Complex Response of the CpxAR Two-Component System to β-Lactams on Antibiotic Resistance and Envelope Homeostasis in Enterobacteriaceae

Abstract

The Cpx stress response is widespread among Enterobacteriaceae We previously reported a mutation in cpxA in a multidrug-resistant strain of Klebsiella aerogenes isolated from a patient treated with imipenem. This mutation yields a single-amino-acid substitution (Y144N) located in the periplasmic sensor domain of CpxA. In this work, we sought to characterize this mutation in Escherichia coli by using genetic and biochemical approaches. Here, we show that cpxA^Y144N is an activated allele that confers resistance to β-lactams and aminoglycosides in a CpxR-dependent manner, by regulating the expression of the OmpF porin and the AcrD efflux pump, respectively. We also demonstrate the effect of the intimate interconnection between the Cpx system and peptidoglycan integrity on the expression of an exogenous AmpC β-lactamase by using imipenem as a cell wall-active antibiotic or by inactivating penicillin-binding proteins. Moreover, our data indicate that the Y144N substitution abrogates the interaction between CpxA and CpxP and increases phosphotransfer activity on CpxR. Because the addition of a strong AmpC inducer such as imipenem is known to cause abnormal accumulation of muropeptides (disaccharide-pentapeptide and N-acetylglucosamyl-1,6-anhydro-N-acetylmuramyl-l-alanyl-d-glutamy-meso-diaminopimelic-acid-d-alanyl-d-alanine) in the periplasmic space, we propose these molecules activate the Cpx system by displacing CpxP from the sensor domain of CpxA. Altogether, these data could explain why large perturbations to peptidoglycans caused by imipenem lead to mutational activation of the Cpx system and bacterial adaptation through multidrug resistance. These results also validate the Cpx system, in particular, the interaction between CpxA and CpxP, as a promising therapeutic target.

Keywords: Enterobacteriaceae; antibiotic resistance; cell envelope; stress response.

FIG 1

cpxA^Y144N is a cpxA* allele. Effects of CpxA, CpxA^Y144N, CpxA^R33C, CpxA^T252P, and NlpE on ppiA expression by assaying β-galactosidase activities of a chromosomal lacZ fusions.

FIG 2

cpxA* mutations confer resistance to β-lactams, aminoglycosides, and fosfomycin. (A) Efficiency-of-plating (EOP) assay on LB agar plates supplemented with 3 μg/ml amikacin (AMK), 2.5 μg/ml gentamicin (GEN), 0.5 μg/ml imipenem (IMP), 0.125 μg/ml ceftazidime (CAZ), or 4 μg/ml fosfomycin (FOF) and grown at 37°C. Susceptibility was determined for the following strains: RAM1292 transformed with pBAD33, pBAD33-cpxA, pBAD33-cpxA^Y144N, pBAD33-cpxA^R33C, or pBAD33-cpxA^T252P. (B) Deletion of cpxR in RAM1292 pBAD33-cpxA^Y144N abolished antibiotic resistance.

FIG 3

The Cpx response regulates genes that impact antibiotic resistance. (A and C) EOP assays. (A) Deletion of the acrD or tolC efflux pump components decreased resistance to aminoglycosides in RAM1292 pBAD33-cpxA^Y144N. (B) OMP expression level is altered by cpxA^Y144N. RAM1292 transformed with pBAD33, pBAD33-cpxA, pBAD33-cpxA^Y144N, pBAD33-cpxA^R33C, or pBAD33-cpxA^T252P was cultured and induced for 2 h with 0.2% l-arabinose. OMPs were detected from whole-cell lysates using polyclonal antibodies directed against OmpF/C/A as described in Materials and Methods. Equivalent of 0.2 OD₆₀₀ units was loaded per well. OmpF/C levels were normalized using the OmpA intensity signal. (C) Plasmid expression of the OmpF ortholog of K. aerogenes Omp35 restores the susceptibility of RAM1292 pBAD33-cpxA^Y144N to β-lactams.

