EvgS/EvgA, the unorthodox two-component system regulating bacterial multiple resistance

ABSTRACT

Two-component systems (TCSs) widely exist in bacteria and assist bacteria in adjusting behaviors to adapt to environmental changes after sensing environmental signals and transducing information into cells. As a member of TCSs in Escherichia coli, after sensing signals, EvgS/EvgA mainly induces multiple resistances, such as acid and multidrug resistance, and increases bacterial adherence. Notably, the EvgS is one of the five unorthodox sensors in E. coli, which has an internal phosphorelay based on the structure with extra domains different from other orthodox sensors. Here, we summarize research on EvgS and EvgA published since 1994. This review includes comprehensive knowledge of EvgS/EvgA and focuses on the EvgS structure and the multiple resistance conferred by EvgS/EvgA. Furthermore, we discuss the unsolved questions of EvgS/EvgA and propose assumptions and inferences on the EvgS structure and the EvgS/EvgA function.

IMPORTANCE

EvgS/EvgA, one of the five unorthodox two-component systems in Escherichia coli, plays an essential role in adjusting bacterial behaviors to adapt to the changing environment. Multiple resistance regulated by EvgS/EvgA endows bacteria to survive in adverse conditions such as acidic pH, multidrug, and heat. In this minireview, we summarize the specific structures and regulation mechanisms of EvgS/EvgA and its multiple resistance. By discussing several unresolved issues and proposing our speculations, this review will be helpful and enlightening for future directions about EvgS/EvgA.

INTRODUCTION

Two-component systems (TCSs) of bacteria transduce signals sensed from the environment and assist in inducing adaptive responses through phosphorylation and phosphate group transferring (1–3). Commonly, proteins in TCSs are divided into histidine kinase (HK) sensors perceiving external and internal stimuli and response regulators (RRs) effecting cellular responses to alter the bacteria’s status (4). The unorthodox sensor contains two histidines that can be phosphorylated instead of one in the orthodox sensor, leading to the distinction in the phosphorelay way (4–6). Five unorthodox HK sensors occur in Escherichia coli, including ArcB, BarA, TorS, YojN, and EvgS (7). In addition, EvgA in this TCS is a typical RR.

Hitherto, EvgS/EvgA can only be found and conserved in E. coli and Shigella (7, 8). The EvgS and EvgA identification is through the homologous proteins BvgS and BvgA in Bordetella pertussis (9). BvgS/BvgA, also belonging to the unorthodox TCSs, modulates the virulence factor expression to control the transition between the virulent and avirulent phases (10–12). Moreover, EvgS/EvgA in E. coli is homologous to KvgS/KvgA in Klebsiella pneumoniae (8). KvgS/KvgA contributes to countering free radical stresses along with sensing iron-limiting conditions and only exists in virulent strains (8, 13).

By regulating multiple genes, activated EvgS/EvgA endows E. coli resistance to survive in adverse environments such as acidic conditions, multidrug use, and heat. Genes induced by EvgS activation mainly depend on phosphorylated EvgA transcription factor activity (14). They are regulated by their direct interaction with EvgA or by other genes regulated by EvgA. The deletion of EvgA is usually concomitant with phenotypes losing acid and multidrug resistance. EvgS/EvgA also modulates genes in establishing connections with other TCSs (15–17). Therefore, this review mainly focuses on the specific structures of EvgS and the multiple resistances induced by EvgS and EvgA.

THE PRODUCTION AND ACTIVATION OF EVGS/EVGA

The evgAS genes encode EvgS and EvgA, and their expressions are regulated by EvgA and other molecules (Fig. 1A). In neutral pH, the unphosphorylated EvgA upregulates the evgAS expression by directly binding its upstream (18). Therefore, an autoregulatory loop is formed. However, the EvgA binding profile might depend on the phosphorylated state of EvgA (19). Burton et al. found that the phosphorylated EvgA has mild repression of its expressions (20). We speculate that the binding differential between EvgA and the phosphorylated EvgA can balance the cellular response regulated by EvgS/EvgA. H-NS, a nucleoid-associated protein regulating acid resistance, represses the expression of evgAS (21) (Fig. 1A). Norepinephrine upregulates their expression levels, leading to an increase in enterohemorrhagic E. coli (EHEC) O157:H7 survival in acidic conditions (22) (Fig. 1A). Yang et al. revealed that nicotinamide upregulates evgAS expression, leading to the promotion of the adherence and colonization of EHEC O157:H7 (23) (Fig. 1A). Carbon monoxide-releasing molecule-2, a novel antibacterial agent, downregulates the evgA expression in multidrug-resistant extended-spectrum β-lactamase-producing uropathogenic E. coli (24) (Fig. 1A). Furthermore, some cellular factors influence the existence or function of EvgS. Lacking YfgM, a periplasmic chaperone relevant to the correctness of membrane protein folding, targeting, or turnover, results in an EvgS decrease (25) (Fig. 1A). Indole can inhibit the EvgS operation by directly or indirectly interacting with EvgS, leading to the abolishment of acid resistance in E. coli (26) (Fig. 1A). In addition, the inhibition can be alleviated by expressing the constitutively active EvgS protein (26). Hence, by regulating the existence or operation of EvgS/EvgA, multiple molecules regulate the acid resistance and adherence phenotype, indicating that EvgS/EvgA plays a critical role in these processes.

