Targeting Type III Secretion System using Salmonella: A Promising Candidate for Innovative Antibacterial Therapy.

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Learn more: PMC Disclaimer | PMC Copyright Notice Indian J Microbiol . 2025 May 16;65(2):898–912. doi: 10.1007/s12088-025-01486-9 Targeting Type III Secretion System using Salmonella : A Promising Candidate for Innovative Antibacterial Therapy Shreya 1 , Tarun Pant 1, # , Swadha Kailoo 1, # , Yatender Kumar 1, ✉ Author information Article notes Copyright and License information 1 Department of Biological Sciences and Engineering (BSE), Netaji Subhas University of Technology (NSUT), New Delhi, 110078 India ✉ Corresponding author. # Contributed equally. Received 2023 Apr 27; Accepted 2025 Apr 25; Issue date 2025 Jun. © Association of Microbiologists of India 2025. Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law. PMC Copyright notice PMCID: PMC12246286  PMID: 40655377 Abstract One of the most significant problems of the twenty-first century is the appearance and rapid evolution of multiple drug-resistant (MDR) bacterial strains. The emergence of resistance towards available drugs poses a growing threat to global public health. Unfortunately, developing new antibiotics is a tedious and difficult challenge, urging the research community to strive for unusual clinical targets with a different mode of action. The antivirulence strategy disarms pathogenic bacteria and attacks the virulence factors. Type III secretion system (T3SS) facilitates virulence in many pathogenic gram-negative bacteria. T3SS is considered an effective needle-syringe mechanism associated with humans, plants, and animal hosts to inject effectors inside the cytosol of host cells. This review focuses on T3SS, its assembly, and visualization, along with presently developed small molecules, peptides, and antibodies to inhibit the complex. Evaluation of the effectiveness of compounds is in progress on animal models, and promising studies are being conducted to develop antibodies and vaccines that target T3SS. Reports published till now specify that T3SS inhibitors can evolve into innovative antibacterial therapy, bringing about a revolution in the pharmaceutical world. Apart from this, to get a better understanding of the mechanism at the molecular level, the functioning of T3SS in Salmonella is also discussed. Keywords: Type III secretion system, Salmonella , Inhibitors, Typhoid, Virulence Introduction Bacterial ribosomes produce approximately 8000 distinct proteins. Nearly half of them are found in a membrane or secreted to the cell wall or the external environment [ 1 ]. Therefore, bacteria secrete many compounds like DNA, proteins and small molecules like enzymes and toxins to establish a network with the external world. To achieve this, they secrete numerous effectors in the extracellular membrane or into the other cells directly. Such a compound allows bacteria to enhance their virulence and antibiotic resistance, scavenge resources in an environmental niche, directly intoxicate target cells, and disrupt their essential tasks. For this purpose, bacteria need an established protein secretion system. The sec and the tat pathway are mainly for gram-positive bacteria with a single membrane. However, Gram-negative bacteria face the challenge of translocating these effector molecules from the cytoplasm across the cell envelope comprised of the inner membrane (IM), the peptidoglycan, and the outer membrane (OM). Therefore, there is a need for an energy-providing system. Many dedicated protein systems provide energy and a passage to secrete a variety of substrates. Bacteria enter the mammalian host through these mechanisms, destroy tissue sites, and inactivate the immune system’s response. These secretion systems are necessary for the growth and development of bacteria by manipulating host mechanisms and establishing their replicative niche. Till now, there are nine secretion systems, Type I to IX, named in the order of their discovery [ 2 ]. Bacterial species can express none, one or more types of secretion systems. Secretion occurs in two ways— 1. The one-step mechanism involves a direct transfer of effectors from the cytoplasm to the extracellular milieu. Example- Type I, III, IV, VI, and VII 2. The two-step mechanism involves the first transfer of effectors in the periplasm via the sec/tat pathway. From there, these are released in the extracellular space using the secretion system. Example- Type II, V, VIII, and IX. The secretion system is vital for virulence and has become an emerging target for the development of novel antibacterial therapeutics [ 3 ] . Most pathogenic gram-negative bacteria contain one or more Type III Secretion Systems. Type III Secretion System Type III secretion system (T3SS) is a protein transfer system commonly used by pathogenic gram-negative bacteria. Through this mechanism, bacteria directly transfer effector proteins into the cytoplasm of the eukaryotic cells. It is a 3.5 MD multi-ring structure embedded in the bacterial envelope [ 4 ] (Fig. 1 ). Fig. 1. Open in a new tab Different parts