Two‐Component System as Drug Target: Is It a Bandwagon or a Breakthrough?

ABSTRACT

MDR ESKAPE pathogens are the leading cause of hospital‐acquired infections (HAIs) that resist most antibiotics and form biofilms. Biofilm formation is dependent on the two‐component system (TCS), which regulates virulence traits including adhesion to host tissues, evasion of innate immunity, the synthesis of exopolysaccharides, and antibiotic resistance. TCS sense environmental stimuli such as pH, osmotic pressure and antimicrobial peptides. They regulate gene expression to promote bacterial survival and persistence during infection. TCS represents a contemporary and novel pathway for the advancement of targeted, adjuvant‐based options to fend off infections caused by ESKAPE pathogens. Targeting TCS may improve drug penetration and reduce resistance pressure, although challenges remain due to their structural conservation, regulatory complexity, and mutation‐driven resistance. However, there are several limitations in targeting TCS for drug development like TCS not always being essential for bacterial viability, structural homologies between TCS are not sufficient for broad‐spectrum inhibitors, sometimes TCS can be involved in complex and essential regulatory networks and can evolve resistance mechanisms by mutations. To further investigate this paradox, we implemented comprehensive computational and phylogenetic analyses of selected ESKAPE TCS. The findings from this investigation form the basis for our in‐depth review of the significance and challenges of TCS as emerging drug targets.

1. Introduction

Biofilms are structured microbial assemblies reflecting a dynamic survival tactic that enables bacteria to persist in hostile environments, evade immune defences, and withstand antimicrobial agents. In biofilms, bacteria are embedded in an extracellular polymeric substance (EPS) matrix that shields them from environmental stresses and promotes their survival on biotic and abiotic surfaces. This protective community structure places bacteria into a metabolic state where they are less active and more resistant to treatment, allowing them to foster chronic infections (Agarwal et al. 2025). According to the CDC (Centers for Disease Control and Prevention), the number of antibiotic‐resistant infections, mostly ESKAPE pathogens in the United States exceeds 2.8 million infections annually, with approximately 35,000 deaths due to these infections (Antimicrobial Resistance Facts and Stats | Antimicrobial Resistance | CDC, n.d.). These organisms can “evade” the actions of many conventional antimicrobial therapies, thereby leading to difficulties in infection control and clinical management worldwide. They are significant contributors to healthcare‐associated infections (HAIs), particularly in intensive care units (ICUs) with immunocompromised and critically ill patients. It accounts for more than 40% of nosocomial infections in the world today. They are responsible for infections such as pneumonia, bloodstream infections, urinary tract infections, and infections from indwelling medical devices (e.g., catheters, ventilators, and all types of prosthetic implants). The emergence of multidrug‐resistant (MDR) strains in ESKAPE pathogens limits treatment options, leading to chronic or recurrent infections, resulting in increased mortality rates, longer hospital stays, and enhanced costs to the healthcare system, estimated to be more than $20 billion every year in the United States (Rather et al. 2021).

At the heart of biofilm formation in these pathogens lies the two‐component system (TCS), a conserved signal transduction mechanism in bacteria that regulates a range of adaptive behaviors in response to environmental stimuli. A typical TCS consists of a sensor Histidine kinase (HK) that detects changes to the environment, and a response regulator (RR) that alters gene expression in response to those signals. They are essential signaling systems that orchestrate multiple physiological activities, particularly those related to virulence, locomotion, and stress resilience. In ESKAPE pathogens, TCS are important regulators of biofilm development, thereby responding to various environmental signals, including nutrient availability, surface adhesion, osmotic pressure, and exposure to antimicrobials. These regulatory pathways regulate patterns of gene expression with genes involved in adhesion, production of EPS, and biofilm maturation, to allow these bacteria to form resilient biofilm communities (Chen et al. 2022; Liu et al. 2019). Thus, targeting the TCS‐regulated pathways can hinder biofilm assembly and help antimicrobial agents work effectively. TCS‐regulated surface proteins that are critical for bacterial adhesion and biofilm assembly can be considered prime therapeutic targets for development into vaccines, monoclonal antibodies, or inhibitors. This review examines TCS pathways and elucidates their role in biofilm formation in ESKAPE pathogens. We have also deliberated on the details of the functional mechanism of TCS. In addition to this, we have discussed environmental stimuli that are sensed by TCS and the effect of TCS on biofilm formation. To further explore this paradox, we employed integrated computational and phylogenetic approaches to analyse selected ESKAPE TCS. Nonetheless, the potential use of TCS as therapeutic drug targets presents a few limitations, such as their dispensability for bacterial life; high structural conservation limiting the specificity of inhibitors; redundancy in the respective signaling networks; and the possibility of the development of resistance by virtue of mutation. There are limitations that we must fundamentally resolve before advancing the clinical usage of TCS‐targeted therapies.

