Deletion of a putative NlpC/P60 endopeptidase BAS1812 affects germination, long-term survival and endospore formation in Bacillus anthracis.

Bacillus anthracis, an aetiologic agent of the zoonotic disease anthrax, encodes a putative NlpC/P60 endopeptidase BAS1812. It harbours a signal peptide, three bacterial SH3 domains and an NlpC/P60 family domain. Previous studies showed that BAS1812 is immunogenic in infected hosts and is a potential biomarker for anthrax treatment. To date, however, little information is known about its function and involvement in anthrax pathogenesis. Here we describe the phenotypic effect of BAS1812 deletion in B. anthracis Sterne strain. Transcriptional analysis showed that BAS1812 expression in a host-like environment was enhanced at the end of log phase, started to diminish after entry to stationary phase and increased again late in stationary phase. The constructed BAS1812 mutant showed impaired long-term survival in the stationary growth phase, less resilience to detergent, lesser endospore formation and delayed germination. The mutant also showed diminished ability to degrade peptidoglycan, but its ability to produce anthrax exotoxins was not affected. We hypothesize that BAS1812 is a cell wall hydrolase involved in biological activities related to maintaining cell wall integrity, sporulation and spore germination.

Palmitoylation controls trafficking of the intracellular Ca2+ channel MCOLN3/TRPML3 to regulate autophagy.

MCOLN3/TRPML3 is a Ca2+-permeable cation channel that is expressed in multiple subcellular compartments with dynamic localization. Our previous studies suggest that upon macroautophagy/autophagy induction MCOLN3/TRPML3 is recruited and provides Ca2+ for the fusion process in autophagosome biogenesis. However, how intracellular trafficking and the Ca2+ channel function of MCOLN3/TRPML3 are related to autophagy are not known. Here we report that MCOLN3/TRPML3 undergoes palmitoylation at its C-terminal region, which is required for dynamic trafficking and cellular function of MCOLN3/TRPML3 in autophagy. Palmitoylation regulated MCOLN3/TRPML3 surface expression and trafficking, but not channel properties or localization and function of intracellular MCOLN3/TRPML3. Activation of intracellular MCOLN3/TRPML3 induced robust Ca2+ release, which solely increased autophagy in Ca2+- and palmitoylation-dependent manners. Palmitoylation regulated not only intracellular MCOLN3/TRPML3 trafficking to autophagic structures but also autophagic flux in induced autophagy. Importantly, nutrient starvation activated MCOLN3/TRPML3 to release Ca2+ and increased the level of MCOLN3/TRPML3 palmitoylation. Disruption of MCOLN3/TRPML3 palmitoylation, however, abolished the starvation-induced MCOLN3/TRPML3 activation without affecting channel activity. These results suggest that trafficking and channel function of MCOLN3/TRPML3 are regulated in the context of autophagy, and palmitoylation is a prerequisite for the function of MCOLN3/TRPML3 as a Ca2+ channel in autophagosome formation by controlling its trafficking between subcellular compartments. Abbreviations: 17-ODYA, 17-octadecynoic acid; 2-BP, 2-bromopalmitate; BFA, brefeldin A; DN, dominant-negative; GPN, glycyl-L-phenylalanine-beta-naphthylamide; HN, hydroxylamine; KD, knockdown; MCOLN3/TRPML3, mucolipin 3; MS, mass spectrometry; PAT, palmitoyl acyltransferase; PM, plasma membrane; WT, wild type; ZDHHC, a zinc-finger motif and an Asp-His-His-Cys sequence.