FIG 4

Enzymatic activities of CpxA^Y144N. (A) CpxA-His and CpxA^Y144N-His exhibit similar autophosphorylation activities. Autophosphorylation was tested by using the ADP-Glo kinase assay on inner membrane fractions as described in Materials and Methods. Reactions were performed with 5 μl IMVs in the presence of 100 μM or 1 mM ATP for 30 min at room temperature. The two-step assay includes the removal of the remaining ATP and then the simultaneous conversion of the ADP produced by the kinase reaction to ATP and into light by a luciferin/luciferase reaction. Bioluminescent signals are proportional to the ADP produced and the activity of the kinase. The amount of ADP produced from each reaction was calculated by using ATP-to-ADP standard curves. (B) CpxA-His and CpxA^Y144N-His exhibit similar in vitro kinase activities on CpxR. CpxA-His or CpxA^Y144N-His in enriched IMVs (5 μl) was phosphorylated under standard conditions and then incubated in the presence of 1.5 μM purified CpxR-His for 30 min. Samples were analyzed by using Phos-tag PAGE fractionation. (C) CpxA-His and CpxA^Y144N-His exhibit similar in vitro phosphatase activities on CpxR. CpxR-His was phosphorylated with acetyl-phosphate as described in Materials and Methods. Phosphorylated CpxR-His was incubated in the presence of CpxA-His- or CpxA^Y144N-His-enriched IMVs (5 μl). Phosphate standards and samples (duplicates) were brought to 200 μl with water in a microplate; 30 μl of phosphate reagent was added to each well, and mixtures were incubated at room temperature in the dark. After 30 min, absorbance at 650 nm was measured. (D) Substitution Y144N in the periplasmic sensor domain of CpxA affects binding to CpxP. Protein-protein interaction analysis was performed by using BATCH. CpxP and the sensor domain of the wild-type CpxA or mutant CpxA^Y144N (CpxA-SD and CpxA^Y144N-SD) were fused to the C- and the N-terminal ends of the T18 and T25 fragments of B. pertussis adenylate cyclase, respectively. E. coli cells DHM1 cotransformed with plasmids encoding the hybrid proteins were grown overnight, spotted on an X-Gal plates, and incubated at 30°C for 24 h. (E) In vivo detection of phosphorylated and nonphosphorylated CpxR. E. coli BL21(DE3) was cotransformed with pET24+-cpxR-His and pBAD33-cpxA or pBAD33-cpxA^Y144N. Cells were grown at 37°C to mid-log phase, and protein expression was induced with 0.2% l-arabinose for 2 h and then with 1 mM IPTG for 3 h. Bacteria were pelleted by centrifugation and lysed with formic acid; samples were rapidly fractionated by Phos-tag PAGE and blotted with anti-His antibodies.

FIG 5

Interplay between the Cpx system, PBPs, and the cell wall-active antibiotic imipenem for β-lactamase induction. (A to D) Nitrocefin degradation assays were performed as described in Materials and Methods. (A) K. aerogenes clinical strain P4 exhibits high β-lactamase activity. Total β-lactamase activity was assessed in a series of clinical strains of K. aerogenes (P1 to P4) sequentially isolated from a patient under treatment with IMP. (B) β-Lactamase activity in P4 is mainly due to AmpC. Inhibition of the β-lactamase activity in K. aerogenes P4 and control strains of E. coli expressing constitutive TEM-3 or AmpC from a plasmid. Tazobactam-clavulanic acid and boronic acid were used as specific inhibitors of class A (e.g., TEM-3) and class C (e.g., AmpC) β-lactamases, respectively. (C) AmpC activity is increased upon the expression of CpxA^Y144N in the absence of IMP. E. coli C41(DE3) was cotransformed with pACYC184-ampRC and pET24a+-cpxA-His or pET24a+-cpxA^Y144N-His. Expression of the CpxA proteins was induced with 1 mM IPTG for 1 h at 30°C, and then cells were exposed or not to IMP (0.32 μg/ml) for 1 h. (D) Functional AmpG and CpxAR are both required to indue AmpC activity in response to IMP exposure. (E) Loss of specific PBPs induces AmpC activity in a Cpx-dependent manner. (D and E) E. coli MC4100, CS109, and derivative mutants were transformed with pACYC184-ampRC, grown, and induced as described in Materials and Methods. **, P < 0.05; ***, P < 0.01 versus wild-type control samples; nd, not determined.

FIG 6

Schematic representation of the interplay between the Cpx system and the chromosomal AmpC β-lactamase regulatory pathway in Enterobacteriaceae. In a wild-type background and under noninducing conditions, the PG is recycled. PG degradation products (disaccharide-peptides [aD-peptides]) are generated in the periplasm by the activity of PBPs. aD-peptides (aD-tripeptides, aD-tetrapeptides, and aD-pentapeptides) are transported into the cytoplasm by the inner membrane permease AmpG and processed by the amidase AmpD into their corresponding monosaccharide-peptides (aM-peptides). aM-peptides are then transformed into UDP-MurNAc-pentapeptides and incorporated to the PG biosynthesis pathway to complete the recycling process. In the cytoplasm, these UDP-MurNAc-pentapeptides interact with AmpR, creating a conformation that represses the transcription of ampC. In addition, normal interactions between CpxP and the periplasmic sensor domain of CpxA keeps the Cpx system off. Strong AmpC inducers such as imipenem simultaneously bind to several PBPs, leading to an increase of aD-pentapeptides in the periplasm (AmpG saturation) and aM-tripeptides (AmpD saturation) in the cytoplasm. In the cytoplasm, the accumulated aM-tripeptides displace UDP-MurNAc-pentapeptides from AmpR, creating a conformation that activates the transcription of ampC. In addition, our data showed that the increased level of aD-pentapeptides in the periplasm is sensed by the Cpx system. The release of CpxP from CpxA-SD by competing interactions with aD-pentapeptides probably switches the Cpx system on. High level of CpxR∼P leads to AmpC overexpression and thus to β-lactam resistance. The proposed feedback loop involving the lytic glycosylase appears in red. It also unknown whether CpxA or CpxP interacts with the putative signaling muropeptide in the periplasm. The cpxA^Y144N mutation alters the interaction between CpxA-SD and CpxP. This activates the Cpx response and leads to ampC overexpression, even in the absence of AmpC inducers. This mutation also acts on other genes of the Cpx regulon that impact antibiotic resistance.