of the type III secretion system complex along with their proteins involved More than 20 proteins make up the whole structure. It is a sec-independent pathway. The first T3SS structure was identified in Salmonella typhimurium and subsequently identified and visualized in many other gram-negative bacteria with a highly conserved structure. T3SS are encoded either in plasmid or in genome or both. For example- Approximately 25 genes located on a large 230 kb plasmid encode Shigella sp. T3SS. This complex is present not only in pathogenic bacteria infecting animals but also in bacterial pathogens infecting plants as well as symbionts for plants or insects. Not only do these deliver proteins into eukaryotic cells, but they also deliver them at the correct location at the right time. Assembly of Injectisome One of the major central components of T3SS is the injectisome, which comprises several protein complexes. Analysis from advanced techniques such as X-ray crystallography and NMR visualization reveals the components of the T3SS complex, providing insights into its formation [ 5 – 7 ]. The injectisome covers two bacterial membranes and one eukaryotic membrane and consists of three different parts, including the extracellular segment, basal body, and cytoplasmic components. The extracellular components connect the bacterial outer membrane to the host plasma membrane, which consists of a needle and the translocator pore. The needle filament present within the injectisome is a key component as it helps in T3SS-mediated infection. The structural presentation of needle filament has exposed to extracellular space and thus appealing to be an essential drug target [ 8 ]. Assembly of injectisome occurs in two distinct phases. In phase I, the formation of the basal body takes place with the help of Sec machinery. After the development of the basal body, the T3SS-dependent phase occurs, which comprises the internal connection of the basal body, needle elongation, and pore formation [ 9 ]. Sec Dependent Phase Formation of Basal Body The basal body is a stack of protein annuli that spans the peptidoglycan layer and double membrane of bacteria. The basal body consists of membrane-inserted rings that firmly connect both the inner and outer membrane, organizing them in a tubular fashion [ 7 ]. The OM ring consists of the secretin protein SctC, which is a 12-m protein [ 10 ]. SctC forms two separate outer rings (OR1 and OR2), while SctD and SctJ together form the distal inner ring (IR1), and the cytoplasmic amino-terminus of SctD creates the innermost ring (IR2) (Fig. 1 ). The N-terminal of SctC extends deeply into the periplasm, forming a channel of 7 nm wide and a length of 16 nm [ 11 ]. Sometimes, pilotin, a small lipoprotein, is used for the oligomerization and stabilization of SctC [ 12 ]. MS ring (supramembrane and the membrane) is made up of inner and outer rings. The inner ring consists of SctJ, which is a 24-mer lipoprotein facing periplasm, whereas the outer ring consists of SctD, which is a 24-mer single transmembrane domain protein. The amino-terminal cytoplasmic domain of SctD is responsible for the formation of the innermost ring of the basal body. Overall, electrostatic interaction between SctJ-SctD stabilizes the MS ring [ 7 ]. Negatively charged SctJ pores act as an adaptor for inner rod protein SctI to anchor [ 10 ]. SctI is believed to produce a cylindrical “inner rod” structure within the central lumen of the basal body annuli, which could sustain the extracellular needle filament. Once the MS ring assembly has been completed, SctD connects both OM and IM via its periplasmic domain. It interacts with the periplasmic domain of SctC, and hence whole basal body structure is stabilized [ 7 , 13 ]. The flexible N-terminal domain of SctD remains in the cytoplasm, acting as a dock for the C-ring [ 14 ]. The export complex consists of five polytopic inner membrane proteins, namely SctR, SctS, SctT, SctV, and SctU. MS ring surrounds this complex. Using cryo-electron tomography and sub-tomogram averaging, in situ structures of Salmonella typhimurium T3SS were observed. It was found that the export apparatus components, SctR/S/T/U, form a funnel-shaped structure that connects to the T3SS needle on its wider end and crosses the inner membrane on its narrow side [ 15 ]. Moreover, Recent studies of SctR, SctS, and SctT complex of Shigella flexneri and needle complex of Salmonella typhimurium perceive similar localization of the complex at the periplasmic space [ 16 , 17 ]. T3SS Dependent Phase Needle Formation Along with Regulation of the Length On completion of basal body formation, the secretion of early substrate SctF and SctI starts. Self-oligomerization of SctF forms an extracellular needle. The needle gets attached to the basal body via SctI, an inner rod protein, and stretches to the external milieu to a length of 80 nm-2 µm [ 18 ]. The foundation of the growing needle is carried by SctI, which is present inside the SctJ channel and forms a cylindrical duct upon oligomerization [ 19 ]. In the end, tip protein gets attached. The tip protein is organized to the