Key roles of two-component systems in intestinal signal sensing and virulence regulation in enterohemorrhagic Escherichia coli

Abstract

Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that infects humans by colonizing the large intestine. Upon reaching the large intestine, EHEC mediates local signal recognition and the transcriptional regulation of virulence genes to promote adherence and colonization in a highly site-specific manner. Two-component systems (TCSs) represent an important strategy used by EHEC to couple external stimuli with the regulation of gene expression, thereby allowing EHEC to rapidly adapt to changing environmental conditions. An increasing number of studies published in recent years have shown that EHEC senses a variety of host- and microbiota-derived signals present in the human intestinal tract and coordinates the expression of virulence genes via multiple TCS-mediated signal transduction pathways to initiate the disease-causing process. Here, we summarize how EHEC detects a wide range of intestinal signals and precisely regulates virulence gene expression through multiple signal transduction pathways during the initial stages of infection, with a particular emphasis on the key roles of TCSs. This review provides valuable insights into the importance of TCSs in EHEC pathogenesis, which has relevant implications for the development of antibacterial therapies against EHEC infection.

enterohemorrhagic Escherichia coli, two-component system, intestinal signal, Shiga toxin, locus of enterocyte effacement, virulence regulation

Introduction

Enterohemorrhagic Escherichia coli infection in humans

Enterohemorrhagic Escherichia coli (EHEC) is a global foodborne pathogen that colonizes the human large intestine (Riley et al. 1983, Lewis et al. 2015). EHEC infection is associated with a wide range of clinical illnesses, including abdominal cramps, bloody diarrhea, hemorrhagic colitis (HC), and the life-threatening complication hemolytic uremic syndrome (HUS) (Riley et al. 1983, Karmali et al. 1985, Nataro and Kaper 1998). Healthy cattle are the primary and natural reservoirs of EHEC (Wells et al. 1991, Chapman et al. 1993), and human infection usually occurs through the ingestion of contaminated food or water and direct contact with infected people or animals (Chapman et al. 1993, Banatvala et al. 1996, National Association of State Public Health Veterinarians et al. 2007). EHEC can infect people of any age, but extraintestinal complications and death occur most frequently in children under 10 years of age and in elderly individuals (Tarr 1995, Heuvelink et al. 2002). Notably, the infectious dose of EHEC is extremely low (∼10–100 cells), resulting in an increased incidence of EHEC infection (Tilden et al. 1996, Sperandio et al. 1999, Schmid-Hempel and Frank 2007).

In 1982, the prototypical EHEC serotype O157:H7 was first identified as the human pathogen associated with two HC outbreaks in Oregon and Michigan, USA (Riley et al. 1983). Since then, multiple large-scale outbreaks caused by EHEC have been recorded in at least 30 countries on six continents, especially in developed countries (Waters et al. 1994). The occurrence of massive outbreaks has made EHEC infection a serious public health concern worldwide. In 2011, one of the largest outbreaks of EHEC occurred in Germany because of the consumption of contaminated fenugreek sprouts (Buchholz et al. 2011). According to a report from the Robert Koch Institute (RKI, the federal institution for disease prevention and control in Germany), this outbreak resulted in a total of 3816 cases, with more than half of these individuals requiring hospitalization, 845 of whom developed HUS, and 54 patients died (Frank et al. 2011). Recent outbreak surveillance data from the CDC's National Outbreak Reporting System (NORS; https://wwwn.cdc.gov/norsdashboard/) revealed that EHEC caused 725 outbreaks in the United States from 2011 to 2021. Among infections, those caused by EHEC have high hospitalization rates, with ∼30% of patients affected during outbreaks requiring hospitalization. The annual economic costs of disease caused by EHEC infections, including costs associated with the loss of productivity, medical care, and premature deaths, are substantial. Therefore, the development of effective prevention and therapeutic strategies for infections caused by this pathogen is an urgent need.

Major virulence factors and pathogenesis of EHEC

EHEC infection is initiated and perpetuated by attachment or adhesion to the intestinal epithelium, as well as by virulence factors released by EHEC. The major virulence factors of EHEC are products of the pathogenicity island termed the locus of enterocyte effacement (LEE; Fig. 1A) and Shiga toxins (Fig. 1B).

Figure 1.

The LEE-encoded T3SS and Shiga toxins are major virulence factors that cause disease in EHEC. (A) Schematic representation of the LEE pathogenicity island and the LEE-encoded T3SS. The LEE pathogenicity island consists of five polycistronic operons (LEE1–LEE5) that contain all the genes encoding the T3SS apparatus. Key genes are noted above their respective LEE operons. The T3SS is a protein appendage that delivers bacterial effector proteins directly into the host cytoplasm, leading to changes in signaling and actin remodeling, ultimately triggering the formation of attaching and effacing lesions. The Tir is exported by the T3SS and integrated into the host cell membrane, where it functions as a receptor for the outer membrane adhesin intimin. Binding of intimin to Tir enables EHEC to establish an intimate attachment to the host epithelium, which is required for successful intestinal colonization. (B) Schematic diagram of the pathogenic mechanisms of Shiga toxin. Shiga toxin is an AB5 toxin consisting of a single enzymatic A subunit that is noncovalently bound to a pentamer of the B subunit. After its secretion from EHEC, the B subunits of Shiga toxin bind to the Gb3 receptor on the surface of the host cell to initiate internalization. After internalization, the Shiga toxin is sequentially transported to the trans-Golgi network and the endoplasmic reticulum, where the A subunit is proteolytically nicked to an enzymatically active A1 subunit and translocated to the cytosol. The A1 subunit exerts RNA N-glycosidase activity that specifically removes an adenine residue from the 28S rRNA of the 60S ribosome, thereby inhibiting protein synthesis and ultimately leading to cell death. IM, inner membrane; OM, outer membrane; EM, eukaryotic membrane; T3SS, type III secretion system; Tir, translocated intimin receptor; Gb3, globotriaosylceramide.

The adherence of EHEC to the host intestinal epithelium and the formation of attaching and effacing (A/E) lesions are critical first steps during EHEC infection (Bardiau et al. 2010, McWilliams and Torres 2014). A/E lesions are pedestal-like structures characterized by the loss of microvilli in epithelial cells and trigger the intimate attachment of EHEC to the host enterocyte membrane (Moon et al. 1983, Frankel et al. 1998). The genetic elements responsible for A/E lesions are located in the LEE pathogenicity island, which consists of five polycistronic operons (LEE1–LEE5) (Fig. 1A) (McDaniel et al. 1995, Elliott et al. 1998, 1999, 2000). The first gene of LEE1 encodes the master LEE regulator Ler, which directly activates gene transcription from LEE2 to LEE5 (Elliott et al. 2000). The remaining genes in LEE1, as well as LEE2 and LEE3, encode the major structural components of a type III secretion system (T3SS) that exports bacterial effector proteins into host cells to subvert host cell signaling pathways during A/E lesion formation (Fig. 1A) (McDaniel et al. 1995). LEE4 encodes additional T3SS structural components and translocator and effector proteins (Naylor et al. 2005). LEE5 encodes the outer membrane adhesin, named intimin, and its translocated receptor Tir, which are necessary for the intimate attachment of EHEC to the host epithelium (Fig. 1A) (Kenny and Finlay 1995, Kenny et al. 1997, Garmendia et al. 2005).

