Involvement of M. avium ssp. paratuberculosis specific genomic regions LSP14/15 in zinc homeostasis

Maintenance of zinc homeostasis is essential for bacterial survival. In the pregenomic era, we identified a 38 kb pathogenicity island in M. avium ssp. paratuberculosis (MAP), which was later shown to be part of the MAP-specific genomic regions LSP14/15. LSP14/15 harbour several predicted metal uptake systems. Due to the presence of binding motifs for the ferric uptake regulator Fur, these systems were initially assumed to support iron homeostasis. In recent years, we could show that these genetic elements were acquired to support MAP zinc homeostasis: Transcriptome analyses of MAP zinc starvation cultures revealed the zinc dependent regulation of 45 genes, 35 of which are presumably regulated by the global zinc uptake regulator Zur. In addition to the widely distributed zinc importer ZnuABC (map_0489-87c) we found two MAP-specific transporters MptABC (map_3736-34c) and ZnuABC-like (map3776-74c) on LSP14/15. We could show that all transporters clearly contributed to zinc uptake in a ZnuABC-deficient M. smegmatis (MSMEG) mutant.
Moreover, by promoter analyses in a MSMEGΔzur mutant we found, that znuABC and mptABC gene expression was regulated by Zur. We also investigated the phagosomal zinc status of MAP-infected macrophages treated with the zinc-specific fluorophore FluoZin3AM by immunofluorescence microscopy. No co-localisation of zinc and MAPCherry could be detected. qRT-PCR experiments of MAP derived from macrophages revealed a significantly induced mptA expression after 24h infection. These findings indicate that MAP in macrophages is subjected to intraphagosomal zinc deprivation. Finally, in infection experiments MAPwt exhibited significantly better survival in macrophages cultured in the presence of zinc than a MAPΔmptA mutant, which lacks a functional zinc uptake system. Overall, we provide strong evidence, that LSP14/15 are important for MAP zinc homeostasis and that ZnuABC, ZnuABC-like and MptABC must be considered as true zinc transporters. This might be of importance in the gut for colonisation and survival in the mucosal environment.