- Research article
- Open Access
The CpxR regulates type VI secretion system 2 expression and facilitates the interbacterial competition activity and virulence of avian pathogenic Escherichia coli
© The Author(s) 2019
- Received: 31 January 2019
- Accepted: 8 May 2019
- Published: 24 May 2019
Systemic infections caused by avian pathogenic Escherichia coli (APEC) are economically devastating to poultry industries worldwide and are also potentially threatening to human health. Pathogens must be able to precisely modulate gene expression to facilitate their survival and the successful infection. The Cpx two-component signal transduction system (TCS) regulates surface structure assembly and virulence factors implicated in Gram-negative bacterial pathogenesis. However, the roles of the Cpx TCS in bacterial fitness and pathogenesis during APEC infection are not completely understood. Here, we show that the Cpx TCS response regulator CpxR is critical to the survival and virulence of APEC. Inactivation of cpxR leads to significant defects in the interbacterial competition activity, invasion and survival of APEC in vitro and in vivo. Moreover, activation of CpxR positive regulates the expression of the APEC type VI secretion system 2 (T6SS2). Further investigations revealed that phosphorylated CpxR directly bound to the T6SS2 hcp2B promoter region. Taken together, our results demonstrated that CpxR contributes to the pathogensis of APEC at least through directly regulating the expression and function of T6SS2. This study broadens understanding of the regulatory effect of Cpx TCS, thus elucidating the mechanisms through which Cpx TCS involved in bacterial virulence.
Systemic infections caused by avian pathogenic Escherichia coli (APEC) are economically devastating to poultry industries worldwide. APEC shares a broad range of virulence factors with human extraintestinal pathogenic E. coli (ExPEC), including uropathogenic E. coli (UPEC) and neonatal meningitis E. coli (NMEC). Moreover, these ExPEC strains can cause cross infections, thus indicating that APEC may be a potential virulence gene reservoir for UPEC and NMEC [1–4]. APEC initially infects poultry via the respiratory tract, then spreads systemically throughout the entire body. Pathogens use several common strategies to increase fitness and facilitate survival and systemic infections [5, 6]. The conserved type VI secretion system (T6SS), present in more than one-fourth of Gram-negative pathogens, is used to deliver effector proteins into eukaryotic or prokaryotic cells. These effectors have roles in a broad variety of functions, including interbacterial competition, stress sensing, biofilm formation and virulence [7–13]. Three distinct T6SSs have been identified in APEC genomes. Among them, the APEC T6SS2, similar to NMEC T6SS, is responsible for the binding and invasion to host cells, survival, interbacterial competition and pathogenesis of APEC and NMEC [11, 14–16]. As contact-dependent pathways, the T6SSs are tightly regulated by various regulatory mechanisms to ensure successful bacterial infection .
When detecting new environmental cues or stresses, bacteria must precisely modulate gene expression to facilitate their survival through complex regulatory networks. The two-component signal transduction systems (TCSs) consist of a histidine kinase sensor in the bacterial plasma membrane and a cytoplasmic response regulator that allows bacteria to cope with changes in the environment [18, 19]. The Cpx TCS of E. coli consists of three proteins, CpxA, CpxR, and CpxP, which mainly mediate the detection of and adaptation to envelope stresses [20, 21]. Many environmental cues leading to Cpx TCS activation have been identified, including envelope protein misfolding, overexpression of the pilus and alkaline pH. In addition, adhesion to hydrophobic surfaces activates the Cpx TCS in an NlpE-dependent manner [22–24]. After an environmental signal is received, the kinase histidine CpxA undergoes autophosphorylation and phosphorylates the response regulator CpxR. Then phosphorylated CpxR binds its regulon and functions as a transcriptional regulator. The third component of the Cpx system is CpxP, a small periplasmic protein thought to negatively regulate the Cpx TCS by binding CpxA and maintaining it in an inactive state [23–25].