top of the needle in pentamer form. The length of the needle is different in different bacteria, and SctP (molecular ruler) controls the length. SctP is present in a partially unfolded state and measures needle length [ 20 ]. The height of the needle is proportional to the extended length of the polypeptide of SctP in each organism [ 20 ]. Translocator Pore Formation Once needle formation is complete, translocator proteins are attached. SctE and SctB are mid-substrates that get secreted and anchored to SctA. SctB and SctE get embedded in the host plasma membrane, where they oligomerize to form a pore [ 21 ]. Four residues of SctE and one of SctB form an internal pore of 10 nm through which the effector’s secretion occurs. C-Ring and ATPase Complex Assembly C-ring consists of a peripheral membrane-linked protein SctQ. SctQ makes a 30 nm circular wide structure [ 14 ]. Upon phosphorylation of SctQ, the C-ring gets embedded into the MS ring placed on the IM base, which is constituted by SctD’s N-terminal cytoplasmic domain. The C-ring behaves as a holder for the ATPase complex [ 22 ]. The SctN ATPase is homologous to F 1 F 0 -ATPase [ 23 ]. SctN stably gets embedded into the membrane through elongated alpha-helical SctL stator and SctO stalk, both connected to SctV’s cytoplasmic domain. Analysis from EM suggests that at the membrane level, six SctL stator units interconnect with SctQ pods, which connect to ATPase [ 24 ]. As the sec-dependant phase gets finished, a highly regulated T3SS-dependent phase comes into play. Switching of Secretion The secretion of protein through T3SS occurs in a back-to-back process, and the switching mechanism determines the secretion hierarchy. The first regulatory switch proposed is SctU. Once the needle is in position by the secretion of SctF, the T3SS complex is all set to secrete all other proteins. The autoproteolysis of the SctU cytoplasmic domain is the signal behind the conformational changes [ 25 ]. Due to this change, alteration occurs in the electrostatic surface of the export apparatus. Gatekeeper SctW mediates the switching of the substrate [ 26 ]. SctW gets positioned at the export apparatus or the C-ring [ 27 ]. It associates with the translocators, sorts them, and encourages preferential secretion. SctW in the cytoplasm blocks the chaperone-effector complexes and prevents their entry into the translocation pore [ 28 ]. Completion of pore formation results in alternation in local potential by the ion flow to the bacterial cytoplasm, leading to SctW detachment from the membrane, switching to the secretion of the late substrate. Visualization and Isolation of Needle Complexes An attempt was made through Cryo-Electron Tomography to visualize the needle complex and isolate them to get detailed knowledge of the proteins involved in it. The amino acid sequence of the target shares homology with the bacterial flagellar export system protein sequences, which signifies that the T3SS of Gram-negative bacteria may have developed from genes related to flagellar export systems [ 29 ]. Kubori et al. [ 4 ] successfully isolated the T3SS complex from S.typhimurium (Fig. 2 ), and further analysis was done by electron microscopy, which demonstrated that the T3SS consists of two crucial parts; one is the needle, which is approximately 80 nm in length. Fig. 2. Open in a new tab A S. typhimurium minicells interacting with the plasma membrane of HeLa cells. Scale bar-200 nm. B High resolution of Cryo-Electron Tomography of Fig. 2A Second is the basal body, approximately 40 nm in width. Like the basal flagellar body, T3SS also contains two sets of rings- upper and lower rings. T3SS structure became more apparent from the osmotically shocked S. typhimurium cells. Electron microscopy showed that the basal body is within the membrane, transversing the outer and inner cell membranes [ 4 ]. The CsCl density gradient method was used to isolate needle complexes. This research gave the limelight to search for the T3SS machinery in other pathogenic gram-negative bacteria. A similar study by Blocker et al. [ 30 ] showed the T3SS complex of S. flexneri 5 M90T strain (Fig. 3 ). Fig. 3. Open in a new tab Electron micrograph of osmotically shocked Shigella M94 cells (open arrow indicates the T3SS complex) Scale bar-100 nm This research provided the supramolecular structure of the needle. It provided a clear interpretation of the host’s contact with the bacteria, which will eventually lead to the growth of the targeted vaccine. Targets of T3SS Management of bacterial infection is complex due to the rise in antibiotic resistance levels and an inadequate pipeline for drug development. Processes that are important for bacterial growth and survival, like DNA replication, protein synthesis, and biosynthesis of the cell wall, are some of the critical targets for classical antibiotics to act. Alternative strategies focus on targeting virulence pathways. There are several virulence factors that play a crucial role in a broad range of pathogenic mechanisms, including systemic infection, adhesion, fimbrial expression, invasion, antibiotic resistance, intracellular