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.