The Cpx TCS affects bacterial virulence through regulating the expression of virulence genes involved in envelope stress relief, biofilm formation, adherence, motility and secretion systems (type III, type IV and type VI) [26–36]. The activation of Cpx TCS in Shigella sonnei, Legionella pneumophila, Xenorhabdus nematophila and Yersinia pestis results in increased expression of virulence genes and contributes to pathogenesis [26, 30, 32, 33, 37, 38]. The Cpx TCS also regulates T6SS expression, and affects the virulence of Citrobacter rodentium [31, 36]. However, constitutive activation of the Cpx TCS results in downregulation of virulence genes and attenuated virulence of Haemophilus ducreyi [39, 40]. In pathogenic E. coli, the Cpx TCS has been implicated in the regulation of pili and the type III secretion system and is responsible for virulence [27, 28, 41–44]. The Cpx TCS has been shown to be involved in the adherence, invasiveness and biofilm formation of APEC through controlling the orientation of the type 1 fimbriae OFF–ON switch. However, the roles of Cpx TCS in the fitness and pathogenesis of APEC during in vivo infection are not completely understood. In this study, we demonstrated that cpxR deletion leads to substantial defects in interbacterial competition activity, invasion and survival, and attenuates the virulence of APEC in vitro and in vivo. Moreover, we provide evidence that CpxR positively regulates the expression and function of APEC T6SS2, which may contribute to the systemic infection and pathogenesis of APEC. This study broadens understanding of the regulatory effect of Cpx TCS, thereby elucidating the mechanisms through which Cpx TCS contributes to virulence.
Bacterial strains, plasmids and growth conditions
Bacterial strains and plasmids used in this study
Strains or plasmids
cpxR gene deletion mutant in DE719
ΔcpxR with plasmid pSTV28-cpxR
DH5α with plasmid pRCL
DH5α with plasmid pRCL-Phcp2B
DE719 with plasmid pBAD/Myc-His
DE719 with plasmid pBAD-nlpE
ΔcpxR with plasmid pBAD/Myc-His
ΔcpxR with plasmid pBAD-nlpE
F-, Δ(lacZYA-argF)U169, recA1, endA1, hsdR17(rk−, mk+), phoA, supE44, λ-
F-, ompT, hsdS (r B − m B − ) gal, dcm (DE3)
Kan, F1 origin, His tag
pET28a(+) carrying cpxR gene
Cm, p15A origin
pSTV28 derivative harboring cpxR gene
Amp, ColE1 derivative cloning vector, pBAD (ara) promoter
pBAD/Myc-His expressing E. coli K12 NlpE-His from the pBAD (ara) promoter
Cm, promoterless lacZ,
pRCL harboring hcp2B promoter
Amp, expresses λ red recombinase
Cm, template plasmid
Cm, Amp, yeast Flp recombinase gene, FLP
Construction of mutant and complemented strains
Primers used in this study
Sequence (5′ to 3′)a
For gene expression, deletion and complementation
Upstream region of cpxR
Downstream region of cpxR
Upstream region of cpxR
Downstream region of cpxR
For lacZ fusion and EMSA
Upstream region of hcp2B
Upstream region of hcp2B
Upstream region of hcp2B
Experimental animal infection
To determine the effect of CpxR on APEC virulence, we intramuscularly injected groups of eight 7-day-old ducks with bacterial suspensions containing 105 Colony-Forming Units (CFUs). The number of CFUs in the injected inoculum was confirmed by plating on LB agar. Ducks inoculated with PBS were used as negative controls. Mortality was monitored daily until 7 days after infection.
The bacterial survival and competitive assays in vivo were measured as described previously [14, 46, 47]. Briefly, 7-day-old ducks were infected intratracheally with 108 CFU bacterial suspensions with wild-type or mutant strains. Bacterial mixtures with equal amounts of wild-type and mutant strains were inoculated into ducks for the competitive assays. After 24 h, the ducks were euthanized and dissected, and the lung, liver and spleen were collected, weighed and homogenized. Serial dilutions of the homogenates were plated onto LB agar with or without chloramphenicol to distinguish the mutant strain or total bacterial loads. The competitive index (CI) was calculated for the mutant by dividing the output ratio (mutant/wild-type) by the input ratio (mutant/wild-type).
Bacterial adhesion and invasion assays
Bacterial adhesion and invasion assays were performed as described previously [14, 47]. Chicken embryo fibroblast DF-1 cell monolayers were washed with Dulbecco’s modified Eagle’s medium (DMEM) without fetal bovine serum (Gibco, Grand Island, NY, USA) and infected with bacteria at a multiplicity of infection of 100 for 2 h at 37 °C under 5% CO2. After being washed with PBS, the cells were lysed with 0.5% Triton X-100, and the bacteria were counted by plating on LB agar plates. For invasion assays, the extracellular adherent bacteria were killed with DMEM containing 100 μg/mL gentamicin for 1 h, then washed and lysed with 0.5% Triton X-100 to enumerate the invasive bacteria.