Another group of main virulence factors of EHEC are Shiga toxins, which are responsible for severe human diseases such as HUS. Shiga toxins are encoded by toxin-converting bacteriophages in the EHEC chromosome (O'Brien et al. 1984). Their genomic location determines that the expression of Shiga toxins is regulated by the phage cycle rather than by the genomic regulatory network employed by most genome-encoded virulence factors (Wagner et al. 2001, Tyler et al. 2013, Kruger and Lucchesi 2015). Shiga toxins are divided into two groups, termed Shiga toxin 1 and Shiga toxin 2, which share 56% amino acid sequence homology (Strockbine et al. 1986, Fraser et al. 2004). All EHEC strains produce one or both of the Shiga toxins. Shiga toxin 2 is more virulent than Shiga toxin 1, and Shiga toxin 2-producing strains are more often associated with HC or HUS in human infections (Ostroff et al. 1989, Boerlin et al. 1999). Shiga toxins belong to the AB5 toxin family and consist of an enzymatic A subunit and five identical receptor-binding B subunits that form a pentamer (Fig. 1B) (Stein et al. 1992, Fraser et al. 1994). The A subunit is an RNA N-glycosidase, and the B subunit is responsible for binding to host receptors (Jacewicz et al. 1986, Endo et al. 1988). During infection, EHEC secretes Shiga toxins into the extracellular milieu, and the B subunits of Shiga toxins bind to the glycolipid receptor globotriaosylceramide (Gb3) on the surface of host cells (Fig. 1B) (Jacewicz et al. 1986). After binding to Gb3, Shiga toxins are internalized via endocytosis pathways, retrogradely sorted into the trans-Golgi network, and further transported into the endoplasmic reticulum (Sandvig et al. 1992, Mallard et al. 1998). The A subunit is further processed by host furin and furin-like proteases into the enzymatic A1 subunit and the linker A2 subunit (Garred et al. 1995, Sandvig and van Deurs 1996). The enzymatically active A1 subunit is then released from the endoplasmic reticulum into the cytoplasm, where it inhibits host protein synthesis by removing a specific adenine residue from the 28S rRNA of the 60S ribosome, ultimately leading to cell death (Fig. 1B) (Endo et al. 1988).

The virulence genes of EHEC are subject to strict control by host intestinal signals

The virulence genes of EHEC are strictly regulated, ensuring that expression occurs only under optimal environmental conditions; this mechanism helps cells avoid unnecessary metabolic costs and/or avoid alerting the host immune system, facilitating successful survival and colonization. The human large intestine, an optimal site for EHEC colonization, is an extremely complex environment containing many metabolites and signaling molecules derived from both the host itself and the commensal microbiota (Hooper et al. 2001, Hughes et al. 2010). EHEC strains have evolved complex regulatory systems that integrate multiple host- and microbiota-derived signals present in the human intestinal tract and precisely coordinate the expression of virulence genes to initiate the disease-causing process in a highly site-specific manner. The intestinal signals that affect EHEC virulence gene expression can be classified into the following main categories: amino acids and their derivatives (d-serine, l-arginine, serotonin, and indole) (Connolly et al. 2015, Kumar and Sperandio 2019, Kumar et al. 2020, Menezes-Garcia et al. 2020), short-chain fatty acids (mainly butyrate) (Nakanishi et al. 2009), mucosal sugars (galactose and fucose) (Pacheco et al. 2012, Garimano et al. 2022), carbon sources (pyruvate, mannose, gluconic acid, malate, and ethanolamine) (Kendall et al. 2012, Carlson-Banning and Sperandio 2016, Liu et al. 2023, Yang et al. 2023a), vitamins (riboflavin, nicotinamide, biotin, and cobalamin) (Yang et al. 2015, 2023b, Cordonnier et al. 2016, Liu et al. 2022), hormones (epinephrine and norepinephrine) (Reading et al. 2009, Moreira et al. 2016), ions (iron, ammonium, and magnesium) (Tobe et al. 2014, Liu et al. 2020, Jia et al. 2021), and intestinal physiology (temperature and pH) (Ebel et al. 1996, Muhldorfer et al. 1996, House et al. 2009).

The precise regulation of EHEC virulence gene expression in response to various intestinal signals is achieved mainly through single regulatory proteins, small regulatory RNAs (sRNAs), and two-component regulatory systems (TCSs). For example, the global regulatory protein H-NS acts as a xenogeneic silencer for adenine-thymine (AT)-rich regions acquired by horizontal gene transfer and mediates the expression of LEE genes in response to culture temperature changes (Lucchini et al. 2006, Oshima et al. 2006, Wan et al. 2016, Shin 2017), whereas the specific regulatory protein ArgR activates the expression of LEE genes and Shiga toxin genes in response to l-arginine (Menezes-Garcia et al. 2020). Two sRNAs with relatively clear roles in intestinal signal sensing and virulence regulation are DicF and Esr055. DicF integrates oxygen sensing to induce LEE gene expression, thereby promoting A/E lesion formation in EHEC (Melson and Kendall 2019), whereas Esr055 senses low DNA concentrations in the large intestine and promotes EHEC colonization by regulating Shiga toxin gene expression (Han et al. 2017). The expression of EHEC virulence genes is temporally and spatially controlled by highly complex regulatory mechanisms during intestinal infection. The complex regulatory network employed by EHEC to control its virulence and pathogenicity has been comprehensively reviewed in the literature (Mellies and Lorenzen 2014, Franzin and Sircili 2015, Gelalcha et al. 2022). In recent years, our team and others have shown that EHEC can sense multiple intestinal signals and coordinate virulence gene expression through TCSs (Hughes et al. 2009, Reading et al. 2009, Pacheco et al. 2012, Gruber and Sperandio 2014, Kumar and Sperandio 2019, Kumar et al. 2020, Liu et al. 2020, 2022, 2023, Feng et al. 2022, Yang et al. 2023a,b). The composition, signal transduction mechanisms, and key roles of TCSs in intestinal signal sensing and virulence regulation are discussed in detail below.

