title: Cyclic AMP-dependent resuscitation of dormant Mycobacteria by exogenous free fatty acids.
abstract: One third of the world population carries a latent tuberculosis (TB) infection, which may reactivate leading to active disease. Although TB latency has been known for many years it remains poorly understood. In particular, substances of host origin, which may induce the resuscitation of dormant mycobacteria, have not yet been described. In vitro models of dormant ("non-culturable") cells of Mycobacterium smegmatis (mc(2)155) and Mycobacterium tuberculosis H37Rv were used. We found that the resuscitation of dormant M. smegmatis and M. tuberculosis cells in liquid medium was stimulated by adding free unsaturated fatty acids (FA), including arachidonic acid, at concentrations of 1.6-10 µM. FA addition enhanced cAMP levels in reactivating M. smegmatis cells and exogenously added cAMP (3-10 mM) or dibutyryl-cAMP (0.5-1 mM) substituted for FA, causing resuscitation of M. smegmatis and M. tuberculosis dormant cells. A M. smegmatis null-mutant lacking MSMEG_4279, which encodes a FA-activated adenylyl cyclase (AC), could not be resuscitated by FA but it was resuscitated by cAMP. M. smegmatis and M. tuberculosis cells hyper-expressing AC were unable to form non-culturable cells and a specific inhibitor of AC (8-bromo-cAMP) prevented FA-dependent resuscitation. RT-PCR analysis revealed that rpfA (coding for resuscitation promoting factor A) is up-regulated in M. smegmatis in the beginning of exponential growth following the cAMP increase in lag phase caused by FA-induced cell activation. A specific Rpf inhibitor (4-benzoyl-2-nitrophenylthiocyanate) suppressed FA-induced resuscitation. We propose a novel pathway for the resuscitation of dormant mycobacteria involving the activation of adenylyl cyclase MSMEG_4279 by FAs resulted in activation of cellular metabolism followed later by increase of RpfA activity which stimulates cell multiplication in exponential phase. The study reveals a probable role for lipids of host origin in the resuscitation of dormant mycobacteria, which may function during the reactivation of latent TB.
authors.affiliation: Bach Institute of Biochemistry Russian Academy of Sciences, Moscow, Russia.
authors.forename: Margarita
authors.initials: M
authors.lastname: Shleeva
authors.affiliation: Bach Institute of Biochemistry Russian Academy of Sciences, Moscow, Russia.
authors.forename: Anna
authors.initials: A
authors.lastname: Goncharenko
authors.affiliation: Bach Institute of Biochemistry Russian Academy of Sciences, Moscow, Russia.
authors.forename: Yuliya
authors.initials: Y
authors.lastname: Kudykina
authors.affiliation: Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, United Kingdom.
authors.forename: Danielle
authors.initials: D
authors.lastname: Young
authors.affiliation: Institute of Biological, Environmental and Rural Sciences, Aberystwyth University, Aberystwyth, United Kingdom.
authors.forename: Michael
authors.initials: M
authors.lastname: Young
authors.affiliation: Bach Institute of Biochemistry Russian Academy of Sciences, Moscow, Russia.
authors.forename: Arseny
authors.initials: A
authors.lastname: Kaprelyants
mesh_terms: D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000154:Aconitate Hydratase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000067956:Adenylyl Cyclase Inhibitors; D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D015169:Colony Count, Microbial; D003470:Culture Media; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology*; D005230:Fatty Acids, Nonesterified / Q000378:metabolism / Q000494:pharmacology*; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects*; D006801:Humans; D055985:Latent Tuberculosis / Q000382:microbiology; D020102:Mycobacterium smegmatis / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D009169:Mycobacterium tuberculosis / Q000187:drug effects* / Q000235:genetics / Q000254:growth & development / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*
chemical_list: D000067956:Adenylyl Cyclase Inhibitors; D001426:Bacterial Proteins; D003470:Culture Media; D005230:Fatty Acids, Nonesterified; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000242:Cyclic AMP; D000154:Aconitate Hydratase; C105962:RpfA protein, bacteria; D000262:Adenylyl Cyclases