2. TCS

A TCS contains two essential components: a sensor kinase (SK) and a RR to transduce external signals into an appropriate cellular response. The SK detects environmental signals and undergoes conformational changes, while the RR alters gene expression by interacting with bacterial DNA. This system is highly conserved and provides bacteria with the ability to rapidly respond to environmental changes, which are crucial for many bacteria to survive and progress as pathogens. Since TCS regulate multiple pathways using the common signal transduction pathway, an understanding of how TCS works will help us understand the many ways in which TCSs contribute to biofilm formation, virulence, stress response, and antibiotic resistance in ESKAPE pathogens.

2.1. Different Elements of a TCS

• SK: SK is a membrane‐linked protein necessary for the bacterial signal transduction process. It has two domains: an external sensor domain to receive stimuli (e.g., antibiotics, osmotic pressure, etc.) and a cytosolic kinase domain, where a conserved histidine is located. When an SK detects the signal, a conformational change occurs, causing autophosphorylation of the histidine, which activates the TCS. With the TCS activated, bacteria can alter their biological processes accordingly to modify or adapt to the original environmental changes (Mascher et al. 2006).

• RR: RRs are usually cytosolic proteins that modulate gene expression from environmental signals and cues. It contains a receiver domain that is phosphorylated by SK, and an effector domain that is associated with specific DNA sequences to regulate transcription. Depending on the system, the RR may act as both an activator and a repressor. The complementary actions of SK and RR ensure accurate genetic responses to changes in the environment to allow survival in changing conditions (Mascher et al. 2006; Stock et al. 2000).

Although the canonical TCS consists of an SK and an RR as core proteins, there are many species of bacteria that have additional regulatory proteins that modulate TCS signaling. The accessory proteins like YycH and YycI in Gram‐positive bacteria regulate the activity of the WalKR (YycFG) SK by modulating WalK SK activity (Gajdiss et al. 2020). Additionally, in Gram‐negative bacteria, PmrD acts as a connector protein between the PhoP/PhoQ and PmrA/PmrB systems for TCS signaling, allowing signal integration under defined conditions (Mao et al. 2025; Nirwan et al. 2021). While these accessory proteins are not universally present in all TCSs, they provide more specificity of TCS signaling, regulate crosstalk between regulatory pathways and enhance bacterial adaptation to environmental and host‐associated stressors.

2.2. Mechanism of TCS Signaling

TCS allow bacteria to sense and respond to their environment through a precise signaling pathway. A membrane‐bound SK senses an external stimulus (e.g., antimicrobials, temperature, and nutrient signals) and undergoes a conformational change and auto‐phosphorylates. It transfers the phosphate from ATP to a conserved histidine residue, followed by the transfer of phosphate to an RR and activates it by phosphorylating a conserved aspartate's side chain. RR is then activated and experiences a conformational change that allows it to bind to DNA at specific promoter regions. Once bound to DNA, either gene expression is activated (e.g., recruiting RNA polymerase to transcribe the genes related to virulence, antibiotic resistance, or metabolism), or transcription is repressed (e.g., blocking the transcriptional machinery). The phosphorylated RR functions molecular switch that converts external signals into changes in the genes that are expressed by the bacterium. This rapid signaling enables the bacterium to adapt to changing environmental conditions without requiring any permanent changes in its genetic material. As a result, bacteria can regulate processes such as biofilm formation, virulence, motility, stress responses, and antimicrobial resistance in a synchronized manner. TCS is a finely tuned system to give bacteria the flexibility to use regulation of gene expression based on environmental signals to adapt to their changing or hostile environment (Stock et al. 2000; Tiwari et al. 2017).