Fundamental composition and signal transduction mechanism of TCSs

Classic TCSs typically consist of an inner membrane-located sensor histidine kinase (HK) and a cytoplasmic response regulator (RR) (Fig. 2). HK contains a receiver domain that detects signals from the environment and a catalytic domain with kinase activity. Typically, RR also has a two-domain structure, consisting of an N-terminal receiver domain with a conserved phospho-accepting aspartate residue and a C-terminal output domain containing a DNA-binding motif. Upon recognition of specific external stimuli, the kinase domain of the HK sensor protein is activated, causing autophosphorylation of the conserved histidine residue. The phosphoryl group is then transferred to the conserved aspartate residue within the receiver domain of the cognate RR. The phosphorylated RR undergoes a conformational change and frequently homodimerizes, enabling it to bind to promoters and regulate the expression of target genes (Fig. 2). Many, but not all, HKs also display phosphatase activity, dephosphorylating their cognate RRs under noninducing conditions (Fig. 2). Dephosphorylation of RR by HK returns the system to its preactivation state, allowing the bacteria to respond again to the same stimulus upon re-exposure. Although most TCSs follow this classic paradigm, the composition and signal transduction mechanisms are more complex and diverse and have been reviewed in detail elsewhere (Stock et al. 2000, West and Stock 2001, Mitrophanov and Groisman 2008, Capra and Laub 2012).

Figure 2.

Schematic representation of the fundamental composition and signal transduction mechanism of classic TCSs. Classic TCSs are composed of a sensor HK and a cognate RR. Upon detection of specific extracellular signals, the HK undergoes autophosphorylation and then transfers the phosphoryl group to the cognate cytoplasmic RR. The phosphorylated RR forms an active dimer that binds to specific promoters, thereby activating or inhibiting the transcription of target genes. Under noninducing conditions, the HK functions as a phosphatase and removes the phosphoryl group from the cognate phosphorylated RR. Therefore, the regulation of gene expression by the phosphorylated RR is terminated, and the system returns to the preactivation state. HK, histidine kinase; RR, response regulator.

EHEC harbors >30 HKs and corresponding RRs, forming a complex regulatory network involved in diverse cellular processes, including survival, growth, metabolism, and motility (Cordeiro et al. 2014, Kurabayashi et al. 2015, Yuan et al. 2017, Han et al. 2023, Yang et al. 2023a). In recent years, critical roles of TCSs in intestinal signal sensing and virulence regulation have also been elucidated in EHEC (Hughes et al. 2009, Reading et al. 2009, Pacheco et al. 2012, Gruber and Sperandio 2014, Kumar and Sperandio 2019, Kumar et al. 2020, Liu et al. 2020, 2022, 2023, Feng et al. 2022, Yang et al. 2023a,b). In this review, we comprehensively and systematically summarize how EHEC detects a wide range of intestinal signals and precisely regulates virulence gene expression through multiple signal transduction pathways during the initial stages of infection, with a particular emphasis on the key roles of TCSs in these signal transduction pathways.

TCS-mediated intestinal signal sensing and virulence regulation during EHEC infection

The PhoPQ TCS senses low magnesium concentrations to regulate EHEC virulence

The PhoPQ TCS consists of the membrane-bound sensor HK PhoQ and the cytoplasmic cognate RR PhoP (Bader et al. 2005). PhoQ is a prototypical homodimeric HK that contains two transmembrane regions; a periplasmic sensor domain (SD); a histidine kinase, adenylyl cyclase, methyl-binding protein, and phosphatase (HAMP) domain; a dimerization and histidine phosphorylation (DHp) domain; and a histidine kinase/Hsp90-like ATPase (HATPase) domain (Fig. 3). PhoP is an OmpR/PhoB family RR with a receiver (REC) domain and a regulatory DNA-binding domain (DBD) (Fig. 3). The PhoPQ TCS is known to respond to stimuli such as low magnesium concentrations, a slightly acidic pH, certain antimicrobial peptides, and high osmolality (Prost et al. 2007, Lippa and Goulian 2009, Yuan et al. 2017). Upon exposure to these external stimuli, PhoQ undergoes autophosphorylation and then transfers its phosphoryl group to PhoP. Phosphorylated PhoP directly regulates the transcription of multiple genes by binding to the conserved motif (T/G)GTTTANNNNN(T/G)GTTTA in promoter regions (Minagawa et al. 2003). In E. coli K12, PhoP regulates the transcription of genes involved in magnesium uptake, acid resistance, antimicrobial peptide resistance, and lipopolysaccharide modification (Kato et al. 1999, Zwir et al. 2005, Moon and Gottesman 2009). In addition, PhoP has been shown to be involved in the regulation of genes related to biofilm formation in avian pathogenic E. coli (Yin et al. 2019).

Figure 3.

Domain structure of HKs and RRs in EHEC. The numbers indicate the full protein length. SD, sensor domain; HAMP, histidine kinase, adenylyl cyclase, methyl-binding protein, and phosphatase domain; HATPase, histidine kinase/Hsp90-like ATPase domain; MASE1, membrane-associated sensor 1 domain; DHp, dimerization/histidine phosphotransfer domain; Hpt, histidine phosphotransfer domain; sCache_3, single cache domain 3; PAS, Per-Arnt-Sim domain; PED, periplasmic domain; SK, sensor kinase domain; HK, histidine kinase domain; ATPase, AAA + ATPase domain; REC, receiver domain; DBD, DNA binding domain; HTH, helix-turn-helix domain.

In EHEC, PhoPQ TCS was shown to sense low magnesium levels and activate LEE gene expression to promote EHEC adherence and colonization in the human large intestine (Fig. 4; Table 1) (Liu et al. 2020). As one of the most essential minerals in the human body, magnesium can only be obtained from external sources through absorption by the small intestine, and the magnesium level in the large intestine is extremely limited (Schweigel and Martens 2000). When EHEC appears in the human large intestine, PhoQ responds to low magnesium concentrations and undergoes autophosphorylation, after which the phosphoryl group is transferred to PhoP (Liu et al. 2020). Phosphorylated PhoP then directly activates the expression of lmiA (encoding low magnesium-induced regulator A) by binding to a specific 19-bp motif (5'-CTTACGTCACGTTTTCAGC-3') within the lmiA promoter. Highly expressed LmiA directly binds to the ler promoter to activate ler expression, and Ler in turn activates other LEE genes to promote EHEC adherence and colonization in the large intestine (Fig. 4; Table 1). In addition to the LEE genes, the stx1 and stx2 genes (encoding Shiga toxin 1 and Shiga toxin 2) have been shown to be positively regulated by PhoP in EHEC; however, the underlying mechanism is still unclear (Fig. 4; Table 1) (Han et al. 2023). Therefore, the PhoPQ TCS senses the low-magnesium status in the large intestine and simultaneously activates the expression of the LEE and Shiga toxin genes, ultimately promoting EHEC intestinal colonization and pathogenesis. Disruption of this PhoPQ-mediated virulence regulatory pathway by the deletion of phoP or phoQ genes significantly decreases EHEC adherence in the mouse intestinal tract, indicating that the PhoPQ TCS is required for the successful colonization of EHEC in the host intestine (Liu et al. 2020).

