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

Brandon Robin, Amélie Dewitte, Véronique Alaimo, Cecile Lecoeur, François

Pierre, Gabriel Billon, Florent Sebbane, Sébastien Bontemps-Gallo

► To cite this version:

Brandon Robin, Amélie Dewitte, Véronique Alaimo, Cecile Lecoeur, François Pierre, et al. The CpxAR signaling system confers a fitness advantage for flea gut colonization by the plague bacillus. Journal of Bacteriology, In press, pp.e00173-24. 10.1128/jb.00173-24. hal-04673165

HAL Id: hal-04673165

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1 The CpxAR signaling system confers a fitness advantage for flea gut colonization

2 by the plague bacillus.

3

4 Brandon Robinª, Amélie Dewitteª, Véronique Alaimoᵇ, Cecile Lecoeurª, François Pierreª,

5 Gabriel Billonᵇ, Florent Sebbaneª, Sébastien Bontemps-Galloª#

6

7 a Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 9017 -

8 CIIL - Center for Infection and Immunity of Lille, Lille, France

9 b Univ. Lille, CNRS, UMR 8516 – LASIRE, Laboratoire de Spectroscopie pour les

10 Interactions, la Réactivité et l'Environnement, Lille, France

11

12 Running Head: *Y pestis*' CpxAR role in flea

13

14 # Address correspondence to Sébastien Bontemps-Gallo, [sebastien.bontemps-](https://sebastien.bontemps-gallo@cnrs.fr)

15 gallo@cnrs.fr

16
17 Abstract

18 The adaptation of *Yersinia pestis*, the flea-borne plague agent, to fluctuating
19 environmental conditions is essential for the successful colonization of the flea vector. A
20 previous comparative transcriptomic analysis showed that the Cpx pathway of *Y. pestis*
21 is up-regulated in infected fleas. The CpxAR two-component system is a component of
22 the envelope stress response and is critical for maintaining the integrity of the cell. Here,
23 a phenotypic screening revealed a survival defect of the *cpxAR* mutant to oxidative
24 stress and copper. Measured copper concentration in the digestive tract contents of fed
25 fleas increased 4-fold during the digestive process. By direct analysis of phosphorylation
26 of CpxR by a Phos-Tag gel approach, we demonstrated that biologically relevant
27 concentrations of copper triggered the system. Then, a competitive challenge
28 highlighted the role of the CpxAR system in bacterial fitness during flea infection. Lastly,
29 an *in vitro* sequential exposure to copper and then H₂O₂ to mimic the flea suggests a
30 model in which, within the insect digestive tract, the CpxAR system would be triggered
31 by copper, establishing an oxidative stress response.

32 **Importance**

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

40 **Keywords**: *Yersinia pestis*, plague, flea, *cpxAR*, fitness
41 INTRODUCTION

42 Plague, caused by the bacterium *Yersinia pestis*, is a devastating infectious disease that
43 has ravaged human populations for centuries (1). Despite the availability of effective
44 antibiotics and improved public health measures, plague remains a serious threat in
45 many parts of the world, particularly in Africa, Asia, and the Americas, where it continues
46 to cause sporadic outbreaks and pose a significant public health challenge (2).
47 Understanding the biology, transmission, and pathology of this fatal disease is critical to
48 prevent and control it.

49 *Y. pestis* is transmitted to mammals by flea bites. During infection of the insect, the
50 bacterium must colonize and obstruct the flea's foregut to increase the likelihood of
51 transmission to a new host (3–7). Shortly after ingestion, *Y. pestis* may elicit a flea
52 response in the proventriculus, leading to the formation of a soft bactericidal mass to
53 which it resists. The plague bacillus then consolidates this mass by secreting poly-β-1,6-
54 GlcNAc, blocking the passage of food and leading to the regurgitation of infected blood
55 during feeding (3, 4, 6, 7). However, the exact mechanism of how *Y. pestis* colonizes the
56 flea digestive tract and forms the bacterial mass is still not fully understood (7, 8).

57 The flea presents an inconstant environment with physicochemical parameters that are
58 gradually or abruptly changing before, during, and after feeding (7, 9–12). Adaptation to
59 these fluctuating conditions is mainly achieved by two-component systems (TCS) or
60 phosphorelays. The CpxAR two-component regulatory system is part of the envelope
61 stress response and is critical for maintaining the integrity of the cell (13). The system
62 consists of the inner membrane histidine sensor kinase CpxA and its cognate
63 cytoplasmic response regulator CpxR (14–16). Upon specific stress, the CpxA
64 autophosphorylates and transfers its phosphate group to CpxR. In *Escherichia coli*, the
phosphorylated regulator modulates dozens of genes involved in protein folding and degradation, chemotaxis, motility, multidrug efflux, and DNA repair (17–19). The system is negatively regulated by the periplasmic protein CpxP, which interacts with CpxA and inhibits its autophosphorylation (20). Previous studies have reported that the CpxAR system enables bacteria to respond to several stresses such as oxidative (21, 22) and osmotic stresses (23) and is involved in virulence (24–27).