2.3. Regulatory Feedback and Adaptation

Feedback loops in TCS are an important mechanism that enables the regulation of signal transduction so that the organism can respond accurately and efficiently to changes in the environment. A negative feedback loop allows an activated RR to attenuate pathway output by limiting further signal propagation. This is typically accomplished by transcriptional regulation to terminate the signal or by facilitating RR dephosphorylation, as described by the best‐known TCS, including PhoP/PhoQ, EnvZ/OmpR, and CpxA/CpxR. This prevents transient or weak stimuli from generating prolonged or energetically costly responses. In contrast, a positive feedback loop involves the RR stimulating the signaling pathway because of a persistent stressor. These two feedback loops allow the cell to quickly adjust to changes in the environment while conserving energy and maintaining homeostasis. Therefore, feedback loops allow for variation in control, enabling bacteria to generate response stimuli at varying levels while balancing the conservation of energy, thereby assisting them in avoiding starvation and creating optimal conditions for adaptation and homeostasis in dynamic environments (Ishikawa et al. 2024; Tiwari et al. 2017). Thus, feedback mechanisms help prevent overactivation of signal transduction pathways. TCS helps bacteria to conserve energy and rapidly adapt, so that they can survive in changing environments.

3. Key TCS of ESKAPE Pathogens

The thorough overview of therapeutically relevant TCS in ESKAPE pathogens is presented in Table 1 and Figure 1a−f. It provides schematic overviews of representative TCS architectures and functional outputs in each organism.

Table 1.

TCS of ESKAPE pathogens.

