title: A partner for the resuscitation-promoting factors of Mycobacterium tuberculosis.
abstract: Many cases of active tuberculosis are thought to result from the reactivation of dormant Mycobacterium tuberculosis from a prior infection, yet remarkably little is known about the mechanism by which these non-sporulating bacteria reactivate. A family of extracellular bacterial proteins, known as resuscitation-promoting factors (Rpfs), has previously been shown to stimulate growth of dormant mycobacteria. While Rpf proteins are clearly peptidoglycan glycosidases, the mechanism and role of Rpf in mediating reactivation remains unclear. Here we use a yeast two-hybrid screen to identify potential binding partners of RpfB and report the interaction between RpfB and a putative mycobacterial endopeptidase, which we named Rpf-interacting protein A (RipA). This interaction was confirmed by in vitro and in vivo co-precipitation assays. The interacting domains map to the C-termini of both proteins, near predicted enzymatic domains. We show that RipA is a secreted, cell-associated protein, found in the same cellular compartment as RpfB. Both RipA and RpfB localize to the septa of actively growing bacteria by fluorescence microscopy. Finally, we demonstrate that RipA is capable of digesting cell wall material and is indeed a peptidoglycan hydrolase. The interaction between these two peptidoglycan hydrolases at the septum suggests a role for the complex in cell division, possibly during reactivation.
authors.affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, MA 02115, USA.
authors.forename: Erik C
authors.initials: EC
authors.lastname: Hett
authors.forename: Michael C
authors.initials: MC
authors.lastname: Chao
authors.forename: Adrie J
authors.initials: AJ
authors.lastname: Steyn
authors.forename: Sarah M
authors.initials: SM
authors.lastname: Fortune
authors.forename: Lynn L
authors.initials: LL
authors.lastname: Deng
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Rubin
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002240:Carbohydrate Sequence; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D010457:Peptidoglycan / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D020798:Two-Hybrid System Techniques
chemical_list: D001426:Bacterial Proteins; D010457:Peptidoglycan; D010450:Endopeptidases