In *Yersinia* species, the system is associated with virulence in *Yersinia enterocolitica* (28, 29) and *Yersinia pseudotuberculosis* (30, 31). Despite the system's involvement in *Y. pestis*' resistance to human neutrophils' bactericidal activity (32), no role in bubonic or pulmonary plague has been found (33). An upregulation of *cpxA* and *cpxP* genes was described in fleas compared to a planktonic culture in a comparative transcriptomic analysis (34), suggesting that the CpxAR system may be activated during insect colonization. However, the role of the CpxAR system in the flea has not yet been investigated. It should be highlighted that the *cpxP* gene is not essential for biofilm formation and flea colonization (34).

Here, we investigated the role of the CpxAR system in flea colonization. We identified that the CpxAR system was involved in the survival of H₂O₂ and copper, two stresses found in the flea. However, the phosphorylation of CpxR is only enhanced upon exposure to copper. Lastly, by co-infecting fleas with the *cpxAR* mutant and the wild-type strain, we revealed a role for CpxAR in bacterial fitness during flea infection.
86 RESULTS

87 The CpxAR system is required for survival to H₂O₂ and copper oxide stresses.

88 During the flea-colonization process, *Y. pestis* forms a biofilm to increase the likelihood
89 of its transmission by the insect (7, 35). However, no evidence was found for a role for
90 CpxAR in biofilm formation (Fig 1A). We therefore investigated whether the system
91 might be involved in adaptation to fluctuating environmental conditions. Indeed, once
92 ingested by the hematophagous insect, *Y. pestis* must adapt to increased osmolarity
93 [300 to 600 mOsM (11)], to a highly oxidizing environment [approximately 10 to 17 mM
94 (36)] and fluctuating metal concentrations (12). Hence, we sought to investigate the
95 system's contribution to coping with these different environmental stresses. Deletion of
96 the *cpxRA* locus did not result in any significant growth defect at various osmolarities
97 (Fig 1B) relevant to those found in the flea gut (11). To assay the susceptibility to ROS,
98 we analyzed the survival of each strain after treatment with hydrogen peroxide (Fig 1C).

99 The Δ*cpxAR* mutant was more susceptible than the wild-type strain. After 1 hour of
100 exposure, only 44% of the population was still viable when plated on LB. The
101 complemented strain displayed a similar level of susceptibility as the wild strain (Fig 1C).
102 To extend our phenotypic analysis of the stresses that *Y. pestis* might encounter within
103 the arthropod's gut, we analyzed whether the CpxAR system was involved in cupric
104 oxide resistance. Indeed, copper is known to be involved in the insect's innate immune
105 system (37–39). No differences in growth could be observed when Cu²⁺ was added up
106 to 0.5 mM (Fig 1D). With the addition of 5 mM Cu²⁺ into the plate, the wild-type strain or
107 the complemented strain showed an increased sensitivity to the presence of copper,
108 while the ΔcpxAR mutant was unable to grow (Fig 1D). These findings suggest that the
109 CpxAR system is involved in oxidative stress and Cu²⁺ resistance in vitro.

110

111 The concentration of copper increases during digestion of the blood meals in the
112 flea's gut.

113 While the H₂O₂ concentration in the flea has been reported previously (36), the
114 concentration of copper in the insect has never been investigated. Therefore, we
115 measured by inductively coupled plasma mass spectrometry (ICP-MS) the concentration
116 of copper in the flea gut at different time points after feeding on mouse blood (12h, 24h,
117 and 6 days) and in the mouse blood used to feed fleas. In the flea gut, the average Cu²⁺
118 concentration increased to 18.0 ± 2.0 µM at 12 h, to 20.1 ± 5.4 µM at 24 h, and reached
119 34.8 ± 4.6 µM 6 days after the blood meal. An average concentration of 7.3 ± 7.0 µM
120 was measured in mouse blood. The 4.7-fold difference in Cu²⁺ concentration between
121 mouse blood and flea strengthens the idea that copper could be a potential signal for the
122 CpxAR system, while *Y. pestis* passes through the insect.

