Cytotoxic Effect Associated with Overexpression of QNR Proteins in Escherichia coli

Introduction

Quinolones are broad-spectrum antimicrobial agents used in the treatment of a variety of infections. Quinolone resistance in Enterobacteriaceae is mainly due to mutations in chromosomal genes encoding type II topoisomerases and, to a lesser extent, altered permeability. In addition, three types of plasmid-mediated quinolone resistance (PMQR) mechanisms have been reported: Qnr proteins, the active efflux pumps QepA and OqxAB, and the acetyltransferase AAC(6′)-Ib-cr variant. ¹ PMQR genes by themselves confer only low-level quinolone resistance. Over the last few years, several new qnr alleles have been discovered worldwide, although their prevalence in Enterobacteriaceae is still low. ²

The existence of chromosomally located qnr-like genes in both gram-negative (such as Citrobacter freundii) and gram-positive bacteria (such as Enterococcus faecalis) has been described. ³ These genes are involved in natural quinolone resistance in these species of bacteria. Both plasmid-encoded Qnr proteins and chromosome-borne Qnr-like proteins bind to essential type II topoisomerases and protect them from quinolone inhibition. In recent years, a cytotoxic effect associated with overexpression of chromosome-borne qnr-like genes in Escherichia coli has been reported.^4–6 In this study, we evaluate the cytotoxic effect produced by overexpression of multiple PMQR mechanisms (qnr and qepA genes) in E. coli and discuss the implications.

Materials and Methods

Coding regions of qnrA1, qnrB1, qnrC, qnrD1, qnrS1, qepA2, and efsqnr (accession number AE016949) genes were amplified by PCR (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/mdr). Additionally, a deficient qnrB1 allelic variant encoding the substitution F111D (located at loop B of this protein and leading to loss of topoisomerase type II protection) was also included as control.^7,8 PCR fragments were cloned into the pCR-Blunt II-TOPO, following the manufacturer's recommendations (Invitrogen, Carlsbad, CA). The recombinant plasmids were digested with XhoI and NdeI (Fermentas, Madrid, Spain) and the purified digested fragments cloned into vector pET29a(+). These constructs were expressed in E. coli BL21. E. coli BL21 with and without empty pET29a(+) vector were used as controls.

Ciprofloxacin (Sigma–Aldrich, Madrid, Spain) susceptibility was determined by microdilution, according to Clinical and Laboratory Standards Institute (CLSI) guidelines, ⁹ in Mueller-Hinton broth (MHB) and Mueller-Hinton broth supplemented with Isopropyl β-D-1-thiogalactopyranoside (IPTG) 0.2 mM.

Gene overexpression was induced by adding 0.2 mM IPTG for 2 hours to exponential cells (OD_600nm 0.6) harboring PMQR genes cloned in the pET29a(+) expression vector (and respective control strains), as previously described. ⁴ Growth in IPTG-free broth was evaluated in parallel as a control. Then, 100 μl of induced and noninduced cultures was plated onto LB agar plates. The noninduced inoculum was ∼5 × 10⁸ colony forming units (CFU)/ml. To support the statistical analysis (Student's t-test), three independent assays were performed. Transmission electron microscopy assays were performed as previously described. ⁵

Results and Discussion

Microdilution results are indicated in Table 1. PMQR increased ciprofloxacin minimum inhibitory concentration (MIC) when Mueller-Hinton broth was supplemented with IPTG, indicating minimum basal expression in the absence of IPTG and overexpression in its presence.

