"Mycobacterium tuberculosis causes tuberculosis, one of the most life-threatening diseases of all time. It infects the host macrophages and survives in its phagosome. The host phagosome is a very hostile environment where M. tuberculosis copes with high concentration of transition metals (Zn2+, Cu2+), low levels of others (Mn2+, Fe2+) and acidic pH. P-ATPases are membrane proteins that transport various ions against their electrochemical gradients utilizing the energy of ATP hydrolysis. Based on their primary sequences; seven of the twelve mycobacterial ATPases are classified as putative heavy metal transporters and a K+-ATPase, while the substrate of four (CtpE, CtpF, CtpH and CtpI) remains unknown. Consistent with their membrane topology and conserved amino acids, CtpE and CtpF are possibly P2 or P3-ATPases that transport alkali metals or protons. We examined the cellular roles of orthologous CtpE and CtpF in M. smegmatis, a non-pathogenic model organism. We hypothesized that these novel P- ATPases play an important role in transporting alkali metals and/or protons. We analyzed growth fitness of strains carrying mutations of the coding gens of these enzymes, in presence of various metals and different pHs, as well as the gene expression levels under different stress conditions. We observed that the M. smegmatis mutant strains, lacking of CtpF or CtpE, are sensitive to high concentrations (mM) of Mn2+. Furthermore, CtpE mutant is sensitive to alkali pH. Our results indicate that CtpE and CtpF might be an Mn2+ or H+-ATPase that are required for cell’s homeostasis sustainability."

"Tuberculosis is a deadly disease caused by bacteria of the genus Mycobacterium. One-third of the world’s population is infected with Mycobacterium tuberculosis. Two million these deaths occur each year in immunocompromised AIDS patients. M. tuberculosis has co-evolved with humans for many thousands of years. The bacillus has developed tactics to overcome the immune defense system and multiply in the macrophage. At the interface of the host and pathogen interactions, there is an interchange of metals and electrolytes. The host on one hand reduces the availability of metals essential for pathogen survival, like manganese and iron, in the macrophage and increases potassium ions which reduces pH in the phagolysosome. The host also generates Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS), to create toxic affects through interactions with metals and metalloproteins. M. tuberculosis copes with the hostile environment in the macrophage by preventing the acidification of the phagolysosome, secreting antioxidant enzymes such as alkylhydroperoxidase (AhpF) and peroxiredoxin (AhpC), superoxide dismutase, SodA and SodC, and catalase KatG through the SecA system. M. tuberculosis contains 28 metal transporters, among them there are 12 unique P-type ATPases. This is an unusually high number of P-type ATPases in an organism. These ATPases transport several monovalent and divalent metals (Cu+, Cu2+, Ag+, Zn2+, Na+, K+, Ca2+, Cd2+, Pb2+, Mn2+, Mg2+, and Co2+) across biological membranes, using energy from ATP hydrolysis. Our analysis has revealed that these P-type ATPases have homologs in other intracellular symbiotic/pathogenic bacteria and certain chemolithotrophic archaea and bacteria. A corelation can hence be drawn among these pumps and the capability of surviving in noxious environments and coping with adverse redox conditions. Possible substrates were identified by determining the consensus sequences in different helices of these ATPases. However, out of the 12 P-type ATPases confirmed, transported substrate could be postulated for four of these proteins; CtpA, CtpB, CtpV and KdpB. Using bioinformatic approaches we have characterized the possible genetic environment of these genes. The transmembrane regions were analyzed for consensus sequences and the N-terminals and C-terminals were scrutinized for metal binding domains, and we were able to categorize these ATPases into P1 type and P2 type ATPases. In an attempt to determine the substrate specificity, two of these ATPases (CtpC and ctpG) were cloned and transformed into Escherichia coli cells. Cells expressing CtpC were grown in different concentrations of metals and pHs. In these experiments CtpC was found to show an interaction with copper and cadmium. Pure protein was obtained by His-tag purification and para-Nitro Phenol Phosphatase (pNPPase) assay was performed with different metals, it was found that copper and zinc activated the phosphatase activity of the enzyme; and cobalt and manganese were inhibitory. Inhibition of the pNPP assay could mean that there would be activation in the ATPase assay, meaning that cobalt and manganese could be possible substrates to this enzyme. "