Pathogen	Two‐component system (TCS)	Function in biofilm formation	Molecular mechanism	Target	Result	References
Enterococcus faecalis	VanS/VanR	Regulates vancomycin resistance and indirectly affects biofilm stability under antibiotic pressure.	Phosphorylation of VanR activates vanA operon, altering peptidoglycan precursors.	vanA operon, cell wall synthesis	Common in vancomycin‐resistant strains.	(Buttner et al. n.d.; Guffey and Loll [2021]; Hong et al. [2008])
	CroRS	Senses cell envelope stress, promoting biofilm formation and resistance to antimicrobial peptides.	CroS phosphorylates CroR, inducing expression of stress‐response and biofilm‐promoting genes.	Genes for cell envelope integrity	Relevant for survival in hostile environments.	(Kellogg et al. [2017]; Rose et al. [2022]; Todd Rose et al. [2023])
	FsrA/FsrC	Regulates gelatinase and serine protease production, enhancing biofilm formation.	Quorum‐sensing system that responds to extracellular signaling peptides (FsrD).	GelE, SprE	Impacts extracellular matrix degradation.	(Pinkston et al. [2011]; Yue et al. [2022])
Staphylococcus aureus	AgrA/AgrC	Controls quorum sensing for biofilm dispersal and virulence factor production.	AgrC senses autoinducing peptides (AIPs), activating AgrA to regulate RNAIII transcription.	RNAIII, toxins, surface adhesins	Plays a dual role in biofilm maturation and dispersal.	(Choudhary et al. [2018])
	SaeS/SaeR	Regulates virulence genes promoting biofilm‐associated infections.	SaeS phosphorylates SaeR, activating genes critical for biofilm survival in infected tissues.	Exoenzymes, adhesins	Crucial for biofilm‐associated tissue infections.	(Liu et al. [2016])
	SrrA/SrrB	Responds to hypoxic conditions in biofilms, enhancing their stability.	Phosphorylation of SrrA under anaerobic conditions activates metabolic adaptation genes.	Genes for anaerobic metabolism	Important for biofilm persistence in oxygen‐limited environments.	(Tiwari et al. [2020])
	WalK/WalR	Regulates cell wall metabolism and biofilm structural integrity.	WalK auto‐phosphorylates and transfers phosphate to WalR, controlling autolysin expression.	LytM, Atl	Essential for biofilm development and antibiotic resistance.	(Delauné et al. [2012]; Takada and Yoshikawa [2018])
Klebsiella pneumoniae	RcsC/RcsB	Regulates capsule synthesis, which is crucial for biofilm matrix formation and host immune evasion.	Phosphorylation cascade from RcsC to RcsB activates cps operon for capsule biosynthesis.	cps operon	Supports biofilm stability and resistance to phagocytosis.	(Liu et al. [2019]; Majdalani and Gottesman [2005])
	PhoP/PhoQ	Responds to magnesium limitation and antimicrobial peptides, enhancing biofilm resilience.	PhoQ senses Mg2+ depletion or antimicrobial peptides, phosphorylating PhoP to induce gene expression.	LPS modification, virulence factors	Relevant for survival under nutrient‐depleted condition.	(Groisman [2001]; Mao et al. [2025])
	CpxA/CpxR	Detects envelop stress and promote biofilm formation.	CpxA phosphorylates CpxR, inducing expression of stress‐related biofilm‐forming genes.	Adhesins, stress response genes	Important for maintaining biofilm structure under stress.	(Kuo et al. [2023]; Raivio et al. [1999])
Acinetobacter baumannii	BfmR/BfmS	Controls genes involved in biofilm initiation, maturation, and antibiotic resistance.	BfmS senses environmental cues and phosphorylates BfmR, regulating biofilm‐related genes.	Outer membrane proteins, pili, efflux pumps	Essential for biofilm‐related antibiotic tolerance.	(Draughn et al. [2018]; Krasauskas et al. [2019]; N et al. [2025]; Raustad et al. [2025])
	AbaI/AbaR	Mediates quorum sensing, regulating biofilm formation and virulence factors.	AbaR detects autoinducer molecules, regulating AbaI expression and downstream biofilm‐associated pathways.	Acyl‐homoserine lactone synthesis	Quorum sensing plays a central role in coordinated biofilm behaviors.	(Pumirat et al. [2024]; Sun et al. [2021]; Tang et al. [2020])
	PmrA/PmrB	Promotes resistance to cationic antimicrobial peptides, indirectly stabilizing biofilms.	PmrB phosphorylates PmrA, activating genes for LPS modification and biofilm resistance.	LPS modification genes	Enhances biofilm protection against antimicrobial agents.	(Chen and Groisman [2013]; Nirwan et al. [2021])
Pseudomonas aeruginosa	GacS/GacA	Regulates small RNAs involved in biofilm formation and repression of acute virulence.	GacS phosphorylates GacA, which controls rsmY and rsmZ small RNAs, repressing acute virulence and enhancing biofilms.	rsmY, rsmZ, exopolysaccharide production	Central to the transition between planktonic and biofilm states.	(Chambonnier et al. [2016]; Ryan Kaler et al. [2021]; Song et al. [2023])
	PhoP/PhoQ	Responds to environmental stress, promoting biofilm formation.	PhoQ phosphorylates PhoP, inducing stress response and biofilm‐enhancing genes.	LPS modification, efflux systems	Supports biofilm resistance under stress.	(Gellatly et al. [2012]; Groisman [2001])
	PilS/PilR	Regulates type IV pili expression, critical for biofilm formation and motility.	PilS phosphorylates PilR, which activates pilin synthesis genes.	Pilin synthesis	Facilitates initial surface attachment and biofilm development.	(Bernard et al. [2009]; Kain et al. [2025]; Ochner et al. [2024])
	RetS/LadS	Balances acute and chronic infection pathways, including biofilm formation.	RetS and LadS regulate exopolysaccharide production, determining biofilm development or acute virulence.	Exopolysaccharides (Pel, Psl), virulence genes	Acts as a switch between virulence and biofilm lifestyles.	(Chambonnier et al. [2016])
Enterobacter species	QseC/QseB	Responds to host‐derived signals (e.g., epinephrine), promoting biofilm formation.	QseC senses host signals and phosphorylates QseB, activating flagellar and biofilm‐related genes.	Flagellar and motility genes	Important for host interaction and colonization.	(Curtis et al. [2014]; Fernandez‐Ciruelos et al. [2023]; Rasko et al. [2008]; Weigel and Demuth [2015]; Zhu et al. [2023])
	PhoP/PhoQ	Enhance biofilm resilience by regulating stress response genes.	PhoQ phosphorylates PhoP in response to low Mg2+ or stress, enhancing biofilm matrix production.	LPS synthesis, efflux pumps	Aids survival in antimicrobial and host defence environments.	(Hong and Ko [2019])

Figure 1.

(A–F) Schematic representation of key two‐component systems (TCSs) in ESKAPE pathogens: (A) E. faecalis‐ FsrC/FsrA, CroS/CroR, VanS/VanR. (B) S. aureus‐ AgrC/AgrA, SrrB/SrrA, SaeS/SaeR, WalK/WalR. (C) K. pneumoniae‐ RcsC/RcsB, PhoP/PhoQ, CpxA/CpxR. (D) A. baumannii‐ BfmR/BfmS, AbaI/AbaR, PmrA/PmrB. (E) P. aeruginosa‐ GacS/GacA, PhoP/PhoQ, PilS/PilR, RetS/LadS. (F) Enterobacter spp.‐ QseC/QseB, PhoP/PhoQ, highlighting their integrated roles in biofilm formation, virulence regulation, and antimicrobial resistance.

