Regulation and inhibition of type III secretion systems in plant pathogenic bacteria

Abstract

Type III secretion systems (T3SS) are syringe-like apparatuses acting as protein transport nanomachines found in most Gram-negative bacterial pathogens. They can inject effector proteins into the host cell cytoplasm, crossing the host cell membrane, and cause infection. Due to their critical role in pathogenicity, T3SS represent attractive targets for vaccinations and disease treatments. This review elucidates the overarching structural framework and operational mechanisms of T3SS apparatuses while also delineating the responsiveness of phytobacterial T3SS to host-derived signals and the nuanced orchestration of their activities by host and environmental stimuli. This discussion encompasses shared features and idiosyncratic attributes among a spectrum of pathogens, including but not limited to Pseudomonas syringae, Ralstonia, Xanthomonas, and Erwinia. Additionally, we scrutinize the contribution of natural products and synthetic chemicals as T3SS inhibitors, elucidating their hallmark and role in the ongoing quest for and design of novel drugs. An in-depth comprehension of T3SS functionality and the modes of action of diverse inhibitors holds promise for developing innovative drugs aimed at swiftly suppressing phyto-pathogenicity elicited by a spectrum of bacterial species.

Background

Most pathogens secrete proteins that play a crucial role in mediating the complex interactions with other organisms in their surrounding environment. These pathogenic bacteria are equipped with specialized excretion systems (type I–VII) to accomplish intricate biochemical tasks of transporting proteins across cellular membranes. Among them, the type III secretion system (T3SS) has been extensively studied in various model organisms (McDermott 2011; Notti et al. 2015; Notti Ryan and Stebbins 2016; Deng et al. 2017). These pathogens are primarily associated with various plant and human diseases (Fig. 1). Pathogenic bacterial strains can deploy T3SS to inject effector proteins into the host cell cytosol to modulate myriads of host cell processes and to establish an intracellular or extracellular niche suitable for differentiation, replication, and dissemination, as illustrated (Fig. 1). The successful establishment of pathogens (extracellular or intracellular) depends on the efficient hijacking of the host immune response, functional machinery, and cytoskeleton. The best-known examples include Escherichia coli (Entero-pathogenic and Entero-hemorrhagic; EPEC and EHEC), Citrobacter rodentium, Chlamydia, Salmonella, and Shigella strains. Because of such invasions, these pathogens can easily invade hosts cells via T3SS. Humans and animals have suffered dysentery and respiratory infections, leading to epidemics of plagues. Similarly, plant hosts resulted in pandemics of canker, blights, and soft rots (Fig. 1; Abby and Rocha 2012).

Fig. 1

Overview of model bacteria endowed with T3SS(s). Particular examples are the pathogenic bacteria identified in humans, mammals, animals, and plants, wildly known to cause epidemics in the world by causing devastating diseases. Data was compiled from Hueck (1998), Abby and Rocha (2012)

A well-documented example is plant-associated Pseudomonas syringae and P. aeruginosa strains, which could show higher virulence. P. syringae have multiple hosts to attack and cause infection by manipulating the plant defense system (immunity) with its T3SS nanomachine. Similarly, P. syringae, Xanthomonas, Ralstonia solanacearum, Rhizobium, and soft rot-causing agents Dickeya and Pectobacterium spp., all have diverse hosts and conserved T3SS to suppress the immune response (Davidson et al. 2013, 2017; Landry et al. 2020; Asif et al. 2024b). T3SS relies on a conserved set of genes to function effectively in these strains. Many structural protein-forming nanomachine apparatus are the same in most of the strains as HrcC forms the outer ring of the T3SS apparatus, while HrcJ anchors the structure to the bacterial cell envelope by contributing to the basal body. HrcR and HrcS (HrpS) regulate the expression of T3SS components and the associated effector proteins. HrcU plays a pivotal role in protein secretion and translocation, while HrcV forms the translocon to ensure that effector proteins are delivered into the host cells. HrcN, an ATPase, provides the energy required for protein translocation through this system. Finally, the HrpB and HrpD families control the expression and secretion of the T3SS machinery in specific bacterial species. Together, this network of genes and proteins coordinates for assembly and regulation of the T3SS, allowing pathogenic bacteria such as P. syringae, R. solanacearum, Xanthomonas spp., and Erwinia spp. to infect their plant hosts. These pathogens inject their effector proteins that manipulate host cells to promote bacterial survival and colonization (Helmann et al. 2019; Teulet et al. 2022). Despite the diversity of the host, there is remarkable conservation in the T3SS apparatus, secretion mechanisms, and their mode of action (Du 2016; Portaliou et al. 2016; Helmann et al. 2019).

Since its discovery in 1994 (Rosqvist et al. 1994; Coburn et al. 2007), the T3SS has been systematically categorized into five primary components: the translocon, the needle complex, the basal body, the transfer apparatus, and the cytoplasmatic complex (Fig. 2). The translocation mechanism is a critical component of the T3SS, facilitating the transfer of proteins from the bacterial cytoplasm to the host cytoplasm (Büttner 2012, 2016; Burkinshaw and Strynadka 2014; Burkinshaw 2015; Sun et al. 2007), and the delivery unit is called as needle complex. This complex includes an ATPase and multiple circular structures (rings) that are intricately integrated into the membrane (Chang et al. 2014; Bernal et al. 2019; Gupta et al. 2024). Contrary to animal pathogens, plant pathogenic bacteria, such as pathovars of P. syringae and Xanthomonas spp., possess an elongated needle, referred to as a pilus, which enables penetration of the thick cell wall and contact with the underlying plasma membrane (Bergeron 2013, 2016). Likewise, EPEC, EHEC, and Citrobacter rodentium possess a filamentous extension (EspA) that exhibits structural similarities to flagellin (Lindeberg et al. 2006; Xin et al. 2018). Interestingly, the T3SS needles of plant pathogens (termed Hrp pili) have neither a tip complex nor an extension, but are considerably longer than the needles of the T3SS of animal pathogens, probably so that they can penetrate the thick cellulose matrix of the plant cell wall. These needle modifications are likely to be evolutionary adaptations for infecting different hosts. The needle tip protein is directly associated with the transport apparatus (Diepold and Wagner 2014; Diepold et al. 2015; Tang et al. 2018). The needle is a cylindrical structure composed of helical protein subunits, with its length determined by the host organism of the pathogen (Fig. 2; Lefebre and Galán 2014; Bergeron 2016; Murillo et al. 2016; Tang et al. 2018).