When the induced cultures were plated onto LB agar plates, vectors harboring PMQR qnr genes produced significant loss of viability in at least 77% of the bacterial population (88% for qnrA1, 77% for qnrB1, 93% for qnrC, 95% for qnrD1, and 97% for qnrS1) compared with the E. coli BL21 pET29a(+) control (Table 1). Loss of viability associated with QnrA and QnrB overexpression was lower than that associated with other Qnr proteins (p < 0.05), but no statistically significant differences in cytotoxic effect were observed between QnrC, QnrD, and QnrS. The effect associated with QnrA was not significantly different from that associated with QnrB (p = 0.08). Loss of viability associated with EfsQnr overexpression was similar to previous reports (97%). ⁴ Of note, deficient QnrB1 variant containing F111D substitution (located at loop B of this protein and leading to loss of topoisomerase type II protection) did not reduce bacterial viability.^7,8 Moreover, no loss of viability was observed in E. coli overexpressing QepA2. Under these conditions, overexpressed PMQR qnr genes specifically produced notable loss of viability (in terms of colony-forming ability).

In transmission electron microscopy assays, signs of cytotoxicity were observed in recombinant E. coli cells expressing EfsQnr, QnrA1, QnrB1, QnrC, QnrD1, and QnrS1 (Fig. 1). The structure of the outer membrane and cell wall was intact, although cells overexpressing Qnr and EfsQnr proteins (30–45% of the bacterial population) frequently showed morphological changes: cytoplasmic retraction at the ends of the cell. These recombinant cells were able to divide, but the daughter cells sometimes appeared to be connected at the extremities. These morphological changes correlated with loss of cell viability, described above. The interior of the cells showed no unusual granules or accumulations of fibrous materials. Morphological changes were observed in less than 5% of the bacterial populations from the control strains and E. coli overexpressing QepA2.

OPEN IN VIEWER

FIG. 1.

Transmission electron microscopy of Escherichia coli BL21 (A), E. coli BL21 harboring the empty vector pET29a(+) (B), E. coli BL21 expressing EfsQnr (C), QnrA1 (D), QnrB1 (E), QnrC (F), QnrD1 (G), QnrS1 (H), and QepA2 (I). Cytoplasmic retraction and loss of cell content are indicated by black arrows in (C–H). E. coli BL21, E. coli BL21 harboring the empty vector pET29a(+), and E. coli BL21 expressing QepA2 did not show morphological alterations (A, B, I).

Overexpression of PMQR qnr genes produces cellular and structural cytotoxic effects in E. coli, the magnitude of which varies depending on the family of Qnr proteins. These data could be related to the observed fitness cost associated with different families of qnr genes. ¹⁰

The cytotoxic effect associated with Qnr overexpression would support the argument that Qnr proteins compete with DNA for binding to type II topoisomerases. If the Qnr protein concentration is low, DNA and Qnr proteins bind to DNA gyrase and topoisomerase IV, but when Qnr protein concentrations are very high, Qnr proteins bind to topoisomerases rather than DNA so that these enzymes are unable to perform their physiological function and DNA does not replicate. ¹¹ In the presence of loss-of-function modifications as observed for F111D (loop B modification), the impact on topoisomerase type II inhibition would be significantly reduced.^7,8 Qnr overexpression would not only produce inhibition of type II topoisomerases but could also inhibit unknown cellular processes, possibly those involved in the final stages of cell division into daughter cells.

The cytotoxic effect associated with overexpression may also explain why qnr genes by themselves confer only low-level resistance as well as the complex regulation of these plasmid determinants. Environmental conditions and stress affect qnr gene expression. qnrA gene expression is induced by cold shock, but not by DNA damage ¹² ; qnrB, qnrD, and qnrS expression is stimulated by DNA damage and different types of stress; qnrB and qnrD allele expression is regulated by the SOS system ¹³ ; and qnrS allele expression on the other hand is stimulated by a mechanism independent of the SOS system. ¹⁴ So, under conditions with no stress, Qnr protein expression is low and type II topoisomerases perform their physiological role. However, under conditions of stress (for example, exposure to quinolones), expression of certain Qnr proteins increases and these interact with the physiological function of type II topoisomerases, leading to protection against the drug or producing loss of cell viability, depending on the Qnr concentration in the cell (a priori this phenomenon would not be expected for QepA protein as observed).

Finally, regulating the expression of plasmid-encoded qnr genes could be a key factor for controlling the potential cytotoxic effect of these genes under overexpression conditions.