Figure 4.

Overview of TCS-mediated intestinal signal sensing and virulence regulation in EHEC. TCSs, including PhoPQ, FusKR, DcuSR, RbfSR, EvgSA, CpxRA, QseEF, QseBC, and BaeSR, sense various intestinal signals to regulate EHEC virulence gene expression. For details, see the main text. Epi, epinephrine; NE, norepinephrine; AI-3, autoinducer-3; Mg²⁺, magnesium; PO₄³⁻, phosphate; SO₄²⁻, sulfate.

Table 1.

TCSs are involved in intestinal signal sensing and virulence regulation in EHEC.

Signals	TCSs	Function	References
Low magnesium	PhoPQ	PhoQ senses low magnesium concentrations to undergo autophosphorylation and then transfers the phosphoryl group to PhoP. Phosphorylated PhoP directly activates the expression of lmiA. LmiA, in turn, directly binds to the ler promoter to activate the expression of ler and therefore of other LEE genes. PhoP also positively regulates the expression of the stx1 and stx2 genes via an unclear mechanism.	Liu et al. (2020), Han et al. (2023)
Fucose	FusKR	FusK senses fucose to undergo autophosphorylation and then transfers the phosphoryl group to FusR. Phosphorylated FusR directly binds to the ler promoter to repress the expression of ler and therefore of other LEE genes.	Pacheco et al. (2012)
l-malate	DcuSR	DcuS senses l-malate to undergo autophosphorylation and then transfers the phosphoryl group to DcuR. Phosphorylated DcuR directly binds to the ler promoter to activate the expression of ler and therefore of other LEE genes. Phosphorylated DcuR also activates the expression of genes involved in anaerobic l-malate transport and metabolism, thereby promoting the utilization of l-malate.	Liu et al. (2023)
Riboflavin/ Mannose	RbfSR	RbfS senses riboflavin/mannose to undergo autophosphorylation and then transfers the phosphoryl group to RbfR. Phosphorylated RbfR directly binds to ler promoter to activate the expression of ler and therefore of other LEE genes. RbfR also directly binds to the lmuX promoter to activate the expression of lmuKAIZYX, thereby promoting mannose utilization.	Liu et al. (2022), Yang et al. (2023a)
Nicotinamide	EvgSA	EvgS senses nicotinamide to undergo autophosphorylation and then transfers the phosphoryl group to EvgA. Phosphorylated EvgA directly activates the expression of ler by binding to the ler promoter, after which Ler activates other LEE genes.	Yang et al. (2023b)
Serotonin/ Indole	CpxRA	CpxA senses serotonin/indole to dephosphorylate itself and CpxR. Dephosphorylated CpxR can no longer bind to the ler promoter, leading to the deactivation of LEE genes.	Kumar and Sperandio (2019), Kumar et al. (2020)
Epinephrine/ Phosphate/ Sulfate	QseEF	QseE senses epinephrine/phosphate/sulfate to undergo autophosphorylation and then transfers the phosphoryl group to QseF. Phosphorylated QseF indirectly regulates the LEE4/LEE5 genes and espFu by activating the expression of the small RNA GlmY. QseF also induces the expression of stx2 by activating the SOS response.	Reading et al. (2007, 2009), Hughes et al. (2009), Gruber and Sperandio (2014, 2015)
Epinephrine/ Norepinephrine/ Autoinducer-3	QseBC	QseC senses epinephrine/norepinephrine to undergo autophosphorylation and then transfers the phosphoryl group to QseB, QseF, and KdpE. Phosphorylated QseB directly activates the expression of the small RNA GlmY that posttranscriptionally regulates the expression of the LEE4/LEE5 genes. Phosphorylated KdpE directly binds to the ler promoter to activate the transcription of ler, and Ler in turn activates the expression of other LEE genes. Phosphorylated QseF regulates the expression of stx2, LEE genes, and espFu, as described for the QseEF TCS.	Hughes et al. (2009), Njoroge et al. (2012), Gruber and Sperandio (2014)
Mechanical signals	BaeSR	BaeS senses mechanical signals to undergo autophosphorylation and then transfers the phosphoryl group to BaeR. Phosphorylated BaeR activates the expression of airA. AirA, in turn, directly binds to the ler promoter to activate the expression of ler and therefore of other LEE genes.	Feng et al. (2022)

The FusKR TCS senses fucose to regulate EHEC virulence

The FusKR TCS consists of the membrane-bound sensor HK FusK and the cytoplasmic cognate RR FusR (Pacheco et al. 2012). FusK consists of eight transmembrane regions, a membrane-associated sensor 1 (MASE1) domain, a DHp domain, and an HATPase domain (Fig. 3). FusR is a LuxR family RR that contains a REC domain and a helix-turn-helix domain (Fig. 3).

Pacheco et al. revealed that the FusKR TCS senses host-derived fucose and inhibits LEE gene expression, thereby regulating EHEC virulence and intestinal colonization (Fig. 4; Table 1) (Pacheco et al. 2012). Fucose is a major component of intestinal mucin glycoproteins (Robbe et al. 2004). As a dominant member of the human intestinal microbiome, Bacteroides thetaiotaomicron is commonly used as a model gut symbiont (Comstock and Coyne 2003, Taketani et al. 2015). Bacteroides thetaiotaomicron produces multiple fucosidases that cleave fucose from the intestinal mucin of the host, resulting in greater fucose availability in the large intestinal lumen (Xu et al. 2003). When EHEC enters the large intestinal mucus layer, FusK senses mucin-derived fucose, undergoes autophosphorylation, and then transfers its phosphoryl group to FusR (Pacheco et al. 2012). Phosphorylated FusR directly binds to a specific 31-bp motif (5'-AAGAGAATAATAACATTTTAAGGTGGTTG-3') within the ler promoter to repress the expression of ler and therefore of other LEE genes (Fig. 4; Table 1). Considering that the mucus layer is close to the lumen but distant from the host cell surface, the authors speculated that the FusKR-mediated inhibition of LEE gene expression in the mucus layer could effectively prevent excessive energy expenditure by EHEC at inappropriate colonization sites. Once EHEC bypasses the mucus layer, the repression of LEE genes by the FusKR TCS is eliminated, promoting EHEC adherence and colonization. Moreover, when EHEC reaches the large intestinal mucus layer, the FusKR TCS also inhibits fucose import and utilization by repressing the expression of fuc genes (Pacheco et al. 2012). By applying this strategy, EHEC can avoid competition with the microbiota for fucose and focus on the efficient utilization of other carbon sources, such as galactose, hexorunates and mannose, which are not utilized by commensal E. coli (Fabich et al. 2008, Pacheco et al. 2012). Therefore, the FusKR TCS plays an important role in precisely controlling virulence and metabolic gene expression at the appropriate time and place during infection.