Across pathogens, resistance‐associated TCS such as VanS/VanR and PmrA/PmrB are typically responsible for regulating remodeling of the cell's envelope, including alterations of peptidoglycan precursors and modifications to Lipid A, which result in lower antibiotic binding (Figure 1a,d) (Guffey and Loll 2021; Hondros et al. 2025; Yamada et al. 2024). Stress‐Responsive systems (e.g. CroR/CroS, CpxA/CpxR, SrrA/SrrB) integrate various signals, such as oxidative, envelope and redox, to allow bacterial survival against host and drug‐mediated stimuli (Figure 1a,c) (Kellogg et al. 2017; Kuo et al. 2023; Tiwari et al. 2020). Biofilm‐linked regulation is mediated by systems such as FsrA/FsrC, BfmR/BfmS, GacS/GacA, and RcsC/RcsB, which coordinate quorum sensing (QS), exopolysaccharide production, and surface adherence (Figure 1b,e) (Chambonnier et al. 2016; Liu et al. 2019; Pinkston et al. 2011; Raustad et al. 2025).

When considering the therapeutic potential of TCS, there are major differences between systems regarding druggability and clinical applicability. Pathway‐specific regulators (WalR/WalK and VanR/VanS) provide direct phenotypic advantages with possible variable essentiality, whereas global regulators such as PhoP/PhoQ and GacS/GacA exert broader control at the cost of redundancy and compensatory signaling (Gellatly et al. 2012; Guffey and Loll 2021; Song et al. 2023; Zheng et al. 2015). Biofilm‐associated TCS are frequently regulated by QS networks, suggesting that antivirulence or combination strategies may be more successful than single target inhibition.

4. Structural and Evolutionary Insights Into TCS Kinase Subunit Across ESKAPE Pathogens

TCS constitutes an attractive target for antimicrobial development. However, the therapeutic potential of TCS depends on structural conservation, their evolutionary constraints, and the druggability of catalytic domains. To support this, we performed an analysis of representative HK sensors conserved across Gram‐positive and Gram‐negative ESKAPE pathogens, focusing on WalK in Enterococcus faecalis and Staphylococcus aureus, PhoQ in Klebsiella pneumoniae, Pseudomonas aeruginosa, and Enterobacter spp., and PmrB in Acinetobacter baumannii.

4.1. From Sequence to Structure: Homology Modeling and Structural Validation

Multiple sequence alignment reveals the highly conserved nature of the dimerization and histidine phosphotransfer (DHp) domains, as well as the catalytic ATP binding (CA) domains, across all TCS sensor proteins, despite the clear divergence between the sensory and linker regions of these proteins. The presence of the phosphorylated histidine and nucleotide‐binding motifs in the TCS sensors of both Gram‐positive and Gram‐negative lineages further supports the fact that there is a common core mechanism for signal transduction (Supporting Information File, Supporting Information S1: Figure 1 and Table 1).

Three‐dimensional structures were predicted using AlphaFold and adopted the canonical HK fold, comprising elongated DHp domains coupled to compact CA domains. Structural quality assessment confirmed model reliability, with over 90% of residues occupying favored or additionally allowed regions in Ramachandran plots. ERRAT analysis yielded high overall quality factors for most models, including Ef WalK (98.03%), Ab PmrB (97.07%), Pa PhoQ (96.89%), Kp PhoQ (94.35%), and Sa WalK (94.24%), while Enterobacter PhoQ showed a lower yet acceptable score (88.39%), with deviations confined to flexible regions. Structural superposition showed conserved kinases across TCS (Figure 2a) (Gulia et al. 2023). This structural conservation highlights the functional importance of TCS HK and supports their potential as common targets for anti‐biofilm and antimicrobial strategies. The high sequence and structural conservation of the CA and DHp domains among bacterial species creates a challenge in drug discovery and development. Inhibitors that target these highly conserved domains may not be pathogen‐specific and may inhibit many TCSs rather than just one target. Furthermore, the CA domain has been shown to have structural similarity to other eukaryotic GHKL‐fold proteins, which may lead to increased risk of off‐target effects and make developing selective inhibitors more difficult (Fihn and Carlson 2021).