Fig. 2

The structural and functional illustrative model of T3SS nanomachine in Salmonella spp. SPI-1. In Salmonella SPI-1 T3SS, the needle complex, basal body, export apparatus, and sorting complex, are shown on the left side. The cross sections of the inner structure of these components are shown on the right side. On the right side, electron microscopy-mediated solved protein structures are compiled to display basic nanomachine orientations. SctC and SctD proteins transfer to the inner rod in the inner membrane and pass to the translocon or needle complex (SctF). Accession numbers from the Protein Data Bank (PDB) were used to search, and carton displays were created in Biorender. The proteins, accession IDs, and their names are listed as Needle complex. SctE (AopB-3WXX; and SipB-3TUL), SctA (SipD; PDB-3NZZ), SctF (PDB-3J0R), SctC; (PDB-5TCQ & 5TCR) (InvG); SctD_N (PDB-3J1W) (InvG), SctD_C; (PDB-5TCP), SctJ (PrgH-PrgK::PrgK; PDB-3J6D, − 5TCP; − 5TCR), SctU (SpaS; PDB-3C01, SpaS_C), SctV (PDB-4A5P), SctO (PDB-3K29), SctN (PDB-2OBM) (Abrusci et al. 2013; Bergeron et al. 2013; 2015; Chatterjee et al. 2011; Fujii et al. 2012; Lorenzini et al. 2010; Worrall et al. 2008, 2016; Zarivach et al. 2007, 2008; Deng et al. 2017)

The ongoing research on specific pathogens involves exploring various functions, regulatory pathways, and inhibitors. However, a comprehensive compilation of comparative data to understand commonalities and specificities among pathogens is still lacking. Such data would clarify the interplay between pathogens, hosts, and environmental cues, particularly regarding the response of phytobacterial T3SS to host signals and host and environmental stimuli regulation. Plant pathogens like Pseudomonas spp., Ralstonia spp., Xanthomonas spp., and Erwinia spp., are crucial subjects for study. In addition, dedicated efforts are required to identify inhibitors of the T3SS from both natural products and synthetic compounds. These investigations should aim for a comprehensive understanding of their mechanisms of action and specific molecular targets within the T3SS machinery of these pathogens. This review provides a comprehensive analysis of the structural framework of the T3SS apparatus, its architectural associations, co-regulatory mechanisms across various species, and the functional roles of its genes. Additionally, we have examined the functional overlap between distinct two-component systems in the species discussed, identified inhibitory compounds, and elucidated the modes of action of diverse inhibitors with potential for novel drug development. Furthermore, we have conducted comparative studies on pathogen infection dynamics and explored bacterial manipulation strategies across different domains.

Regulation of T3SS in plant pathogenic bacteria

In Gram-negative bacteria, T3SS encoded by hrp/hrc (HR and Pathogenicity; HR and Conserved) cluster is a prevalent feature observed in various genera including Erwinia, Pseudomonas, Ralstonia, and others (Alfano et al. 2000; Andrade et al. 2014). The majority of hrp genes are situated within chromosomes, forming pathogenic islands, although a subset of hrp genes, such as hrpM in P. syringae, is dispersed throughout the chromosome. Typically, the hrp gene cluster size ranges from 18 to 40 kb, encompassing approximately 20–25 genes, among which highly conserved genes are denoted as hrc genes. Meanwhile, a functional common nomenclature includes “secretion and cellular translocation” (Sct) as a unified system for conserved components of T3SS for all species is developed, widely adopted and recently expanded (Gazi et al. 2012; Peeters et al. 2013; Diepold and Wagner 2014; Portaliou et al. 2016; Gaytán et al. 2016; Portaliou et al. 2016). The T3SS spans the bacterial inner and outer membranes, and the host cell membrane, comprising several substructures: a cytosolic ATPase complex, C-ring, inner membrane export apparatus, basal body, needle, and translocation pore in the host membrane (Fig. 2). The basal body is composed of inner membrane rings (SctJ, SctD) and an outer membrane ring (SctC), with the export apparatus formed by SctR, SctS, SctT, SctU, and SctV. Below this, the C-ring (SctQ) and ATPase complex (SctN, SctO, SctL, SctK) form the sorting platform for secretion (Schraidt et al. 2010; Schraidt and Marlovits 2011; Burkinshaw and Strynadka 2014; Hu et al. 2015; Makino et al. 2016; Portaliou et al. 2016). The inner rod anchors the needle, which is capped by the SctA tip complex and connects to host cells through translocators SctE and SctB. The length of the needle is regulated by SctP (Journet et al. 2003; Thomas et al. 2012; Wee and Hughes 2015), while SctU and SctW control the secretion hierarchy (Büttner 2012). A functional T3SS, containing the basal body and needle, is referred to as the ‘needle complex’, analogous to the flagellar hook-basal body structure (Fig. 2; Macnab 2003; Schraidt and Marlovits 2011). Sct hereafter is referred to as conserved hrp/hrc locus for P. syringae nomenclature. HrcV exhibits significant homology with their counterparts in Yersinia (Xin et al. 2018). The T3SS is the main virulence determinant in most pathogenic Xanthomonas species, encoded by the conserved hrp/hrc locus similar to Pseudomonas T3SS, having 20 proteins with multiple copies to develop the major T3SS subassemblies: the sorting platform (HrcQ, HrcL, HrcN, and HrpB7), the export apparatus (HrcR, HrcS, HrcT, HrcV, and HrcU), the needle complex (HrcD, HrcJ, HrcC, and HrpE), and the translocon (HrpF) (Alvarez-Martinez et al. 2020).

Based on the genetic similarity within the hrp cluster, it consists of two distinct groups (Baltrus et al. 2017; Wang et al. 2018). The first group, “Hrp1”, includes E. amylovora, P. syringae, and Pantoea spp. (Baltrus et al. 2017), and the second group, “Hrp2”, comprises R. solanacearum, Xanthomonas spp., and Acidovorax citrulli (Clarke et al. 2010; Abby and Rocha 2012). The primary disparity between these two groups lies in their transcriptional regulation systems. The first group, including P. syringae, regulates the T3SS expression using extra-cytoplasmic function sigma (σ) factor rpoN and alternative sigma factor hrpL transcriptionally (Fig. 3a), while both Erwinia spp., and Pseudomonas spp., also utilize the upstream transcriptional regulator HrpS alone or together with HrpR to control the Hrp genes expression. In addition, Hrp2 species use the transcriptional regulator HrpR to modulate the HrpS expression, subsequently activating the functional action of hrpL to control T3SS activity (Hutcheson et al. 2001; Jovanovic et al. 2014; Wang et al. 2018). In the second group, members of the AraC family of proteins regulate transcriptional activity associated with T3SS (Chang et al. 2014; Costa et al. 2015; Waite et al. 2017).

Fig. 3

The pathogenicity island and basic master regulators of T3SS genes. a The pathogenicity islands present in the Pseudomonas syringae pv. actinidae M228, compared to gene orientation in P. syringae pv. tomato DC3000 and P. savastanoi pv. phaseolicola 1448a. This orientation shows the similarity and uniqueness of the three strains and their evolutionary aspects. For the orientation of the T3SS cluster of P. syringae pv. tomato DC3000 and P. savastanoi pv. phaseolicola 1448a was adapted from Xie et al. (2019). b HrpL master regulator influenced by different factors, especially HrpX (HrpG, V, U), TCS, and RSM system, and ultimately infects the host plant. c the master regulator hrpG regulated by different sigma factors (PhoPQ) and other factors in P. aeruginosa. The display data was compiled from Anantharajah et al. (2016), Xie et al. (2019)

R. solanacearum and Xanthomonas spp. synthesize their T3SS using about 20–22 hrp/hrc genes, which contain tight regulatory control. It is unique in linking T3SS gene expression directly to plant host contact, using effectors to alter host defense and metabolic pathways (Rico and Preston 2008; Xian et al. 2020). Expression of hrp/hrc genes is triggered when the outer membrane receptor PrhA detects an unidentified cell wall component, initiating a signaling cascade. HrpG, an OmpR-type regulator, activates HrpB, which directly regulates T3SS genes and effectors, likely by binding to the hrp box motif (Occhialini et al. 2005; Coll and Valls 2013). HrpG and HrpB have homologs in Xanthomonas and Burkholderia species (Li et al. 2011; Lipscomb and Schell 2011), but PrhA and its upstream regulators are not conserved in other species. Moreover, the Xanthomonas T3SS cluster facilitates the translocation of type III secreted effectors into plant host cells (White et al. 2009). These effectors, known as Xops (Xanthomonas outer proteins), are crucial for Xanthomonas pathogenicity. The T3SS cluster is uniquely positioned in X. campestris pv. campestris, suggesting independent acquisition as indicated by its chromosomal location, distinct from other group 2 species (Jacob et al. 2015; Merda et al. 2017). However, further studies related to pathogenicity-related genes in R. solanacearum and their mechanisms of regulation are extensively reviewed elsewhere (Peeters et al. 2013; Lowe-Power et al. 2018; Vailleau and Genin 2023).

