The CpxAR signaling system confers a fitness advantage for flea gut colonization by the plague bacillus

Abstract

The adaptation of Yersinia pestis, the flea-borne plague agent, to fluctuating environmental conditions is essential for the successful colonization of the flea vector. A previous comparative transcriptomic analysis showed that the Cpx pathway of Y. pestis is up-regulated in infected fleas. The CpxAR two-component system is a component of the envelope stress response and is critical for maintaining the integrity of the cell. Here, a phenotypic screening revealed a survival defect of the cpxAR mutant to oxidative stress and copper. The measured copper concentration in the digestive tract contents of fed fleas increased fourfold during the digestive process. By direct analysis of phosphorylation of CpxR by a Phos-Tag gel approach, we demonstrated that biologically relevant concentrations of copper triggered the system. Then, a competitive challenge highlighted the role of the CpxAR system in bacterial fitness during flea infection. Lastly, an in vitro sequential exposure to copper and then H₂O₂ to mimic the flea suggests a model in which, within the insect digestive tract, the CpxAR system would be triggered by copper, establishing an oxidative stress response.

Importance: The bacterium Yersinia pestis is the agent of flea-borne plague. Our knowledge of the mechanisms used by the plague bacillus to infect the flea vector is limited. The up-regulation of the envelope stress response under the control of the Cpx signaling pathway was previously shown in a transcriptomic study. Here, our in vivo and in vitro approaches suggest a model in which Y. pestis uses the CpxAR phosphorelay system to sense and respond to the copper present in the flea gut, thereby optimizing the flea gut colonization. In other words, the system is essential for bacterial fitness in the flea.

Keywords: Yersinia pestis; cpxAR; fitness; flea; plague.

Fig 1

Phenotypic characterization of the ΔcpxAR mutant in flea-mimicking conditions. (A) Cristal violet assay showing biofilm formation of the wild-type (WT) and the ∆cpxAR strain. Data are quoted as the mean ± SD from at least three independent experiments. (B) Assessment of the WT and the ∆cpxAR strain growth in LB at 300 mOsM and 600 mOsM by measuring optical density at 600 nm (OD600nm) over time. (C) Survival of Y. pestis WT, the ∆cpxAR, and the complemented strain after exposure to H₂O₂ (20 mM) for 1 h. The mean percentage of survival ± SD values from three independent experiments is shown. Statistical analyses were performed with GraphPad Prism V10.0.2 using the Kruskal-Wallis test with Dunn’s correction for multiple comparisons (*P < 0.05). (D) Photos showing the growth of the WT, ∆cpxAR, and complemented strain on LB agar plates containing different concentrations of CuSO₄. For each strain, different quantities of bacteria (8 × 10⁶ CFU/mL to 4 × 10⁵ CFU/mL) from three different cultures were spotted.

Fig 2

The copper is a cue for CpxR activation. Assessment of CpxR phosphorylation with or without exposure to oxidant stresses. (A) CpxR and CpxR~P proteoforms were separated by Phos-Tag acrylamide gel after extraction from the bacterial pellet and were revealed by western blot. This result is representative of three independent experiments. (B) Box-and-whisker plots (Tukey) show the percentage of the CpxR~P proteoform/CpxR total in the WT strain. Data are the results from at least three independent experiments. Statistical analysis was performed with GraphPad Prism V10.0.2 using the Mann-Whitney test (*P < 0.01).

Fig 3

The CpxAR system provides a fitness advantage in the flea gut. Bacterial loads immediately after and 13 days after the infectious meal. Experiments were performed with Y. pestis ∆cpxAR mutant and Y. pestis WT Kim6+ or Y. pestis-complemented strain. The data correspond to samples from two independent experiments (white diamond: first experiment, black circles: second experiment). Each symbol indicates the ratio within an individual flea. The horizontal lines indicate the median CFU per flea. Statistical analyses were performed using the Kruskal-Wallis test and a Conover post-hoc test with a Benjamini-Hochberg adjustment for multiple testing. Any test with a P value < 0.05 is declared as significant.

Fig 4

Copper-activated CpxAR protects Y. pestis against oxidative stress. (A) In vitro LB co-cultures did not display any bacterial fitness defect of the ΔcpxAR compared with the WT strain. The co-culture growth curve is represented by linked half-black and half-white dots. The number of CFU/mL of the total population and the mutant was determined by enumeration on LB plates and LB plates, respectively, containing trimethoprim at 0, 3, and 8 h of culture. (B) In vitro assessment of the copper pre-exposed Y. pestis survival to 1 h and 3 h of H₂O₂ stress treatment in HPLM and the control conditions (untreated, H₂O₂, or copper). Statistical analysis was performed with GraphPad Prism V10.0.2 using a two-way ANOVA multiple comparison test (*P < 0.05).