Calcium transport by the Mycobacterium tuberculosis PE15/PPE20 proteins

Many aspects of eukaryotic physiology are regulated by calcium ions (Ca2+). Whether bacteria have similar Ca2+ systems for transport, storage, binding, and response to Ca2+ is not well understood. To identify components of Ca2+ signaling in Mycobacterium tuberculosis, we determined its transcriptional response to Ca2+. Overall, only few genes changed expression, suggesting a limited role of Ca2+ as a transcriptional regulator. However, two of the most strongly downregulated genes were the pe15 and ppe20 genes that code for members of a large family of proteins that localizes to the outer membrane. PE15 and PPE20 formed a complex and PPE20 directly bound Ca2+. Ca2+-associated phenotypes such as an increase in ATP consumption and increase in biofilm formation were reversed in a pe15/ppe20 knockout strain, suggesting a direct role in Ca2+ homeostasis. To test whether the complex has a role in Ca2+ transport across the outer membrane, we created a fluorescence resonance energy transfer (FRET)-based Ca2+ reporter strain. A pe15/ppe20 knockout in the FRET background showed a specific and selective loss of Ca2+ influx that was dependent on the presence of an intact outer cell wall. These data show that PE15/PPE20 form a Ca2+-binding protein complex that selectively imports Ca2+ and support the emerging idea of a general family-wide role of PE/PPE proteins in transport across the outer membrane.