of qualitative and quantitative expression analysis of total proteins under two different conditions. The second analyzes the three-dimensional conformation and structural complexities of functional proteins. This strand makes it possible to identify all the proteins of a complex system and characterize the possible interactions of these proteins and protein complexes. Functional proteomics reveals the function of proteins based on their interactions with specific protein complexes and the detailed description of cell signaling pathways to which they are involved [66].

The most used methods for the identification and quantification of proteins are those based on mass spectrometry (MS). This technique allows the detection of compounds by the separation of the ions through mass-charge ratio. As each compound has a unique fragmentation pattern, the samples are ionized and separated with further identification of this pattern. Generally, two MS-based methods are currently most commonly used. The first method involves two-dimensional electrophoresis (2-DE) followed by staining, selection and MS. The other method involves isotopic markers to label proteins, separation by multidimensional liquid chromatography and MS analysis [65, 67, 68].

both surface and extracellular proteome of two *C. ulcerans* strains, where NanoLC-MS/MS was performed to analyze the set of proteins, which were similar expression patterns of puta-

The Genus *Corynebacterium* in the Genomic Era http://dx.doi.org/10.5772/intechopen.80445 69

The mapping of the extracellular proteome of *C. diphtheriae* through 2-DE and MALDI-TOF-MS detected proteins present in pathogenicity islands. According to these tests, possibly, the exoproteome of this pathogen is constituted of two distinct classes. The first involves molecules that have functions in the cytoplasm related with cell viability, such as protein synthesis and folding and detoxification mechanisms. The second class appears to be actively secreted and includes iron transporters and possible virulence factors that can be used in new

The exoproteome of *C. pseudotuberculosis* has also been extensively characterized in the past years [74]. The use of transposon-binding proteins was investigated through a method of data-independent LC-MS acquisition (LC-MSE), used for proteins identification and quantification that was applied to compare the exoproteome of two biovar *ovis C. pseudotuberculosis* strains, C231 and 1002, where there were found 44 presents in both isolates in a total of 93

Further, the combination of different proteomic methodologies as the 2-DE along with MALDI-TOF/TOF allowed the finding of 11 novel molecules in the *C. pseudotuberculosis* exoproteome, noncharacterized on the first comparative work [76]. The integration with *in silico* approaches also gives important insights about the behavior of the exoproteome. Pan-genome analysis can be performed to predict the set of exported proteins present in a large number of

The proteomic map of a *C. jeikeium* strain was examined through 2-DE and MALDI-TOF-MS, through peptide mass fingerprinting (PMF), a high throughput protein identification methodology in which a protein is digested with endoprotease to produce the small constituent peptides. In this investigation, most spots were associated with functions essential for cell viability, such as protein synthesis and energy production, as carbohydrate, lipid and nucleotide metabolism. The surface proteins SurA and SurB, the adhesin CbpA and Che cholesterol esterase, known to act as virulence factors were also identified in the extracellular

In addition to these efforts, structural characterization methods for protein elucidation have also been used. The DtxR repressor is activated by transition metal ions and acts on the modulation of *tox* gene expression in *C. diphtheriae*. Through X-ray crystallography, it was possible to determine the general architecture of this biologically active Ni(II) bound protein with a resolution of 2.4 Å [79]. In *C. pseudotuberculosis*, the ArgR protein that acts as a regulator of arginine biosynthesis, an important metabolic pathway for bacteria, had the C-terminal domain crystal structure determined from X-ray diffraction with a resolution of 1.9 Å. The interest in this molecule lies in the fact that it participates in a pathway that is absent in its hosts, which makes it a potential target for the design of new

tive virulence factors [72].

extracellular proteins [75].

genomes available on public databases [77].

vaccines [73].

proteome [78].

drugs [80].

A typical proteomic experiment involves the step of preparing the sample, consisting of separating and isolating the proteins from the cell lysates, followed by separation of the protein mixture, and then the individual portions can be analyzed. Analysis may involve the bottomup strategy and the top-down strategy. The first involves obtaining the peptides by enzymatic digestion of protein solutions and the subsequent separation of these peptides by liquid chromatography and MS analysis. In contrast, the top-down strategy involves the analysis of intact proteins by MS. For the quantitative determination of proteins two approaches are most commonly used: two-dimensional electrophoresis followed by staining, selection and identification by MS; and isotopic markers followed by protein separation by multidimensional liquid chromatography and MS [68, 69].