Transcriptional and post-transcriptional regulation of T3SS genes

The major transcriptional regulatory control of the T3SS cascade is under the HrpL protein belonging to the ECF family. This protein, along with its alternate sigma factor (σ⁵⁴), interacts with a conserved “hrp-box; GGAACC-_N15-16-CCACNNA” located in the promoters of certain genes involved in the T3SS regulation (Fig. 3b; Fouts et al. 2002; Xiao et al. 2004; Chang et al. 2014). HrpL facilitates the transcription of the hrp/hrc and other T3SS effector genes with the assistance of RNA polymerase, resulting in improved pathogenicity (Hutcheson et al. 2001; Zwiesler-Vollick et al. 2002; Schechter et al. 2006; Ferreira et al. 2006; Huang et al. 2022; Zhao et al. 2022). Many studies are inclined to propose that HrpL-mediated regulation and coordination with other T3SS-encoding genes are achieved in a well-organized pattern. It precisely controls the expression and timely induction of associated genes in the pathway (Ferreira et al. 2006; Thwaites et al. 2004; Waite et al. 2017). The interruption of the hrpL gene during the natural or synthetic process of evolution results in impaired hrpL promoter activity, transcriptional activity, and subsequent base mutation in the promoter region, resulting in the loss of pathogenicity in P. syringae pv. actinidae (Xie et al. 2023).

In addition to the σ⁵⁴ sigma factor (RpoN), two other proteins HrpR and HrpS, also play a crucial role in the transcription of hrpL regulation and start the T3SS gene expression (Ortiz-Martín et al. 2010a; Wang et al. 2018). These two proteins are found in a HrpRS locus present inside the hrp/hrc cluster (Hendrickson et al. 2000; Wang et al. 2018). Moreover, the HrpR is required for hrpS transcription, and this interaction with an upstream regulatory sequence known as the Hrp-box was described in halo-blight bacterium NPS3121 (Ortiz-Martín et al. 2010a; Jovanovic et al. 2011). The oligomerization of HrpR and HrpS results in the formation of a heterohexameric complex, essential for the complete activation of the hrpL gene. However, it should be noted that HrpS (Hutcheson et al. 2001), alone can interact with hrpL promoter to activate it. Still, the potential expression would be weak when the other partner, hrpR, is not present (Jovanovic et al. 2011). In addition to the activation of hrpL, hrpS is responsible for regulating the activation of several genes that are not dependent on T3SS throughout the entire genome (Lan et al. 2006, 2007; Wang et al. 2018). A study on three deficient mutants of hrpL, along with hrpRS genes in P. syringae DC3000, revealed rapidly accumulated gene expression of different housekeeping genes other than T3SS. This transitional switching off of T3SS activity to a widespread increase of the normal metabolic processes indicated a tradeoff function in P. syringae (Lan et al. 2006).

Moreover, HrpA (or HrpA1) is known to form pilus protein and exert its transcriptional regulatory effect on the T3SS pathway (Wei et al. 2000). The hrpA gene is located on the upstream region of hrpRS and hrpL in the P. syringae DC3000 and is necessary for the release of different substrates primarily as harpins (Abrusci et al. 2013; Waite et al. 2017). HrpA is a gene regulated by HrpL and can be activated by soluble signals from plant cells (Wei et al. 2005; Waite et al. 2017). HrpA1 is necessary for the induction of hrpL and hrpS transcription in P. syringae pv. phaseolicola 1448a in the leaves of host bean plants (Ortiz-Martín et al. 2010b). To activate the HrpZ operon expression, P. syringae pv. phaseolicola and syringae DC3000 show the same upregulated expression of hrpL, hrpRS, and hrcC mRNA transcripts upon hrpA mutation compared to wild-type control, upon contact with the plant, suggesting the presence of an important feedback mechanism to control T3SS by hrpA (Wei et al. 2000; Ortiz-Martín et al. 2010b). Although HrpA can autoactivate the T3SS pathway, negative self-regulation prevents excessive production of T3SS-related proteins. An additional promoter region containing hrp-box is also situated in the hrpJ gene, and the promoter activity of hrpL is downregulated by HrpL itself through its binding to that box. This HrpL-T3SS signaling cascade exhibits robust signal amplification, while negative autogenous regulation prevents excessive accumulation of T3SS substrates (Waite et al. 2017). Similarly, higher expression of hrpL transcripts increased the extracellular accumulation of HrpA1, but the increased level of T3SS harpin and effector proteins were not secreted out of the cell. The intracellular accumulation of these compounds in P. syringae modulated the T3SS in two distinct phases: First, HrpL-dependent expression of T3SS genes and secretion of HrpA1, and second, secretion of downstream T3SS substrates, which appears to be regulated post-translationally. The exact mechanism triggering the secretion of these substrates remains unknown, but it is likely linked to the maturation of the HrpA1 and its contact with the host plasma membrane (Waite et al. 2017).

The HrpRS heterodimer, which plays a role in the transcriptional activation of hrpL, undergoes translational control by two other small proteins HrpV and HrpG (Fig. 4a). Accordingly, HrpV has a direct interaction with HrpS, resulting in a constraint on the binding capacity of HrpS to HrpR (Jovanovic et al. 2011, 2014). HrpV can interact with HrpG (a chaperone protein) linked to the T3SS basal body located at the plasma membrane (Jovanovic et al. 2011). It is postulated that HrpV binds to the cellular membrane by interacting with HrpG during stimulated T3SS expression. This spatial contact hinders the connection of HrpV with bacterial DNA and, therefore, inhibits the blockage effect of HrpS (Charova et al. 2018). The HrpVG complex still can interact with HrpJ in the inner membrane, but the HrpJ role within the cellular environment of plants is unidentified other than inhibiting the immune response of the host (Crabill et al. 2012). Before its translocation, HrpJ blocks the inner channel of the T3SS by creating a ternary complex resembling a gatekeeper, together with HrpVG (Cherradi 2013; Charova et al. 2018). In the inner core membrane of T3SS, another binding complex is formed by the interaction between HrpG and HrcU (Charova et al. 2018). During the infection of P. syringae inside the host plant, HrpJ acts as a facilitator for the complete functionality of the T3SS to secrete translocator proteins and harpins (Fu et al. 2006; Ramos et al. 2007; Crabill et al. 2012), depending on signals that induce secretion (Crabill et al. 2012).

