As there are well-known disparities between cells in exponential and in stationary growth phases at transcriptomic level, it is crucial to monitor and to compare the changes in the proteome profile of M. avium through these two different growth phases. Former studies revealed remarkable growth phase associated alterations in protein expression within M. smegmatis (26) and M. tuberculosis (27). In our study, mono-dimensional electrophoresis for the proteins in two phases, not unexpectedly, showed variations in some band intensities between samples as same protein could be identified in 2 different growth phases and up/ or down regulated according to the phase. Simultaneously, two-dimensional gel electrophoresis coupled with mass spectrometric and bioinformatic analysis, as a gel-based proteomics approach, showed that expression of 12 proteins altered in the stationary phase.
As expected we found that elongation factor Tu was down regulated in the stationary growth phase. Notoriously, this protein has translation elongation activity and plays a significant role in cell energy metabolism which is not a critical point in the stationary phase (28). Elongation factor Tu has been known for its roles in protein synthesis, in adherence, and in immune regulation (29). In a similar fashion, a protein which is related to the transferase activity has been declined in the stationary phase, since it is known that the transferase activity is needed for maintaining energy inside the bacterial cell which is again less necessarily during this growth phase (28) (30). Our results were coincidently with what was previously established for E. coli, that proteins associated with energy metabolism and phosphotransferase proteins are down-regulated through the stationary growth phase relative to the exponential growth phase (31).
On the other side, eight proteins were up-regulated in the stationary growth phase; some of these proteins were involved in maintaining osmotic balance of the living cells under stress such as glucose-methanol-choline and dibenzothiophene desulfurization enzyme C which have oxidoreductase activity. This result was previously confirmed at the transcriptomic level of E. coli that revealed up-regulation of genes that are involved in survival during osmotic stress (32).
Another up-regulated protein that was identified in our proteomic analysis was ATP-dependent Clp protease, suggesting its contribution to stationary phase survival by controlling protein quality. ATP-binding protein that is pertinent to the anaerobic respiration process is required after entering the stationary growth phase. This is concordant with a previous study performed on M. smegmatis showing that genes involved in anaerobic respiration have been up-regulated in the stationary phase (28). Another protein obliquely involved in the anaerobic respiration and noted to be up regulated in our study is 6-phosphofructokinase 1 which aids in the conversion of fructose 6-phosphate and ATP to fructose 1,6-bisphosphate and ADP as a critical step of glycolysis that is deemed the base for both anaerobic and aerobic respiration. Interestingly, we found that one of the up-regulated proteins in the stationary phase, LprG protein, was associated with bacterial virulence and antibiotic resistance (33) and this is unsurprisingly since the secreted virulence factors of most human pathogens are often increased in the stationary phase of growth (34). It has been proved that LprG involved in triacylglyceride levels, growth rate, and virulence regulation (35). Its implication in mycobacterium virulence has been appeared through the induction of mitochondrial fission, interference with complex I and complex II respiration, and modification of mitochondrial calcium uptake, which in turn proposing that LprG-stimulated cells are in a lower bioenergetics state, which could support its immunosuppressive capacity in infection (36). The identification of LprG protein in M. tuberculosis was previously confirmed as P27 in the Mycobacterium tuberculosis complex and its gene is conserved across several pathogenic and nonpathogenic Mycobacterium species (37).
Aspartyl aminopeptidase, a protein that has a role in the protein turnover, was up regulated in the stationary phase in our study. It is established that during the exponential phase, bacteria degrade l-2% intracellular protein whilst during stationary phase, it degrades 5–12% (38). These alterations could be attributed to the nutrient limitation during the stationary phase thus the degradation of certain proteins becomes a demand in order to confer the amino acids required for the syntheses of new protein (38).
A recent study has sequenced a highly transformable virulent MAH approving novel genes that are essential for infection establishment; from these genes some involve in the growth and others in the virulence of the bacteria (39). katG and pfkA were of these genes that encodes for catalase peroxidase and phosphofructokinase; respectively which were down- and up-regulated in the stationary phase in our proteomic analysis; respectively. Catalase peroxidase has a critical role in mycobacterial pathogenesis, it is countering the phagocyte oxidative burst and it has been shown to protect M. tuberculosis against the reactive oxygen intermediates in macrophage since catalase and peroxidase activities are associated with survival inside macrophages (40). On the other hand, phosphofructokinase implicated in the first committing step of glycolysis which irreversibly catalyzes the phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate. Phong et. al., demonstrated that phosphofructokinase is essential for mycobacterial growth on glucose as sole carbon source, and is responsible for the total phosphofructokinase activity in M. tuberculosis(41).
These changed protein expression levels that have been proved in our study between the two growth phases might, for most of our identified proteins, underlie functional adaptations of the cells such as raising in cell synthesis and metabolism during the exponential phase while elevating in the virulence and increasing adaptation as well as survival in the stationary phase. Even though, there were some controversial results. Thus, we performed the transcriptomic analysis to unravel the discrepancy.
Previous transcriptomic studies of M. avium have focused on the transcriptome during infection [10] or during the mid-exponential growth phase [9]. We explored the expression of the dys-regulated proteins that detected by proteomic analysis between the 2 phases at the transcriptional level. Our results showed a down-regulation of elongation factor and universal stress protein coherently with the proteomic results. Strikingly, universal stress protein was down- regulated at both levels, although previous study proved the up-regulation of genes that are implicated in responses to stress in E. coli in stationary growth phase (32). This could be attributed to the notion that responses to alterations in resource availability could vary between species (42). Alternatively, the transcription of acetyl-CoA acetyltransferase in the stationary phase was down- regulated even it is 2.2 fold increase at the proteomic level. Forbye, ATP-dependent Clp protease ATP-binding protein and dibenzothiophene desulfurization enzyme C were down- regulated at the transcriptomic level during the stationary growth phase although they were up- regulated at the proteomic level. These differences between transcriptome and proteome over the two growth phases could be attributed to several factors like the extremely low abundance of transcripts and the poor recovery of proteins due to its low solubility or the membrane attachment. Additional factor is the fact that a single transcript can be translated several times to protein, and protein is more stable than transcript, thus accumulating more than transcripts (43). Another likely explanation for the transcriptome and proteome differences is post-transcriptional regulation. The weak correlation between the transcriptome and the proteome implies a major role for regulation at the post-transcription level in growth phase adaptation(43).