Proteomics studies involving the genus *Corynebacterium* mainly comprise studies with *C. glutamicum*, due to, in large part, its industrial importance in the production of amino acids. This species is investigated in relation to its genetics and physiology and, consequently, a diversity of information about its molecular biology and biochemistry available, including a variety of proteomic techniques. In consequence of the membrane organization with high concentration of mycolic acids, *C. glutamicum* has been used as a model for development and new proteomic technologies [70].

Proteomics analysis have also been used as an alternative to traditional molecular methods for the characterization of poorly known bacteria, especially those of clinical interest, by reason of the ability of these methodologies to provide a fast and reliable identification of these species. The matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) technique was able to detect strains of *Corynebacterium argentoratens, Corynebacterium confusum, Corynebacterium coyleae, Corynebacterium imitans, Corynebacterium kroppenstedtii, Corynebacterium mucifaciens, Corynebacterium riegelii* and *Corynebacterium ureicelerivorans* isolated from different clinical samples, such as blood, wounds and abscesses, also from respiratory, genitourinary, digestive tract, among others. These analyses show that there is a tendency for clinical laboratories to integrate proteomics in order to obtain faster and more sensitive results for the diagnosis of infections caused by rare bacteria [71].

In the case of pathogenic corynebacterial species, the repertoire of secreted and surfaceexposed proteins, the exoproteome, have been documented because of their potential to act as antigenicity and virulence factors, since these molecules are promptly exposed to the host cells, making they are suitable for the use of vaccine and drug targets. A recent study investigated both surface and extracellular proteome of two *C. ulcerans* strains, where NanoLC-MS/MS was performed to analyze the set of proteins, which were similar expression patterns of putative virulence factors [72].

The most used methods for the identification and quantification of proteins are those based on mass spectrometry (MS). This technique allows the detection of compounds by the separation of the ions through mass-charge ratio. As each compound has a unique fragmentation pattern, the samples are ionized and separated with further identification of this pattern. Generally, two MS-based methods are currently most commonly used. The first method involves two-dimensional electrophoresis (2-DE) followed by staining, selection and MS. The other method involves isotopic markers to label proteins, separation by multidimensional

A typical proteomic experiment involves the step of preparing the sample, consisting of separating and isolating the proteins from the cell lysates, followed by separation of the protein mixture, and then the individual portions can be analyzed. Analysis may involve the bottomup strategy and the top-down strategy. The first involves obtaining the peptides by enzymatic digestion of protein solutions and the subsequent separation of these peptides by liquid chromatography and MS analysis. In contrast, the top-down strategy involves the analysis of intact proteins by MS. For the quantitative determination of proteins two approaches are most commonly used: two-dimensional electrophoresis followed by staining, selection and identification by MS; and isotopic markers followed by protein separation by multidimensional

Proteomics studies involving the genus *Corynebacterium* mainly comprise studies with *C. glutamicum*, due to, in large part, its industrial importance in the production of amino acids. This species is investigated in relation to its genetics and physiology and, consequently, a diversity of information about its molecular biology and biochemistry available, including a variety of proteomic techniques. In consequence of the membrane organization with high concentration of mycolic acids, *C. glutamicum* has been used as a model for development and new proteomic

Proteomics analysis have also been used as an alternative to traditional molecular methods for the characterization of poorly known bacteria, especially those of clinical interest, by reason of the ability of these methodologies to provide a fast and reliable identification of these species. The matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS) technique was able to detect strains of *Corynebacterium argentoratens, Corynebacterium confusum, Corynebacterium coyleae, Corynebacterium imitans, Corynebacterium kroppenstedtii, Corynebacterium mucifaciens, Corynebacterium riegelii* and *Corynebacterium ureicelerivorans* isolated from different clinical samples, such as blood, wounds and abscesses, also from respiratory, genitourinary, digestive tract, among others. These analyses show that there is a tendency for clinical laboratories to integrate proteomics in order to obtain faster and more sensitive results for the diagnosis of infections

In the case of pathogenic corynebacterial species, the repertoire of secreted and surfaceexposed proteins, the exoproteome, have been documented because of their potential to act as antigenicity and virulence factors, since these molecules are promptly exposed to the host cells, making they are suitable for the use of vaccine and drug targets. A recent study investigated

liquid chromatography and MS analysis [65, 67, 68].