survival, magnesium and iron uptake, and toxin production. Most of these factors are primarily located in Salmonella pathogenicity islands (SPIs) [ 31 ] T3SS machinery is an interesting target responsible for the translocation of virulence proteins into the lumen of eukaryotic cells. So, it can be stated that anti-virulence blockers can serve as alternatives to antibiotics. Till now, there is no antibiotic available in the market that inhibits the T3SS machinery explicitly. Identification and characterization of T3SS inhibitors have taken place in recent years, but there is no evidence for their commercial use. Recently, Harmine, Quercitrin, Terazosin, Atenolol, and Prazosin have been developed as efficient T3SS inhibitors that are one of the most promising compounds for Salmonella infection treatments [ 32 – 36 ]. The whole complex of T3SS consists of many proteins, so disruption of any aspect can abolish its pathogenicity. Due to this reason, T3SS acts as a potential anti-virulence target. Turn off the Action of ATPase The ATPase provides energy to the secretion of effector protein, performs several cellular functions, and is responsible for different human diseases. So, they act as an attractive target for drugs. Bacterial strains lacking this ATPase cannot secrete T3SS substrates and are significantly diminished in vivo, providing a clear-cut idea to be a useful target to limit T3SS. Many ATPase inhibitors are already present in the market, for example, Prilosec (omeprazole) and Cipro (ciprofloxacin) [ 37 ]. There are many ATPase inhibitors in clinical trials, like XR9576 [ 38 ]. To prevent gastric ulcers, an H + -ATPase inhibitor is used [ 37 ] (Fig. 4 a). Fig. 4. Open in a new tab Potential targeting strategies of the T3SS complex Target the Basal Body The basal body comprises inner and outer rings that are embedded in the cell membrane of gram-negative bacteria. 2-imino-5-arylidene thiazolidinone TTS29 targets the basal body. The outer membrane ring of the basal body consists of secretin that oligomerizes and leads to pore formation. Type II and IV secretion systems also contain secretin, which was also inhibited by TTS29 treatment. It was, therefore, figured out that TTS29 disrupted secretin components [ 39 ] (Fig. 4 b). Target T3SS Needle Apparatus The needle apparatus comprises approx. Twenty-five different proteins assemble to facilitate effector secretion inside the host cells. There are two ways to target the needle apparatus: first, it could block protein–protein interactions, and second, it could prevent the interaction between tip-translocons (Fig. 4 c). Target the Tip Complex and Translocon At the end of the T3SS needle, the tip complex assembles. On host cell contact, the formation of a translocon pore takes place, and the subsequent transfer of the effector takes place. The blockage of TC prevents contact between host cells, making it an outstanding molecular target. In the literature study, the relative invasion was studied; it was seen that Caco-2 cells with no serum showed 100% invasion, and when antibodies against SipD were used, there was a significant decrease in the invasion level [ 40 ]. Antibodies against LcrV ( Yersinia sp. ) and PcrV ( Pseudomonas sp. ) prevent hemolysis in the erythrocyte membrane by inhibiting translocon formation. Administration of anti-LcrV protects mice from Y. pestis infection as well as bubonic plague and pneumonia [ 41 ]. Several single-domain antibodies from camelid species were used against IpaD ( Shigella sp. ) to inhibit pore formation [ 42 ]. Thus, the tip complex is an essential target in blocking host–pathogen contact (Fig. 4 d, e). Chaperones and Effectors T3SS plays a significant role in pathogenicity by secreting T3ES (Type III Effectors), which overthrow the defense mechanism of the host [ 43 ]. T3SS effectors have a broad spectrum of functions. The deletion of effectors diminishes the virulence so they can act as an essential drug target [ 44 ]. To prevent the secretion of effector protein, disruption of chaperone and effector interaction can be done. However, there is no such positive result that supports this mode of target strategy (Fig. 4 f, g). T3SS Gene Transcription T3SS is organized either in plasmids or in the genome, and transcription factors tightly regulate their gene expression. Sukhan et al. [ 9 ] demonstrated that deleting T3SS transcription factors leads to the disappearance of needle apparatus on the bacterial surface, as most of the essential constituents are not being expressed (Fig. 4 h). The Literature of T3SS Inhibitors Salicylidene Acylhydrazides The first class of small molecule used as a T3SS inhibitor is Salicylidene acyl hydrazides. Through luciferase reporter gene assay in viable Yersinia pseudotuberculosis , Kauppi et al. [ 45 ] used a reporter of effector protein YopE combined with luciferase in Yersinia through a whole-cell assay. Screening of 9400 compounds resulted in several salicylidene acyl hydrazide derivatives, such as INP0007 and INP0010. At low micromolar concentrations, these derivatives significantly suppressed the YopE promoter-reporter