The DcuSR TCS senses l-malate to regulate EHEC virulence

The DcuSR TCS consists of the membrane-bound sensor HK DcuS and the cytoplasmic cognate RR DcuR (Abo-Amer et al. 2004). DcuS consists of two transmembrane regions, a single cache domain 3 (sCache_3), a Per-Arnt-Sim (PAS) domain, and an HATPase domain (Fig. 3). DcuS essentially exists as a dimer in the bacterial inner membrane, and the transmembrane helices of the DcuS homodimer can be linked via cysteine residues (Monzel and Unden 2015). DcuR is a CitB family RR that consists of a REC domain and a regulatory DBD domain (Fig. 3). Upon phosphorylation, DcuR is converted from a monomer to a dimer (Scheu et al. 2010). The DcuSR TCS is known to respond to physiological and nonphysiological C4-dicarboxylates, including fumarate, succinate, malate, aspartate, tartrate, and maleate, as regulatory signals (Janausch et al. 2002, Abo-Amer et al. 2004, Gencheva et al. 2022). In E. coli K12, DcuR controls the expression of genes encoding carriers and enzymes for the catabolism of externally supplied C4-dicarboxylates (Zientz et al. 1998, Davies et al. 1999, Golby et al. 1999, Janausch et al. 2002).

We recently showed that the DcuSR TCS senses high l-malate concentrations and activates the expression of ler and other LEE genes to promote EHEC adherence and colonization in the human large intestine (Fig. 4; Table 1) (Liu et al. 2023). L-Malate, a C4-dicarboxylate, is a key substrate for the aerobic and anaerobic growth of E. coli and other enteric bacteria (Kay and Kornberg 1969, Macy et al. 1976, Schubert and Unden 2022). The human large intestine contains high levels of l-malate derived from both host cells and the microbiota (Liu et al. 2023). When EHEC reaches the human large intestine, DcuS responds to host- and microbiota-derived l-malate and undergoes autophosphorylation, after which the phosphoryl group is transferred from DcuS to DcuR (Liu et al. 2023). Phosphorylated DcuR directly activates the expression of ler by binding to a specific 18-base pair motif (5ʹ-TTATCTCACATAATTTAT-3ʹ) within the ler promoter, after which Ler activates other LEE genes to promote EHEC T3SS-dependent adherence to host epithelial cells (Fig. 4; Table 1). Moreover, during infection, EHEC also utilizes l-malate as an essential nutrient to drive fumarate respiration, thereby promoting its own growth and colonization in the host large intestine. The infant rabbit is a readily available animal model in which EHEC colonizes the infant rabbit intestine, leading to the formation of A/E lesions and the development of severe diarrhea and intestinal inflammation (Ritchie et al. 2003, Moreira et al. 2016). The deletion of dcuS or dcuR genes significantly reduces the colonization efficiency of EHEC in the rabbit intestinal tract, indicating that the DcuSR TCS is crucial for EHEC survival and colonization in the host large intestine (Liu et al. 2023).

The RbfSR (LmvKR) TCS senses riboflavin/mannose to regulate EHEC virulence

The RbfSR TCS consists of the membrane-bound sensor HK RbfS and the cytoplasmic cognate RR RbfR (Liu et al. 2022, Yang et al. 2023a). RbfS contains two transmembrane regions, a HAMP domain, a DHp domain, an HATPase domain, and a REC domain (Fig. 3). RbfR is an OmpR/PhoB family RR with a REC domain and a regulatory DBD domain (Fig. 3).

The RbfSR TCS was recently found to sense microbiota-produced riboflavin (vitamin B2) and increase LEE gene expression, thereby promoting EHEC colonization in the human large intestine (Fig. 4; Table 1) (Liu et al. 2022). Riboflavin is abundant in the human large intestine and is produced mainly by commensal bacteria such as Lactobacillus and Bifidobacterium species (Thakur et al. 2016, Levit et al. 2017, Yoshii et al. 2019). When EHEC enters the human large intestine, RbfS senses microbiota-produced riboflavin, undergoes autophosphorylation, and then transfers its phosphoryl group to RbfR (Liu et al. 2022). Phosphorylated RbfR directly activates ler expression by binding to a specific 21-base pair motif (5ʹ-TCTCACATAATTTATATCATT-3ʹ) in the ler promoter region. Ler in turn induces the expression of other LEE genes and increases the adherence of EHEC to host epithelial cells (Fig. 4; Table 1). The RbfSR TCS is present in EHEC O157:H7 and EHEC O145:H28 but is absent from other EHEC strains. Introducing rbfSR into EHEC serotypes without native rbfSR significantly increased the ability of the strains to express LEE genes and to adhere to host epithelial cells (Liu et al. 2022). Citrobacter rodentium is a natural murine intestinal pathogen that shares key LEE-dependent pathogenic mechanisms with EHEC and is therefore widely used as a surrogate model for studying the molecular mechanism of EHEC infection in vivo (Petty et al. 2010, Collins et al. 2014). The introduction of rbfSR into C. rodentium, which does not carry native RbfSR, increased bacterial intestinal colonization, resulting in increased disease severity in mice. Therefore, lateral transfer of rbfSR has the potential to increase bacterial virulence, and the acquisition of rbfSR may represent an important step in the evolution of EHEC (Liu et al. 2022).

Similarly, Yang and coworkers reported that the RbfSR TCS (which they renamed LmvKR) also senses host-derived mannose as a signal to directly activate LEE gene expression during infection (Fig. 4; Table 1) (Yang et al. 2023a). Mannose is a preferred mucin-derived sugar that provides nutrients to the commensal microbiota and enteric pathogens (Fabich et al. 2008). Mannose can be released from N-linked glycosylated glycoproteins in mucus and is therefore abundant in the large intestine (Hattrup and Gendler 2008). In addition to mediating mannose-induced LEE gene expression, the RbfSR TCS also activates the expression of lmuKAIZYX (encoding mannose transporters and metabolic enzymes) by directly binding to the lmuX promoter, thereby promoting mannose utilization (Yang et al. 2023a). Therefore, the RbfSR TCS integrates two intestinal signals (riboflavin and mannose) to coordinately control virulence and metabolic gene expression accordingly, ultimately leading to successful colonization by EHEC in the host large intestine.