68 Basic Biology and Applications of Actinobacteria

liquid chromatography and MS [68, 69].

technologies [70].

caused by rare bacteria [71].

The mapping of the extracellular proteome of *C. diphtheriae* through 2-DE and MALDI-TOF-MS detected proteins present in pathogenicity islands. According to these tests, possibly, the exoproteome of this pathogen is constituted of two distinct classes. The first involves molecules that have functions in the cytoplasm related with cell viability, such as protein synthesis and folding and detoxification mechanisms. The second class appears to be actively secreted and includes iron transporters and possible virulence factors that can be used in new vaccines [73].

The exoproteome of *C. pseudotuberculosis* has also been extensively characterized in the past years [74]. The use of transposon-binding proteins was investigated through a method of data-independent LC-MS acquisition (LC-MSE), used for proteins identification and quantification that was applied to compare the exoproteome of two biovar *ovis C. pseudotuberculosis* strains, C231 and 1002, where there were found 44 presents in both isolates in a total of 93 extracellular proteins [75].

Further, the combination of different proteomic methodologies as the 2-DE along with MALDI-TOF/TOF allowed the finding of 11 novel molecules in the *C. pseudotuberculosis* exoproteome, noncharacterized on the first comparative work [76]. The integration with *in silico* approaches also gives important insights about the behavior of the exoproteome. Pan-genome analysis can be performed to predict the set of exported proteins present in a large number of genomes available on public databases [77].

The proteomic map of a *C. jeikeium* strain was examined through 2-DE and MALDI-TOF-MS, through peptide mass fingerprinting (PMF), a high throughput protein identification methodology in which a protein is digested with endoprotease to produce the small constituent peptides. In this investigation, most spots were associated with functions essential for cell viability, such as protein synthesis and energy production, as carbohydrate, lipid and nucleotide metabolism. The surface proteins SurA and SurB, the adhesin CbpA and Che cholesterol esterase, known to act as virulence factors were also identified in the extracellular proteome [78].

In addition to these efforts, structural characterization methods for protein elucidation have also been used. The DtxR repressor is activated by transition metal ions and acts on the modulation of *tox* gene expression in *C. diphtheriae*. Through X-ray crystallography, it was possible to determine the general architecture of this biologically active Ni(II) bound protein with a resolution of 2.4 Å [79]. In *C. pseudotuberculosis*, the ArgR protein that acts as a regulator of arginine biosynthesis, an important metabolic pathway for bacteria, had the C-terminal domain crystal structure determined from X-ray diffraction with a resolution of 1.9 Å. The interest in this molecule lies in the fact that it participates in a pathway that is absent in its hosts, which makes it a potential target for the design of new drugs [80].

One of the greatest challenges of the postgenomic era is the amount of data generated through the different approaches, as well as the functional characterization of proteins. In this context, the analysis of protein-protein interaction networks (PPI) has been used for the identification of essential proteins and discovery of new therapeutic targets. This computational method is based on biological data topology information according to known interaction patterns to predict new interactions between molecules, where nodes represent proteins and the edges represent the predicted interactions [81].

**Author details**

Joana Marques1

Minas Gerais, Brazil

2018 Apr 19]

2012;**13**:141

**References**

Carlos Leonardo Araújo1

, Jorianne Alves1

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Federal University of Pará, Belém, Pará, Brazil

\*Address all correspondence to: carneiroar@gmail.com

, Alyne Lima1

1 Laboratory of Genomics and Bioinformatics, Center of Genomics and System Biology,

2 Department of General Biology, Federal University of Minas Gerais, Belo Horizonte,

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The Genus *Corynebacterium* in the Genomic Era http://dx.doi.org/10.5772/intechopen.80445

,

71

The inter-specific PPI networks of *C. pseudotuberculosis* were constructed from proteins conserved in multiple pathogens, such as *M. tuberculosis, Y. pestis, E. coli, C. diphtheriae* and *C. ulcerans*, where the interaction network of the protein acetate kinase (Ack) was indicated as a possible new broad-spectrum therapeutic target [82]. Later, another study involving the interactome of *C. pseudotuberculosis* was developed, where the networks were constructed, revealing nonhomologous proteins to humans, cattle, goats, sheep and horses. The fact that such proteins predicted by the PPI result are essential to the pathogen, but not to the hosts, makes them important candidates for use as targets for the synthesis of new drugs [83].
