Targeting QseC signaling and virulence for antibiotic development

Abstract

Many bacterial pathogens rely on a conserved membrane histidine sensor kinase, QseC, to respond to host adrenergic signaling molecules and bacterial signals in order to promote the expression of virulence factors. Using a high-throughput screen, we identified a small molecule, LED209, that inhibits the binding of signals to QseC, preventing its autophosphorylation and consequently inhibiting QseC-mediated activation of virulence gene expression. LED209 is not toxic and does not inhibit pathogen growth; however, this compound markedly inhibits the virulence of several pathogens in vitro and in vivo in animals. Inhibition of signaling offers a strategy for the development of broad-spectrum antimicrobial drugs.

Fig. 1. LED209 inhibits EHEC virulence traits in vitro

(A) Schematic of the autophosphorylation of QseC in response to signals and phosphotransfer to QseB. (B) QPCR of LEE1 gene ler, fliC, and stx2A in wild-type (WT) EHEC with self-produced AI-3 (black bars) and AI-3 plus 50 µM epinephrine (white bars), and the qseC mutant with self-produced AI-3 (dark gray bars) and AI-3 plus 50 µM epinephrine (light gray bars). (C) Colonization of the colon of infant rabbits by WT EHEC, the qseC and qseB mutants (CFUs/gram of tissue). (D) Chemical structure of LED209. (E) Binding of QseC to 5 µM tritiated NE and to 5 µM tyrosine (tyrosine is a negative control, not a signal to QseC). Binding of NE to QseC is inhibited by phentolamine (PE) but not propranolol (PO); 5 pM LED209 inhibits binding of 5 µM NE to QseC, but 5 fM LED209 does not. (F) QseC autophosphorylation in the absence of signal (NT, not treated), with 50 µM epinephrine (Epi) and with 50 µM epinephrine and 5 pM LED209. (G) QPCR of ler without signals (white bar), with 50 µM epinephrine (black bar), and with 50 µM epinephrine plus 5 pM LED209. (H) QPCR of LEE genes ler and eae, flagella genes flhDC and stx2A in WT EHEC with self-produced AI-3 (black bars) and AI-3 plus 5 pM LED209 (white bars). (I) Western blot of secreted proteins of EHEC (EspA and EspB) with 50 µM epinephrine, 50 µM epinephrine plus 5 nM LED209, and 50 µM epinephrine plus 5 pM LED209 (cross-reactive band as loading control). (J) Inhibition of the AE lesions by LED209 (5 µM and 5pM). Cell nuclei andbacterial cells are stained red (propidium iodide) and the cytoskeleton is stained green (fluorescein isothiocyanate–phalloidin). EHEC forms AE lesions atop the green-stained pedestals. There are no pedestals with LED209. * P < 0.05; ** P < 0.001; *** P < 0.0001.

Fig. 2. LED209 inhibits S. typhimurium and F. tularensis virulence in vivo and in vitro

(A) Survival plot of mice (strain 129×1/SvJ) on intraperitonial infection with 10⁸ CFUs of WT S. typhimurium strain SL1344, oral treatment with LED209 (20mg/kg) alone, and intraperitonial infection with 10⁸ CFUs of S. typhimurium strain SL1433 plus LED209 (20 mg/kg), and the qseC isogenic mutant. (B) CFUs of S. typhimurium harvested from liver and spleens of mice infected intraperitonially with 10⁸ CFUs of S. typhimurium strain SL1344 and infected intraperitonially with 10⁸ CFUs of S. typhimurium strain SL1433 plus LED209 (20mg/kg) 48 hours after infection. (C) QPCR of sifA in vitro in WT and a qseC mutant (ΔqseC) of S. typhimurium in the absence and presence of NE (50 µM). (D) QPCR of expression of the flagella regulator (flhDC) and sifA in vitro in the absence (black bars) and presence (5 pM) of LED209 (white bars). (E) The E. coli fliC::lacZ fusion was introduced into WT E. coli, the qseC E. coli mutant, and the qseC E. coli mutant complemented with F. tularensis qseC (qseC pFTQseC) in the absence and presence (5 pM) of LED209. The F. tularensis QseC was his-tagged and the inset shows that the F. tularensis QseC is expressed in the E. coli qseC mutant. (F) Infection of J774 murine macrophages with F. tularensis SCHU S4 in the absence and presence (5 nM) of LED209. (G) QPCR of F. tularensis virulence genes in the absence (gray bars) and presence (white bars) of LED209 (5 pM). (H) QPCR measuring expression of qseC in SCHU S4 during growth in vitro and in vivo (spleen, liver, and lungs). These data were collected from five C3H HeN mice intranasally infected with 30 CFUs of SCHU S4. QPCR of qseC was normalized against rpoA. (I) Survival plot of mice (C3H HeN) upon oral treatment with LED209 (20 mg/kg) alone, intranasal infection with 30 CFUs of SCHU S4, and intranasal infection with 30 CFUs of SCHU S4 plus LED209 (20 mg/kg). * P < 0.01; ** P < 0.001; *** P < 0.0001.