123

124 Phosphorylation of CpxR increases in the presence of copper.

125 CpxAR is needed for resistance to H₂O₂ and Cu²⁺ *in vitro*, two fluctuating parameters in
126 the flea compared to the mammalian host. Therefore, we next wondered whether H₂O₂
127 and Cu²⁺ are a stimulus triggering the *Y. pestis* CpxAR system. To test this hypothesis,
128 we assayed the ratio of CpxR phosphorylated form (CpxR~P)/CpxR total in the
129 presence of H₂O₂ or Cu²⁺ using a polyacrylamide Phos-Tag approach (40). The Phos-
130 tag molecule reversibly binds the phosphate group (-PO₃²⁻) attached to CpxR and
consequently slows the migration of the CpxR phosphorylated form compared to the non-phosphorylated form (Fig 2A). The (CpxR~P)/CpxR ratio in *Y. pestis* exposed or not to 20 mM H₂O₂ was about 2,5% (Fig 2B). In contrast, the ratio was ~34,5% (14-fold increase) when the bacterium was exposed to 50 µM Cu²⁺. This result supports the idea that copper could be a triggering signal for the CpxAR system within the flea.

136

## 137 Loss of the CpxAR system led to a competitive fitness defect in the flea

Given that the CpxAR system is required *in vitro* for survival under two stresses encountered by the bacterium in the flea gut (Fig. 1C & 1D), and that copper triggers the system (Fig 2), we sought to determine the role in the production of a transmissible infection in flea. Noting that the system is not required for biofilm formation *in vitro* (Fig. 1A) and that the *cpxP* mutant (which is unable to inhibit CpxAR) has no impact on flea blockage, (34), we wondered whether the CpxAR phosphorelay might play a role in bacterial fitness within the insect gut. To investigate this hypothesis, a competitive co-infection of the *ΔcpxAR* mutant with the wild-type strain was performed. Cohorts of fleas were co-infected in a 1:1 ratio of either *ΔcpxAR/wild-type* or *ΔcpxAR/ΔcpxAR* complemented (Fig 3). During the co-infection with the wild-type and the *ΔcpxAR* strain, the number of *ΔcpxAR* bacteria significantly decreased at 13 days post-infection. A similar observation was obtained when the fleas were co-infected with the *ΔcpxAR* mutant and its complemented strain (Fig 3).

151

## 152 The CpxAR system promoted survival to coexposure to H₂O₂ and copper.
To shed light on the role of the CpxAR system during flea infection, we tested whether the competitive defect in flea of the *cpxAR* mutant compared to the WT strain was due to a slight growth defect of the mutant *in vitro*. To this, culture media were inoculated in a 1:1 ratio of the *ΔcpxAR* mutant and the wild-type strain (Fig. 4A). No significant difference was observed between both strains after 3h and 8h of culture (Fig. 4A). Therefore, the role of *cpxAR* in the flea does not appear to be the result of a general growth defect. In the flea, the bacterium is co-exposed to oxidative stress (36) and copper (the present study), two stresses sensed and inducing a bacterial response via CpxAR (41). It is therefore possible that the exposure to each stress or both stresses simultaneously could explain the growth defect of the *cpxAR* mutant in the flea compared to the WT strain. To test this hypothesis, we measured the bacterial survival after pre-incubation of the bacteria with (or without) copper for 1h (to trigger the CpxAR system), then exposure to 20mM H₂O₂ for 1h and 3h; as control bacteria were also exposed to copper for 3 hours (Fig. 4B). Regardless of the genetic background and time of exposure, copper had no deleterious effect. In contrast, H₂O₂ kills the *ΔcpxAR* mutant, as expected. Interestingly, the incubation of the bacteria with H₂O₂ after exposure to copper decreased the viability of both the WT and *ΔcpxAR* mutant. However, 3h after treatment, the wild-type strain had recovered, while the *ΔcpxAR* mutant no longer appeared viable. Lastly, regardless of the treatment used and the time of exposure to the stress, the complemented strain showed a similar profile as the wild-type strain. These results suggest that triggering the CpxAR system can improve *Y. pestis* survival when the bacterium is co-exposed to biologically relevant concentrations (i.e. found in the flea) of copper and H₂O₂.
176

177 DISCUSSION

178 Over the last thirty years, the role of the CpxAR system in virulence has been
179 investigated in various pathogens. In *Y. pestis*, this system has been studied for its
180 contribution to the bubonic and pulmonary plague forms in rodents (33), but its role in
181 the arthropod part has not been explored. While no role in rodents could be observed
182 (33), our study identified a fitness-competitiveness defect against the wild-type strain
183 (Fig. 3). Furthermore, neither *in vitro* growth nor biofilm was affected by the deletion of
184 the *cpxRA* operon (Fig. 1A & 1B). These data are comparable to those observed with
185 *Vibrio cholerae*, the pathogen responsible for cholera. In *V. cholerae*, *cpxP* expression is
186 increased in the mouse small intestine, but deletion of the *cpxAR* system does not lead
187 to a growth defect or loss of virulence (42). With a few exceptions where the system is
188 crucial for virulence [*Dickeya dadantii* (27), *Citrobacter rodentium* (24), *E. coli* UPEC
189 (43)], our data, combined with the literature, confirm that *cpxAR* is more a virulence
190 lifestyle gene than a true virulence gene per se (44), as has been observed in *Legionella*
191 *pneumophila* (25), *Salmonella enterica* (26) and *Y. enterocolitica* (28). In other words,
192 the CpxAR system is involved in host colonization, rather than being required for direct
193 interaction with the host.

