T3SS Inhibitor Research Articles

Design and Synthesis of 6-amido-3-carboxypyridazine Derivatives as Potent T3SS Inhibitors of Salmonella enterica Serovar Typhimurium.

Salmonella enterica (S. enterica) serovar Typhimurium, an anaerobic enteric pathogene, could cause human and animal diseases ranging from mild gastroenteritis to whole body serious infections. The goal of this paper was to synthesize new 6-amido-3-carboxypyridazine derivatives with different lengths of side chains with the aim of getting potent antibacterial agents. Synthesized compounds were analyzed by analytical techniques, such as 1H NMR, 13C NMR spectra, and mass spectrometry. We designed a series of novel 6-amido-3-carboxypyridazines using FA as the lead compound with the scaffold hopping strategy and their inhibitory activity against the effectors of type III secretion system (T3SS) using SDS-PAGE and western blot analysis for two rounds. Also, the preliminary mechanism of action of this series of compounds on T3SS was performed using real-time qPCR. Nine 6-amido-3-carboxypyridazines was synthesized. The inhibitory activities evaluated showed that compound 2i was the most potent T3SS inhibitor, which demonstrated potent inhibitory activities on the secretion of the T3SS SPI-1 effectors in a dose-dependent manner. The transcription of SPI-1 may be affected by compound 2i through the SicA/InvF regulatory pathway. The novel synthetic 6-amido-3-carboxypyridazines could act as potent leads for the development of novel antibacterial agents.

Design and Synthesis of Aryl Amide Derivatives Containing Thiazole as Type III Secretion System Inhibitors against Pseudomonas aeruginosa.

To identify potent inhibitors of the type III secretion system (T3SS) in the foodborne pathogen Pseudomonas aeruginosa, we synthesized 35 thiazole-containing aryl amides by merging salicylic acid with various heterocycles through active splicing. Screening for exoS promoter activity led to the discovery of a highly effective T3SS inhibitor from these 35 compounds. Through subsequent experiments, it was confirmed that compound II-22 specifically targeted the T3SS of P. aeruginosa. Additionally, compound II-22 inhibited the secretion of the effector protein ExoS by modulating the CyaB-cAMP/Vfr-ExsA and ExsCED-ExsA regulatory pathways. Furthermore, compound II-22 suppressed the transcription of genes involved in the needle complex assembly, leading to reduced bacterial virulence. Further validation through inoculation tests using Galleria mellonella larvae demonstrated the strong in vivo efficacy of compound II-22. The study also revealed that compound II-22 enhanced the bactericidal activity of antibiotics, such as CIP (ciprofloxacin) and TOB (tobramycin). These results could help develop novel antimicrobial drugs to reduce bacterial resistance.

PurA is the main target of aurodox, a type III secretion system inhibitor

Anti-microbial resistance (AMR) is one of the greatest threats to global health. The continual battle between the emergence of AMR and the development of drugs will be extremely difficult to stop as long as traditional anti-biotic approaches are taken. In order to overcome this impasse, we here focused on the type III secretion system (T3SS), which is highly conserved in many Gram-negative pathogenic bacteria. The T3SS is known to be indispensable in establishing disease processes but not essential for pathogen survival. Therefore, T3SS inhibitors may be innovative anti-infective agents that could dramatically reduce the evolutionary selective pressure on strains resistant to treatment. Based on this concept, we previously identified a polyketide natural product, aurodox (AD), as a specific T3SS inhibitor using our original screening system. However, despite its promise as a unique anti-infective drug of AD, the molecular target of AD has remained unclear. In this paper, using an innovative chemistry and genetic biology-based approach, we show that AD binds to adenylosuccinate synthase (PurA), which suppresses the production of the secreted proteins from T3SS, resulting in the expression of bacterial virulence both in vitro and in vivo experiments. Our findings illuminate the potential of PurA as a target of anti-infective drugs and vaccination and could open a avenue for application of PurA in the regulation of T3SS.

Erwinia amylovora Type III Secretion System Inhibitors Reduce Fire Blight Infection Under Field Conditions.