Fig. 4

The identified factors influencing the de-repression and suppression of T3SS regulation in terms of HrpRS-HrpL pathway in P. syringae. a HrpL transcription is regulated by different proteins that include AefR, PsrA (indirect positive activators), and CorR upstream binding agent (binds directly to HrpL and activates early transcription). HrpJ established a binding complex with hrpL promoter to prevent the formation of HrpRS complex, which creates a negative feedback loop to negatively regulate hrpL activity. Then, HrpS protein itself can stimulate the T3SS effector proteins. AlgU and HrpA induce the overexpression of the hrpRS locus in a direct manner. In GacSA TCS, phosphorylated-gacA binds indirectly to the IR (inverted repeats) region of hrpR and hrpL to induce their expression. CvsR has a direct regulatory effect on the activation of hrpRS, which is expressed in the presence of calcium ion Ca²⁺ as an external environmental stimulus and ultimately sustains the T3SS-dependent virulence in P. syringae. In a higher level of c-di-GMP, HrpR/HrpL expression is repressed. In addition, HrpV blockage is removed by HrpG activity and it frees the HrpS as well as HrpJ makes a complex with both (HrpJG/V) to allow the induction of the expression of T3SS. Thus, HrpG is the anti-repressor of an anti-activator HrpV. The bonding of HrpG with HrpF exerts a negative regulatory effect on T3SS by allowing the formation of the HrpRSVG circuit. P-RhpR independently activates T3SS effector genes like hopR1. The chaperone protein RhpC directs metalloprotease RhpP to the periplasm. In the absence of RhpC, RhpP decreases the expression of HrpL. Induction of hrpRS and hrpL transcription requires (p)ppGpp. b P. syringae cultured in Kings B (nutrient-rich condition, KB) resulting in suppression of its T3SS; hrpL gene allows HrpT to bind its promoter and motif which indicates an indirect regulation by HrpT as shown by dotted lines, The PRhpR starts to pile-up and its abundance leads to the suppression of hrpRS to check its low expression. HrpV influences the binding affinity of HrpS protein to prevent the oligomerization of HrpRS; HrpRS heterodimer destruction and negative regulation is described with red dotted arrow. Phosphorylation of RhpR is mediated by another TCS unit kinase RhpS, and helps in a sustained phosphorylated state. To achieve the phosphorylated RhpR abundance, another pathway is activated using its own promoter with an inverted repeat (IR) element, and their covalent bonding leads to producing more PRhpR. Unphosphorylated-RhpR has a positive regulatory effect on three genes a) functional accumulation of c-type cytochrome, b) regulate alcohol dehydrogenase synthesis, and c) positive modulation of hemB and negative effect on protease production in KB medium. RhpR also functions as a negative regulator of anthranilate synthase activity. On the other hand, phosphorylated RhpR inhibits swimming motility but shows a positive effect on the induction of twitching motility. Additionally, in vivo, P-RhpR functions as a negative regulator of the biofilm (algD) and c-di-GMP levels while improving lipopolysaccharide production in a positive manner. P-RhpR has a positive effect on LonB protein, which results in the breakdown of HrpR protein and prevention of HrpRS oligomerization. Gene orientations and scheme of events adapted with permission from Xie et al. (2019)

Although the HrpA1 protein undergoes rapid degradation inside the bacterial cell, it can remain intact by forming a complex with the T3SS regulatory protein HrpF (Haapalainen et al. 2009; Huang et al. 2022). The importance of HrpF is well-established in disease pathogenesis and for the functional T3SS development in different P. syringae strains (Huang et al. 2016; Deng et al. 2017). This hrpF interaction may result in the de-repression of HrpS and the enhancement of the expression of the T3SS signaling cascade (Huang et al. 2016, 2022). In addition, elucidating the possible interactions of HrpF, HrpA1, and HrpJ-VG circuits could reveal their crucial role in intracellular dynamics and establish the regulatory link between the expression and secretion of the T3SS (Fig. 4). The HrpA1 protein is accumulated in the extracellular space before the secretion of other substrates of T3SS (Haapalainen et al. 2009; Waite et al. 2017). Overall, the pilus synthesis mediated by the hrpA1 gene and subsequent interaction with host cells might potentially initiate the secretion process (Waite et al. 2017). However, the exact mechanisms behind the activation of downstream T3SS substrate secretion remain unknown.

The hrp genes in Xanthomonas spp. and R. solanacearum were classified with hrp group 2; they are different from E. amylovora and P. syringae in some aspects. Xanthomonas spp. and R. solanacearum use HrpG regulon to regulate it by HrpX, which interacts with a cis‐element within the promoter region of hrp genes, referred to as plant‐inducible promoter (PIP)‐box, which is also present in the promoter of many T3 effectors ( Fan et al. 2017). In addition to HrpG and HrpX, HrpD6 was reported as a hrp regulator in Xanthomonas spp, responsible for regulating multiple hrp genes (Li et al. 2011; Fan et al. 2017). In addition to conserved genes, some extra regulatory and control genes, sigma factors are underway to discover.

Activity of T3SS in artificial medium in relation to environmental cues

The T3SS-mediated virulence of Hrp1 species plays a crucial role, with strains investing substantial cellular resources in synthesizing the T3SS apparatus (Sturm et al. 2011). This allocation aims to quickly initiate the mechanism to counteract the host’s immune system. Thus, the interaction between bacterial T3SS and the host’s immunity is crucial in the initial stages of plant infection (Xin et al. 2018). Thus, the initial period during infection is critical in determining disease outcome and severity. To comprehend this critical phase of infection, especially in P. syringae, it is necessary to identify the specific signal from the pathogen that initiates T3SS activity. The hrp/hrc genes are generally repressed when the pathogen is grown in nutrient-rich environments. Nevertheless, a low level of expression may persist in these conditions due to the presence of genes such as hrpV and hrpT in T3SS cluster (O’Malley and Anderson 2021). The dissociation of the anti-repressor and anti-terminator complexes may occur under nutrient-deprived conditions at the hrp/hrc genes, facilitating a rapid increase in T3SS activity. However, additional evidence is required to support this hypothesis, providing opportunities for further investigation.

The expression of the T3SS-oriented hrp/hrc genes in P. syringae can be evaluated during growth in nutrient-deprived media (O’Malley and Anderson 2021). These nutrient-poor formulations, known as HIM (hrp-inducing minimal medium) and HDM (hrp-de-repressor medium), are designed to promote higher expression levels. The main difference lies in the carbon, nitrogen, and sugar sources (fructose or mannitol) and their slightly acidic pH. In addition, for Xanthomonas spp., hrp-inducing medium XOM2 is used to trigger T3SS activity. This environmental condition primes P. syringae for T3SS activation, highlighting the importance of understanding the nutritional cues that modulate T3SS expression in bacterial pathogenesis. It can be said that the expression of T3SS-associated genes in P. syringae and Xanthomonas may be influenced by broader environmental variables during infection, independent of specific plant metabolites acting as signals.

Positive and negative regulation of T3SS levels by plant-derived signals

Similar to synthetic media conditions, studies have shown elevated expression levels of T3SS during infection in planta (Haapalainen et al. 2009), suggesting the existence of distinct signals in the rhizosphere microenvironment that trigger the production of the T3SS in bacteria. During initial investigations, it was observed that the series of T3SS genes in a cluster, especially hrpL expression was increased when two different host plants (susceptible and resistant) were infiltrated with P. syringae (Xiao et al. 2007). In addition to susceptible and resistant varieties, non-host species exposed to similar treatment revealed improved T3SS expression, suggesting metabolite-based signals are standard in different plants instead of being specific to the host plant. Previously, a study stated a significant ten-times rise in mRNA levels of hrpA1 in a minimum medium supplemented with tomato exudates (soluble signals) (Haapalainen et al. 2009). Subsequently, the exudates obtained from Arabidopsis-based extracted suspension cultures without cells also induced the RNA levels of hrpL and levels of avrPto effector protein (Yan et al. 2019). In addition, the presence of plant cells in the suspension increased the proliferation of P. syringae DC3000 using the “infection-in-a-flask” approach. It indicates that the signals originating from plants boosted bacterial proliferation by augmenting the deployment of the T3SS (Yan et al. 2019).