194 Gahlot and collaborators have reported that in *Y. pseudotuberculosis*, the species from
195 which *Y. pestis* emerged, the CpxAR system is involved in biofilm formation (30).
196 Considering that the CpxAR system is identical between *Y. pseudotuberculosis* and *Y.
197 pestis*, they assume that CpxAR would play a similar role in *Y. pestis* (30). However, our
198 data do not support this idea (Fig. 1A). There are at least two issues that need to be
199 raised. Firstly, Gahlot and colleagues used the highly biofilm-producing *Y*.
200 *pseudotuberculosis* strain YPIII (serotype O3), whereas *Y. pestis* appears to be a clonal
201 emergence from the mild biofilm-producing *Y. pseudotuberculosis* serotype O:1b (45,
202 46). Secondly, the example of the OmpR-EnvZ or RcsCDB two-component systems
203 highlight the fact that, during its emergence, *Y. pestis* rewrote its regulatory networks to
204 support its new skills (11, 47, 48). Since the CpxA-CpxR protein sequences are identical
205 between *Y. pseudotuberculosis* and *Y. pestis* (30), only the environmental component in
206 the equation 'Phenotypic traits = Genetic + Environment' can explain the phenotypic
difference. In other words, it's the selection pressure of the environment (i.e. different
207 envelope composition, stresses encountered) that has led to a shift in the output of the
208 CpxAR system, and so its role. Therefore, our study confirms that even if the protein
209 sequences of CpxA-CpxR are conserved between *Y. pestis* and *Y. pseudotuberculosis*,
210 their implications in physiology and regulatory networks are tailored to the life cycle of
211 each species. This observation reinforces the idea that it is not possible to extrapolate
212 from studies carried out on *Y. pseudotuberculosis* to *Y. pestis*.

214 The role of the CpxAR system of *Y. pestis* in the bacterial fitness during the flea
215 colonization suggests a role in bacterial population dynamics to cope with changing
216 environmental conditions. This idea is reinforced by the system's involvement in survival
217 under oxidative stress and Cu²⁺ *in vitro*. The relationship between these two stresses
218 and the CpxAR system had already been observed previously in other species such as
219 *S. enterica* serovar Typhimurium (41, 49). In *Y. pestis*, while the Δ*cpxAR* mutant survival
220 was impacted *in vitro* by H₂O₂ oxidative stress (Fig. 1C) at a concentration similar to that
221 measured in the flea (36), this stress does not serve as a signal for the CpxAR system
222 (Fig. 2). Interestingly, Cu²⁺ at biologically relevant concentrations does not cause growth
defects (Fig. 1D and Fig. 4B) but can trigger the CpxAR system (Fig. 2) and could be a physiological cue in *Y. pestis* as it is case for *Salmonella enterica* serovar Typhimurium (41). In addition, when *Y. pestis* is pre-incubated *in vitro* with copper and then exposed to oxidative stress, the absence of the CpxAR system was deleterious for bacterial survival (Fig. 4B). Yet it should be borne in mind that the *ΔcpxAR* mutant was still fully able to form a biofilm (Fig. 1A), and therefore to cluster in a matrix. Biofilms have been described as a protective environment against stress (50). This could explain why the *ΔcpxAR* mutant is not cleared in the flea. Our results suggest a model in which copper triggers the CpxAR system, thereby contributing to the oxidative stress resistance of *Y. pestis* during flea colonization.