Fire blight, caused by Erwinia amylovora, is an economically important disease in apples and pears worldwide. This pathogen relies on the type III secretion system (T3SS) to cause disease. Compounds that inhibit the function of the T3SS (T3SS inhibitors) have emerged as alternative strategies for bacterial plant disease management, as they block bacterial virulence without affecting growth, unlike traditional antibiotics. In this study, we investigated the mode of action of a T3SS inhibitor named TS108, a plant phenolic acid derivative, in E. amylovora. We showed that adding TS108 to an in vitro culture of E. amylovora repressed the expression of several T3SS regulon genes, including the master regulator gene hrpL. Further studies demonstrated that TS108 negatively regulates CsrB, a global regulatory small RNA, at the posttranscriptional level, resulting in a repression of hrpS, which encodes a key activator of hrpL. Additionally, TS108 has no impact on the expression of T3SS in Dickeya dadantii or Pseudomonas aeruginosa, suggesting that its inhibition of the E. amylovora T3SS is likely species specific. To better evaluate the performance of T3SS inhibitors in fire blight management, we conducted five independent field experiments in four states (Michigan, New York, Oregon, and Connecticut) from 2015 to 2022 and observed reductions in blossom blight incidence as high as 96.7% compared with untreated trees. In summary, the T3SS inhibitors exhibited good efficacy against fire blight.

Derivative of cinnamic acid inhibits T3SS of Xanthomonas oryzae pv. oryzae through the HrpG-HrpX regulatory cascade

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv. oryzae (Xoo) has a significant impact on rice yield and quality worldwide. Traditionally, bactericide application has been commonly used to control this devastating disease. However, the overuse of fungicides has led to a number of problems such as the development of resistance and environmental pollution. Therefore, the development of new methods and approaches for disease control are still urgent. In this paper, a series of cinnamic acid derivatives were designed and synthesized, and three novel T3SS inhibitors A10, A12 and A20 were discovered. Novel T3SS inhibitors A10, A12 and A20 significantly inhibited the hpa1 promoter activity without affecting Xoo growth. Further studies revealed that the title compounds A10, A12 and A20 significantly impaired hypersensitivity in non-host plant tobacco leaves, while applications on rice significantly reduced symptoms of bacterial leaf blight. RT-PCR showed that compound A20 inhibited the expression of T3SS-related genes. In summary, this work exemplifies the potential of the title compound as an inhibitor of T3SS and its efficacy in the control of bacterial leaf blight.

Novel pyrimidin-4-one derivatives as potential T3SS inhibitors against Xanthomonas campestris pv. campestris.

Cruciferous black rot is caused by Xanthomonas campestris pv. campestris (Xcc) infection and is a widespread disease worldwide. Excessive and repeated use of bactericide is an important cause of the development of bacterial resistance. It is imperative to take new approaches to screening compounds that target virulence factors rather than kill bacterial pathogens. The type III secretion system (T3SS) invades a variety of cells by transporting virulence effector factors into the cytoplasm and is an attractive antitoxic target. Toward the search of new T3SS inhibitors, an alternative series of novel pyrimidin-4-one derivatives were designed and synthesized and assessed for their effect in blocking the virulence. All of the target compounds were characterized by proton (1 H) nuclear magnetic resonance (NMR), carbon-13 (13 C) NMR, fluorine-19 (19 F) NMR and high-resolution mass spectrometry (HRMS). All compounds were evaluated using high-throughput screening systems against Xcc. The results of the biological activity test revealed that the compound SPF-9 could highly inhibit the activity of xopN gene promoter and the hypersensitivity (HR) of tobacco without affecting bacterial growth. Moreover, messenger RNA (mRNA) level measurements showed that compound SPF-9 inhibited the expression of some representative genes (hrp/hrc genes). Compound SPF-9 weakened the pathogenicity of Xcc to Raphanus sativus L. Compound SPF-9 has good potential for further development as a novel T3SS inhibitor against Xcc. © 2023 Society of Chemical Industry.

Harmine, an inhibitor of the type III secretion system of Salmonella enterica serovar Typhimurium.

