The Yersinia pestis siderophore, yersiniabactin, and the ZnuABC system both contribute to zinc acquisition and the development of lethal septicaemic plague in mice

Abstract

Bacterial pathogens must overcome host sequestration of zinc (Zn(2+) ), an essential micronutrient, during the infectious disease process. While the mechanisms to acquire chelated Zn(2+) by bacteria are largely undefined, many pathogens rely upon the ZnuABC family of ABC transporters. Here we show that in Yersinia pestis, irp2, a gene encoding the synthetase (HMWP2) for the siderophore yersiniabactin (Ybt) is required for growth under Zn(2+) -deficient conditions in a strain lacking ZnuABC. Moreover, growth stimulation with exogenous, purified apo-Ybt provides evidence that Ybt may serve as a zincophore for Zn(2+) acquisition. Studies with the Zn(2+) -dependent transcriptional reporter znuA::lacZ indicate that the ability to synthesize Ybt affects the levels of intracellular Zn(2+) . However, the outer membrane receptor Psn and TonB as well as the inner membrane (IM) ABC transporter YbtPQ, which are required for Fe(3+) acquisition by Ybt, are not needed for Ybt-dependent Zn(2+) uptake. In contrast, the predicted IM protein YbtX, a member of the Major Facilitator Superfamily, was essential for Ybt-dependent Zn(2+) uptake. Finally, we show that the ZnuABC system and the Ybt synthetase HMWP2, presumably by Ybt synthesis, both contribute to the development of a lethal infection in a septicaemic plague mouse model.

Fig. 1

The structures of Ybt, micacocidin (A) and pyochelin (B). The asterisks indicate Fe³⁺ chelation sites in Ybt. The pentyl chain (in grey) of micacocidin is the only structural difference with Ybt (A). Pyochelin (B) lacks the malonyl linker and second thiazoline ring present in YbtA (A).

Fig. 2

Growth and virulence of a Y. pestis Pgm^- Znu^- mutant. A. Cells were grown at 37°C in cPMH2 supplemented with 0.6 μM ZnCl₂; strains: KIM6+ (Pgm⁺ Znu⁺); KIM6 (Pgm^- [Δpgm] Znu⁺); KIM6-2077+ (Pgm⁺ Znu^- [ΔznuBC]); KIM6-2077 (Pgm^- [Δpgm] Znu^- [ΔznuBC]). Growth curves shown are from one experiment that is representative of two or more independent experiments. B. Average time-to-death (percent survival) analyses for two independent septicemic Y. pestis infections. Strains: KIM5-2077(pCD1Ap) (Pgm^- [Δpgm] Znu^- [ΔznuBC]); KIM5(pCD1Ap) (Pgm^- [Δpgm] Znu⁺); KIM5-2077.10(pCD1Ap) (Pgm^- [Δpgm] Znu^-/+ [ΔznuBC/znuABC⁺). The average infectious doses are indicated in parentheses.

Fig. 3

The Y. pestis HMWP2^- Znu^- mutant has a severe growth defect at 37°C in cPMH2 supplemented with 0.6 μM ZnCl₂ and 1.0 μM FeCl₃. A: Strains – KIM6+ (Ybt⁺ Znu⁺); KIM6-2046.1 (HMWP2^- [irp2∷kan] Znu⁺); KIM6-2077+ (Ybt⁺ Znu^- [ΔznuBC]); KIM6-2077.7 (HMWP2^- [irp2∷kan] Znu^- [ΔznuBC]). B: strains – KIM6-2046.3(pBGL2) (HMWP2^- [Δirp2] Znu⁺); KIM6-2077.8 (Δirp2 ΔznuBC) carrying pBGL2 (HMWP2^- Znu^-) or pIrp2 (HMWP2^-/+ Znu^-). An irp2 mutant cannot express the Ybt synthetase HMWP2 and thus cannot synthesize the Ybt siderophore (Perry and Fetherston, 2011). To complement the in-frame Δirp2 mutation (KIM6-2077.8), the WT irp2 gene with its native promoter was cloned into the pBGL2 vector plasmid generating pIrp2. Growth curves shown are from one experiment that is representative of two or more independent experiments.

Fig. 4

An irp2 mutation in Y. pestis results in lower intracellular Zn²⁺ levels (A and B) and increases the concentration of exogenous Zn²⁺ required to stimulate growth (C). The β-galactosidase activities shown (A and B) are averages (with standard deviations) of replicate samples from two independent cultures. Statistical significances were calculated using the Student's two tailed t-test; p = <0.001 - ***; p = < 0.005 - **. Growth curves (C) shown are from one experiment that is representative of two or more independent experiments. Strains were grown in cPMH2 at 37°C unsupplemented (A) or with increasing levels of ZnCl₂ (B and C). cPMH was also supplemented with 1.0 μM FeCl₃ (B) or indicated FeCl₃ concentrations (C). Strains: KIM6+ (Ybt⁺ Znu⁺); KIM6-2046.1 (HMWP2^- [irp2∷kan] Znu⁺); KIM6-2077+ (Ybt⁺ Znu^- [ΔznuBC]); KIM6-2077.7 (HMWP2^- [irp2∷kan] Znu^- [ΔznuBC]). In A and B, all strains carry pEUZnu1 which encodes the znuA∷lacZ transcriptional reporter.

