A Novel Calcium Uptake Transporter of Uncharacterized P-Type ATPase Family Supplies Calcium for Cell Surface Integrity in Mycobacterium smegmatis

Abstract

Ca2+ plays an important role in the physiology of bacteria. Intracellular Ca2+ concentrations are tightly maintained in the nanomolar range. Molecular mechanisms of Ca2+ uptake in bacteria remain elusive. Here we show that CtpE is responsible for Ca2+ uptake in Mycobacterium smegmatis. It represents a previously uncharacterized P-type ATPase family in bacteria. Disruption of ctpE in M. smegmatis resulted in a mutant with impaired growth under Ca2+-deficient conditions. The growth defect of the mutant could be rescued by Ca2+ or by ectopic expression of ctpE from M. smegmatis or the orthologous gene (Rv0908) from Mycobacterium tuberculosis H37Rv. Radioactive transport assays revealed that CtpE is a Ca2+-specific transporter. Ca2+ deficiency increased expression of ctpE, resulting in increased 45Ca2+ accumulation in cells. ctpE is a gene that is part of an operon, which is negatively regulated by Ca2+. The ctpE mutant also showed hypersensitivity to polymyxin B, increased biofilm formation, and higher cell aggregation, indicating cell envelope defects. Our work establishes, for the first time, the presence of Ca2+ uptake pumps of the energy-dependent P-type ATPase superfamily in bacteria and also implicates that intracellular Ca2+ is essential for growth and cell envelope integrity in M. smegmatis.

Role of Dietary Micronutrients on Gut Microbial Dysbiosis and Modulation in Inflammatory Bowel Disease

“…[15] In bacteria, Ca is required for many cellular processes such as gene regulation, enzymatic activity, biofilm formation, signaling and maintenance of cell wall structural integrity. [195,196] Overall, the literature on Ca-dependent growth is scarce. The addition of Ca did not affect the growth rate of LAB and Shiga toxin-producing E. coli.…”

The P-type ATPase CtpF is a plasma membrane transporter mediating calcium efflux in Mycobacterium tuberculosis cells

“…However, critical physiological processes such as cellular growth, motility, quorum sensing, sporulation, and the development of different bacterial structures are regulated by the cytosolic Ca 2þ concentration in bacteria [21]. All forms of life, even mycobacteria, developed mechanisms such as passive and active transporters, ion channels, and non-protein channels to regulate calcium homeostasis [21], including Ca 2þ -ATPases that mediate calcium homeostasis [22,23].…”

Mycobacterium tuberculosis Calcium Pump CtpF Modulates the Autophagosome in an mTOR-Dependent Manner

“…Surge in calcium levels seen in macrophages after 1 and 2 h of infection with Mtb ( Figure 4 ) and lack of such increase in ctpFCKD, indicates the importance of CtpF-mediated calcium flux from the pathogen to the host to modulate downstream events. The resultant higher calcium milieu in the macrophages may be responsible for negative regulation of ctpE , as seen earlier in transcriptomic survey of intracellular mycobacteria (Botella et al, 2011 ) and calcium mediated negative regulation of ctpE in in vitro cultures (Gupta et al, 2017 ). The lower calcium milieu in macrophages infected with ctpFCKD, could induce ctpE -mediated calcium uptake into Mtb from host intracellular stores, restoring the calcium levels after 4 h of infection (shown in Figure 4 ).…”

“…Similarly, CtpV which exports Cu 2+ , enables bacterial survival in such conditions (Ward et al, 2010 ; Wolschendorf et al, 2011 ). An unusual ATPase, CtpE is shown to be involved in uptake of Ca 2+ ions by M. smegmatis (Gupta et al, 2017 ). Being a P2A ATPase, CtpF is predicted to be involved in alkali/alkaline earth metal transport (Novoa-Aponte et al, 2012 ).…”