New therapeutic strategies for clinical Salmonella enterica serovar Typhimurium (S. Typhimurium) infection are urgently needed due to the generation of antibiotic-resistant bacteria. Inhibition of bacterial virulence has been increasingly regarded as a potential and innovative strategy for the development of anti-infection drugs. Salmonella pathogenicity island (SPI)-encoded type III secretion system (T3SS) represents a key virulence factor in S. Typhimurium, and active invasion and replication in host cells is facilitated by the secretion of T3SS effector proteins. In this study, we found that harmine could inhibit T3SS secretion; thus, its potential anti-S. Typhimurium infection activity was elucidated. Harmine inhibits the secretion and expression of T3SS effector proteins and consequently attenuates the S. Typhimurium invasion function of HeLa cells. This inhibition may be implemented by reducing the transcription of pathogenesis-related SPI-1 transcriptional activator genes hilD, hilC, and rtsA. Harmine improves the survival rate and bacterial loads of mice infected with S. Typhimurium. In summary, harmine, an effective T3SS inhibitor, could be a leading compound for the development of treatments for Salmonella infection.

Structural optimization of natural product fusaric acid to discover novel T3SS inhibitors of Salmonella

Type III secretion system (T3SS) plays a critical role in host cell invasion and pathogenesis of Salmonella. We recently identified the mycotoxin fusaric acid (FA) as a T3SS inhibitor of Salmonella. Herein, twenty-two diphenylsulfane derivatives were designed and synthesized using FA as a lead compound through scaffold hopping. Among them, SL-8 and SL-19 possessing strong anti-T3SS and anti-invasion activity were identified as T3SS inhibitors with improvement in potency as compared to FA. The inhibitory mechanisms on SPI-1 did not depend on the HilD–HilC–RtsA–HilA or PhoP-PhoQ pathway or the assembly of T3SS needle complex. Accordingly, we proposed that the inhibitory effects of SL-8 and SL-19 on SPI-1 probably influence the formation of SicA/InvF-effector complex or other related proteins.

Fluorothiazinon, a small-molecular inhibitor of T3SS, suppresses salmonella oral infection in mice.

Therapeutic strategies that target bacterial virulence have received considerable attention. The type III secretion system (T3SS) is important for bacterial virulence and represents an attractive therapeutic target. Recently, we developed a new small-molecule inhibitor belonging to a class 2,4-disubstituted-4H-[1,3,4]-thiadiazine-5-ones, Fluorothiazinon (FT-previously called CL-55). FT effectively suppressed T3SS of Chlamydia spp., Pseudomonas aeruginosa, and Salmonella without affecting bacterial growth in vitro. FT was previously characterized by low toxicity, stability, and therapeutic efficacy in animal models. Salmonella T3SS inhibition by FT was studied using in vitro assays for effector proteins detection and estimation of salmonella replication in peritoneal macrophages. The antibacterial effect of FT in vivo was investigated in murine models of salmonella chronic systemic and acute infection. Oral administration of the virulent strain of Salmonella enterica serovar Typhimurium to mice-induced chronic systemic infection with the pathogen persistence in different lymphoid organs such as spleens, Peyer's plaques, and mesenteric lymph nodes. We found that FT suppressed orally induced salmonella infection both with therapeutic and prophylactic administration. Treatment by FT at a dose of 50 mg/kg for 4 days starting from day 7 post-infection (therapy) as well as for 4 days before infection (prevention) led to practically complete eradication of salmonella in mice. FT shows a strong potential for antibacterial therapy and could be used as a substance in the design of antibacterial drugs for pharmaceutical intervention including therapy of antibiotic-resistant infections.

Myricetin inhibits the type III secretion system of Salmonella enterica serovar typhimurium by downregulating the Salmonella pathogenic island I gene regulatory pathway

Based on the in-depth study of type III secretion systems (T3SS) in pathogenic bacteria, approaches targeting T3SS have become new alternative strategies to combat drug-resistant bacterial infections. As an important food-borne pathogen, Salmonella enterica serovar Typhimurium (S. Typhimurium) injects effector proteins into host cells through the T3SS to disrupt cell signaling and host responses. In this study, myricetin was screened for its ability to block the translocation function of effector proteins (SipA/SipB) using cell biology and molecular biology methods. It exerted strong effects on inhibiting the expression of Salmonella pathogenicity island 1 (SPI-1)-associated effector proteins without affecting S. Typhimurium growth and thus prevented S. Typhimurium from invading HeLa cells and ultimately inhibited S. Typhimurium-mediated cell damage. In an animal experiment, myricetin comprehensively protected mice from death and pathological damage. A further analysis of the mechanism of action showed that myricetin interfered with the regulatory network of SPI-1-related genes, resulting in a significant decrease in the levels of key effector proteins, and thus inhibited T3SS-mediated virulence. In summary, this study provides a solution for clinical resistance to S. Typhimurium infection and potential candidate compounds. Myricetin, a potential T3SS inhibitor, possesses effective biological activity and exerts protective effects in vitro and in vivo. Myricetin will likely be developed as a novel type of antibiotic targeting S. Typhimurium infections in the future.