Bacterial competition assays in vitro
Bacterial competition assays were performed as described previously [11, 48] with some modifications. In brief, fresh donor and recipient strains were adjusted to an OD600nm of 0.5 and mixed at a 5:1 ratio. This mixture was spotted on LB low-salt plates with nitrocellulose membranes for 6 h at 30 °C. Bacterial spots were collected, diluted and spotted onto LB plates with or without antibiotics for the selection of donor or recipient strains. Then the competition outcomes were calculated as the ratio of the donor strain to the recipient strain.
Quantitative real-time reverse transcription PCR (qRT-PCR)
The expression of genes was investigated with qRT-PCR, as described previously [14, 46]. Briefly, total RNA was extracted from bacteria with TRIzol reagent (Invitrogen), and residual genomic DNA was removed with a Turbo DNase kit (Life Technologies, Carlsbad, CA, USA). cDNA synthesis was performed with a PrimeScript RT reagent kit (TaKaRa) according to the manufacturer’s protocol. qRT-PCR was conducted with SYBR Premix Ex Taq (TaKaRa) and gene-specific primers (Table 2), and the data were normalized to the expression of the housekeeping gene dnaE. The relative fold change was calculated via the ΔΔCT method .
Antibody production and Western blotting
Polyclonal anti-Hcp2B serum was raised in New Zealand white rabbits through subcutaneous immunization, as described previously . For Western blotting, bacterial samples were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Amersham Pharmacia Biotech, Piscataway, NJ, USA) as described previously [14, 47]. The proteins were reacted with the primary antibodies anti-Hcp2B or anti-DnaK (Enzo Life Sciences, Farmingdale, NY, USA), followed by horseradish peroxidase conjugated goat anti-mouse or goat anti-rabbit IgG secondary antibodies. The antigen–antibody complexes were visualized with chemiluminescence substrate (Amersham Pharmacia Biotech).
To confirm the promoter activity of the hcp2B gene, we performed β-galactosidase assays as described previously . Briefly, the Phcp2B-lacZ fusions were constructed by cloning the Phcp2B promoter into the promoterless plasmid pRCL. Then, the bacteria containing the Phcp2B-lacZ fusion plasmid or promoterless plasmid pRCL were collected and resuspended in Z buffer. The β-galactosidase activity was quantified with ortho-nitrophenyl-β-galactoside as the substrate. This assay was performed three times in triplicate.
Electrophoretic mobility shift assay (EMSA)
The cpxR gene was cloned into the pET28a(+) plasmid (Novagen, Madison, WI, USA), and the recombinant proteins were expressed in E. coli BL21 (DE3) cells by addition of 1 mM isopropyl-β-d-thiogalactopyranoside. The purification of CpxR fusion protein was performed with a HisTrap high-performance column (GE Healthcare, Little Chalfont, Buckinghamshire, UK) as previously described . The CpxR protein was phosphorylated with acetyl phosphate (Sigma, St. Louis, MO, USA) as previously described . Then, EMSA were performed to determine the binding of phosphorylated CpxR (CpxR-P) to the hcp2B promoter. Briefly, the sequence of the hcp2B promoter region with or without the putative CpxR binding site was amplified and labeled with biotin. The biotin-labeled DNA probe (40 ng) was incubated with increasing concentrations of CpxR-P protein in EMSA binding buffer (10 mM Tris, 50 mM KCl, 5 mM MgCl2, 1 mM dithiothreitol, 0.1 mM MnCl2, 2.5% glycerol and 50 ng/μL poly[dI-dC]). After incubation for 30 min at room temperature, the reactions were subjected to electrophoresis and transferred to a nylon membrane. The biotin-labeled DNA was detected with a chemiluminescent substrate (Amersham Pharmacia Biotech). A competitive EMSA was performed by simultaneously incubating the biotin-labeled and unlabeled hcp2B promoter region with CpxR-P protein.
Statistical analyses were conducted with the GraphPad Software package. One-way analysis of variance (ANOVA) was used to analyze the results of the adhesion, invasion and bacterial competition assays. Two-way ANOVA was used to analyze the qRT-PCR data. Analysis of the animal infection study results was performed with the non-parametric Mann–Whitney U-test. P < 0.05 was considered statistically significant.