Figure 2.

Structural and evolutionary analysis of sensor kinases in ESKAPE pathogens. (A) Structural comparison of WalK (E. faecalis, light blue; S. aureus, cyan), PmrB (A. baumannii, light magenta), and PhoQ (K. pneumoniae, lavender; P. aeruginosa, mustard yellow; Enterobacter spp., salmon red). (I) Superposition highlighting conserved kinase architecture. (II) Three‐dimensional structures with active sites indicated. (B) Phylogenetic tree depicting evolutionary relationships among six bacterial genera (ESKAPE). Bootstrap values represent node support, and the scale bar (0.2) indicates genetic distance.

4.2. Mapping Evolutionary Lineages: Phylogenetic and Comparative Analysis

The Phylogenetic analysis was done using the Maximum Likelihood (ML) method implemented in MEGA version 12. The LG amino acid substitution model was applied based on ML method and 1000 ultrafast bootstrap replicates were selected (Kumar et al. 2008). It revealed two distinct clades of TCS sensors: Gram‐positive Kinases and Gram‐negative Kinases. WalK proteins clustered tightly, consistent with conserved regulatory roles, while the Gram‐negative sensors were divided into PhoQ and PmrB sub‐clusters, which reflect the functional specialization of these kinases with respect to environmental sensing and antimicrobial resistance (Figure 2b). High bootstrap support was seen on all internal nodes, demonstrating that the inferred topology is reliable.

In summary, these data illustrate that through their separate evolutionary paths, the TCS SK have continued to maintain highly conserved catalytic architectures for furthering the development of structure‐based inhibitors while peripheral divergence offers opportunities for pathogen‐selective targeting (Figure 2b).

5. Emerging Inhibitors of TCS in ESKAPE Pathogens: Promise and Pitfalls

TCS are appealing options for antimicrobial therapy as they control important aspects of virulence, biofilm formation, antibiotic resistance, and evasion of host immunity in ESKAPE pathogens. Numerous natural products, synthetic compounds, and repurposed drugs have been documented to disrupt TCS. For example, waldiomycin inhibits the WalK/WalR TCS, a conserved regulator of cell wall metabolism in Gram‐positive bacteria (Igarashi et al. 2013). Savirin and RNAIII‐inhibiting peptide (RIP) derivatives suppress Agr signaling in S. aureus, with savirin being reported to inhibit AgrA–DNA binding. Ajoene has been shown to reduce QS–regulated virulence and biofilm formation. LED209 blocks QseC‐mediated virulence signaling in Enterobacteriaceae (Baëtz et al. 2021; Curtis et al. 2014; Nakayama et al. 2009; Pant et al. 2022). In total, these TCS inhibitors demonstrate the promise of treating infections related to TCS, including reversing resistance, preventing biofilms, reducing virulence, enhancing immune clearance, and working in synergy with existing antibiotics. TCS inhibitors encompass diverse chemical classes and mechanisms, including interference with HK activity (e.g., WalK) or RR–DNA interactions (e.g., AgrA), rather than universally competing for ATP binding in specific SK. The different binding sites and mechanisms of action point to a degree of diversity, as well as an ongoing challenge for optimizing TCS inhibitors for clinical applications. Despite promising In vitro efficacy, many TCS inhibitors are often weakened due to pathway redundancy or compensatory signaling within many global pathway regulators (e.g., PhoP/PhoQ, GacS/GacA). In contrast, pathway‐restricted systems such as VanR/VanS offer clearer phenotypic readouts but a limited spectrum. These differences likely explain the uneven translational progression of TCS‐directed strategies.

Nonetheless, there are still serious limitations. Many inhibitors are only effective in In vitro assays or animal models and have not progressed to human clinical trials. The structural homology of HK complicates specificity which may reduce efficacy, hygienic redundancy allows compensatory pathways to bypass inhibition, and several inhibitors have poor bioavailability or have been toxic (Figure 3) (Goulian 2010). Additionally, resistance can still arise through mutations in components of the TCS. While TCS inhibitors hold promise as a combination therapy or novel antimicrobial therapy, being developed into clinically infectious drugs remains a very challenging task. TCS inhibitors hold therapeutic potential; however, the lack of clinical translation raises the relevant question of whether it could be a therapeutic breakthrough or simply the next bandwagon in antimicrobial drug discovery efforts (Guffey and Loll 2021; Igarashi et al. 2013; Jakobsen et al. 2012; Mitrophanov and Groisman 2008; Rasko et al. 2008; Stock et al. 2000; Sully et al. 2014) (Table 2).