Fluorescence Detection of Type III Secretion Using a Glu-CyFur Reporter System in Citrobacter rodentium.

Enteropathogenic Escherichia coli (EPEC) is a major cause of infantile diarrhea worldwide. EPEC and the closely related murine model of EPEC infection, Citrobacter rodentium, utilize a type III secretion system (T3SS) to propagate the infection. Since the T3SS is not essential for the bacteria to survive or propagate, inhibiting the virulence factor with a therapeutic would treat the infection without causing harm to commensal bacteria. Studying inhibitors of the T3SS usually requires a BSL-2 laboratory designation and eukaryotic host cells while not indicating the mechanism of inhibition. We have designed a BSL-1 assay using the murine model C. rodentium that does not require mammalian cell culture. This CPG2-reporter assay allows for more rapid analysis of secretion efficiency than Western blotting and is sensitive enough to differentiate between partial and total inhibition of the T3SS. Here we present our method and the results of a small collection of compounds we have screened, including known T3SS inhibitors EGCG, regacin, and aurodox and related quorum sensing inhibitors tannic acid and ellagic acid. We have further characterized EGCG as a T3SS inhibitor and established its IC50 of 1.8 ± 0.4 μM. We also establish tannic acid as a potent inhibitor of the T3SS with an IC50 of 0.65 ± 0.09 μM.

Myricanol Inhibits the Type III Secretion System of Salmonella enterica Serovar Typhimurium by Interfering With the DNA-Binding Activity of HilD.

The type III secretion system (T3SS) consists of a syringe-like export machine injecting effectors from the bacterial cytosol directly into host cells to establish infection. This mechanism is widely distributed in gram-negative bacteria and can be targeted as an innovative strategy for the developing of anti-virulence drugs. In this study, we present an effective T3SS inhibitor, myricanol, inspired by the use of folk medicinal plants traditionally used against infections. Myricanol is a cyclic diarylheptanoid isolated from the medicinal plant Myrica nagi, which is found in South and East Asia. Bioassay-guided fractionation revealed that myricanol inhibited not only the secretion of type III effector proteins of Salmonella enterica serovar Typhimurium UK-1 χ8956 (S. Typhimurium) but also the invasion of S. Typhimurium into mammalian cells, but showed no toxicity to bacterial growth or the host cells. RNA-Seq data analysis showed that the transcription of the pathogenesis-related SPI-1 gene was significantly inhibited by myricanol. Further study demonstrated that myricanol binds physically to HilD and interferes with its DNA-binding activity to the promoters of the hilA and invF genes. In conclusion, we propose that myricanol is responsible for the anti-infectious properties of M. nagi and is a novel T3SS inhibitor of S. Typhimurium through a previously unappreciated mechanism of action.

Novel S-Thiazol-2-yl-furan-2-carbothioate Derivatives as Potential T3SS Inhibitors Against Xanthomonas oryzae on Rice.

Bacterial leaf blight (BLB) caused by Xanthomonas oryzae pv oryzae (Xoo) is considered as the most destructive disease of rice. The use of bactericides is among the most widely used traditional methods to control this destructive disease. The excessive and repeated use of the same bactericides is also becoming the reason behind the development of bactericide resistance. The widely used method for finding the new antimicrobial agents often involves the bacterial virulence factors as a target without affecting bacterial growth. Type III secretion system (T3SS) is a protein appendage and is considered as having essential virulence factors in most Gram-negative bacteria. Due to the conserved construct, T3SS has been regarded as an important mark for the blooming of novel antimicrobial drugs. Toward the search of new T3SS inhibitors, an alternative series of 1,3-thiazole derivatives were designed and synthesized. Their structures were characterized and confirmed by 1H NMR, 13C NMR, MS, and elemental analysis. All the title compounds inhibited the promoter activity of hpa1 gene significantly. Eight of them showed better inhibition than our previous T3SS inhibitor TS006 (o-coumaric acid, OCA). The treatment of Xoo with eight compounds significantly attenuated HR without affecting bacterial growth. The mRNA levels of some representative genes (hrp/hrc genes) were reduced up to different extents. In vivo bioassay results showed that eight T3SS inhibitors could reduce bacterial leaf blight and bacterial leaf streak symptoms on rice, significantly.