Deletion of cpxR attenuates APEC virulence in ducks
CpxR provides the colonization and competition fitness for APEC during infection in vivo
To determine whether the decreased mortality was associated with altered bacterial colonization and survival capacity, we investigated the bacterial loads of APEC strains in the lung, liver and spleen at 24 h post-infection. The CFUs of the mutant strain ΔcpxR recovered from the lung, liver and spleen were significantly less than those of the wild-type and complemented strains in the noncompetitive assays (P < 0.05; Figures 1B–D). Bacterial pathogens compete with other bacteria, thus facilitating their replication and survival, and systemic infection . Thus, the effects of CpxR on interbacterial competition fitness and pathogenesis in vivo were further examined by inoculation with equal amounts of mutant and wild-type strains. Similarly, the mutant strain ΔcpxR was strongly outcompeted by the wild-type strain in the tested organs (Figure 1E). These results indicated that CpxR potentiates the effective colonization and increases the competition fitness of APEC during infection in vivo.
The role of CpxR in the invasion of APEC to DF-1 cells
CpxR facilitates the interbacterial competition of APEC in vitro
Previous studies have indicated that overproduction of the outer membrane lipoprotein NlpE upregulates the Cpx pathway in E. coli [21, 22]. Thus, to further demonstrate the roles of CpxR in interbacterial competition, we overexpressed NlpE in wild-type and mutant strains, and measured the interbacterial competition activity of these strains. As expected, the overexpression of NlpE significantly increased the interbacterial competition activity in the wild-type strain (P < 0.01). However, no difference in interbacterial competition activity was observed for the mutant strain ΔcpxR overexpressing NlpE (Figure 3C). Collectively, these observations indicated that activation of CpxR facilitates the interbacterial competition activity of APEC.
CpxR regulates the expression of T6SS2 in APEC
To further demonstrate that activation of CpxR promotes T6SS2 expression, we overexpressed NlpE in wild-type and mutant strains. The overexpression of NlpE significantly upregulated the transcription of hcp2B, vgrG and xmtU genes in the wild-type strain (P < 0.001) but not the mutant strain ΔcpxR (Figure 4C). Similarly, Hcp2B production increased with CpxR activation in the wild-type strain compared with the mutant strain ΔcpxR (Figure 4D). These data indicated that CpxR positively regulated the expression of T6SS2 hcp2B operon in APEC.
CpxR protein directly binds the hcp2B promoter region
Bacterial virulence is determined by the expression of virulence factors, and pathogens must be able to precisely modulate gene expression to facilitate their adaptation and survival in the local microenvironment or host cells. The infection process requires rapid adaptation to the host environment through various regulatory mechanisms. TCSs are activated in response to changing environmental cues, thus representing one mechanism enabling bacterial adaptation through transcriptional regulation of gene expression [18, 19]. The Cpx TCS responds to cell envelope stress, which is used by bacteria to maintain cell envelope integrity [23, 58]. Increasing studies implicate the Cpx TCS in the virulence of numerous Gram-negative bacteria [26–35, 41, 44, 52, 53]. These findings prompted us to determine the regulatory roles of the Cpx TCS in the fitness and virulence of APEC. This study provided evidence that the regulator CpxR contributes to the interbacterial competition activity, survival and virulence of APEC at least through directly regulating the expression and function of T6SS2.
By using a duck systemic infection model, we demonstrated that inactivation of the cpxR gene attenuates the virulence of APEC. Moreover, the complemented strain showed recovered virulence. Thus, we concluded that CpxR is necessary for full virulence of APEC. APEC initially infects poultry via the respiratory tract, then spreads systemically throughout the entire body, thus resulting in subsequent bacteremia and death. Colonization and invasion are important virulence parameters in APEC infection [6, 59]. A previous study has indicated that the Cpx TCS affects virulence features including the adherence, invasiveness, motility and biofilm formation of APEC through the direct binding of CpxR-P to the fimA promoter . Indeed, type 1 fimbriae are filamentous surface organelles known to contribute to adherence, invasiveness and biofilm formation [6, 60]. Our results indicated that inactivation of CpxR does not affect the adherence ability of APEC, in agreement with findings from a previous study. This observation might have been because CpxR affects the ON/OFF orientation on the fimA promoter . In contrast, the mutant strain ΔcpxR showed a significantly decreased ability to invade into host cells. The decreased capacity of the mutant strain to invade DF-1 cells might contribute to the inability of this mutant to effectively colonize, survive in and infect ducks. Similarly, decreased colonization and virulence have been observed for the cpx mutant strain in different Gram-negative pathogens [31, 61–63].
