Structure and Function of RNase AS, a Polyadenylate-Specific Exoribonuclease Affecting Mycobacterial Virulence In Vivo

Abstract

The cell-envelope of Mycobacterium tuberculosis plays a key role in bacterial virulence and antibiotic resistance. Little is known about the molecular mechanisms of regulation of cell-envelope formation. Here, we elucidate functional and structural properties of RNase AS, which modulates M. tuberculosis cell-envelope properties and strongly impacts bacterial virulence in vivo. The structure of RNase AS reveals a resemblance to RNase T from Escherichia coli, an RNase of the DEDD family involved in RNA maturation. We show that RNase AS acts as a 3'-5'-exoribonuclease that specifically hydrolyzes adenylate-containing RNA sequences. Also, crystal structures of complexes with AMP and UMP reveal the structural basis for the observed enzyme specificity. Notably, RNase AS shows a mechanism of substrate recruitment, based on the recognition of the hydrogen bond donor NH2 group of adenine. Our work opens a field for the design of drugs able to reduce bacterial virulence in vivo.

Enantioselective Preparation of δ‐Valerolactones with Horse Liver Alcohol Dehydrogenase

“…These genes fall into diverse functional categories such as conserved hypotheticals ( MSMEG_4245, MSMEG_0973 ), transporters ( MSMEG_5693 , nudC ), lipid metabolism genes ( pmp1A, mas ), regulatory genes ( glnR, MMAR_5206 ), and cell division gene ftsX (Table 1). Finally, we also identified RNase AS ( MSMEG_4245 and MMAR_3223 ; Table 1), which had already been described by us in more detail in a recent study 26. Because these genes were not previously linked to or known to be involved in mycobacterial capsular α‐glucan biogenesis, our study reveals new insights into elements of capsular α‐glucan biogenesis.…”

“…We investigated in detail several mutants, glgB::Tn , glgC::Tn (data not shown), rnase_AS::Tn 26 and pstS::Tn (unpublished data) in which alterations in the α‐glucan levels were gene‐specific. We identified diverse genes found in well‐known biosynthesis pathways of glycogen, such as glgB and glgC , that would also be expected to be involved in capsular α‐glucan production,15 and this thereby validates our screening approach (Table 1).…”

Nuclease Activity of Legionella pneumophila Cas2 Promotes Intracellular Infection of Amoebal Host Cells

“…However, other types of bacterial cytoplasmic nucleases have been associated with virulence and/or regulatory events. These include the CvfA endoribonuclease of S. pyogenes, the RNase AS exoribonuclease of Mycobacterium tuberculosis, and the Cas9 nuclease of F. novicida, which, along with the tracrRNA and scaRNA from the Francisella CRISPR-Cas locus, degrade the mRNA for a lipoprotein that is recognized by the innate immune system (43,49,50). Perhaps most relevant to our current study are the VapDlike proteins, which exist in a range of bacteria and exhibit sequence similarity to Cas2 proteins (23,32,44,51,52).…”

A new dehydratase conferring innate resistance to thiacetazone and intra‐amoebal survival of Mycobacterium smegmatis

“…M. tuberculosis mc 2 6230 (Table S5) was grown with the presence of 24 μg ml −1 pantothenate. The M. smegmatis transposon library was described previously (Romano et al, 2014). Mycobacterial strains were transformed and selected on Middlebrook 7H10 agar supplemented with oleic-albumin-dextrose-catalase enrichment (OADC, Difco) containing 25 μg ml −1 kanamycin, 50 μg ml −1 hygromycin or at 50 μg ml −1 apramycin, when required.…”