FIG 1.

The structure and production of EvgS/EvgA. (A) Factors regulate EvgS/EvgA in a positive or negative way. (B) The structure and transphosphorylation of EvgS/EvgA. (C) Genes containing the EvgA-binding motif can interact with the phosphorylated EvgA directly.

EvgS senses signals such as alkali metals, the redox state in the mild acidic condition (pH 5.0–6.0), and nicotinamide, thereby activating EvgS/EvgA and triggering the subsequent reaction (8, 23, 27) (Fig. 1B). The optimum pH for EvgS/EvgA activation is 5.5–5.7 (20, 27). In pH 5.5, the exponential-phase cells activate EvgS/EvgA in the minimal medium but not in the rich medium (20, 28, 29). No activation was found at the same pH until adding the alkali metals such as K⁺ and Na⁺ (27) (Fig. 1B). However, under anaerobic conditions, EvgS/EvgA is inactive, and the overproduction of EvgA activates EvgS/EvgA, indicating that EvgS responds to the redox state and that its activation requires oxidation (8). Ubiquinone (UQ) is one of three kinds of quinones (the other two are demethylmenaquinone and menaquinone) (8). Notably, the effect of UQ on EvgS is controversial. In 2002, using EvgS without the periplasmic and transmembrane regions in in vitro phosphorylation assays, Bock and Gross found that oxidized ubiquinone-0 can significantly inhibit the EvgS autophosphorylation activity, which decreases the HK activity instead of intrinsic phosphatase activity (30). However, research in 2021 showed that UQ is required for EvgS activation (8) (Fig. 1A). Inada et al. found that EvgS in ΔubiA (ubiA is responsible for UQ biosynthesis) occurs without activation under any redox conditions (8). Also, with the evgS overexpression, it can be activated without UQ, therefore presumably requiring other quinones, for it is still inactive under anaerobic conditions (8). Research in 2023 reported that nicotinamide, derived from microbiota and sensed by EvgS, activates EvgS/EvgA to upregulate virulence gene expressions, helping EHEC O157:H7 colonize in the mouse intestinal tract (23) (Fig. 1B). Hence, exploring whether other substances in the intestine might be sensed by EvgS is required. Signals activating EvgS in different bacteria are listed in Table 1.

TABLE 1.

Signals activating the EvgS in different bacteria

Organism	Signal	Reference
Escherichia coli	Low pH	(20, 29)
	Alkali metals (K+, Na+)	(27)
	The redox state	(8)
	Nicotinamide	(23)
Shigella flexneri	Low pH; K+	(31)

THE STRUCTURE OF EVGS

EvgS, which forms a dimer, consists of a rather large N-terminal periplasmic region, a C-terminal cytoplasmic region, as well as a transmembrane region connecting these two parts (5, 7, 9, 32) (Fig. 1B). In the cytoplasmic region, compared to the typical sensor with one HK domain, EvgS additionally includes a Per-ARNT-Sim (PAS) domain between the transmembrane part and the HK domain, a receiver, and an output domain containing the his-containing phosphotransfer module (HPt) domain (8, 33–35) (Fig. 1B). Here, we divide these domains into two parts depending on their function.

Domains related to signal recognition and EvgS activation

In EvgS, the periplasmic region, transmembrane helix, and PAS domain are related to the signal recognition and activation of EvgS (7, 8, 27, 36).

The periplasmic region participates in sensing low pH and alkali metals. Johnson et al. first proved that this region participates in the EvgS response to low pH by removing 148 amino acids (7). Eguchi and Utsumi found that L152, which is highly conserved among E. coli and Shigella, is required for the EvgS activation because the EvgS with L152Y shows no activation in KCl-supplemented M9 medium (pH 5.5) (27) (Table 2). To further investigate the signal response in this region, Sen et al. built a high-fidelity model of this region based on the BvgS (36). Similar to BvgS, the periplasmic region in EvgS contains two tandem Venus flytrap (VFT) domains, one of which is a bacterial periplasmic solute-binding protein domain belonging to the periplasmic sensor domain (27, 36, 37) (Fig. 1B). With two jaws opening and closing, the VFT domain binds the specific ligand to import material or transduce signals (38–40). It can be inferred that the VFT domains in EvgS are responsible for signal sensing. Based on this model, Sen et al. found amino acids in EvgS related to its activation (36). In the EvgS dimer, H226 from one protomer is close to P522, which is in another protomer (36). In EvgS with H226Q and P522A, no activation of EvgS is detected under pH 5.6 (36) (Table 2). Furthermore, they found that VFT1 in EvgS is unlikely to bind amino acid-like ligands (36). Interestingly, H63 is responsible for targeting EvgS to the membrane; EvgS with H63A cannot be detected in the membrane fraction (36) (Table 2). Sen et al. mainly focused on VFT1 in EvgS rather than VFT2, predicted to be a close conformation (36). However, in BvgS, VFT2 can bind nicotinate or related molecules, and the dynamics of VFT1 are responsible for the BvgS kinase activity (38, 41–43). Hence, further research on the VFT2 function in EvgS is needed.