Fig. 5

Transcription from the irp2 promoter is affected by Zn²⁺. The β-galactosidase activities from the irp2∷lacZ transcriptional reporter carried by KIM6+ (Znu +) or KIM6-2077+ (Znu -) are averages (with standard deviations) of replicate samples from two independent cultures. Strains were grown in cPMH2 at 37°C unsupplemented or with ZnCl₂ or FeCl₃ added to 10 μM. Statistical significances were calculated using the Student's two tailed t-test; p = <0.001 - ***. As previously demonstrated (Perry et al., 2003a), transcription from the irp2∷lacZ reporter in both strains was repressed by FeCl₃ supplementation (p = <3 × 10^-7; not shown on the graph for clarity).

Fig. 6

Ybt-dependent Zn²⁺ acquisition does not require the OM Ybt receptor Psn or any other TonB-dependent OM receptor. Cells were grown at 37°C in cPMH2 supplemented with 0.6 μM ZnCl₂ and 1.0 μM FeCl₃ or 1.0 μM FeCl₃ alone. Strains: KIM6-2045.1 (Psn^- [Δpsn] Znu⁺); KIM6-2077+ (Ybt⁺ Znu^- [ΔznuBC]); KIM6-2077.7 (HMWP2^- [irp2∷kan] Znu^- [ΔznuBC]); KIM6-2077.12+ (Znu^- [ΔznuBC] TonB^-[tonB∷kan] HasB^- [ΔhasB]); Psn^- Znu^- (KIM6-2077.9 [psn∷kan ΔznuBC], ▼ and KIM6-2077.14 [Δpsn ΔznuBC];◆). Psn is the OM receptor for Ybt and HasB is a second TonB-like protein in Y. pestis (Perry and Fetherston, 2011; Perry et al., 2003b). Growth curves shown are from one experiment that is representative of two or more independent experiments.

Fig. 7

A YbtQX^- Znu^- mutant (open symbols) has a severe growth defect and requires additional Zn²⁺ supplementation to restore growth, compared to single Znu^- and YbtQX^- mutants (closed symbols). Cells were grown at 37°C in cPMH2 with 1.0 μM FeCl₃ and increasing levels of ZnCl₂. Strains: KIM6-2066 (YbtQX^- [ΔybtQX]); KIM6-2077+ (Znu^- [ΔznuBC]); KIM6-2077.13 (YbtQX^- [ΔybtQX] Znu^- [ΔznuBC]). YbtQ, but not YbtX, is part of the IM ABC transporter (YbtPQ) required for the use of Fe³⁺ from Ybt (Perry and Fetherston, 2011). Growth curves shown are from one experiment that is representative of two or more independent experiments.

Fig. 8

YbtX is required for Ybt-dependent Zn²⁺ uptake. Cells were grown at 37°C in cPMH2 with 1.0 μM FeCl₃ and 0.6 μM ZnCl₂. A. The growth of KIM6-2077.13 (YbtQX^- [ΔybtQX] Znu^- [ΔznuBC] is restored when carrying pYbtPQX, which expresses ybtPQX⁺ (YbtQX^-/YbtPQX⁺) but not when carrying pYbtPQ, which expresses only ybtPQ⁺ (YbtQX^-/YbtPQ⁺). B. A plasmid (pYbtX) expressing only ybtX⁺ restored the growth of KIM6-2077.13. KIM6-2077+ (Ybt⁺ Znu^- [ΔznuBC]) and strains carrying the vector plasmid pACYC184 were used as controls. Growth curves shown are from one experiment that is representative of two or more independent experiments.

Fig. 9

Addition of apo-Ybt or culture supernatants containing Ybt stimulates the growth of the ΔznuBC Δpsn irp2∷kan mutant. A. After acclimation to growth at 37°C in cPMH2 supplemented with 0.6 μM ZnCl₂ and 1.0 μM FeCl₃, cultures were then back diluted to an OD₆₂₀ of ∼0.1 in a 1:1 mixture of the same medium with culture supernatants. B. Alternatively, similarly grown cultures of the ΔznuBC Δpsn irp2∷kan mutant were back diluted to an OD₆₂₀ of 0.1 and incubated with apo-Ybt (μM Ybt) or ethanol solvent (0 μM Ybt). Culture optical densities were measured after overnight incubation at 37°C. Strains KIM6-2077.18 (ΔznuBC Δpsn irp2∷kan; labeled YbtX⁺) and KIM6-2077.19 (ΔznuBC Δpsn irp2∷kan ΔybtX; labeled YbtX^-) were tested for growth with supernatants from KIM6+ (Ybt +) and KIM6-2046.1 (irp2∷kan; Ybt -) (Panel A and B) or apo-Ybt (Panel B). Addition of apo-Ybt to a final concentration of 1.7 μM is equivalent to the Ybt present in cultures with at a 1:1 mixture with supernatant from KIM6+. NA – not applicable. Data presented for the YbtX⁺ strain are averages from 10 independent experiments with 6 independent culture supernatants (panel A) or 3 independent experiments (panel B). Data presented for the YbtX^- strain are averages from two or more independent experiments supplemented with 2 independent culture supernatants (panel A). Error bars represent standard deviations while asterisks with brackets indicate statistical significances calculated using the Student's two tailed t-test (p = <0.001 - ***; p = <0.05 - *).

Fig. 10

A proposed model of Zn²⁺ uptake in Y. pestis. Fe³⁺ uptake via the Ybt system as well as Zn²⁺ uptake is shown. Dashed arrows represent putative steps. Our results suggest passage through both the OM and IM when exogenous Ybt is supplied.