How copper can activate the CpxAR system and what bacterial determinants are deployed to protect the cell from oxidative stress remain to be elucidated. In *Salmonella enterica*, the CpxAR system protects against high copper concentrations (1mM) and oxidative stress (5/6mM) (41). Pre-incubation with copper also confers a protective effect against oxidative stress. The CpxAR system regulates the *scsABCD* locus encoding proteins with homologies to thioredoxin-type proteins. Lopez *et al*. propose that regulation of the *ScsABCD* system by the CpxAR system restores the redox balance of the *Salmonella* envelope (41). Yet it remains unclear how copper triggers the system. A recent study by Andrieu *et al*. suggests that the CpxAR system can detect specific oxidative stress (49). In their study, they reported that *N*-chlorotaurine oxidative stress directly triggers the activation of the CpxAR system. Interestingly, other oxidative stress such as $H_2O_2$, did not elicit the system (49). Here, the presence of copper could lead to a type of oxidative stress different (51) from what is encountered when using $H_2O_2$.
247 MATERIALS AND METHODS

248 Strains, mutants, plasmids and growth conditions

249 The bacterial strains and plasmids used in this study are listed in Table 1. The mutant
250 strain was generated using the lambda red recombinase-based method (52) with some
251 modifications (6, 53). The coding sequence of the *cpxRA* locus was replaced by a
252 trimethoprim resistance cassette flanked by flippase recognition target (FRT) sites that
253 had been amplified from the pEP1087 (Table 1). The mutant strain was complemented
254 with the mini-Tn7 (54) containing a WT copy of the *cpxRA* locus under the control of its
255 own promoter (Table 1). Mutations were confirmed by PCR with the primers sets given
256 in the Table 1 and sequencing following the method described previously (11).

257 The strains were grown in lysogeny broth (LB, 240230, BD), Brain Heart Infusion (BHI,
258 211059, BD), or Human Plasma-Like Medium (HPLM, A4899101, Gibco) at 21°C or
259 28°C. When needed, agar (15 g.L⁻¹), kanamycin (50 µg.mL⁻¹), and trimethoprim (25
260 µg.mL⁻¹) were added to obtain solid and/or selective media. The LB osmolality is 345
261 mOsM (55). To obtain a high-osmolarity medium, NaCl was added. The HPLM
262 concentration of copper was determined on three different lots by ICP-MS and was 0.9 ±
263 3.3*10⁻² µM. To compare the growth rate of different strains, bacteria were cultured at
264 21°C. The growth in LB was monitored by measuring the OD₆₀₀nm.

265

266 Assessment of biofilm formation in vitro

267 Biofilms were assayed as previously described (11). Briefly, bacteria were incubated in
268 LB supplemented with 4 mM MgCl₂ and 4 mM CaCl₂ for 48 h at 21°C with 180 rpm
269 shaking in a 24-well plate (1x10⁷ bacteria in 1 mL). Then, the medium was removed, and
270 the attached bacterial biofilm was stained with a crystal violet dye solution (0.01%) for 15
271 min at room temperature. Next, the wells were rinsed three times with ddH₂O. The dye
272 attached to the biofilm was released with the addition of an ethanol-acetone (80:20)
273 solution. Lastly, the solution was transferred to a 96-well plate, and the absorbance of
274 each well was measured at 542 nm (ELx800 microplate reader, BioTek).

275

276 Survival assays

277 To evaluate bacterial survival to H₂O₂, *Y. pestis* KIM6+ WT, the Δ*cpxAR*, and the
278 complemented strains were grown to the mid-log phase and were harvested by
279 centrifugation (4,000×g, 5 min). Bacteria were resuspended and incubated in one
280 volume of 1X PBS supplemented with 20 mM H₂O₂ for 1 h at 21 °C under 180 rpm. After
281 incubation, bacteria were plated on LB agar and enumerated after 48h incubation at
282 28°C.

283 To evaluate bacterial survival to copper, overnight cultures of *Y. pestis* KIM6+ WT, the
284 Δ*cpxAR* and the complemented strains were diluted in fresh LB to 8×10⁶ CFU/mL. Then,
285 bacteria were two-fold serial diluted to 4×10⁵ CFU/mL and spotted on LB agar plates
286 supplemented with 50 µM, 500 µM or 5 mM CuSO₄. Bacterial survival was determined
287 after 48 h incubation at 28°C.

288 To evaluate bacterial survival to H₂O₂ after copper exposition, overnight cultures of *Y.
289 pestis* KIM6+ WT, the Δ*cpxAR* and the complemented strains were thricely washed in
290 HPLM medium and diluted in fresh HPLM medium to 1x10⁸ bacteria/mL supplemented
291 with 50 µM of CuSO₄. After 1h pre-incubation at 21°C, bacteria were exposed to 20 mM
292 H₂O₂ for up to 3h. After 1h and 3h incubation to oxidative stress, bacteria were plated on
293 LB agar and enumerated after 48h incubation at 28°C.