TABLE 2.

Amino acids and their function in EvgS/EvgA

Proteins	Amino acids	Function	Reference
EvgS	H63	Targeting of EvgS to the membrane	(36)
	H226	Involved in EvgS activation
	P522	Transduction of the inducing signal across the membrane
	L152	Involved in EvgS activation	(27)
	R564	Related to acid and heat resistance	(44)
	C671	Related to EvgS activation in redox conditions	(8)
	C683
	H721	Three phosphorylation sites in EvgS	(45, 46)
	D1009
EvgA	D52	Receiving the phosphoryl group from EvgS and related to thermal resistance	(45, 47)

Signal sensing in EvgS requires the transmembrane region. Eguchi and Utsumi found that the periplasmic and/or transmembrane regions play a role in sensing K⁺ (27). Sen et al. discovered that P522 is likely to cap the predicted transmembrane helix, and EvgS with P522A leads to no activation under low pH, suggesting that the transmembrane region contributes to transducing the information to the cytoplasmic region (36) (Table 2). In 2021, Inada et al. found that the transmembrane region is essential for sensing redox signals; the cytoplasmic region, without anchoring to the membrane, cannot sense the change in the redox state (8).

The PAS domain in the EvgS cytoplasmic region is critical in sensing redox state and low pH. Nearly 33% of HK sensors contain at least one PAS domain having structural plasticity and an inclination for protein-protein interaction (4). EvgS is predicted to have a PAS fold similar to BvgS (7). It has been reported that the PAS domain senses changes such as redox potential, oxygen, and light (4, 48). In 2021, Inada et al. found key mutations (C671 and C683) in the PAS domain related to EvgS activation in redox conditions (8). C671 and C683 are conserved in E. coli and Shigella, which do not exist in the same position in BvgS (8) (Table 2). However, Sen et al. found that the PAS domain in EvgS also participates in the response to both external and internal low pH (36). It modulates the EvgS dimer from the tight, inactive status to the weak, active status (36). This result is consistent with previous research conducted by Johnson et al., who found that EvgS becomes active as an autokinase by altering the EvgS dimer interaction during activation (7).

Since regulating the EvgS activity can be achieved by modulating the dimerization through the PAS domain, mutations in this domain are more likely to form EvgS constitutive mutants at pH 7.0 (7, 36, 49). In the evolutionary experiment, spontaneous mutations in exponential-phase E. coli survived at pH 2.5 and are in this domain (7). In 2022, Van Riet et al. found that a mutant lacking R564 within the PAS domain survived cid shock (pH 2.5, 1 h) (44) (Table 2). Although EvgS is activated at pH 5.0–6.0, these results indicate that the PAS domain in EvgS connects with the evolution of E. coli to adapt to the more robust acidic condition.

Currently, most research on the signal sensing and activation of EvgS is done through mutating analyses. Mechanisms of these domain sensing signals and their dynamics are waiting to be explored.

Domains related to the EvgS phosphorelay

In the cytoplasmic region, the HK domain, receiver, and Hpt domain contribute to the EvgS phosphorelay. Here, we introduce their own specificities. The HK domain in EvgS contains an ATP-binding subdomain (CA domain) and a dimerization-inducing subdomain (DHp domain) with a His residue (45) (Fig. 1B). The receiver contains an Asp in EvgS and lacks phosphatase activity, which is different from the homologous BvgS receiver (50, 51). In addition, the Hpt domain possesses specificity (52). It has the ability to distinguish between the different RRs and confer specificity to the system (50). These domains all contribute to the EvgS phosphorelay, which is different from the regular sensor kinases and is illustrated in the later section (Fig. 1B).

THE STRUCTURE OF EVGA

EvgA contains an N-terminal receiver and a DNA-binding domain called the output domain in the C-terminal (9, 53) (Fig. 1B). However, controversy exists about whether EvgA forms a dimer. In 1998, Tanabe et al. found that the signals of the interaction between EvgA and EvgA were not detected in surface plasmon resonance, indicating that both unphosphorylated and phosphorylated EvgA do not interact with each other in a multimeric way (15). In contrast, Perraud et al. found that the unphosphorylated proteins EvgA form dimers in the solution, as proven by the analytical sedimentation equilibrium ultracentrifugation, the native polyacrylamide gradient gels, and gel permeation experiments in 2000 (54) (Fig. 1B). Interestingly, this phenomenon is different with most RRs, which form dimer after phosphorylation (4).