In another study, Arabidopsis seedlings immersed in water (exudates) resulted in significant AvrPto accumulation and enhanced hrpL gene expression level in P. syringae pv. tomato DC3000 (Anderson et al. 2014). Most importantly, citric acid and aspartic acid were identified as the bioactive molecules responsible for triggering the T3SS, as they are present in significant quantities in the tissues of plants. This finding further reinforces prior discoveries that T3SS is activated by common characteristics of the host signals instead of signals unique to a particular host (Rico and Preston 2008; Kumar et al. 2017; Yan et al. 2019). Although all bioactive substances retain a carboxyl group to exhibit acidity, not all can induce T3SS. For instance, amino acids such as leucine and valine may trigger T3SS, suggesting a certain degree of selectivity to detect these compounds. It also indicates that optimal biological activity mediated by acidic metabolites in triggering T3SS is dependent on the existence of an essential sugar like fructose. The generation of phytotoxins, such as syringomycin, in P. syringae pv. syringae is modulated by specific plant-derived signals, with the syrB gene playing a pivotal role in both syringomycin production and full virulence during plant pathogenesis. Research has demonstrated that phenolic glucosides, such as arbutin, phenyl-β-D-glucopyranoside, and salicin, act as strong inducers of syrB transcription, while sugars like D-fructose and sucrose enhance the pathogen’s sensitivity to these phenolic signals, thereby amplifying toxin biosynthesis. These findings suggest that the synthesis of phytotoxins like syringomycin is closely linked to the pathogen’s ability to detect and respond to plant metabolites, which may complement T3SS-mediated infection strategies during pathogenesis (Mo and Gross 1991).

In a quest to find a specific signal exuded from plants in regulating the effects of P. syringae infection, model organism Arabidopsis was mutated to impair the production of immunological regulator mitogen-activated protein kinases MAPK phosphatase 1 (MKP1) (Andrade et al. 2014). The mkp1 mutant exhibited improved immune responses to pathogenic elicitors and increased resistance against P. syringae DC3000 infection. Moreover, in the context of mkp1 infection, DC3000 exhibited a diminished capacity to stimulate T3SS-associated genes and transport effectors molecules (Anderson et al. 2011; 2014). Analysis of mkp1 mutant exudates via GC–MS revealed decreased concentrations of several metabolites that induce T3SS, such as citric acid and aspartic acid. The complemented MKP1 plant with these metabolites fully reestablished DC3000’s capacity to transfer effectors, thereby regenerating susceptibility to DC3000 infection (Anderson et al. 2011; 2014). Overall, it can be inferred that the virulence-inducing signals depend on the plant host’s genetic makeup during infection.

Auxin, also known as indole-3-acetic acid (IAA), is a plant-derived chemical capable of suppressing the production of P. syringae’s T3SS (Anderson et al. 2011; McClerklin et al. 2018; Djami-Tchatchou et al. 2020). As the infection progresses, P. syringae reallocates cellular resources from T3SS development to the other secretion system due to higher bacterial density in the apoplast, favoring microbial competition (McAtee et al. 2018; Djami-Tchatchou et al. 2020). Sulforaphane, a glucosinolate synthesized in Arabidopsis and other Brassicaceae family members, also suppresses T3SS gene expression by modifying the T3SS master regulator HrpS through covalent interactions, thereby enhancing plant defense (Wang et al. 2020). Meanwhile, the Arabidopsis plant lines unable to synthesize glucosinolate showed more susceptible action towards the infection of P. syringae, indicating that sulforaphane-mediated T3SS suppression contributes significantly to host defense mechanisms (Wang et al. 2020). Additional substances originating from plants, such as plant flavonoids and other phenolics (Vargas et al. 2013; Lee et al. 2015; Kang et al. 2020; Zhi et al. 2022), together with several synthetic chemicals, have shown inhibitory effects on the P. syringae T3SS (Ma et al. 2019; Puigvert et al. 2021). Considering the fact that the energy expenditure associated with the production and upkeep of the T3SS is substantial (Sturm et al. 2011), it is plausible that cells exhibiting low levels of T3SS expression, commonly referred to as “cheater” cells, can allocate their energy resources towards proliferation or other cellular activities. This mechanism may improve the pathogen population’s adaptability and overall fitness (Xie et al. 2023). To fully grasp the impact of tactics on P. syringae’s virulence, it is crucial to comprehensively characterize the precise patterns responsible for initiating and sustaining T3SS bistability in the host environment.

Phenolic caffeic acid, secreted in increased amounts by tobacco roots following R. solanacearum inoculation, reduces disease symptoms when applied exogenously in pot and field experiments (Li et al. 2021). Other plant-derived natural products, such as coumarins, also exhibit antibacterial activity. For instance, 7-methoxycoumarin mitigated tobacco bacterial wilt in pot experiments (Han et al. 2021). Additionally, salicylic acid inhibits R. solanacearum growth and suppresses several bacterial wilt virulence factors (Lowe-Power et al. 2016). Plant signals as essential oils from aromatic plants contain various volatile molecules with protective effects against many phytopathogens, including R. solanacearum (Pradhanang et al. 2003; Deberdt et al. 2018). These oils have been studied for over 20 years for their efficacy against this bacterium (Raveau et al. 2020).

In Ralstonia and Xanthomonas, the enzymatic functions of some type III effectors (T3Es) include acetyltransferases, proteases, and ubiquitin ligases, with established repertoires of plant proteome interactors (González-Fuente et al. 2020). T3Es can have multiple functions depending on the host; for example, RipAB downregulates calcium signaling in potatoes (Zheng et al. 2019) and inhibits TGA transcription factors in Arabidopsis (Qi et al. 2022). Furthermore, screening of various plant-based chemicals against these strains resulted in the identification of salicylidene acylhydrazides as T3SS inhibitors, which limits bacterial growth in plants (Puigvert et al. 2021).

Host-derived amino acids and sugars, especially in the apoplast which serves as a battleground where pathogens attempt to breach the plant’s defenses and establish infection in leaves (Rico and Preston 2008; Anderson et al. 2014), are the first compounds encountered by P. syringae throughout the process of infection. These plant exudates contain organic acids and amino acids that optimally trigger T3SS genes after their inclusion in the induction medium (minimal medium). In order to understand the regulatory T3SS pathway in response to these organic acids and amino acids, a notable gene was identified by the genetic mutations known as SetA that induce the T3SS genes in response to sugars, and is a putative transcription factor of the DeoR-type (Turner et al. 2020). The gene SetA alone has been implicated in partially increasing the expression levels of the effector genes avrRpm1 and hrpL in DC3000 upon exposure to sucrose, fructose, and mannitol. In Arabidopsis leaves, it was identified as an essential factor for achieving the highest hrpL expression (Turner et al. 2020). A mutant strain of DC3000 lacking the setA gene exhibited reduced growth capability, accompanied by impaired expression of hrpL. Furthermore, the observed activity of hrpRS and rpoN was quite like the wild type, suggesting a role for SetA in directly regulating hrpL induction or post-translational feedback modulation of unaffected genes (Turner et al. 2020).

