POST TRANSLATION MODIFICATION MEDIATED REGULATION OF BACILLUS ANTHRACIS AND MYCOBACTERIUM TUBERCULOSIS METABOLISM

Abstract

As a pathogen, the success of B. anthracis depends on the spore‟s ability to develop into vegetative cells. Upon sensing the muropeptides under favorable conditions, spore initiates its metabolism and develops into a fully functional cell (Setlow et al.,2008). The metabolic checkpoints and energy reserves in spore provide stimulus at an early time point and ensures the success of developmental program. Such molecular program that leads to successful dormancy, helps in awakening and coordinating the outgrowth was not known for long. In this study, we discovered the role of metabolic enzyme Eno in spore germination and pathogenesis of B. anthracis. Our results show that Eno is expressed at higher levels in vegetative cells and is also expressed at albeit lower levels in the spores of B. anthracis. This made us inquisitive to investigate its role in breaking down the dormancy. In view of this, we tried to metabolically reprogram B. anthracis cells by increasing the levels of Eno and two other glycolytic enzymes Pgm and Pgk. Surprisingly, manipulation of glycolytic pathway revealed impairment in the germination ability of spores, with elevated levels of Eno causing the germination efficiency to drop down to a level of nearly 25%. Intracellular signaling proteins regulate the transition of B. anthracis from dormancy to vegetative state where the bacteria start to express virulence factors (Bryant-Hudson et al., 2011, Sajid et al., 2015). There is a growing body of evidence supporting the notion that Ser/Thr protein kinase PrkC plays an important role in spore dormancy exit program (Pompeo et al., 2016, Shah et al., 2008, Setlow et al., 2008). Thus, we tried to understand the contribution of signaling in maintaining the metabolic state of spore. Previous large-scale phosphoproteomic analysis in B. anthracis and B. subtilis suggested that Eno is phosphorylated (Arora et al., 2017, Rosenberg et al., 2015). Confirming that data, we show the reversible phosphorylation-dependent regulation of Eno by PrkC and cognate phosphatase PrpC. Interestingly, Eno was found to be phosphorylated in spores and germinated cells by PrkC, which regulates the expression, activity and cell surface localization of Eno. This mode of regulation decreases the Mg2+ binding with Eno, thus affecting its enzymatic activity. Bas∆prkC spores were found to have 2-3-fold higher expression of Eno as compared to Bas-wt which showed that the protein levels are progressively diluted through PrkC as vegetative cells continue to form spores and PrkC controls a yet to discover transcriptional factor that Summary and Conclusions

86 regulates Eno expression. In-depth phosphorylation analysis through mass spectrometry revealed that PrkC phosphorylates internal hydrophobic Ser/Thr residues of Eno along with residues present on outer exposed surface. Out of nine identified residues, Ser336 and Thr363 are present on the antiparallel beta sheets, thus forming hydrogen bonds and Ser367 interacts with Lys340 and Ile339, which are involved in the catalytic activity of the protein. Thus, substituting all these three residues reduced the overall activity of the protein with a significant loss in phosphorylation. Eno is present on the cell surface with fibrinolytic activity (Agarwal et al., 2008). It has also been found to be secreted in the host simulated conditions and is recognized by the sera of cutaneous anthrax patients (Delvecchio et al., 2006, Kudva et al., 2005, Walz et al., 2007). Whereas, PrkC did not affect the secretory profile of Eno, it influences Eno positioning towards the cell surface/membrane. This suggests the role of Eno in virulence since its increased expression in spores enhances its uptake by macrophages to upto 2-times but remained less infective due to lower germination. The higher expression and Eno activity in Bas∆prkC strains with reduced pathogenicity could explain the compromised virulence effect observed for PrkC null mutant in earlier studies (Shakir et al., 2010). We further show the protective efficacy of immunization with Eno alone or in combination with PA followed by challenge with Sterne and clinical strains of B. anthracis. This showed Eno as an important immunogen for more efficacious vaccine formulations which can be helpful in the development of spore detection and combating strategies. Bacteria masters its art of surviving in harsh conditions by keeping an alternate source of energy, 3-PGA in the spore, which is utilized by the bacteria during the early events of germination (Ghosh et al., 2015). A balanced ratio of 3-PGA and 2-PGA is maintained at the time of spore formation by keeping the spore metabolically dormant. Further, dehydrated acidic spore core diminishes the inside metabolic activity to maintain the 3-PGA reserve (Sunde et al., 2009, Driks et al., 2002). Eno stands at an important metabolic junction where it may help in regulating the level of crucial metabolite, 3-PGA. It is possible that an upsurge in Eno activity may generate few water molecules that let spore regain its metabolic status. Thus, PrkC-mediated Eno phosphorylation can be a bacterial way of regulating the spore metabolism. This Summary and Conclusions

87 hypothesis explains the significance of Eno phosphorylation by PrkC and highlights the role of PrkC as a dominant germination regulator. However, limitations on our ability to measure the different metabolites in spore on Eno overexpression so far has been a serious drawback and the possible consequences of Eno as a memory to link sporulation and germination is still unclear. During spore germination, PrkC is known to play an important role by activating protein translation machinery (Pereira et al., 2015). Our results indicate that PrkC initiates a molecular program in mother cell that controls overall Eno levels in spore and ensures success of spore germination. Also, a regulation at the level of expression, activity, localization, Mg2+ binding and catalysis showed PrkC as a master regulator of Eno. These steps are vital to ensure the success of spore germination as moderate change in Eno expression abrogates spore germination capacity and therefore, pathogenesis. Thus, here we identify Eno playing an important role in host-pathogen interaction during spore germination. In conclusion, we establish a key link between how infection specific kinase PrkC mediated regulation of a metabolic enzyme, Eno helps in spore germination and pathogenesis. Further, homocysteine is an important metabolite in the one-carbon metabolic pathway as its elevated levels can interfere with the SAM-dependent methylation reactions (Skovierova et al., 2016). Mycobacterial cording and biofilm formation are two such virulence associated phenotypes that help the bacteria to evade host immune responses. Biofilm was previously known to be regulated through autoinducers which is generated by LuxS through an intermediate, DPD. However, complementation of a metabolic enzyme, SahH, in LuxS deleted cells regains the cellular biofilm forming ability. Thus, we checked for the involvement of AMC in the biofilm formation in mycobacteria. SahH forms homocysteine through SAH hydrolysis and thus raising the levels of homocysteine affects biofilm formation by M. smegmatis in a concentration dependent manner. Mycobacterial lipid rich cell wall constitutes glycopeptidolipids and mycolic acids which play a key role in biofilm formation and cell morphology (Recht et al., 2001). Methylation of mycolic acids and glycopeptodilipids are important for their integration into cell wall (Takayama et al., 2005). Thus, the effects of homocysteine upregulation on different mycobacterial cellular processes could be a result of perturbation in methylation reactions.