The receiver of EvgA contributes to the specific interaction between EvgA and EvgS (50). In addition, the receiver alone can transfer the phosphoryl group by acting as an active enzyme, and the D52 of this domain is responsible for receiving the phosphoryl group from EvgS (15, 46, 55). D52 can also be phosphorylated by acetyl phosphate (45, 56) (Fig. 1A). Specific interactions between the output domain and the receiver are possibly required in the activation of EvgA (55). The linker between the two domains is sensitive to the thermolysin and extends from L127 to S133, which resembles the linker part in NarL (55, 57). NarL is a nitrate-dependent RR in the NarX/NarL TCS (58). Based on DNA-binding motifs, EvgA is classified into the NarL family, which contains eight other RRs such as FimZ, NarL, and RcsB (59). Genes containing the EvgA-binding motif are regulated by directly binding with EvgA (Fig. 1C). The affinity and specificity of the bound DNA are increased after the EvgA phosphorylation, even using acetyl phosphate as a phosphoryl donor (55, 60). The structure of the putative positive transcription regulator (sensor EvgS) from Shigella flexneri is released in the RSCB PDB (https://www.rcsb.org/structure/5F64). The crystal structure of the N-terminal domain of the positive transcription regulator EvgA from E. coli is also released (https://www.rcsb.org/structure/3F6C).

THE MULTISTEP TRANSPHOSPHORYLATION OF EVGS/EVGA

Through phosphotransfer from the HK sensor to the RR, TCS helps bacteria adapt to the environment after sensing signals (4). EvgS has a unique phosphorelay resulting from three phosphorylation sites in the cytoplasmic region rather than one in the typical sensor. EvgS belongs to the tripartite sensor kinases (TSK), indicating that EvgS has a His-Asp-His phosphorelay pattern (61, 62) (Fig. 1B). This sensor contains three phosphorylation sites on H721 in the DHp domain, D1009 in the receiver, and H1137 in the HPt domain (45, 46) (Fig. 1B) (Table 2). The activating CA domain catalyzes the transfer of the ATP γ-phosphate to H721 (45) (Fig. 1B). Then, the phosphoryl group is transferred to the D1009 and, after that, to the H1137 (45) (Fig. 1B). Besides, D1009 can be directly autophosphorylated by acetyl phosphate (45) (Fig. 1B). The three-step phosphorelay of EvgS above proceeds in an intramolecular reaction in cis-cis-cis mode, despite the autophosphorylation mode of BvgS being a trans mode (45, 62, 63) (Fig. 1B). Autophosphorylation modes are different in TSKs depending on the need to form a homodimer, which usually contains phosphate group transfer in the dimer (64). However, the EvgS autophosphorylation deviates from the usual model, which does not involve the interaction in the homodimer (62) (Fig. 1B).

In EvgS/EvgA, the HK sensor and the RR form a complex to function in bacteria, and the phosphoryl group delivered by the H721-D1009-H1137 way in the HPt domain of EvgS is transmitted to the D52 of EvgA (46, 50) (Fig. 1B) (Table 2). The receiver containing D52 acts like the phosphotransferase and catalyzes phosphorylated H1137 to obtain the phosphoryl group (45, 55). EvgA forms as a dimer to interact with EvgS in biosensor experiments (54) (Fig. 1B). EvgS and EvgA directly interact with each other, and the arrest of the phosphorylation of the functional domains disrupts the formation of the EvgS/EvgA complex (15). In multistep transphosphorylation, the intermediate steps in HK signaling show ultrasensitivity, which leads to reaching the same output levels and responding to signals in a tighter range than in common two-step transphosphorylation (5, 65). Therefore, we speculate that the multistep transphosphorylation of EvgS/EvgA results in responding to the slight pH change (from neutral to 5.0–6.0).

EVGS/EVGA REGULATES BACTERIAL ADAPTABILITY TO MULTIPLE ENVIRONMENTS

Living in conditions with rapidly changing characteristics, bacteria develop various TCSs to sense signals and adjust their behavior to adapt to the environment (4). Here, we illustrate that EvgS/EvgA regulates multiple gene expressions to improve their adaptability.