gene signal and secretion of effector protein without hampering bacterial growth. INP0007 blocked T3SS secretion in Yersinia , and no invasion was seen in HeLa cells by bacteria. Gu et al. [ 46 ] incubated these uninfected HeLa cells with INP0007 (50 µM) and showed reduced cytotoxic reactions in cells caused by YopE. Keyser et al. [ 47 ] showed that INP0010 blocked Yops microinjection into the target cell’s cytosol and inhibited Chlamydia replication in Hep-2 cells. INP0400 inhibited Chlamydia trachomatis intracellular replication and infectivity. Thiazolidinediones Thiazolidinones are another class of T3SS inhibitors. Felise et al. [ 39 ] used a reporter construct of the effector SipA fused to Yersinia phospholipase YplA to identify thiazolidinone from a whole-cell screen assay against protein secretion in Salmonella . The reporter construct’s cleavage of PED6 resulted in measurable fluorescence proportional to the secreted SipA amount. Screening of 92,000 compounds resulted in the identification of 2-imino-5-arylidene thiazolidinone as a promising candidate [ 48 ]. It also inhibits the T3SS of Yersinia , T2SS in Pseudomonas , and T4SS of Francisella. Benzimidazoles N-hydroxybenzimidazole derivatives disrupt the ability and work of DNA-binding MAR proteins in several organisms and could thus be successful T3SS inhibitors. Kim et al. [ 49 ] synthesized a new T3SS inhibitor N-hydroxybenzotriazole derivative, and using a primary cell-free LcrF-DNA binding assay, they found this compound as a potent inhibitor of LcrF and prevention of T3SS-mediated cytotoxicity during Y. pseudotuberculosis. Octaketides Cytosporone is a fungal metabolite. It comes under octahedrite antibiotics. Csn-B and their derivatives showed an inhibitory effect on S. typhimurium . Csn-B exposure decreased SPI-1-encoded effector T3SS without major bacterial toxicity and prevented the bacterial invasion of cultured epithelial cells by SPI-1-media. On further analysis, Li et al. [ 50 ] showed that Csn-B probably inhibited T3SS by increasing the transcription of HNA and hns , two proteins that negatively control the transcription of the main SPI-1 T3SS regulator HilA. Compounds in the Human Body Many substances suppress T3SS in the human body. Ochoa and Clearly [ 51 ] , in their study, showed that lactoferrin, part of the innate human defense, binds and proteolytically cleaves the EPEC translocator protein EspB and inhibits T3SS. Polyol Products A complex glycolipid, caminoside A, was isolated from the marine sponge Caminus sphaeroconia [ 52 ]. Caminoside A showed significant results by inhibiting the T3SS expression with an IC 50 value of 20 µM without harming Enteropathogenic E. coli . Caminosides B, C, and D (IC 50 = 20 µM) show inhibitory activity against EPEC T3SS. Isolation of a linear polyketide aurodox from Streptomyces sp. hindered the EPEC effectors and their translocation but showed no effect on bacterial growth [ 53 ]. Aurodox administration protected mice from a lethal dose of C. rodentium , a model bacterium for human pathogens, such as EPEC and T3SS-mediated colonic hyperplasia. Table 1 depicts the small molecule and its target, and Table 2 represents antibodies and vaccines against T3SS. Table 1. Small molecule and their target Compound Organism Function Affects bacterial growth? References SAH C1-INP0007 SAH C11-INPO403 Salmonella enterica Inhibition of secretion and invasion No [ 48 ] C20 Y. pseudotuberculosis P.aeruginosa Inhibition of translocation No [ 54 ] Thiazolidinones S.enterica P.aeruginosa Yersinia Pseudomonas Inhibition of transcription and secretion, as well as minimizing needle complex formation No [ 39 ] WEN05-03 EPEC Inhibition of ATP hydrolysis, Reduction of toxicity in infected HeLa cells No [ 55 ] Obovtal S.enterica Minimized hemolysis of red blood cells of sheep No [ 56 ] Quercitrin Salmonella typhimurium T3SS Inhibits the effector SipA translocation to eukaryotic host cells of S. typhimurium T3SS No [ 34 ] Terazosin Salmonella typhimurium Reduce biofilm formation and adhesion of S. typhimurium No [ 33 ] Atenolol Salmonella typhimurium Diminish the biofilm formation of S. typhimurium No [ 36 ] Prazosin Salmonella enterica It affects the expression of qseC, sdiA, qseE, and T3SS encoding genes and reduces biofilm formation No [ 32 ] Fisetin Salmonella typhimurium Hinder the interaction of the transcriptional regulator HilD with its respective promoters. Thus, regulates the expression of T3SS No [ 57 ] 6-amido-3-carboxy pyridazine Salmonella typhimurium Inhibit secretion of T3SS effectors No [ 58 ] Open in a new tab Table 2. Antibodies and vaccines against T3SS components Class Organism Target Function Result References MEDI3902 (Bispecific Antibody) P.aeruginosa PcrV-tip PsI-exopolysaccharides In vivo protection from acute pneumonia model in mice Presently in clinical trial phase II [ 59 ] Single VHH Domain Antibodies Shigella flexneri IpaD-tip A decrease in hemolysis of red blood cells in sheep – [ 42 ] Vaccine S.enterica PrgI-needle, SipD-tip Prevents mice from infection – [ 60 ] Polypeptide S.enterica, S.flexneri SipB, IpaB-translocon Prevent the