Discovery of Ethyl 2-Nitro-3-Arylacrylates Molecules as T3SS Inhibitor Reducing the Virulence of Plant Pathogenic Bacteria Xanthomonas.

Xanthomonas oryzae pv. oryzae (Xoo) is a gram-negative pathogen which causes leaf blight disease. Known traditional bactericides are not much more effective in inhibiting this bacteria than before. Selecting the virulence factor of the bacteria as the target without affecting their growth has been considered as a novel method for developing new anti-microbial drugs. Type III secretion systems (T3SS) are one of the important and highly conserved virulence factors in most gram-negative pathogens, which has been considered as an effective target to develop new anti-microbial drugs. In order to discover potential anti-microbial drugs against Xoo pathogens, a series of ethyl 2-nitro-3-arylacrylates compounds were screened. Among them, the compounds I-9, I-12, and I-13 could highly inhibit the promoter activity of a harpin gene hpa1, which were used to further check for the influence on bacterial growth and on the hypersensitive response (HR) caused by Xoo bacteria on non-host plants. The results showed that above compounds could reduce HR without affecting bacterial growth and survival. Moreover, qRT-PCR analysis indicated that treatment with the three inhibitors (I-9, I-12, and I-13) could suppress the expression of the Xoo T3SS in different extent. The mRNA levels of representative genes in the hrp cluster, including the key regulatory genes hrpG and hrpX, were decreased. Last but not least, in vivo test ensured that the above compounds reduced the disease symptoms of Xoo on the rice and Xcc on the Chinese radish.

Identification of a potent inhibitor of type II secretion system from Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic pathogen infecting human population. The pathogen is becoming a serious health problem due to its ability to evade normal immune response of the host and multiple drug resistance to many antibiotics. The pathogen has 2 major virulence systems of which the type III secretion system (T3SS) is of major concern to humans. A third system, type 2 secretion system (T2SS), is common to bacteria and used to secrete exotoxin A (ExoA) responsible for human cell destruction. To help bypass the drug resistance, a strategy to block the T2SS based on a low similarity between human ATPases and the essential ATPases of the T3SS and T2SS of P. aeruginosa, was used. An in silico-optimized inhibitor of T3SS, made directly from the computer-optimized of previously published compounds and their combinatorial libraries, showed IC50 = 1.3 ± 0.2 μM in the T2SS ExoA secretion blocking test. The compound was non-toxic to human lung epithelial cell line A549 and could block cellular destruction of those cells in a cell infection model at 200 μM for at least 24 h. The compound could be a lead candidate for the development of T2SS virulence blockers of Pseudomonas aeruginosa.

Identification of the Targets of Type III Secretion System Inhibitors.

A type III secretion system (T3SS) Inhibitor can be utilized for study in the research lab but also progressed into drug development. Since many pathogenic Gram-negative bacteria utilize this highly conserved system as a virulence factor, the prospect of the T3SS as a drug target is promising. To effectively move a T3SS inhibitor into the route of either research or pharmaceuticals an understanding of the target and mechanism of the inhibitor is required. Several methods can be utilized to identify the target. Included here is the use of knockout mutations, tagged inhibitor pull-down assays, and targeted identification methods.

Lon Protease Is Involved in RhpRS-Mediated Regulation of Type III Secretion in Pseudomonas syringae.