EvgS/EvgA confers acid resistance on bacteria

The capacity to grow under acidic conditions is essential for enteric bacteria to colonize mammals (66). EvgS/EvgA mainly confers acid resistance in the exponential-phase cells (14, 67) (Fig. 2). After exposure to the pH 2.5 medium for 1 h, the survival rates of mutants that overexpress evgA or constitutively express evgS are 100-fold higher than those of wild-type cells (14, 67). Although EvgS is activated in pH 5.0–6.0, responses induced by EvgS and EvgA confer bacteria’s ability to survive under pH 2.5. Generally, EvgS/EvgA alone and its connection with the PhoQ/PhoP TCS participate in the acid resistance (AR) system 2, requiring glutamic acid to improve the adaptability of bacteria in acidic conditions. It also confers acid resistance in an amino acid-independent way (67). Genes induced by EvgS activation mainly depend on EvgA (14). EvgA upregulates the expression levels of gadA, gadBC, gadE, ydeO, hdeAB, and ydeP (67) (Fig. 2).

FIG 2.

Acid resistance is conferred by EvgS and EvgA. yfdXWUVE encodes YfdW (a formyl-CoA:oxalate CoA-transferase), YfdU (an oxalyl-CoA decarboxylase), and YfdE belonging to a class III CoA-transferase, inducing the oxalate tolerance (68, 69).

Four AR systems are responsible for the survival of E. coli at pH 2.5 (29, 70). The AR system 2 has the major responsibility for the acid resistance in E. coli (71). EvgS/EvgA only participates in AR system 2, requiring glutamic acid by upregulating the expression of genes related to the system (29). It indicates that EvgS/EvgA is crucial to the survival of E. coli under acidic conditions. GadA and GadBC function in AR system 2 (72). GadA and GadB are isozymes of glutamate decarboxylase (GAD) (73). GadC is the glutamate/γ-aminobutyric acid antiporter on the membrane (70, 72). GadE is the activator of the gadA and gadBC expressions (74, 75) (Fig. 2). Moreover, YdeO, directly regulated by EvgA, is a transcriptional regulator belonging to the AraC/XylS family (18, 76) (Fig. 2). EvgA, YdeO, and GadE are able to directly bind the GadE promoter (29) (Fig. 2). Furthermore, PhoQ/PhoP can raise the amount of a stress-induced sigma factor called RpoS, which can initiate gadE to enhance acid resistance (77, 78) (Fig. 3). Through the membrane protein SafA (formerly named B1500), PhoQ/PhoP can be activated by EvgS/EvgA (16) (Fig. 3). The specific mechanism is illustrated in the following context. Notably, the GAD production mainly depends on the EvgA-YdeO-GadE circuit and the connection between EvgS/EvgA and PhoQ/PhoP by SafA (29, 77) (Fig. 2 and 3). Besides, both GadE and PhoP can initiate hdeAB (20, 77, 79) (Fig. 2 and 3). HdeA and HdeB are periplasmic chaperones that can prevent the proteins in the periplasm from aggregating in acidic conditions (80) (Fig. 2). In acid-sensitive strains with YdeO and SafA intact loci, evgAS form E. coli K-12 can confer E. coli Nissle 1917 and UTI89 resistance to acid shock (17, 20, 77, 79). However, E. coli MP1 without ydeO and safA loses acid resistance in the exponential phase, even after adding evgAS (17). It indicates that YdeO and SafA are essential for the acid resistance conferred by EvgS and EvgA.

FIG 3.

Signal transduction between EvgS/EvgA and other TCSs. Through SafA, EvgS/EvgA connects with PhoQ/PhoP to regulate downstream genes (16). EvgS/EvgA contributes to bacterial osmoregulation through the cross-talk between EvgS and OmpR (15).

In an amino acid-independent way, EvgA directly regulates YdeP (an oxidoreductase), and this process does not involve AR system 2 (18, 20) (Fig. 2). YdeP might contribute to the survival of E. coli under pH 2.4 for 2 h (29). In addition, E. coli with YdeP losing the oxidoreductase domain cannot develop constitutive resistance to acid pH (7). In S. flexneri, inhabiting the ydeP expression by repressing evgA is related to acid sensitivity (81). In addition, EvgA upregulates the transcription of yfdXWUVE, inducing the acid tolerance response of oxalate (18) (Fig. 2).

EvgS/EvgA confers multidrug resistance on bacteria

In E. coli, among open reading frames that developed single-drug or multidrug resistance, the most significant drug resistance to multiple compounds was conferred by the evgA expression (82, 83). When evgA is overexpressed, the minimum inhibitory concentration (MIC) of multiple drugs, such as doxorubicin, erythromycin, and benzalkonium, is approximately increased by 4, even 100-fold (83). In addition, evgS overexpression confers resistance to ciclopirox (84, 85). The MIC of sodium deoxycholate in evgS constitutively active mutants is eightfold higher than that in the wild type (86). In 2022, Naha and Ramaiah found that the evgS deletion results in interrupting the interaction of genes in antimicrobial resistance networks because EvgS has the highest number of interactions (71.6%) in the networks (87). These results indicate that EvgS/EvgA has a critical role in the multidrug resistance of E. coli.