294
295 Phos-Tag™ and SDS-PAGE immunoblot analysis

296 Gel retardation of the phosphorylated form of CpxR was performed as described
297 previously (27, 40) with some modifications. Briefly, 2×10⁸ bacteria harvested by
298 centrifugation were suspended and lysed with 20 µL of 1 M formic acid and vortexed
299 thoroughly. Acid was neutralized by 5 µL of 5 N NaOH and then 6 µL of 5× SDS-PAGE
300 loading buffer (composition: 250 mM Tris-HCl pH 6.8, 4% (w/v) SDS, 30% (w/v) glycerol,
301 20% (v/v) β-Mercaptoethanol, 1 mM MnCl₂, 0.02% (w/v) bromophenol blue) were added.
302 The sample was loaded onto Phos-tag™ acrylamide gels composed of a 12.5%
303 resolving solution [12.5% (w/v) 37.5:1 acrylamide/N,N'methylenebisacrylamide, 375 mM
304 Tris (pH 8.8) and 0.1% (w/v) SDS, 35 µM Phos-tag™ acrylamide and 70 µM MnCl₂] and
305 a 4% stacking solution [4% (w/v) 37.5:1 acrylamide/N,N'methylenebisacrylamide, 125
306 mM Tris (pH 6.8) and 0.1% (w/v) SDS]. The gels were run at 4°C under constant voltage
307 (130 V) with fresh running buffer (0.1% (w/v) SDS, 25 mM Tris and 192 mM glycine pH
308 8.8).
309 Then, a 15-minute wash with transfer buffer (25 mM Tris and 192 mM glycine) supplied
310 with 2 mM EDTA, followed by two 15-minute washes without EDTA were performed
311 before transferring the gel to 0.22 µm PVDF membrane using a Trans-Blot® Turbo™
312 Blotting system (Bio-Rad) with a pre-programmed protocol (2.5 A, up to 25 V, 7 min).
313 Western blotting against CpxR was performed using standard protocols with the rabbit
314 anti-CpxR polyclonal antibodies (27) at a dilution of 1:1 000 and anti-rabbit secondary
315 antibody coupled to horseradish peroxidase at a dilution of 1:5 000. Blots were imaged
316 by chemiluminescent detection (SuperSignal™ West Pico PLUS from Thermo Scientific
317 and Amersham ImageQuant 800 imaging system from Cytiva).
318
319 **Flea infection**

320 Flea infection was assayed as previously described (56, 57). Starved *Xenopsylla cheopis* rat fleas were allowed to feed on heparinized mouse blood containing 5x10<sup>8</sup> bacteria per mL (grown at 37 °C overnight in BHI), using an artificial apparatus (feeder) for 1 h. For co-infection assay, equivalent amounts of each strain were added to the blood and then placed into the feeder. Then, cohorts of 100 female fleas were collected.

325 Fleas were allowed to feed mouse blood at 2-, 6-, 9-, and 13-days post-infection. To determine *Y. pestis* competitive fitness in fleas, groups of 20 females were collected at different times post-infection to individually plate them (after trituration) on LB agar plates containing 1 µg/mL irgasan, 10 µg/mL hemin and either kanamycin or trimethoprim to select for the mutant strains. The CFUs were counted after 48 h at 28°C.

330

331 **ICP-MS analysis**

332 The midgut content from fed *X. cheopis* was collected using the following methods.
333 Intact digestive tracts lacking their Malpighian tubule system were collected from fleas
334 under a dissecting microscope. The midgut epithelium was pierced, and the midgut
335 contents were carefully expressed. Six to nine midgut contents were pooled and diluted
336 5-fold in H₂O to provide sufficient samples for subsequent analyses. Mouse blood was
337 collected in a heparinized tube (Becton Dickinson). 50 µL of mice blood or 45 µL of fleas
338 gut was treated as follows: in the first step, the samples were attacked with a mixture of
339 100 µL of H₂O₂ (Promochem, 30% high purity) and 200 µL of HNO₃ (Fisher Scientific,
340 67-69%, Optima, and trace metal grade) at room temperature during 24 hours to extract
341 Cu from the matrixes. The samples were then diluted with Milli-Q water (18.2 MΩ.cm) to
342 get 10 mL of solution. Before ICP-MS analysis, these solutions were systematically
343 filtrated at 0.45 µm (Sartorius, acetate cellulose membrane). Copper analyses (⁶³Cu
344 isotope chosen) were performed with an inductively coupled plasma-single quadrupole
345 mass spectrometer (ICP-MS 7850, Agilent Technologies) using He as collision gas and
346 Kinetic Energy Discrimination (KED) to eliminate spectral interferences. To take into
347 account the potential drift of the spectrometer, an internal standard (⁷²Ge) was added to
348 the samples. To control the quality of the results, the water SLRS-6 certified standard
349 was also analyzed. The certified values were 24.0 +/-1.8 µg/L and our experimental
350 result was 25 +/- 1 µg/L. Finally, the limit of detection was estimated to be 0.02 µg/L in
351 this run of analysis and the average blank values (including all the steps of sample
352 preparation) did not exceed 10% of the sample's values. Blank values were therefore
353 neglected for the calculations.