The EvgSA TCS senses nicotinamide to regulate EHEC virulence

The EvgSA TCS consists of the membrane-bound sensor HK EvgS, which is present in the inner membrane as a homodimer, and the cytoplasmic cognate RR EvgA (Itou et al. 2009, Zhang and Wang 2023). EvgS contains a single transmembrane region, two periplasmic domains, a DHp domain, an HATPase domain, a REC domain, and a histidine phosphotransfer domain (Fig. 3). EvgA is a LuxR family RR that contains a REC domain and a helix-turn-helix domain in the C-terminal region (Fig. 3). The known environmental signals to which EvgS responds include mild acidity (pH), high potassium or sodium concentrations, and oxidative stress (Bock and Gross 2002, Eguchi and Utsumi 2014, Sen et al. 2017). EvgA has been shown to modulate the expression of multiple genes related to acid resistance, multidrug resistance, and the osmotic stress response in E. coli K12 (Kato et al. 2000, Nishino and Yamaguchi 2001, 2002, Masuda and Church 2002, Eguchi et al. 2003, Zhang and Wang 2023).

We recently reported that the EvgSA TCS senses high nicotinamide levels as an important signal for activating LEE gene expression to promote EHEC colonization in the human large intestine (Fig. 4; Table 1) (Yang et al. 2023b). Nicotinamide is the amide form of vitamin B3 (niacin) (Weiss et al. 2015). The human large intestine is rich in nicotinamide, which is derived primarily from the commensal microbiota (Begley et al. 2001, Kurnasov et al. 2003). EvgS responds to high concentrations of microbiota-derived nicotinamide in the large intestine via autophosphorylation, followed by phosphotransfer to EvgA (Yang et al. 2023b). Phosphorylated EvgA activates the expression of evgS and evgA via positive autoregulation and directly activates the expression of ler by binding to the ler promoter. Ler then activates other LEE genes, promoting EHEC adherence and colonization in the large intestine (Fig. 4; Table 1). Disruption of this EvgSA-mediated virulence-regulating pathway by the deletion of evgS or evgA genes substantially attenuates EHEC virulence both in vitro and in vivo, indicating that the EvgSA TCS is essential for the virulence and colonization of EHEC in the host large intestine (Yang et al. 2023b).

The CpxRA TCS senses serotonin/indole to regulate EHEC virulence

The CpxRA TCS consists of the membrane-bound sensor HK CpxA and the cytoplasmic cognate RR CpxR (Keller et al. 2011). CpxA is a homodimeric HK in which each subunit consists of two transmembrane domains, a PED domain, a HAMP domain, a DHp domain, and an HATPase domain (Fig. 3). CpxR is an OmpR family RR consisting of a REC domain and a regulatory DBD domain (Fig. 3). CpxA responds to envelope stressors, such as an alkaline pH, the overproduction of secreted proteins, and envelope perturbation by ethylenediaminetetraacetic acid (EDTA) (Jones et al. 1997, Danese and Silhavy 1998, DiGiuseppe and Silhavy 2003, Mitobe et al. 2005, Bury-Mone et al. 2009, Raivio 2014). In E. coli K12, CpxR regulates the expression of genes involved in multidrug resistance, adherence, biofilm development, and the envelope stress response (Danese et al. 1995, Danese and Silhavy 1997, Wang et al. 2021).

The CpxRA TCS was recently found to sense host-derived serotonin and microbiota-derived indole as signals to regulate EHEC colonization and pathogenesis in the host intestine (Fig. 4; Table 1) (Kumar and Sperandio 2019, Kumar et al. 2020). Serotonin is a tryptophan derivative synthesized and secreted by intestinal enterochromaffin cells (O'Hara et al. 2006, Esmaili et al. 2009). In the host intestine, indole is a tryptophan derivative that is produced mainly by microbes such as Bacteroidetes and Proteobacteria species (Kumar and Sperandio 2019). When EHEC enters the large intestinal lumen, CpxA senses serotonin and indole as signaling molecules, leading to CpxA dephosphorylation, which can lead to the dephosphorylation of CpxR (Kumar and Sperandio 2019, Kumar et al. 2020). In the phosphorylated state, CpxR directly binds to the ler promoter to activate the expression of ler and therefore other LEE genes, whereas dephosphorylated CpxR can no longer bind to the ler promoter, leading to the deactivation of LEE genes (Fig. 4; Table 1). When EHEC is in close contact with the epithelial cell surface, where the serotonin and indole levels are low, the repression of the CpxRA TCS by these two signals is eliminated, thereby activating LEE gene expression to promote EHEC adherence and colonization.

The QseEF TCS senses epinephrine/phosphate/sulfate to regulate EHEC virulence

The QseEF TCS consists of the membrane-bound sensor HK QseE, which exists as a dimer in the bacterial inner membrane, and the cytoplasmic cognate RR QseF (Reading et al. 2007, 2009). QseE contains two transmembrane domains, a periplasmic SD domain, a HAMP domain, a DHp domain, and an HATPase domain (Fig. 3). QseF is a CheY family RR that contains a REC domain and an AAA + ATPase (ATPase) domain (Fig. 3).

In EHEC, the QseEF TCS senses epinephrine, phosphate, and sulfate as signals and regulates the expression of virulence genes (Fig. 4; Table 1) (Reading et al. 2009). Epinephrine is a host hormone synthesized in the central nervous system and the adrenal medulla that reaches the intestine after being released into the bloodstream (Furness 2000). Phosphate and sulfate are important dietary micronutrients that are obtained primarily from external sources through intestinal digestion and absorption in humans (Markovich 2014, Yee et al. 2021). QseE senses the presence of epinephrine, phosphate, and sulfate, undergoes autophosphorylation, and then transfers its phosphoryl group to QseF (Reading et al. 2009). Phosphorylated QseF directly activates the expression of the small RNA GlmY, which posttranscriptionally regulates the expression of LEE4/LEE5 genes, and espFu (encoding an effector directly involved in A/E lesion formation) (Reading et al. 2007, 2009, Gruber and Sperandio 2014, 2015). In addition, QseF also induces the expression of stx2 by activating the SOS response (Fig. 4; Table 1) (Hughes et al. 2009). Upon triggering the SOS response, the bacteriophage enters its lytic cycle, during which RecA is produced and activated (Muhldorfer et al. 1996). Activated RecA cleaves the λ CI repressor through its coprotease activity, allowing stx2 expression (Fuchs et al. 1999, Galkin et al. 2009, Colon et al. 2016). Therefore, the QseEF TCS initiates complex signaling cascades to sense multiple intestinal signals and coordinately regulate the expression of various virulence-related genes during infection (Reading et al. 2007, 2009, Hughes et al. 2009, Gruber and Sperandio 2014, 2015).