Pathogens have developed diverse attack strategies to efficiently compete with other bacteria for limited resources and facilitate survival and infection . Our results showed that the interbacterial competition capacity of the mutant strain ΔcpxR was much lower than that of the wild-type strain in vitro. Moreover, the mutant strain ΔcpxR was significantly outcompeted by the wild-type strain in ducks. These results indicated that CpxR contributes to competition and survival in vivo, and might be responsible for the systemic infection and virulence of APEC. Consistently with these results, CpxRA influences the initial colonization and outgrowth of Xenorhabdus nematophila during infection through regulation of the nil locus . In addition, the Cpx system may affect bacterial virulence by modulating surface characteristics and serum resistance of pathogens .
The E. coli CpxR regulon contains hundreds of genes, according to genome-wide screens for the putative CpxR binding sequence [56, 57]. In addition to regulating protein folding and degradation, CpxR also plays roles in regulating T6SS expression in Citrobacter rodentium . T6SSs are a common strategy used by many pathogens to mediate successful infections in hosts [7–14, 16]. The APEC T6SS2 functions in the invasion, survival, interbacterial competition and pathogenesis of APEC and NMEC [11, 14–16, 55]. In this study, we observed that CpxR influenced the expression of T6SS2; we then sought to investigate the mechanism of CpxR mediated T6SS2 regulation in APEC. We found that inactivation of the regulator CpxR significantly downregulated the transcription of the T6SS2 hcp2B operon. Although the fold-change in the Hcp2B protein was less than the changes at the transcription level, it was also decreased in the mutant strain ΔcpxR. Previous study showed that the hcp2B operon is necessary for the interbacterial competition activity of T6SS2 in APEC . Moreover, our results showed that the decreased expression of Hcp2B protein was in good agreement with the interbacterial competition activity for mutant strain ∆cpxR. Our previous study has also found different fold-changes in the transcription and expression levels , possibly because many factors affect protein expression. Collectively, these results suggested that CpxR promotes the expression of APEC T6SS2. This result is consistent with the regulatory role in Citrobacter rodentium . Additional, the decreased expression of the T6SS2 hcp2B operon, including the interbacterial effector encoding the gene xmtU, might be responsible for the decreased invasion, diminished survival and impaired competition of the mutant strain ΔcpxR. Many environmental conditions upregulate the Cpx pathway. The lipoprotein NlpE has been shown to activate the Cpx pathway when it is overproduced [21–23, 58, 65, 66]. We observed that NlpE overexpression in the wild-type strain DE719 increased the T6SS2 hcp2B operon expression and subsequently significantly enhanced interbacterial competition. In contrast, no regulatory effect was found for the mutant strain ΔcpxR overexpressing NlpE. An in silico analysis identified a putative CpxR binding site in the hcp2B promoter region. Furthermore, we provided evidence that CpxR-P binds the promoter region of the hcp2B operon, thus suggesting that CpxR directly regulates T6SS2 hcp2B expression. Taken together, these results indicated that the regulation of T6SS2 expression and function by CpxR may contribute to the infection and pathogenesis of APEC.
We thank the staff of Shanghai Tolo Biotechnology Co. Ltd for helps in Electrophoretic mobility shift assay (EMSA).
This work was supported by the National Natural Science Foundation of China (Grant Number 31572523) and the National Key Research and Development Program of China (Grant Number 2016YFD0500800). The funders had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.
SW designed the research studies. ZY, DW, SX and DZ performed the experiments. SW, ZY, DW, TL, MT, JQ, CD and SY analyzed the data and discussed the results. ZY, SW and SY wrote the manuscript. All authors read and approved the final manuscript.
Ethics approval and consent to participate
All animal experiments were conducted in strict accordance with the Guidelines on the Humane Treatment of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of the Shanghai Veterinary Research Institute (Permit No: Shvri-Po-0080).
The authors declare that they have no competing interests.
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