354

355 **Ethics statement**

356 The animal experiments were carried out following the European directive 2010/63/EU
357 and the French governmental decree 2013-118, and all protocols had been approved by
358 the French Ministry of Higher Education, Research and Innovation, and the Ethics
359 Committee in Animal Experimentation. All animal husbandry adhered to the current
360 European and French regulations.

361
362 ACKNOWLEDGMENTS

363 The authors thank Pr. Jean-Marie Lacroix for the gift of reagents (antibodies and
364 plasmids) and insightful discussions, Thierry Chassat for providing mouse blood and the
365 staff of the PLETHA animal facility. All experimental data pertinent is accessible for
366 review in the manuscript.

367

368 FUNDING

369 This work was supported by the French National Centre for Scientific Research (CNRS),
370 the National Institute for Health and Medical Research (INSERM), the Institute Pasteur
371 of Lille, the University of Lille, the European Regional Development Fund as part of the
372 *Action de recherche concertée d'initiative régionale* program (grant 10090077-Présage
373 35712 to F. S.), grants of the Agence National de la Recherche (07-MIME-017-01
374 IVOTIMP to F. S. and ANR-21-CE15-0047 to S.B.G.) and a grant of the Investments for
375 the Future program managed by the Agence National de la Recherche (ANR-16-IDEX-
376 0004 ULNE to S.B.G.). ICP-AES and ICP-MS measurements were performed on the
377 Chevreul Institute Platform (U-Lille / CNRS). The Region Hauts de France and the
378 French government are warmly acknowledged for the co-funding of these apparatus.
379 The funding bodies were not involved in the study design, data collection, and analysis,
380 decision to publish, or preparation of the manuscript.

381

382 Author contributions

383 S.B.G. conceived and supervised the project.

384 B.R., A.D., V.A., G.B., F.S., and S.B.G. contributed to the study design.

385 B.R., A.D., V.A., and S.B.G. performed the experiments.
386 B.R., A.D., V.A., G.B., F.S., and S.B.G analyzed the data.

387 C.L. performed statistical analysis.

388 F.P. reared the mice and the fleas

389 S.B.G. wrote the manuscript with inputs from all authors.

390

391 **CONFLICT OF INTEREST**

392 The authors declare no conflict of interest.

393
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562

563
566 **Figure 1. Phenotypic characterization of the ΔcpxAR mutant in flea-mimicking**
567 **conditions.**

568 (A) Cristal violet assay showing biofilm formation of the WT and the ΔcpxAR strain. Data
569 are quoted as the mean ± SD from at least three independent experiments. (B)
570 Assessment of the WT and the ΔcpxAR strain growth in LB at 300 mOSM and 600
571 mOsM by measuring OD₆₀₀ over time. (C) Survival of Y. pestis WT, the ΔcpxAR, and the
572 complemented strain after exposure to H₂O₂ (20 mM) for one hour. The mean
573 percentage of survival ± SD values from three independent experiments are shown.

574 Statistical analyses were performed with GraphPad Prism V10.0.2 using the Kruskal-
575 Wallis test with Dunn's correction for multiple comparisons (*: p<0.05). (D) Photos
576 showing the growth of the WT, ΔcpxAR and complemented strain on LB agar plates
577 containing different concentrations of CuSO₄. For each strain, different quantities of
578 bacteria (8x10⁶ CFU/mL to 4 x 10⁵ CFU/mL) from three different cultures were spotted.

580 **Figure 2. The copper is a cue for CpxR activation.**

581 Assessment of CpxR phosphorylation with or without exposure to oxidant stresses. (A)
582 CpxR and CpxR~P proteoforms were separated by Phos-Tag™ acrylamide gel after
583 extraction from the bacterial pellet and were revealed by Western blot. This result is
584 representative of three independent experiments. (B) Box-and-whisker plots (Tukey)
585 show the percentage of the CpxR~P proteoform/CpxR total in the WT strain. Data are
586 the results from at least three independent experiments. Statistical analysis was
587 performed with GraphPad Prism V10.0.2 using the Mann-Whitney test (*: p<0.01).
588

Figure 3. The CpxAR system provides a fitness advantage in the flea gut.

590 Bacterial loads immediately after and 13 days after the infectious meal. Experiments 591 were performed with *Y. pestis* ΔcpxAR mutant and *Y. pestis* WT Kim6+ or *Y. pestis* 592 complemented strain. The data correspond to samples from two independent 593 experiments (white diamond: first experiment, black circles: second experiment). Each 594 symbol indicates the ratio within an individual flea. The horizontal lines indicate the 595 median CFU per flea. Statistical analyses were performed using the Kruskal-Wallis test 596 and a Conover post-hoc test with a Benjamini-Hochberg adjustment for multiple testing. 597 Any test with a p-value < 0.05 is declared as significant. 598

599

Figure 4. Copper-activated CpxAR protects *Y. pestis* against oxidative stress.