T3SS regulatory pathway in response to environmental stimuli

The regulation of T3SS by host signals is intricate, involving multiple sensory and signaling pathways in combination that detect the signals from the host and respond to environmental stimuli, ultimately impacting T3SS deployment (Ma et al. 2019). According to recent advances, HrpL and its associated co-regulating components, HrpRS, are used to sense the environmental cues in P. syringae, resulting in a canonical cascade (Khokhani et al. 2013). Its network tightly regulates the T3SS due to its shared nature among Gram-negative pathogens. However, different pathogens of various types use unique master regulators of T3SS. For example, comparatively, Hrp1-type pathogens initiate the T3SS pathway from HrpS, while in Hrp2-type, T3SS is initiated by HrpG (Fig. 4b). Further research has expanded our knowledge of the Hrp1 and Hrp2 regulatory networks, revealing their modulation by key regulatory pathways such as the Gac-Rsm pathway, the C-di-GMP second messenger pathway, and quorum sensing (Fig. 4b). For instance, the global regulatory factor ExsA is influenced by factors like cAMP, Gac-Rsm, and RpoS (sigma factor), independent of T3SS, exerting direct or indirect regulatory effects on T3SS transcription and function (Fig. 4; Anantharajah et al. 2016). However, our understanding of the sensory domains and the environmental stimulus inducing T3SS in P. syringae is limited.

The involvement of the two-component systems (TCSs) in regulating bacterial virulence in the environment is a regular occurrence (Beier and Gross 2006; Tang et al. 2006). Bacteria predominantly use TCSs for signaling and communication, which consist of a membrane-integrated histidine kinase that perceives a stimulus and a cytoplasmic response regulator. The exchange of information by histidine kinase (sensory unit) and a response regulator through conserved phosphorylation and dephosphorylation reactions (Jung et al. 2012; Sankhe et al. 2023). After the perception of stimulus by the sensory first component, it is transferred to the cognate transcriptional factor and response regulator to regulate gene expression (West and Stock 2001). A couple of years ago, the two-component system AauSR was discovered by the Tn5-mediated genetic screening of DC3000. This research further revealed that the identified TCS could utilize amino acids, thus designating it as an Amino-Acid Utilization Sensor and Response Regulator (AauSR). This nutrient-sensing system also comprises a histidine kinase (AauS) and response regulator AauR and TCS was primarily responsive to aspartic acid and fructose, resulting in augmented expression of T3SS genes (Yan et al. 2020). These two genes were located inside the aat/aau locus, widely conserved in Pseudomonas strains. This locus is also comprised of genes encoding the ABC transporter complex AatQMP linked to another protein, AatJ, present in the periplasm. Previously, this locus’s roots were identified and described in nonpathogenic P. putida (Sonawane et al. 2006; Singh and Röhm 2008). Aspartic acid and glutamic acid consumption in P. putida is facilitated by the proteins AatQMP and AatJ. Additionally, the protein AauS is responsible for sensing and transporting the substrates that have accumulated in cells. The aatJ gene was supposed to have a binding site regarded as AauR binding motif (Rbm). Upon exposure to aspartic acid or glutamic acid, a series of events used to happen in a sequence that includes AauR finds its binding site in the aatJ gene followed by activation of ABC co-transporter locus aatJQMP, which leads to improvement and enhanced hrpRS and hrpL gene expression (Deng et al. 2014; Yan et al. 2020). The genome mining of DC3000 for Rbm sequences revealed an extra Rbm sequence located upstream of hrpRS, suggesting a potential direct influence of AauR on the production of hrpRS (Fig. 4).

Deletion of the Rbm upstream of hrpRS exhibited reduced T3SS gene activity levels in response to aspartic acid and glutamic acid, suppressing virulence of DC3000 in Arabidopsis plants (Deng et al. 2014; Yan et al. 2019). The presence of Rbm in the hrpRS promoter is conserved among 38 P. syringae isolates with the canonical tripartite pathogenicity island, suggesting a common regulatory function of AauSR towards HrpRS (Fig. 4). A similar universal nature of the motif of each locus was observed in P. syringae B728a, where aauR essentially increased T3SS gene activity, resulting in heightened strain virulence in bean plants (Vinatzer et al. 2006; Kumar et al. 2017; Yang et al. 2017). All these events proved that the Rbm incorporation happened earlier than hrpRS motif in P. syringae during evolution, likely playing a key role in the initial development of P. syringae virulence (Kumar et al. 2017).

Redundancy in different global regulatory pathways on T3SS regulation

Three different TCS localized in the periplasmic space to the inner membrane are identified and widely described in P. syringae strains. One of the two-component systems, GacSA, is responsible for responding to an unidentified stimulus and subsequently activating the cytoplasmic response regulator GacA through a process known as phosphorelay (Latour 2020). Earlier studies on TCS in P. syringae initially designated the gacS gene as lemA (lesion manifestation). However, It was a difficult task for authors to pinpoint the associated response regulator gacA essentially present in the vicinity of the response regulator, despite the common occurrence of two units being co-located in the genome (Koretke et al. 2000). This system bears a resemblance to the BarA/UvrY TCS found in E. coli (Pernestig et al. 2001). In the γ-proteobacteria, the gacA is located distant from gacS and co-located with uvrC in an operon. The uvrC gene encodes a component of the nucleotide excision repair complex (Heeb and Haas 2001; Heeb et al. 2002).

Certain studies described a positive effect of GacSA on T3SS expression (Chatterjee et al. 2003, 2007; Vargas et al. 2013; Ferreiro et al. 2018), while others have documented a negative regulation by GacSA and suggested that it is not essential to initiate T3SS expression in planta conditions (Marutani et al. 2007). For instance, GacSA suppresses the T3SS in P. aeruginosa (Brencic et al. 2009; Valentini et al. 2018). In another study focused on investigating the molecular mechanisms behind the regulation of virulence by GacSA in P. syringae (O’Malley et al. 2019), an AC811 mutant strain exhibited reduced virulence and a decreased T3SS activity in the plant microenvironment. This model has been represented in several contemporary publications (Brencic and Winans 2005; Tang et al. 2006; Moll et al. 2010), as well as in different predictive biological models of virulence regulation in P. syringae (MacLean and Studholme 2010).

In addition, the deactivation of gacA revealed increased expression in an AC811 strain rather than a reduction of T3SS activity (O’Malley et al. 2019). The diminished virulence of an AC811 mutant strain was ascribed to a secondary alteration in anmK, encoding an enzyme implicated in the reprocessing of the bacterial cell wall. It showed adverse effects on uvrC`s transcriptional levels, contributing to the observed attenuation. However, the association of P. syringae virulence with anmK and uvrC has not been previously established. Thus, a gacA mutation can polarize the downstream expression of uvrC, suggesting a similar adverse impact on the virulence of P. aeruginosa infections in mammals (Ferreiro et al. 2018; Qin et al. 2022).