To develop multidrug resistance in E. coli, EvgS/EvgA activates katE and efflux-related genes including emrKY, mdtEF (formerly named yhiUV), tolC, acrAB, and mdfA in direct or indirect manners through mediators such as other proteins or TCSs (77, 83, 86, 88) (Fig. 4). In addition, it requires phosphorylated EvgA (83).

FIG 4.

Resistance to multidrug and host immune responses conferred by EvgS/EvgA in E. coli and S. flexneri. The regulation mechanism by which EvgS/EvgA endows the ability of bacteria to survive in macrophages and multidrug resistance to bacteria.

EvgS/EvgA upregulates the expression of multidrug efflux pump genes. EmrKY, which is directly induced by EvgA, effluxes sodium deoxycholate and eliminates toxic metabolites produced by DNA damage in E. coli (86, 88, 89) (Fig. 1C and 4). In E. coli OCL62, EmrKY and EvgS/EvgA contribute to antibiotic resistance during the biofilm formation process (90, 91). Moreover, EvgA induces mdtEF expression through YdeO (67) (Fig. 4). The mdtEF gene encodes the multidrug resistance pump (92) (Fig. 4). Interestingly, the resistance induced by the evgA overexpression is similar to the mdtEF overexpression, indicating that mdtEF is essential for the multidrug resistance developed by EvgA (83). Notably, both EmrK and MdtE are members of the membrane fusion protein family, and their normal function requires the outer membrane channel TolC (83, 93–95) (Fig. 4). TolC production can be increased by the evgA overexpression and the signal transduction between EvgS/EvgA and PhoQ/PhoP (88) (Fig. 3). Furthermore, EvgA upregulates the acrAB and mdfA expressions in an indirect way (88) (Fig. 4). MdfA, a multidrug transporter, recognizes an exceedingly broad spectrum of drugs (96, 97). In E. coli, AcrAB and TolC compose a multidrug efflux transporter, contributing to the resistance to β-lactams (98, 99).

In addition, katE, which encodes a catalase HPII repressing ROS, is upregulated by the connection between EvgS/EvgA and PhoQ/PhoP (77, 100) (Fig. 3). Gallium nitrate is an antimicrobial agent that disrupts the iron metabolism in bacteria (101). In research conducted in 2021, EvgS constitutively active mutants induce a reduction in gallium nitrate killing rate without changing the MIC, raising the tolerance to gallium nitrate (102). The tolerance is developed by initiating the EvgA-YdeO-GadE circuit and KatE, then suppressing the surge of intracellular ROS mediated by gallium (102) (Fig. 3 and 4).

The multidrug resistance and acid resistance phenotypes occur at the same time when EvgS/EvgA influences the regulation network of multiple genes (28).

EvgS/EvgA confers resistance to host immune responses on bacteria

In S. flexneri, EmrKY induced by EvgS/EvgA contributes to the infection of macrophages and responds to mildly acidic conditions with high-level K⁺ concentrations (31) (Fig. 4). It might be because the intracellular condition of macrophages turns into an environment that coincides with the signals activated by EvgS in E. coli after the S. flexneri infection (103). Furthermore, in adherent invasive E. coli strain LF82, knockouts of evgAS, phoP, and ydeO lead to the proportion of viable bacteria in macrophages decreasing by half or more (104). Also, in 2020, Fanelli et al. found that MdtEF contributes LF82 to surviving in macrophages, where the expression is increased (105).

EvgS/EvgA confers heat resistance on bacteria

EvgS/EvgA is also related to the heat resistance of bacteria, which might become a potential risk to food processing. LHR is a locus conferring exceptional heat resistance to E. coli, whose expression depends on the evgA chromosomal copy (106–108) (Fig. 5). In E. coli W3100, EvgA contributes to significantly enhancing the thermal resistance to temperatures higher than 50℃ (47). Compared to E. coli K-12, EvgS constitutively active mutant obtains a slight but reproducible improvement in surviving at 51°C (7). Under heat shock (52°C, 20 min), the absence of R564 in EvgS also imported resistance to heat in E. coli K-12 (44) (Table 2). In the evgS^Δ564 mutant, the deletion of safA, a membrane protein connecting EvgS/EvgA and PhoQ/PhoP, results in retrieving the sensibility to heat (44).

FIG 5.

Other resistance and functions conferred by EvgS/EvgA. EvgS/EvgA endows the bacteria with heat resistance, adherence, and other functions.

EvgS/EvgA is related to high osmolarity resistance in bacteria

The expression of treA (also called osmA) and osmC, which products respond to the osmotic stress, can be induced by EvgA overproduction (109–111) (Fig. 5). ompC, expressed in high osmolarity, is regulated by the cross-talk between EvgS/EvgA and the EnvZ/OmpR TCS, which is explained in the following section (15, 112) (Fig. 3).