Pseudomonas syringae depends on the type III secretion system (T3SS) to directly translocate effectors into host cells. Previously, we reported a nonpathogenic rhpS mutant, suggesting that the two-component transduction system rhpRS is an important regulator of T3SS in P. syringae. rhpRS regulates itself and a variety of downstream genes under an inverted repeat element promoter in a phosphorylation-dependent manner. Here, we identify lon as a suppressor of the rhpS mutant through transposon screening. A lon/rhpS double mutant restored the phenotypes of the rhpS mutant. The expression level of lon was higher in rhpS and other T3SS-deficient mutants than the wild-type strain, suggesting a negative feedback mechanism between lon and T3SS genes. lon was also induced by a novel T3SS inhibitor, acetate, which substantially compromises the activation of T3SS genes in minimal medium and bacterial growth in host plants.

Oleanolic Acid Induces the Type III Secretion System of Ralstonia solanacearum.

Ralstonia solanacearum, the causal agent of bacterial wilt, can naturally infect a wide range of host plants. The type III secretion system (T3SS) is a major virulence determinant in this bacterium. Studies have shown that plant-derived compounds are able to inhibit or induce the T3SS in some plant pathogenic bacteria, though no specific T3SS inhibitor or inducer has yet been identified in R. solanacearum. In this study, a total of 50 different compounds were screened and almost half of them (22 of 50) significantly inhibited or induced the T3SS expression of R. solanacearum. Based on the strong induction activity on T3SS, the T3SS inducer oleanolic acid (OA) was chosen for further study. We found that OA induced the expression of T3SS through the HrpG-HrpB pathway. Some type III effector genes were induced in T3SS inducing medium supplemented with OA. In addition, OA targeted only the T3SS and did not affect other virulence determinants. Finally, we observed that induction of T3SS by OA accelerated disease progress on tobacco. Overall our results suggest that plant-derived compounds are an abundant source of R. solanacearum T3SS regulators, which could prove useful as tools to interrogate the regulation of this key virulence pathway.

The type III secretion system (T3SS) of Chlamydophila psittaci is involved in the host inflammatory response by activating the JNK/ERK signaling pathway.

Chlamydophila psittaci (C. psittaci) is a human zoonotic pathogen, which could result in severe respiratory disease. In the present study, we investigated the role and mechanism of the type III secretion system (T3SS) of C. psittaci in regulating the inflammatory response in host cells. C. psittaci-infected THP-1 cells were incubated with the specific T3SS inhibitor INP0007, inhibitors of ERK, p38, or JNK, and the levels of inflammatory cytokines were analyzed using Q-PCR and ELISA. The levels of ERK, p38, and JNK phosphorylation were analyzed by Western blot. Our results verified that INP0007 inhibited chlamydial growth in vitro, but the coaddition of exogenous iron completely reversed the growth deficit. INP0007 inhibited the growth of C. psittaci and decreased the levels of IL-8, IL-6, TNF-α, and IL-1β. Exogenous iron restored the chlamydial growth but not the production of inflammatory cytokines. These results demonstrated that the expression of inflammatory cytokines during infection was associated with the T3SS which reduced by incubation with ERK and JNK inhibitors, but not with p38 inhibitor. We concluded that the T3SS elicited inflammatory responses by activating the JNK or ERK signaling pathways in the infection of C. psittaci.

Fusaric acid modulates Type Three Secretion System of Salmonella enterica serovar Typhimurium

Natural small-molecule products are promising lead compounds for developing a generation of novel antimicrobials agents to meet the challenge of antibiotic-resistant pathogens. To facilitate the search for novel anti-virulence agents, we chose a virulence factor of Type Three Secretion System (T3SS) as a drug target to screen candidates from a small-molecule library in our laboratory. This study demonstrated fusaric acid had dramatically inhibitory effects on secretion of Salmonella island 1 (SPI-1) effector proteins and invasion of Salmonella into HeLa cells. Moreover, fusaric acid had no inhibitory effects on bacterial growth and viability of host cells. Protein HilA is a key regulator of SPI-1 in Salmonella, which affects transcription of SPI-1 effectors and SPI-1 apparatus genes. In this study, fusaric acid (FA) did not affect secretion of SPI-1 effectors in HilA over-expressed strain, suggesting it did not affect the transcription of SPI-1. In addition, fusaric acid did not affect the protein level of apparatus protein PrgH in SPI-1 needle complex. As a result, we proposed fusaric acid had an inhibitory effect on SPI-1 probably depending on its influence on SicA/InvF. In summary, fusaric acid is a novel inhibitor of T3SS with potential for further developing novel anti-virulence agents.