Moreover, in contrast to the deployment of T3SS, the presence of GacA was necessary for the cell motility of DC3000, suggesting an inverse regulatory relationship between the T3SS and motility (O’Malley et al. 2019). Frequent cell movement (motility) is essential for P. syringae attachment with the host to enter the intercellular space via stomatal openings in the surface of leaves (Chieda et al. 2005). Consequently, this process may enhance the ability of P. syringae to survive on the surface of plants until enter the internal tissues of leaves. The GacSA system is subsequently inactivated, leading to the de-repression of T3SS at the apoplast (Fig. 4a). The precise mechanism(s) underlying the impact of GacSA on the deployment of T3SS remains uncertain, but it is established that GacSA exerts regulatory control over the RsmA proteins.

Another TCS, RhpSR, is responsible for regulating the expression of T3SS in P. syringae pv. phaseolicola 1448a. The response regulator RhpR undergoes phosphorylation to exert repression on T3SS through its interaction with an IR element located in the promoters of hrpR and/or hopR1 (Xiao et al. 2007; Deng et al. 2010). Furthermore, the phosphorylated form of RhpR can activate the promoter activity of the Lon protease gene, leading to the degradation of HrpR (Xiao et al. 2007; Deng et al. 2010; Xie et al. 2019). The sensor histidine kinase, RhpS, can show autokinase activity, including kinase and phosphatase activity towards RhpR (Deng et al. 2010; 2014). During the abundance of nutrients in a medium, the protein RhpS acts as a kinase to facilitate the suppression of T3SS. In contrast, RhpS exhibits phosphatase activity towards RhpR for the induction of T3SS in minimal nutrition. This activity leads to a decrease in the activity of RhpR at specific target promoters (Xiao et al. 2007; Xin et al. 2016, 2018; Xie et al. 2019). RhpR can undergo phosphorylation in addition to RhpS, perhaps by the action of physiological phosphor-donors like acetyl phosphate, and/or by sensor kinases from other TCSs (Deng et al. 2017). This regulatory process is achieved through a negative feedback loop (Deng et al. 2010; Xin et al. 2018). The RhpSR system may play a role in the co-regulation of T3SS and cellular maintenance, potentially enabling a broader interchange of metabolism and pathogen virulence in response to environmental factors, such as variations in nutrition accessibility (Zhou et al. 2015; 2016; Xie et al. 2019). Nevertheless, the precise environmental stimuli that regulate the activity of RhpSR have yet to be determined.

Another well-documented regulatory pathway, the calcium-induced TCS CvsSR can influence the T3SS genes (Fishman and Filiatrault 2019). The CvsSR expression was significantly accumulated in DC3000 when exposed to exudes derived from the leaves of tomatoes and by a calcium (Ca²⁺) cation (Fishman et al. 2018). The plant apoplast contains plenty of Ca²⁺ ions, and it goes on increasing during bacterial infection in plants (Stael et al. 2011). Moreover, the phosphorylated response regulator CvsR binds to the inverted repeat element of hrpR and hrpS and some other regions inside the T3SS effector genes in the presence of calcium supplementation in T3SS-inducing minimal media and activates T3SS expression. The strain impaired in cvsS/cvsR resulted in reduced proliferation and disease symptoms on host plants, suggesting that CvsSR has a direct role in the virulence of Pseudomonas strains (Fishman et al. 2018; Fishman and Filiatrault 2019). The CvsSR system has been found to indirectly inhibit the expression of AlgU, which was responsible for the positive regulation of the T3SS. The specific method by which CvsSR affects algU expression remains unknown, suggesting that the impact of CvsSR on T3SS dynamics may be intricate and multifaceted (Stael et al. 2011).

Inhibitors of the T3SS genes in pathogenic bacteria

T3SS inhibitors primarily consist of small molecular compounds that specifically interfere with the structural synthesis or functional expression of T3SS (Grier et al. 2010). Unlike traditional antibiotics, which target pathogen growth, T3SS inhibitors act specifically on the T3SS, significantly reducing the selective pressure driving pathogen resistance (Slepenkin et al. 2011). Through extensive research on T3SS structure, expression regulation, and virulence (Asif et al. 2024a), a variety of plant extracts and small molecule T3SS inhibitors are reported (Yang et al. 2008; Puigvert et al. 2021; Wang et al. 2020). Screening T3SS inhibitors on plant pathogens has yielded analogs of some inhibitors found in animal pathogens and novel plant-derived and chemically synthesized compounds (Slepenkin et al. 2011). Initial screening efforts were focused on animal pathogens such as P. aeruginosa and Yersinia species, and their efficacy was proven for other pathogenic strains (Kowal 2013; Aguilera-Herce et al. 2023).

In the search for effective inhibitors targeting the T3SS in plant pathogens, several promising compounds have been identified. Salicylidene acylhydrazides, for instance, inhibit T3SS gene expression in R. solanacearum, reducing its virulence (Puigvert et al. 2021). Phenolic compounds like caffeic acid, released by plant roots in response to pathogen attacks, have also shown efficacy in reducing disease symptoms by affecting T3SS regulation in R. solanacearum (Li et al. 2021). Coumarins, such as 7-methoxycoumarin, mitigate tobacco bacterial wilt caused by R. solanacearum through their impact on T3SS (Han et al. 2021). Additionally, salicylic acid has been shown to inhibit T3SS in R. solanacearum by repressing several virulence factors (Lowe-Power et al. 2016). Cyclohexanone derivatives interfere with T3SS in X. campestris, reducing pathogenicity, while quinazolinone derivatives prevent T3SS assembly in Xanthomonas species (Yang et al. 2017). Moreover, 2-imino-5-arylidene thiazolidinone inhibits T3SS in E. amylovora, a pathogen causing fire blight in apple and pear trees, by blocking the secretion of T3SS effectors (Anantharajah et al. 2017). These inhibitors offer promising leads for developing new control strategies against bacterial plant diseases by targeting T3SS, a critical virulence factor in many plant pathogens.

Further research is required to elucidate the mechanism of action of these compounds and explore their potential value for preventing and controlling animal and plant diseases. Different inhibitors have different targets (Table 1), but we focused on three categories: inhibiting T3SS regulatory factors genes, inhibiting T3SS structural genes, and inhibiting T3SS effector proteins.

Table 1 Reported chemicals involved in inhibiting type III secretion systems

T3SS regulatory factor inhibitors

Plants can produce various secondary metabolites to resist pathogen infection. Most of these metabolites are toxic to pathogenic bacteria (Wang et al. 2020). Sulforaphane (SFN) crude extracts was tested against DC3000 which damaged the expression of effector protein AvrPto, ultimately inhibits its T3SS expression (Table 1; Wang et al. 2020). Further analysis revealed that SFN directly acted on the 209 amino acids of HrpS in DC3000, and competitively bind to HrpS to suppress the expression of T3SS. N-dihydroxybenzenes were obtained by screening the interaction between E. coli AraC family regulatory factors (such as ExsA) and DNA binding regions. N-dihydroxybenzene binds to the C-terminal of P. aeruginosa ExsA, thereby preventing the binding of ExsA to the DNA promoter region. The biological activities of N-dihydroxybenzenes (Table 1) include reducing T3SS gene expression and T3SS-mediated in vitro virulence (Grier et al. 2010; Marsden et al. 2016).

