2.3.1. Subcellular Localization

The tracing of the location of essential proteins is an important facet to understand the functions of proteins in their suitable cell compartments. It is important to know the localization of a drug target in order to optimize the mode of action of the drug for its specific target. The prediction of sub-cellular localization of the essential non-homologous protein sequences was achieved by a computational tool called PSORTb. The results depict that approximately 48% of proteins resided in the cytoplasm of each strain. A proportion of 23% was distributed in the cytoplasmic membrane. The rest of the proteins were present in di fferent regions, including ~1% of proteins in the extracellular region, >1.5% proteins in the periplasm and very few proteins in the outer membrane of each of the strains. Despite these results, some fractions were considered "unknown" due to the tool's prediction of proteins in multiple sites simultaneously. The distribution of proteins by PSORTb is graphically shown for each strain in Figure 1.

**Figure 1.** Sub-cellular localization of non-homologous essential proteins. The outermost circle refers to strain MAH-TH135, the middle circle represents strain OCU-466 and the inner circle denotes strain A5.

### 2.3.2. Functional Family Classification

The functional families of protein sequences were also determined using the Support Vector Machine of Proteins (SVM-Prot) tool. Only the sequences whose functions were not known earlier were submitted to this tool. Hence, only uncharacterized sequences were retrieved from the non-homologous essential proteins' sequences. About 193, 119 and 187 uncharacterized sequences of TH135, OCU466 and A5 strains, respectively, were predicted by the SVM-Prot method. The results of the SVM-Prot tool are depicted in Figure 2. The proteins were broadly classified based on their molecular and biological functions and were further sub-divided into several protein classes, i.e., enzymes, transporters, trans-membranes, zinc or magnesium binding or other elements, DNA condensation, repair, etc. Complete information on classes with their strains is summarized in Supplementary Table S1.

**Figure 2.** Functional family prediction of *M. avium* subsp. *Hominissuis* (MAH) strains by the SVM-Prot method. The x-axis reports the frequency of each protein family.

### 2.3.3. Metabolic Pathway Analysis via KEGG

The KEGG database provides a network of metabolic pathways with their complete annotation. It helps to predict which protein sequences are essential in playing a unique role in metabolism. This step predicts the potential drug target based on the pathogen's unique metabolism. Metabolic pathways analysis was carried out for the essential protein sequences using the KEGG database. The DEG's results were subjected to the KEGG database via the KEGG Automated Annotation Server (KAAS). Briefly, out of 675 protein sequences of the MAH-135 strain, 72, 70, 29, 16 and 103 proteins were found to take part in carbohydrate metabolism, energy metabolism, lipid metabolism, nucleotide metabolism and amino acid metabolism, respectively. For OCU-466, 76 were involved in carbohydrate metabolism, while 69, 30 and 15 took part in energy metabolism, lipid metabolism and nucleotide metabolism, respectively, whereas the A5 strain possessed 93 proteins that majorly contributed to amino acid metabolism. The distribution of proteins in different metabolisms is presented in Figure 3a–c. Details are provided in Supplementary Tables S2–S4.

(**b**) 

**Figure 3.** *Cont*.

**Figure 3.** Percent distribution of non-homologous essential proteins involved in different metabolic pathways of the (**a**) MAH-TH135, (**b**) MAH-OCU466 and (**c**) MAH-A5 strains.

### *2.4. Discussion of Significant Unique Metabolic Pathways (UMPs) of the Pathogens*

Bacterial metabolism refers to the collection of the biochemical reactions required for bacterial survival and growth, which mainly includes respiration (aerobic and anaerobic) and fermentation. Bacteria, as a pathogen to humans, conduct all the same types of basic biochemical reactions a human cell performs. However, bacteria may have several types of energy generating metabolisms that do not exist in human or eukaryotic cells. This diversity of energy generation and metabolism allows bacteria to survive in a variety of habitats and flourish in otherwise not-suitable conditions. On the other hand, these differential metabolic pathways make bacteria susceptible by serving as an ideal target for antibiotics. Metabolic pathways that exist only in pathogens are called unique metabolic pathways (UMP). These UMPs are listed in Supplementary Table S5. We provide brief information on some bacterial UMPs and their significance as an antibiotic target.