EvgS/EvgA contributes to E. coli adherence and colonization

Under the nicotinamide treatment, only half the amounts of ΔevgS and ΔevgA are recovered in the Hela cell adhesion assay, in contrast with the EHEC O157:H7 wild type (23). In ΔevgS and ΔevgA, the levels of proteins for intimate adherence are significantly decreased (23).

Through phosphorylated EvgA directly binding with ler, EvgS/EvgA upregulates the expression of the locus of enterocyte effacement (LEE) genes (23). LEE with the ler promoter encodes components in the type III secretion system (T3SS) and adhesin-related proteins, contributing to the attaching and effacing lesions of EHEC O157:H7 (113). These results indicate that EvgS/EvgA plays a critical role in promoting EHEC O157:H7 adherence and colonization by upregulating LEE expression in the presence of nicotinamide (23) (Fig. 5).

Interestingly, EvgS/EvgA has a contradictory influence on T3SS. Except for upregulating the LEE expression, the evgA overexpression might relate to upregulating T3SS by inducing the dctR expression, for DctR upregulates the core gene expression of T3SS in avian pathogenic E. coli (114, 115) (Fig. 5). However, in enteropathogenic E. coli, the EvgS/EvgA activation results in T3SS inhibition by the coaction of YdeO and YdeP, both activated by EvgA, inhibiting the expression of T3SS genes in direct or indirect manners (116).

Other bacterial behaviors regulated by EvgS/EvgA

The evgA overexpression slows down the bacterial growth rate and increases the yield of the heterologous polyketide 6-deoxyerythronolide B (18, 87, 89, 117). It can also induce hdeD and slp expression (114) (Fig. 5). HdeD reduces the flagellar filaments by activating the lrhA promoter (118) (Fig. 5). Slp, a carbon starvation-inducible lipoprotein, maintains the outer membrane’s stability (119). Furthermore, YdeO indirectly enhances poly-β-1,6-N-acetylglucosamine synthesis, contributing to biofilm formation (120). In addition, the ydeO expression level is different between species depending on their differential biofilm formation ability (121).

THE SIGNAL TRANSDUCTION BETWEEN EVGS/EVGA AND OTHER TCSS

In previous content, EvgS/EvgA confers multiple resistances on bacteria by connecting with other TCSs. Notably, the relationship between EvgS/EvgA and RcsC/RcsD/RcsB, a three-component system participating in GadE-related acid resistance, is interesting (122). EvgS/EvgA cannot be activated in ΔrcsB, and the process of activating EvgS/EvgA by RcsB (RR) does not require the HK sensors (RcsC and RcsD) (27). Here, we focus on two types of signal transduction between EvgS/EvgA and other TCSs.

EvgS/EvgA connects with other TCSs through SafA

EvgS/EvgA and PhoQ/PhoP are connected by the small inner membrane protein SafA (16) (Fig. 3). In the EvgS/EvgA constitutively active mutant, 14 genes upregulated by the PhoQ/PhoP two-component system are activated through PhoP enhanced by the evgA overexpression (123, 124) (Fig. 3). It can even activate genes at high Mg²⁺ levels, which usually inactivate PhoQ and PhoP (123). All these genes cannot directly bind with EvgA. Among the 14 genes, nine of them belong to the Mg²⁺ stimulon, and the mgtA expression is enhanced by the evgS overexpression but not the phoP expression (125). Notably, Eguchi et al. identified that EvgA can activate safA expression through direct binding with the promoter of safA that composes an operon with ydeO (14, 16, 18) (Fig. 1C and 3).

SafA directly interacts with PhoQ (126). The R53 in SafA and the D179 in the internal cavity of the PhoQ sensory domain are essential for the PhoQ activation by SafA (126, 127). The periplasmic part of SafA alone is able to activate PhoQ by interacting with a cavity in the central core of PhoQ in a way that is different from the PhoQ activation through Mg²⁺ (126, 127). In this process, signal transduction is functioning in the EvgS/EvgA-SafA-PhoQ/PhoP-regulated genes (16). Interestingly, through PhoQ/PhoP, SafA confers acid resistance by enhancing the transcription of the gadE promoter, which is related to the RR RssB (77) (Fig. 3). PhoQ/PhoP activated by SafA can be connected with RssB by activating the production of IraM, an anti-adaptor of RssB (77, 78, 128). The RssB bond with IraM loses the ability to interact with RpoS, resulting in increasing RpoS stability (78) (Fig. 3). In addition, RpoS initiates gadE and katE, indicating that EvgS/EvgA enhances multidrug resistance by connecting with multiple TCSs (77, 129) (Fig. 3). Furthermore, because the normal function of EmrK and MdtE requires TolC (83, 93–95), EvgS/EvgA-SafA-PhoQ/PhoP can contribute to multidrug resistance by regulating tolC; the tolC promoter contains the binding box of PhoP (16, 88) (Fig. 3). Also, in E. coli ATCC 25922, the colistin resistance may be developed by EvgS/EvgA-SafA-PhoQ/PhoP (130).