T3SS regulatory pathway inhibitors

Most of the T3SS inhibitors are hydroxycinnamic acid and its derivatives. Using the hrpA promoter reporter vector (pHrpA) and GFP-gene, hydroxycinnamic acid compounds from plant metabolites were screened by flow cytometry. Among them, o-coumaric acid and p-coumaric acid reduce the expression of T3SS by inhibiting related genes in the HrpX-HrpY pathway in E. amylovora (Li et al. 2015; Fan et al. 2017), while 4-methyl cinnamic acid and trans-2-methyl cinnamic acid (Table 1) subdued HrpS-HrpL pathway in X. oryzae pv. oryzae and E. amylovora, respectively. They can also inhibit T3SS expression related to the HrpG linked to HrpX pathway in rice pathogen (Fan et al. 2017, 2019; Shi et al. 2023). Benzoic acid and ethyl-2-nitro-3-acrylates affect the HrpS-HrpL pathway in the Xanthomonas strain (Khokhani et al. 2013; Jiang et al. 2019).

Transcriptional and post-transcriptional pathway inhibitors affecting T3SS

Salicylidene acetylhydrazides (SA) compounds are the class of T3SS inhibitors widely reported, explored, and first identified against the pathogenic bacterium Y. pseudotuberculosis. It can help mice resist the infection of Y. pseudotuberculosis and EHEC. SA compounds directly inhibit the activity of three secondary metabolic synthase enzymes, WrbA, Tpx, and FolX, which indirectly affect the transcription of flagellar synthesis genes and T3SS regulatory genes, thereby exerting the function of inhibiting T3SS (Slepenkin et al. 2011; Anantharajah et al. 2017). Trans-4-hydroxycinnamic acid (TMCA), screened from plant phenols, inhibits GacSA, RsmYZ-RsmA, and ExsA mediated regulatory pathway (Table 1). These compounds induce and regulate the expression of small RNA genes rsmY and rsmZ, which compete for RsmA to inhibit the expression of ExsA in P. aeruginosa. Subsequent studies have found that TMCA can also inhibit plant pathogens D. dadantii, and the RsmB-RsmA-HrpL regulatory pathway in E. amylovora extends the inhibitory range of TMCA from animal pathogens to plant pathogens (Khokhani et al. 2013).

The needle/pilus structure and base protein formation inhibitors

The needle structure of T3SS is crucial for its functional performance, and the scale protein SctP can detect needle length during the synthesis process of needle-like structures. In plant pathogens, poorly developed needle-like structures can affect the pathogen’s contact with the host’s cytoplasm, thereby affecting the transport of effectors. The most direct ways to suppress needle-like structures include subunit polymerization, inducing incorrect subunit folding, physically hindering the binding between subunits, or altering subunit polymerization. SA compounds also show inhibitory effects on the T3SS of S. typhimurium, Shigella spp., EHEC, X. oryzae, and E. amylovora (Anantharajah et al. 2017; Fan et al. 2017; Tao et al. 2019). SA did not affect the expression level of needle-like structure synthesis subunits in T3SS, thus suggesting that the assembly of T3SS was affected. Subsequent observations under electron microscopy indicated that the number of T3SS injection devices per cell decreased by 30.40% after SA treatment to Shigella spp., and the number of poorly developed needle-like structures significantly increased. Therefore, SA is considered a class of inhibitors that affect the assembly of needle-like structures (Tao et al. 2019). The T3SS base attaches needle-like structures to bacterial cell membranes with two main parts: a lower ring connected to the inner membrane and an upper ring connected to the outer membrane through the periplasm. Due to the conserved nature of these base proteins across secretion systems and flagellar structures, targeting T3SS base proteins for inhibitors could lead to unintended effects (Abby and Rocha 2012; Diepold et al. 2015). Thiazolidinones, particularly 2-imino-5-arylidene thiazolidinones (Table 1), interact with SctC, inhibiting base proteins in S. typhimurium T3SS and reducing effector secretion (Felise et al. 2008). This inhibition is achieved by targeting the HBF1 homologous protein of SctC, affecting the effectors’ excretion, like SipA and SspH1, without impacting flagella motility (Kline et al. 2009). Considering their pedestal-like structure and conservation, HBF1 family proteins are promising targets for broad-spectrum T3SS inhibitors. The compound MBX2359, identified using a luxCDABE reporter system and the ExoT effector gene, and phenoxyacetamides discovered through screening over 80,000 compounds exemplify promising avenues for T3SS inhibitor development (Aiello et al. 2010).

Future prospects

Over the last 25 years since its discovery, significant studies have been undertaken to unravel the intricacies of the T3SS nanomachine. Despite the advancements, challenges and gaps in knowledge that exist, future research endeavours should prioritize unravelling the molecular mechanisms controlling the priority of secretion processes within a biological system, understanding the perception of secreted signals, and establishing a unified model for exploring the functions of effectors in relation to pathogenesis. Small genetic changes can fundamentally alter T3SS control, leading to diverse variations adapted to the specific lifestyles of different pathogens. Current research advancements highlight the incomplete understanding of T3SS regulation, emphasizing the need to explore molecular mechanisms, including regulatory RNAs, under different infection conditions. Additional fundamental questions left unanswered include: Is the concept of an effector network universally applicable among pathogens utilizing a T3SS? Future research directions in understanding bacterial pathogenesis could focus on elucidating the essential cellular processes targeted for sustained infection and identifying potential targets within these pathways. Investigating how molecular mechanisms identified in one bacterial strain extend to other strains, related species, or across bacterial families would provide insights into common virulence strategies and potential vulnerabilities. The study of post-transcriptional control elements, such as sensory and regulatory RNAs, and their adaptation to intrinsic changes and regulatory rewiring is particularly intriguing. Understanding how these elements contribute to strain-specific variations advantageous for bacteria in diverse host niches could unveil novel targets for intervention and offer strategies for combating bacterial infections effectively.

Certain other basic trending questions include: Can artificial intelligence be implied to predict the essential cellular processes regarding T3SS? What are the molecular targets of current T3SS inhibitors, and how do they interact with these targets? Can inhibitors’ specificity, potency, and efficacy be enhanced by leveraging our understanding of T3SS function? How quickly will resistance to T3SS inhibitors emerge, and can it be managed through dose optimization, combination therapy, or the development of multi-target molecules similar to antibiotic strategies? What are the cellular repercussions when T3SS is inhibited? Does inhibition impose fitness costs, disrupt other virulence factors, trigger stress responses, interfere with quorum sensing, or induce resistance mechanisms?

Conclusions

The T3SS protein transports nanomachines found in most Gram-negative bacterial pathogens, which transport effector proteins into the host cell cytoplasm, spanning the host cell membrane and causing infection. Many plant pathogens, like P. syringae, Ralstonia spp., and Xanthomonas spp., possess these T3SS apparatuses, and their associated diseases are the cause of severe economic losses. A wide diversity of the hosts is affected by the T3SS-possessing pathogen, and their T3SS apparatus and secretion mechanisms are remarkably conserved. The probable interaction of the pathogens with the host and environmental cues, particularly regulate the response of phytobacterial T3SS. Different studies found conflicting results regarding GacSA’s effect on T3SS expression, especially in P. syringae. Certain studies described it as a positive regulator, while others indicated it as a negative regulator. TCS-oriented complications can be resolved by species and strain-specific studies. In addition to pesticides and antibiotics, T3SS inhibitors precisely target the T3SS instead of disturbing pathogen growth and cause no risk for resistance development. Different inhibitors have different targets, especially inhibiting regulatory factors genes, damaging functionality of structural genes forming proteins, and suppressing effector protein secretion. These target sites could be useful to develop more novel drugs to inhibit T3SS.