The QseBC TCS senses epinephrine/norepinephrine/autoinducer-3 to regulate EHEC virulence

The QseBC TCS consists of the membrane-bound sensor HK QseC and the cytoplasmic cognate RR QseB (Sperandio et al. 2002). QseC consists of two transmembrane regions, a sensor kinase (SK) domain, a DHp domain, and an HATPase domain (Fig. 3). QseB is an OmpR/PhoB family RR that comprises a REC domain and a regulatory DBD domain (Fig. 3). The QseBC TCS is a key component of the bacterial quorum sensing regulatory cascade (Sperandio et al. 2002, Clarke et al. 2006). In E. coli K12, QseB regulates the expression of multiple genes involved in biofilm formation and antibiotic resistance (Gonzalez Barrios et al. 2006, Li et al. 2020). In addition, QseB controls the expression of conserved metabolic genes involved in nucleic acid metabolism, amino acid metabolism, and the tricarboxylic acid cycle in uropathogenic E. coli (Hadjifrangiskou et al. 2011).

The QseBC TCS was also found to sense host-derived epinephrine/norepinephrine and microbiota-derived autoinducer-3 to modulate virulence gene expression in EHEC (Fig. 4; Table 1) (Hughes et al. 2009, Gruber and Sperandio 2014). Like epinephrine, norepinephrine is a host hormone synthesized by adrenergic neurons of the enteric nervous system and is present in the gastrointestinal tract (Moreira and Sperandio 2016). Autoinducer-3 is a hormone-like compound that is produced by the commensal microbiota and released into the intestine (Clarke et al. 2006). Upon sensing these signals, QseC phosphorylates its cognate RR, QseB, and noncognates RRs, QseF and KdpE (Hughes et al. 2009). Phosphorylated QseB indirectly regulates the LEE4/LEE5 genes by affecting the expression of the small RNA GlmY (Hughes et al. 2009, Gruber and Sperandio 2014). Phosphorylated QseF regulates the expression of stx2, LEE genes, and espFu, as described above. KdpE is an OmpR/PhoB family RR that consists of a REC domain and a regulatory DBD domain (Narayanan et al. 2014). Phosphorylated KdpE activates the transcription of ler by directly and specifically binding to the ler promoter region, and Ler in turn activates the expression of other LEE genes (Fig. 4; Table 1) (Njoroge et al. 2012). The qseC mutant exhibited attenuation in both an infant rabbit EHEC infection model and a C. rodentium mouse infection model, indicating that QseC is necessary for EHEC to activate its virulence program and successfully colonize the host intestine (Rasko et al. 2008, Moreira et al. 2016).

The BaeSR TCS senses mechanical signals to regulate EHEC virulence

The BaeSR TCS consists of the membrane-bound sensor HK BaeS and the cytoplasmic cognate RR BaeR (Yao et al. 2015). BaeS is an active dimer, and each monomer contains two transmembrane regions, a HAMP domain, a DHp domain, and an HATPase domain (Fig. 3). BaeR is an OmpR/PhoB family RR that consists of a REC domain and a regulatory DBD domain (Fig. 3). BaeS senses membrane-damaging compounds and bacterial envelope perturbants, such as misfolded, mislocalized, or aggregated proteins (Raffa and Raivio 2002). However, little is known about the specific signals received by BaeS. In E. coli K12, BaeR binds as a dimer to the promoters of target genes at the site sequence motif 5ʹ-TTTTTCTCCATDATTGGC-3ʹ (where D is G, A, or T) (Nishino et al. 2005) and regulates the expression of genes involved in envelope stress responses, multidrug transport, flagellum biosynthesis, chemotaxis, and maltose transport (Hirakawa et al. 2005, Nishino et al. 2005, Leblanc et al. 2011).

The BaeSR TCS was recently found to respond to mechanical signals generated from initial adherence to the host cells and activate LEE gene expression to promote EHEC colonization in the human large intestine (Fig. 4; Table 1) (Feng et al. 2022). When EHEC initially adheres to intestinal epithelial cells, the BaeSR TCS senses mechanical signals and activates the expression of airA (encoding adherence-inducible regulator A); AirA subsequently activates ler expression by directly binding to a specific 17-bp motif (5ʹ-TGCAATGAGATCTATCT-3ʹ) in the ler promoter region and therefore regulates the expression of other LEE genes (Fig. 4; Table 1). Furthermore, the outer-membrane lipoprotein NlpE is involved in surface sensing and is required for BaeSR-mediated LEE induction in response to initial adherence to host cells (Feng et al. 2022). However, the exact mechanism by which NlpE signaling is induced and the signal is transduced to the BaeSR TCS has not been fully elucidated.

Concluding remarks

EHEC is an important human pathogen that specifically colonizes the large intestine to cause disease (Lewis et al. 2015). The human large intestine is an extremely complex environment containing numerous metabolites and signaling molecules derived from the host itself, the intestinal microbiota, and dietary sources. Currently, >20 different intestinal signals, most of which exert their regulatory effects in a dose-dependent manner, have been shown to be involved in EHEC virulence regulation. For example, the ability of EHEC to adhere to HeLa cells and form A/E lesions is not affected by low levels of nicotinamide (<20 μM) but is significantly increased in the presence of high nicotinamide concentrations (above 50 μM) (Yang et al. 2023b). Notably, the large intestine environment is highly dynamic and changes with shifts in the composition of the intestinal microbiome, dietary components, disease states, and other factors (Di Vincenzo et al. 2024). To establish successful colonization in such a variable and dynamically changing intestinal environment, EHEC strains have evolutionarily acquired the ability to simultaneously employ multiple TCSs to sense and integrate various intestinal signals and coordinately control virulence genes expression. In addition, different TCSs exhibit extensive cross-regulatory interactions and cross-phosphorylation. For example, the EvgSA TCS triggers the expression of the safA gene, which encodes a small membrane protein that directly interacts with PhoQ to activate the PhoPQ TCS, thereby bridging the EvgSA and PhoPQ TCSs (Eguchi et al. 2011, 2012, Roggiani et al. 2017). Phosphorylated QseC can phosphorylate the noncognate RRs QseF and KdpE and promote cross-regulation, bypassing their cognate HKs and thus initiating complex signaling cascades (Hughes et al. 2009, Njoroge and Sperandio 2012). This cross-regulatory network enables EHEC to integrate various intestinal signals more efficiently and adapt its virulence program more rapidly toward successful host infection. Additional research efforts are needed to elucidate the extensive interactions of TCSs in intestinal signal sensing and virulence regulation and establish a complete TCS-mediated virulence regulatory network for EHEC, which will provide a comprehensive understanding of the pathogenic mechanisms and infection site recognition mechanisms of EHEC.