invasion of bacteria into host cells Not used for therapeutic purposes because polypeptide size is too large [ 61 ] Peptides EHEC Citrobacter rodentium Esp A-tip Prevent mice from colon damage after the C. rodentium challenge – [ 62 ] Open in a new tab Fusaric Acid Fusarium oxysporum, a common fungus present in the soil, is responsible for constructing a toxin called Fusaric acid [ 63 ]. Analysis of SDS-PAGE and western blot shows that effector proteins produced from SPI-1 were inhibited when treated with 100 μm of fusaric acid, while no effect on cell growth was observed. Also, Fusaric acid significantly reduces infection of Salmonella in Hela cells without causing harm to cells. Thymol Deng’s lab discovered that thymol can inhibit T3SS [ 64 ] . Thymol is found in plants of the genus Thymus [ 65 ] . Thymol was shown to have an inhibitory concentration of 0.2 mm, whereas 0.6 mm was found to be harmful to Hela cells. In the gentamycin protection experiment, 90% depletion of Salmonella infection was observed while treated with 0.2 mm thymol. When mice were treated with 50 mg/kg of thymol along with the lethal dose of Salmonella , the survival rate observed was 100% [ 66 ]. Sanguinarine Chloride Sanguinarine chloride is produced by Sanguinaria canadensis (bloodroot plant) [ 67 ]. After observation of the anti-inflammatory properties of Sanguinarine chloride, a study was performed against gingivitis [ 68 ] . It was detected that T3SS in Salmonella is inhibited by Sanguinarine chloride [ 64 ] . Myricetin Myricetin acts on SPI-1 of Salmonella typhimurium and prevents HeLa cells from Salmonella infection. Myricetin also protects infected mice from demise. It is responsible for inhibiting the action of effector proteins SipA and SipB and thus inhibits the virulency caused by Salmonella [ 69 ] . Cinnamaldehyde Cinnamaldehyde was found as an SPI-1 inhibitor in this study, which inhibits the release of many SPI-1-associated effector proteins and, as a result, inhibits SPI-1-mediated invasion of HeLa cells. Further research demonstrated that cinnamaldehyde inhibited the transcription of some SPI-1 genes present in S. typhimurium , including SipA and SipB, via impacting multiple SPI-1 regulator genes [ 70 ] . To get a better understanding of the mechanism of T3SS and its functioning, we took Salmonella as our model organism, as this exhibits two T3SS systems during its course of infection. Salmonella sp. -Model Organism for T3SS Salmonella enterica is a facultative gram-negative enteropathogenic, anaerobic bacteria with a dimension of 0.3–1.5 μm by 1.0–2.5 μm, sharing homolog with Escherichia , Yersinia, and Shigella . It has a circular chromosome with 4.7Mbp and a GC content of 52%. Six subspecies are present under Salmonella : salamae, diarizonae, houtenane, arizonae, indica, and enteric . Not every Salmonella causes serious health consequences, as there is significant genetic diversity among 2600 serovars within the genus. Numerous genetic factors that control virulence are linked to this variation between Salmonella serovars. Factors governing the type of disease include serovar of the infecting Salmonella , species, and host’s immunological status. Salmonella entrica infects a wide variety of hosts, including cold-blooded vertebrates (lizards and turtles) and warm-blooded animals (chickens and humans). For association, Salmonella has developed an extraordinarily excellent mechanism to infect vertebrate hosts. At the cellular and molecular level, this functional attachment exhibits characteristics like sensing the host’s environment, engaging the innate immune system, and modifying cellular processes like actin cytoskeleton and intracellular cell trafficking. Salmonella enterica contains two T3SS systems that are important for pathogenicity in distinct regions of its chromosome (pathogenic islands). T3SS1 is present at centisome 63 of the chromosome within Pathogenicity Island 1 (SPI-1) [ 71 ], and T3SS2 is present at centisome 31 within pathogenicity island 2 (SPI-2) [ 72 ]. These systems were most probably inherited through horizontal gene transfer, as indicated by their G + C content, which deviates significantly from the rest of the bacterial chromosome. Both systems are independent of one other and express at different infection levels. SPI-1 is required for initial interaction with the intestinal epithelial cells, while SPI-2 is essential for systemic infection. Expression of SPI-2 comes into play once the bacteria have gained proper control over host cells. Because of the ease of genetic modification and the existence of biological assays, Salmonella is an ideal model species for T3SS study. Salmonella Pathogenicity Island-1 Galan and Curtiss [ 73 ] identified Salmonella T3SS encoded in SPI-1 during a lookup for the Salmonella genes associated with bacterial entry into non-phagocytic cells. Further analysis showed that this system is present within pathogenicity islands [ 74 ]. The regulators help the bacteria detect the perfect environmental conditions for the proper invasion and expression of SPI-1 genes. SPI-1 conditions mimic the host intestinal lumen