EvgS/EvgA connects with other TCS by cross-talk

In E. coli, EvgS/EvgA connects with EnvZ/OmpR through cross-talk (15) (Fig. 3). EnvZ/OmpR regulates ompC expressed in high osmolarity (112). However, in ΔenvZ with a multicopy plasmid expressing EvgS/EvgA, the ompC expression was induced and regulated by MgSO₄, low temperature, and nicotinic acid (9). Furthermore, EvgS can interact with OmpR, as proven by surface plasmon resonance, indicating that EvgS is able to transduce the signals to OmpR acting on the ompC expression (15, 131) (Fig. 3).

CONCLUSION AND FUTURE PROSPECTS

Multiple resistances and main proteins regulated by EvgS and EvgA are summarized in Fig. 6. Recently, EvgS and EvgA have been found to sense nicotinamide derived from the intestinal microbiota and upregulate bacterial adherence (23). It is reasonable to infer that more potential signals in intestine sensing by EvgS and more bacterial survival behaviors regulated by EvgS/EvgA are waiting to be explored.

FIG 6.

The signal transduction process and the main genes and proteins regulated by EvgS/EvgA. (A) EvgS/EvgA conferred multi-function through the signal transduction system. (B) EvgS/EvgA directly or indirectly regulates various genes and proteins to function.

The specific mechanism by which EvgS detects the environmental alternation is still unknown. To verify the VFT2 simulated as a close conformation, the specific structure needs to be further analyzed. In BvgS activated by molecules with a planar aromatic ring structure and carboxylate group, VFT2 can bind nicotinate but not nicotinamide (23, 38, 43, 132, 133). Due to the structural similarity between EvgS and BvgS, we speculate that VFT2 in EvgS might relate to binding nicotinamide. Interestingly, BvgS/BvgA is active until binding nicotinate because VFT2 is close and active without binding with a ligand (38). However, EvgS is activated after sensing signals, indicating the activation mechanism of EvgS would be different from BvgS. Notably, the interaction between signals and EvgS demands direct evidence by conducting further experiments. Moreover, the biological significance of the fact that EvgSs tend to self-associate and cluster with each other in the cell is still unknown (134).

In EvgS autophosphorylation after sensing signals, EvgS with three phosphorylation sites transfers the group in a three-step way. Although the unactive EvgSs form a homodimer, the three phosphorylation steps in EvgS are conducted in cis-cis-cis mode, indicating that the active EvgS works in a monomeric way (62). We speculate that the autophosphorylation of two EvgS in one dimer might be together or separately, and different signals might lead to a different phosphorylation mode.

To colonize in the intestine, E. coli demands abilities to adapt to multiple conditions in the digestive tract and survive in the stomach with acidic pH. Benefiting from the multistep transphosphorylation, EvgS/EvgA can respond to pH changes in a tight range (pH 5.0–6.0) and activate multiple genes to help the bacteria survive in pH 2.5. It indicates that bacteria might sense the possibility of environmental deterioration and prepare for it. Furthermore, exposure to acid shock can lead to acid resistance through spontaneous mutations in EvgS (7, 44). It suggests that EvgS/EvgA plays a major role in conferring acid resistance on bacteria and the evolution of E. coli in acid resistance is related to EvgS/EvgA.

In addition, belonging to multidrug resistance genes, EvgS, which has numerous copies in some rickettsiae, shows a high prevalence in the industrial park wastewater and gut microbial communities of various animals, such as sea turtles and agricultural animals (135–139). Also, as time goes by, EvgS and EvgA can accumulate in the intestinal microorganisms of seagulls (140). Furthermore, exposure to phorate (an organophosphorus insecticide) significantly upregulates evgS expression in the intestinal microbiota of mice (141). All of the above indicate that EvgS and EvgA are prevalent in the environment and are a potential threat to public health and rising global antibiotic resistance. Combining its significant impact on multiple resistance, adherence, and colonization of bacteria, it is constructive to consider EvgS and EvgA as drug targets.

EvgS/EvgA also regulates other TCSs through proteins or cross-talk (15, 16, 78). It indicates that EvgS/EvgA is a part of the complex signal transduction regulatory network composed of TCSs and regulated genes. In addition, we speculate that EvgS/EvgA is related to the QseC/QseB TCS. QseC/QseB is able to sense norepinephrine produced by the adrenergic neurons in the gut (142, 143). In EHEC O157:H7, norepinephrine can induce acid resistance by increasing evgAS expression (22). We infer that upregulating their expressions mediated by norepinephrine is done through QseC/QseB. Therefore, QseC/QseB may participate in the process of regulating bacterial behaviors by EvgS/EvgA with multiple TCSs. Further research can focus on the regulatory relationship between QseC/QseB and EvgS/EvgA.