Structural analysis of the N‐acetyltransferase Eis1 from Mycobacterium abscessus reveals the molecular determinants of its incapacity to modify aminoglycosides

Abstract

Enhanced intracellular survival (Eis) proteins belonging to the superfamily of the GCN5related N-acetyltransferases play important functions in mycobacterial pathogenesis. In Mycobacterium tuberculosis, Eis enhances the intracellular survival of the bacilli in macrophages by modulating the host immune response and is capable to chemically modify and inactivate aminoglycosides. In non-tuberculous mycobacteria (NTM), Eis shares similar functions. However, Mycobacterium abscessus, a multi-drug resistant NTM, possesses two functionally distinct Eis homologues, Eis1 Mab and Eis2 Mab. While Eis2 Mab participates in virulence and aminoglycosides resistance, this is not the case for Eis1 Mab, whose exact biological function remains to be determined. Herein, we show that overexpression of Eis1 Mab in M. abscessus fails to induce resistance to aminoglycosides. To clarify why Eis1 Mab is unable to modify this class of antibiotics, we solved its crystal structure bound to its cofactor, acetyl-CoA. The structure revealed that Eis1 Mab has a typical homohexameric Eis-like organization. The structural analysis supported by biochemical approaches demonstrated that while Eis1 Mab can acetylate small substrates, its active site is too narrow to accommodate aminoglycosides. Comparison with other Eis structures showed that an extended loop between strands 9 and 10 is blocking the access of large substrates to the active site and movement of helices 4 and 5 reduces the volume of the substrate-binding pocket to these compounds in Eis1 Mab. Overall, this study underscores the molecular determinants explaining functional differences between Eis1 Mab and Eis2 Mab, especially those inherent to their capacity to modify aminoglycosides.

Biochemical, structural, and functional studies reveal that MAB_4324c from Mycobacterium abscessus is an active tandem repeat N‐acetyltransferase

“…We next tested substrates suited for numerous GNAT and all possessing primary amines notably histamine, tyramine, spermine, and spermidine (Fig. 1B) [14,40]. This screening identified only spermine and spermidine as substrates of MAB_4324c, with a slightly better apparent activity for spermidine (Fig.…”

“…Moreover, Eis2 confers high‐level resistance to aminoglycosides in Mab [12–14]. A very close homologue of Eis2, named Eis1, is neither required for intracellular survival [19] nor aminoglycoside resistance, suggesting that it possesses another function that remains to be defined [12,14]. Together, these findings exemplify the need to experimentally address the substrate specificity and biological function of GNAT proteins despite their high homology levels with well‐described homologues.…”

“…The poor efficacy of drug treatments is correlated with the intrinsic resistance of Mab to most common antibiotics, including first-line anti-tubercular isoniazid and rifampicin [1,7]. In addition to the low permeability of the mycobacterial cell wall which limits the penetration of small molecules, Mab has developed multiple resistance mechanisms that include drug efflux activity mediated by MmpS/MmpL transporters [8,9], modifications or hydrolysis of the drug [7,[10][11][12][13][14][15], and/ or alteration of the drug target [16,17] ultimately incapacitating drug activity.…”

“…In addition, due to their N-acetyltransferase activity, Eis proteins can modify aminoglycoside antibiotics through acetylation, thereby impairing their binding to the ribosomes and leading to drug resistance [22,23]. Moreover, Eis2 confers high-level resistance to aminoglycosides in Mab [12][13][14]. A very close homologue of Eis2, named Eis1, is neither required for intracellular survival [19] nor aminoglycoside resistance, suggesting that it possesses another function that remains to be defined [12,14].…”

Virulence-Associated Secretion in Mycobacterium abscessus

“…Eis1 seems to be the closest homologue to Mtb’s Eis by Bidirectional Best Hit (BBH) search ( 91 ). However, it was shown that Mabs’ Eis1 does not modify aminoglycosides, and the basis for this lack of activity was pinpointed to structural reasons, where the active site is too narrow to accommodate large substrates like these antibiotics ( 92 ). Curiously, Mabs Eis2 deletion mutant ( Δeis2 ) was dramatically attenuated in murine macrophages, showing increased ROS levels.…”