environment, like low oxygen and high osmolarity. Thirteen proteins secretion is governed by SPI-1 T3SS, namely as AvrA, SipA, SipB, SipC, SipD, SlrP, SopA, SopB/SigD, SopD, SopE, SopE2, SptP, and SspH1. SpaO, InvJ, PrgJ, and PrgI form the needle complex and mediate protein secretion. SipB, SipC, and SipD mediate protein translocation across the eukaryotic cell membrane along with additional functions. The rest of the proteins carry out effector functions inside the host and are hence termed effector proteins. The needle complex encodes in prg/org and inv/spa operons, while the effector, along with the translocon, encodes in the sic/sip operon. Upon contact with the epithelial surface, 10–100 assembled T3SS come into play to release a pool of soluble effector molecules [ 4 ]. Recent studies on real-time imaging have shown that the stored pools of two, SipA and SopE, are completely translocated within 80–200 s [ 75 ]. Translocation pore mediates transfer within the target plasma, which consists of SipB and SipC. Salmonella enterica usually penetrates the non-phagocytic cell-like intestinal epithelium [ 76 ] (Fig. 5 ). Fig. 5. Open in a new tab Schematic diagram depicting infection in epithelial cells of the lower intestine. a On entering the distal intestinal lumen, Salmonella moves across M cells of Peyer’s Patch and invades epithelial cells by secreting effector proteins through SPI-1, b Once the epithelial barrier passes, Salmonella infects macrophages and secretes SPI-2 effectors, c Proliferation takes place inside the SCV, d Salmonella escapes as macrophages undergo apoptosis and reinvade epithelial cells, harming host innate immunity Penetrance is an essential step towards pathogenicity because it helps bacteria to stay away from the defense mechanisms of the host, break the intestinal epithelium to infect inner tissues, alter gene expression, and invade the host cell to establish a niche to facilitate its proliferation and replication. Takeuchi [ 76 ] reported the activities involving the internalization of Salmonella into an infected animal’s intestinal epithelial cells. He found a remarkable change in the plasma membrane on reaching the close vicinity of the brush border of the intestinal epithelium, which results in microvilli disruption. Immediately, Salmonella could be seen in a membrane-bound pocket inside the intestinal epithelial cells. The following changes are reversible. Therefore, despite the existence of many internalized bacteria, the brush boundary of the intestinal epithelium has regained its natural structure. The differences observed are now understood to be the result of actin cytoskeleton rearrangements induced by Salmonella at the point of contact with bacteria-host cells regulated by SPI-1 effectors (Fig. 6 ). Fig. 6. Open in a new tab Invasion of eukaryotic cells in the T3SS1-dependant manner. T3SS1 effectors SipA, SipC, and SopB cause Salmonella internalization into host cells, causing the ruffling of the membrane at the bacteria entry site ( a , b ). SopE/SopE2 activates acts as GEF and activates Cdc42, RhoG, Rac1 ( c ). Host factors mediate the remodeling of actin. SptP reverts the process ( d ) Salmonella Pathogenicity Island-2 (SCV Formation) Salmonella entrance into host cells leads to the development of membrane-bound containing internalized bacteria called SCVs ( Salmonella Containing Vacuole). After exposure to the host cell, Salmonella releases effector proteins. After induced internalization, Salmonella exists in a vacuolar-shaped compartment termed SCV [ 77 ]. SPI-2 is present in a 40-kb pathogenicity island directly adjacent to a tRNA gene at centisome 31 of the Salmonella chromosome. SPI-2 shows its presence as soon as the bacteria enter the macrophages or epithelial cells, and it is expressed continuously throughout the whole process of infection. The conditions for expression of SPI-2 include those that are similar to the intracellular environment like low osmolarity, increase in acid content, and low calcium level. Shea et al. [ 78 ]showed mutations in SPI-2 T3SS, which resulted in the attenuation of Salmonella virulence in an infected mouse model independent of the inoculation route. SPI-2 is separated into two parts. The minor part includes the ttrRSBCA operon regulating tetrathionate reduction, and seven open reading frames (ORFs) with unknown functions are present. The larger part of the island helps Salmonella to sustain within the host cell in the SCV. SPI-2-mediated events are carried out by the T3SS-2 effector protein. SPI-2 encodes four types of genes necessary for virulence: ssr (encodes regulator), ssa (genes for T3SS-2 apparatus) , ssc (encodes chaperons), and sse (encodes effectors) [ 79 ]. SCV biogenesis and maturation lead to the formation of various types of tubules that emerge and attach to SCV. During the SCV maturation, the generation of various types of the tubular network takes place, for example, Salmonella- induced filaments (SIFs), Salmonella -induced secretory carrier membrane protein 3 (SCAMP3), Salmonella -induced secretory tubules (SISTs), and LAMP1-negative tubules (LNTs). In