EHEC harbors >30 TCSs, forming a complex signal transduction network that responds to diverse environmental changes with coordinated changes in genes expression. Among these TCSs, nine (PhoPQ, FusKR, DcuSR, RbfSR, EvgSA, CpxRA, QseEF, QseBC, and BaeSR) are involved in intestinal signal sensing and EHEC virulence regulation (Fig. 4; Table 1). Specifically, all nine of these TCSs are involved in regulating EHEC adherence to host intestinal epithelial cells and the formation of A/E lesions. Furthermore, three TCSs, PhoPQ, QseBC, and QseEF, contribute to Stx prophage induction and the subsequent upregulation of Shiga toxin gene expression by triggering the SOS response or other unknown mechanisms. The abundance of TCSs in EHEC, coupled with the ability of TCSs to regulate virulence in response to host intestinal signals, highlights the importance of these signaling systems in host‒microbe interactions. However, the effects of the remaining TCSs on EHEC virulence and intestinal colonization remain largely unknown. Furthermore, research on the regulation of EHEC virulence by TCSs has focused mainly on how TCSs affect the expression of LEE pathogenicity island genes, whereas the understanding of how Shiga toxin genes are regulated by multiple TCS pathways is relatively limited. Therefore, additional efforts are needed to further systematically elucidate the key roles of TCSs in intestinal signal sensing and virulence regulation, especially the effects of TCSs on Shiga toxin genes. Comprehensive functional analyses of these TCSs and the construction of TCS-based virulence regulatory networks will be highly important for understanding the pathogenesis of EHEC infection.

EHEC TCSs are encoded by genes located in either stable genomic regions termed the core genome or variable genomic regions acquired via horizontal transfer (Perna et al. 2001). Among the nine TCSs involved in intestinal signal sensing and EHEC virulence regulation, PhoPQ, DcuSR, EvgSA, CpxRA, QseEF, QseBC, and BaeSR are core genome-encoded TCSs, and FusKR and RbfSR are laterally acquired TCSs. Bioinformatics analysis revealed that the TCSs encoded by the EHEC core genome are generally highly conserved and widely distributed among various E. coli strains with different pathotypes and other human intestinal pathogens, such as Salmonella, Vibrio cholerae, and Shigella. Whether the TCS-mediated virulence regulatory pathways identified in EHEC are also exploited by other intestinal pathogens to modulate bacterial virulence in response to different intestinal signals requires further investigation. In contrast to core genome-encoded TCSs, laterally acquired TCSs are generally present in only a few specific hypervirulent EHEC serotypes but are absent in other E. coli pathotypes and human intestinal pathogens. For example, the FusKR TCS is present in EHEC O157:H7, whereas the RbfSR TCS is present in EHEC O157:H7 and EHEC O145:H28 (Pacheco et al. 2012, Liu et al. 2022). The horizontal acquisition of these TCSs during evolution enables EHEC to sense additional host intestinal signals and more precisely mediate bacterial virulence, which contributes greatly to the generation of highly pathogenic EHEC serotypes. In the future, other E. coli pathotypes and intestinal pathogens may acquire these TCSs through lateral gene transfer to increase their pathogenicity, which represents a potential risk to public health.

Since EHEC was first identified in the United States, diarrheal diseases caused by EHEC have become a major public health problem worldwide. EHEC outbreaks occur in countries worldwide and cause many deaths in both developing and developed countries (Lopez et al. 1989, Waters et al. 1994, Rubino et al. 2011, Tau et al. 2012). Although several therapeutic strategies, including the use of antibiotics and vaccinations, have been developed, currently, no effective treatment is available for EHEC infection (Proulx et al. 1992, Bosworth et al. 1996). The use of antibiotics is also contraindicated, as antibiotic treatment may lead to severe dysbiosis of the intestinal microbiota and the expansion of antibiotic-resistant EHEC strains (Jernberg et al. 2007, MacLean and San Millan 2019). In addition, antibiotics may also induce bacteriophages to enter the lytic cycle, which increases the production and release of Shiga toxins, ultimately leading to serious complications such as HUS (Walterspiel et al. 1992, Grif et al. 1998, Slutsker et al. 1998, Aragon et al. 2000, Zhang et al. 2000, Tyler et al. 2013). Therefore, alternative strategies are needed to develop safe and effective antibacterial therapies to treat EHEC infection. Currently, much research is focused on the development of antibacterial agents that aim to disrupt the intercellular signaling networks necessary for pathogenic bacteria to colonize the host and induce disease (LaSarre and Federle 2013). One of the most difficult challenges of such research is identifying optimal therapeutic targets within these complex networks to inhibit bacterial virulence and pathogenic processes (Ellermann and Sperandio 2020). Our group and others have demonstrated that the deletion of several key TCS genes, including phoQ/phoP, fusK, dcuS/dcuR, rbfS/rbfR, evgS/evgA, and qseC, severely reduces intestinal colonization by EHEC in different animal models (Rasko et al. 2008, Pacheco et al. 2012, Liu et al. 2020, 2022, 2023, Yang et al. 2023b). Therefore, these TCS genes may serve as potential therapeutic targets, and antibacterial agents designed to block signal binding or inhibit the gene expression and/or protein activity of these TCSs may effectively prevent and treat EHEC infections. Furthermore, TCSs are critical for the survival and colonization of EHEC in cattle, which are the natural reservoir of EHEC (Audia et al. 2001, Nguyen and Sperandio 2012, Sharma and Casey 2014, Sharma et al. 2022, Han et al. 2023). Therefore, antibacterial agents targeting TCSs may help reduce EHEC carriage in cattle and, subsequently, shedding and contamination of EHEC in the environment, thus preventing human infection. Considering that orthologs of TCS genes do not exist in humans or other mammals, agents that target bacterial TCSs would specifically combat EHEC without damaging host cells. Indeed, over the last 20 years, many compounds targeting TCSs have been tested as antibacterial agents, some of which have yielded positive results in drug development. For example, through a high-throughput screen, Rasko et al. identified the small molecule LED209 that inhibits the binding of signals to QseC, preventing its autophosphorylation and consequently inhibiting QseC-dependent virulence gene activation (Rasko et al. 2008). Additionally, LED209 has been shown to inhibit the biofilm formation, virulence, and motility of EHEC, as well as several other gram-negative pathogens (Curtis et al. 2014, Yang et al. 2014, Li et al. 2016). Undoubtedly, a greater understanding of the role and underlying mechanisms of TCSs in EHEC pathogenesis will facilitate the rational development and optimization of such new antibacterial agents.

Conflict of interest

None declared.

Funding

This work was supported by the National Natural Science Foundation of China (NSFC) Program [grant number 32270191] and the Fundamental Research Funds for the Central Universities [grant number 980-63243161].

Author notes

Hongmin Sun, Di Huang, Yu Pang, Jingnan Chen, and Chenbo Kang contributed equally to this work