Figure 3.

Illustration of major obstacles in using TCSs as antimicrobial targets: non‐essentiality, conserved structures, compensatory pathways, mutation‐driven resistance, incomplete characterization, and low screening efficiency.

Table 2.

Therapeutic strategies targeting TCSs in ESKAPE pathogens.

Therapeutic strategy	Targeted TCS (Pathogen)	Inhibitor (s)	Structural class	Binding site/pathway	Mechanism	Limitations
Counteracting resistance	WalK/WalR (Gram‐positive bacteria, incl. S. aureus, B. subtilis)	Waldiomycin	Aromatic polyketide (natural product)	Histidine kinase (WalK)	Inhibits WalK autophosphorylation; disrupts cell wall regulatory signaling	In vitro only, no clinical validation
	PhoP/PhoQ (K. pneumoniae, P. aeruginosa)	PMBN (Polymyxin B nonapeptide)	Cyclic peptide (polymyxin derivative)	Outer membrane/PhoQ modulation	Sensitizes bacteria to polymyxins by altering membrane signaling	Adjunctive; not a direct TCS inhibitor
	PmrA/PmrB (A. baumannii)	Artemisinin derivatives	Sesquiterpene lactones	Lipid A modification pathway	Reduces lipid A modification and colistin tolerance	In vitro, pharmacokinetics unresolved
Disrupting biofilms	FsrA/FsrC (E. faecalis)	Ambuic acid	Polyketide	QS‐linked TCS signaling	Inhibits gelatinase expression and biofilm formation	In vitro only
	AgrA/AgrC (S. aureus)	Savirin, RIP peptides	Small molecule/cyclic peptide	AgrA–DNA interaction/QS signaling	Suppresses toxin production and biofilm formation	Animal models promising, not clinical
	— (QS‐regulated biofilm pathways) (A. baumannii, P. aeruginosa)	Ajoene	Organosulfur natural compound	QS and biofilm regulatory networks	Reduces QS‐regulated virulence and biofilm; enhances antibiotic penetration	Poor bioavailability
	GacS/GacA (P. aeruginosa)	Salicylic acid	Phenolic compound	HK sensor modulation	Weakens small‐RNA signaling and biofilm formation	Non‐specific; moderate efficacy
Antivirulence	SaeS/SaeR (S. aureus)	2‐Aminoimidazole derivatives	Synthetic heterocycles	RR‐mediated transcription	Downregulate toxin gene expression	Early preclinical
	RetS/LadS (P. aeruginosa)	Phenothiazines (e.g., chlorpromazine)	Repurposed drugs	Sensor kinase signaling interface	Shifts virulence from acute to chronic phenotype	Off‐target effects
	QseC/QseB (Enterobacteriaceae)	LED209	Synthetic small molecule	QseC allosteric pocket	Blocks host‐signal sensing and virulence activation	Effective in animal models
Enhancing immune clearance	CroRS (E. faecalis)	NDGA	Polyphenolic lignan	Stress‐response regulation	Sensitizes bacteria to oxidative stress	In vitro, cytotoxicity concerns
	SrrA/SrrB (S. aureus)	Flavonoids (apigenin, luteolin)	Natural flavones	Redox‐sensing HK	Reduce hypoxic survival and virulence gene expression	Low stability
	CpxA/CpxR (K. pneumoniae)	Tannic acid	Polyphenol	Envelope stress response	Attenuates stress adaptation	Poor drug‐likeness
Synergy with antibiotics	PhoP/PhoQ + polymyxins (K. pneumoniae, P. aeruginosa)	PMBN + colistin	Cyclic peptide + antibiotic	Membrane/lipid A signaling	Potentiates polymyxin activity	Adjunctive only
	Biofilm‐regulatory pathways + aminoglycosides (ESKAPE spp.)	Ajoene + gentamicin	Organosulfur + aminoglycoside	Biofilm disruption + ribosomal inhibition	Improves biofilm penetration and killing	Bioavailability limits

Note: The table lists representative inhibitors, their structural class, binding site, mechanism, outcomes, and limitations.

