Evolution for improved secretion and fitness may be the selective pressures leading to the emergence of two NDM alleles

Highlights

• NDM-1 has an inefficient signal peptide for secretion to the periplasm.

• NDM-1 is cleaved by signal peptidase I as verified by mass spectrometry.

• Recently evolved signal peptide variants in NDM-1 have better secretion.

• NDM-2 forms a new signal peptide I cleavage site.

• NDM-2 has better secretion and fitness in E. coli compared to NDM-1.

Abstract

The New Delhi metallo-β-lactamase (NDM-1) mediates resistance to β-lactam antibiotics. NDM-1 was likely formed as the result of a gene fusion between sequences encoding the first six amino acids of cytoplasm-localised aminoglycosidase, AphA6, and a periplasmic metallo-β -lactamase. We show that NDM-1 has an atypical signal peptide and is inefficiently secreted. Two new bla_NDM-1 alleles that have polymorphisms in the signal peptide; NDM-1(P9R), a proline to arginine substitution, and NDM-2, a proline to alanine substitution (P28A) were studied. Here, we show that both the P9R and P28A substitutions improve secretion compared to NDM-1 and display higher resistance to some β-lactam antibiotics. Mass spectrometry analysis of these purified NDM proteins showed that the P28A mutation in NDM-2 creates new signal peptide cleavage sites at positions 27 and 28. For NDM-1, we detected a signal peptide cleavage site between L21/M22 of the precursor protein. We find no evidence that NDM-1 is a lipoprotein, as has been reported elsewhere. In addition, expression of NDM-2 improves the fitness of E. coli, compared to NDM-1, in the absence of antibiotic selection. This study shows how optimization of the secretion efficiency of NDM-1 leads to increased resistance and increased fitness.

Introduction

A new class of metallo-β-lactamase (MBL) was described in 2009, from a patient in New Delhi [1]. NDM-1 (New Delhi metallo-β-lactamase), has subsequently spread worldwide, and is responsible for clinical treatment failures for many bacterial pathogens, including Escherichia coli [2], Pseudomonas aeruginosa [3], Klebsiella pneumoniae [1], Proteus mirabilis [4] and Acinetobacter baumanii [5].

Efficient secretion of a β-lactamase to the periplasm of Gram-negative bacteria is essential to confer resistance to β-lactam antibiotics that target periplasm-localized peptidoglycan biosynthesis [6]. When originally described, mature NDM-1 protein was reported to be 28 kDa in size, with a predicted 19 amino acid signal peptide [1]. Toleman et al. suggested that the NDM-1 gene (bla_NDM-1) was likely created as the result of a gene fusion between the aphA6 gene, which encodes a cytoplasmic aminoglycoside phosphotransferase, and a preexisting A. baumanii MBL gene [7]. This gene fusion likely mobilized bla_NDM-1, bringing it under the control of a new promoter from an insertion sequence ISAba125 allowing successful transfer and expression in many bacterial species [7]. The gene fusion altered the start of the protein, by adding six new amino acids to the N-terminus of the ancestral MBL. The start of the protein changed from MHPVAKL-to MELPNIMH- (see Fig. 1B).

Several signal peptide cleavage sites have been reported for NDM-1. Thomas et al., who purified the NDM-1 protein with a C-terminal his-tag, reported four different signal peptide cleavage sites by mass spectrometry (MS) analysis [8]. Due to the many reported signal peptide cleavage sites, many studies have expressed mature versions of NDM-1 lacking a signal peptide region in order to purify and characterize NDM-1 [[9], [10], [11], [12], [13], [14]]. Adding to the unusual nature of the signal peptide, there is a strong lipobox signal [11]. A subsequent study reported that NDM-1 is a lipoprotein, cleaved by signal peptidase II at position 25, before the presumed, lipid-modified cysteine at position 26 [15]. In total, seven different secretory signal peptide lengths (15, 17, 18, 25, 26, 28 and 35) have been reported for NDM-1. Hence, there is no consensus in literature as to the signal peptidase cleavage site, which is essential information required to define the NDM-1 secretory signal sequence, and thereby whether it is cleaved by signal peptidase I or signal peptidase II and modified to become a lipoprotein.

In this study we examine NDM-1 secretion and determine the NDM-1 signal peptidase I cleavage site, thereby defining the signal peptide. We examine recently arisen NDM alleles with signal peptide polymorphisms to determine their impact on secretion efficiency antibiotic resistance and fitness.