600 (A) *In vitro* LB co-cultures did not display any bacterial fitness defect of the ΔcpxAR 601 compared to the WT strain. The co-culture growth curve is represented by linked half- 602 black and half-white dots. The number of CFU/mL of the total population and the mutant 603 was, respectively, determined by enumeration on LB plates and LB plates containing 604 trimethoprim at 0, 3, and 8h of culture. (B) *In vitro* assessment of the copper pre- 605 exposed *Y. pestis* survival to 1h and 3h of H₂O₂ stress treatment in HPLM and the 606 control conditions (untreated, H₂O₂, or copper). Statistical analysis was performed with 607 GraphPad Prism V10.0.2 using a two-way ANOVA multiple comparison test (*: p<0.05). 608

609
<table><caption>609 Table 1: Strains, vectors and primers used in this study.</caption><thead><tr><th>Strains, plasmids or primers</th><th>Description*</th><th>Reference</th></tr></thead><tbody><tr><td><strong><em>Y. pestis</em></strong></td><td></td><td></td></tr><tr><td>KIM6+ (WT)</td><td>Wild-type strain pYV- negative strain</td><td>(58)</td></tr><tr><td>Δ<em>cpxAR</em></td><td>Δ<em>cpxAR</em>::<em>dfrB</em>, <em>Tmp</em><sup>R</sup></td><td>This study</td></tr><tr><td>Δ<em>cpxAR</em> Comp.</td><td>Δ<em>cpxAR</em>::<em>dfrB</em>, miniTn7::<em>cpxRA-aphA3</em>'<br>Kan<sup>R</sup></td><td>This study</td></tr><tr><td><strong>Vectors</strong></td><td></td><td></td></tr><tr><td>pKD4</td><td>ori6K vector, Kan<sup>R</sup> cassette flanked by FRT sites</td><td>(52)</td></tr><tr><td>pEP1087</td><td>derived from pKD4, <em>Tmp</em><sup>R</sup> cassette flanked by FRT sites</td><td>(53)</td></tr><tr><td>pEP1013</td><td>vector encoding the Red recombinase, <em>Amp</em><sup>R</sup></td><td>(53)</td></tr><tr><td>pEP1436</td><td>pEP1013 encoding the endonuclease I-Sce I</td><td>(6)</td></tr><tr><td>pUC18R6K-mini-Tn7</td><td>oriR6K vector bearing empty R. mini-Tn7T, <em>Amp</em><sup>R</sup></td><td>(54)</td></tr><tr><td>pUC18R6K-mini-Tn7::<em>cpxRA-aphA3</em>'</td><td>pUC18R6K-mini-Tn7 encoding the fusion <em>cpxRA-aphA3</em>'</td><td>This study</td></tr><tr><td><strong>Primers</strong></td><td><strong>Sequences (5'-3')</strong></td><td></td></tr><tr><td>CpxAR-F</td><td>ACGGTATCGAAACCTTGAAAGAATTACGCCAACACCATCAG<br>ACCCCGGTGGTGTAGGCTGGAGCTGCTTC</td><td>This study</td></tr><tr><td>CpxAR-R</td><td>GACGCCCGATCTCGGGCTTCATCTGTACGATAAAATGGCCG<br>GAATATTTGCATATGAATATCCTCCTTAG</td><td>This study</td></tr><tr><td>CpxAR-FC-PstI</td><td>TATCTGCAGGTGCATTGCCTCGCGATCTT</td><td>This study</td></tr><tr><td>CpxAR-RC-EcoRV</td><td>TATGATATCGCATTATCGGGTTACCGTTG</td><td>This study</td></tr><tr><td>CpxAR-check-F</td><td>CTATTAGTTGATGATGACCG</td><td>This study</td></tr><tr><td>CpxAR-check-R</td><td>AAGATTTAAGCGGATGCAGC</td><td>This study</td></tr><tr><td>CpxARi-CF</td><td>AGTTCACCCTGCTCTATCTG</td><td>This study</td></tr><tr><td>CpxARi-CR</td><td>GCGATAACACCAGCAAGTCA</td><td>This study</td></tr></tbody></table>

610 *: <em>Tmp</em><sup>R</sup> (<em>dfrB</em>), <em>Kan</em><sup>R</sup> (<em>aphA3</em>'), <em>Amp</em><sup>R</sup> (<em>bla</em>), resistance to trimethoprim, kanamycin, and

611 ampicillin respectively

612