6. The Prevailing Enigma of TCS: Bandwagon or Breakthrough?

TCS are the most important regulators of the virulence, biofilm formation, and antibiotic resistance of bacteria; however, their therapeutic potential remains debated. Their absence in humans and central role in virulence support a breakthrough narrative, whereas redundancy, non‐essentiality, and the lack of clinical candidates argue for a bandwagon interpretation. Preclinical studies report that inhibiting TCSs such as VanS/VanR, PhoP/PhoQ, and BfmR/BfmS can reduce antibiotic resistance or biofilm formation by 90% (Guffey and Loll 2021; Lu et al. 2023; Paharik and Horswill 2016; Sharma et al. 2023). Inhibitors have different scaffolds also, from polyketides (waldiomycin) to cyclic peptides (RIP) to small molecules (LED209), but they inhibit a common site as competitive inhibitors (e.g., the ATP‐binding pocket or the sensor‐regulator interface). Depending on the type of TCS, the inhibitor may disrupt kinase activity, inhibit active phosphorylation, block through allosteric inhibition and disrupt QS, which highlights versatility and the challenge of achieving drug‐like properties.

Despite promising In vitro results, TCS inhibitors face major limitations. First, many TCSs are not necessary for bacterial viability under standard laboratory conditions. However, they play important role in virulence, biofilm development, stress adaptation, QS and persistence during host infection. Therefore, inhibiting TCS may not directly kill the bacterial cell but will most likely reduce or eliminate the ability of the bacteria to cause disease. Thus, it provides the basis for developing anti‐virulence therapies which will exert lower selective pressure on antimicrobial resistance than conventional antibiotics (De Gaetano et al. 2023). The therapeutic potential of TCS inhibitors can be improved by the co‐administration of TCS inhibitors with existing antibiotic therapies to enhance therapeutic effectiveness, targeting pathogen‐specific or conditionally essential TCS (e.g., those that become essential during an infection). These combinations often provide significantly greater reductions in biofilm formation, bacterial persistence and tolerance to antibiotics than using only one of the treatments, therefore indicating the potential of utilizing TCS inhibitors as an adjunct to current therapeutic strategies rather than in isolation (Chen et al. 2022). Second is the structural conservation between HK and RR complicates selective targeting. Lastly, pathway redundancy and compensatory signaling can attenuate inhibitor efficacy, and mutations may compromise durability. These three factors seem to explain the low conversion of TCS inhibitor candidates from preclinical models to clinical practice. To demonstrate how structural information can inform rational inhibitor design, we focused on conserved SK components of TCS, specifically WalK found in Gram‐positive pathogens and PhoQ/PmrB found in the ESKAPE gram‐negative pathogens. These SK regulators are not like traditional housekeeping proteins; they are at the center of the signal transduction pathways that control virulence, stress adaptation, and antimicrobial resistance. The conserved kinase core of the cytoplasm provides a solid foundation to evaluate the potential of these targets and allows for structure‐based identification of pathway‐specific as well as broad‐spectrum TCS inhibitors.

In summary, TCS inhibitors offer a paradoxical profile. They have a high potential for success in preclinical studies, but also present substantial challenges related to redundancy, structural constraints and limited clinical evidence for their use. Current evidence supports cautious optimism, encouraging further research through properly conducted comparative clinical studies, which will be needed to identify those TCS inhibitors that should proceed to clinical application.

7. Conclusion

TCS plays a central role in the survival strategies of the MDR ESKAPE pathogens governing biofilm formation, virulence, stress adaptation, and resistance. Disruption of TCS, especially PhoP/PhoQ, BfmR/BfmS, VanS/VanR, and QseC/QseB, can disrupt biofilms, restore drug susceptibility, and diminish pathogenicity. In many cases, TCS inhibitors act in synergy with existing antibiotics. However, functional redundancy, structural similarity, and poor clinical translation continue to limit progress. Rational structure‐based drug design has started to demonstrate advancements in this field as it relates to proteins that hold potential for development, as in the case of WalK, PhoQ and PmrB. TCS inhibitors remain in a state of flux between bandwagon and breakthrough; their therapeutic advancement is dependent on overcoming key translational hurdles.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Supporting File

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.