culture models, matured SCV becomes a replicative niche for Salmonella . SCV serves as a potential source of nutrients for the pathogen (Fig. 7 ). Fig. 7. Open in a new tab Biogenesis of SCV. Inside the host cell, Salmonella forms a modified phagosome known as SCV. The initial step in SCV formation (0-1 h) is regulated by SopB and SptP (T3SS1 effectors) along with the formation of tubules (SVATs and SNX3). During the intermediate phase (1-4 h), the SCV movement takes place to a juxtanuclear position. A large number of effectors are involved during the final stage of SCV maturation and maintenance. Replication starts 4-6 h postinvasion, and simultaneously formation of tubules (SIFs, SISTs, and LNTs) starts. In the maturation phase, the bacteria interact with host endocytic and secretory pathways Conclusion T3SS is an essential mechanism in several human pathogens that have become resistant to antibiotics. Therefore, it is a potent therapeutic target. Work on T3SS inhibitors aims at finding safe, reliable therapies that disable T3SS and nullify these pathogens without causing resistance. Since the T3SS is not necessary for growth, resistance through high selective pressure is negligible. T3SS virulence blockers would not interfere with the host microbiome, which is essential for human health and pathogen colonization, so, as a whole, T3SS virulence blockers can treat many human diseases caused by Salmonella sp., enterohemorrhagic E. coli , etc . Ideally, T3SS virulence blockers would be useful if given before infection, as prophylaxis, and during infection as a therapeutic. Nonetheless, most studies have tested the effectiveness of inhibitors when applied to bacteria or cultured mammalian cells before infection. Future work should check T3SS inhibitors during infection. The therapeutic use of small molecule bacterial virulence inhibitors as an anti-infective need further investigation. Anti-infective agents will not affect bacterial growth, so the removal of bacteria occurs by other means, such as the immune system of the host. These agents can enhance the memory of the immune cells. Antivirulence drugs, in combination with antibiotics, help to clear off bacteria from the body. The identification and validation of small molecule inhibitors targeting T3SS indicate that it is a promising strategy that will be applied to future clinical settings. Many laboratories are extensively working to discover new T3SS inhibitors as potential anti-infective agents. Still, there is a long way to go before these inhibitors can serve as successful therapies. One major problem is that the specific mechanisms of action remain unclear. Minimal potency, with fewer IC 50 values in micromolar concentrations, i.e., single digits, are significant drawbacks of current small-molecule T3SS inhibitors. The first successful discovery was the S. typhimurium needle atomic structure, and the second significant advancement will be the complete structural interpretation of all T3SS structural proteins, effector proteins, and chaperones. If this happens, then there will be specific goals for T3SS inhibitors within the scope, which will help to develop small-molecule T3SS inhibitors with greater ease, which will be more precise and effective. Acknowledgement Authors acknowledge the Department of Biological Sciences and Engineering (BSE), Netaji Subhas University of Technology (NSUT), New Delhi-110078, INDIA for technical support. Abbreviations T3SS Type III secretion system SPI-1 Salmonella pathogenicity island 1 SPI-2 Salmonella pathogenicity island 2 SCV Salmonella containing vacuole EHEC Enterohemorrhagic E. coli EPEC Enteropathogenic E. coli SIFs Salmonella- induced filaments SCAMP3 Salmonella -induced secretory carrier membrane protein 3 SISTs Salmonella -induced secretory tubules LNTs LAMP1-negative tubules SVATs Spacious vacuoles associated tubules SNX1 Sorting nexin-1 SNX3 Sorting nexin-3 PI(4,5)P 2 Phosphatidylinositol 4,5-bisphosphate PI3P Phosphatidylinositol 3-phosphate Author Contributions Writing-original draft preparation, Shreya, T.P, S.K.; review and editing, Y.K..; supervision, Y.K. “I certify that the above information is true and correct. All the authors contributed to the study of the manuscript. If the manuscript is accepted for publication, I agree to transfer all copyright ownership of the manuscript to the World Journal of Microbiology and Biotechnology, which covers the rights to use, reproduce, or distribute the article. Funding No funding was received by the authors for the project. Declarations Conflict of interest I, as the corresponding author, declare, on behalf of all authors of the paper, that no financial conflict of interest exists about the work described. Consent for Publications I, as the corresponding author, declare that the results/data/figures in this manuscript have not been published, nor are they under consideration for publication elsewhere. Footnotes Publisher's Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Tarun Pant and Swadha Kailoo have contributed equally to this work. References 1. 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