The Mycobacterium tuberculosis PE15/PPE20 complex transports calcium across the outer membrane

The mechanisms by which nutrients traverse the Mycobacterium tuberculosis (Mtb) outer membrane remain mostly unknown and, in the absence of classical porins, likely involve specialized transport systems. Calcium ions (Ca(2+)) are an important nutrient and serve as a second messenger in eukaryotes, but whether bacteria have similar Ca(2+) signaling systems is not well understood. To understand the basis for Ca(2+) transport and signaling in Mtb, we determined Mtb’s transcriptional response to Ca(2+). Overall, only few genes changed expression, suggesting a limited role of Ca(2+) as a transcriptional regulator. However, 2 of the most strongly down-regulated genes were the pe15 and ppe20 genes that code for members of a large family of proteins that localize to the outer membrane and comprise many intrinsically disordered proteins. PE15 and PPE20 formed a complex and PPE20 directly bound Ca(2+). Ca(2+)-associated phenotypes such as increased ATP consumption and biofilm formation were reversed in a pe15/ppe20 knockout (KO) strain, suggesting a direct role in Ca(2+) homeostasis. To test whether the PE15/PPE20 complex has a role in Ca(2+) transport across the outer membrane, we created a fluorescence resonance energy transfer (FRET)-based Ca(2+) reporter strain. A pe15/ppe20 KO in the FRET background showed a specific and selective loss of Ca(2+) influx that was dependent on the presence of an intact outer cell wall. These data show that PE15/PPE20 form a Ca(2+)-binding protein complex that selectively imports Ca(2+), show a distinct transport function for an intrinsically disordered protein, and support the emerging idea of a general family-wide role of PE/PPE proteins as idiosyncratic transporters across the outer membrane.