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.

Anti-Pathogenic Properties of the Combination of a T3SS Inhibitory Halogenated Pyrrolidone with C-30 Furanone.

Antimicrobial resistance is one of the current public health challenges to be solved. The World Health Organization (WHO) has urgently called for the development of strategies to expand the increasingly limited antimicrobial arsenal. The development of anti-virulence therapies is a viable option to counteract bacterial infections with the possibility of reducing the generation of resistance. Here we report on the chemical structures of pyrrolidones DEXT 1-4 (previously identified as furan derivatives) and their anti-virulence activity on Pseudomonas aeruginosa strains. DEXT 1-4 were shown to inhibit biofilm formation, swarming motility, and secretion of ExoU and ExoT effector proteins. Also, the anti-pathogenic property of DEXT-3 alone or in combination with furanone C-30 (quorum sensing inhibitor) or MBX-1641 (type III secretion system inhibitor) was analyzed in a model of necrosis induced by P. aeruginosa PA14. All treatments reduced necrosis; however, only the combination of C-30 50 µM with DEXT-3 100 µM showed significant inhibition of bacterial growth in the inoculation area and systemic dispersion. In conclusion, pyrrolidones DEXT 1-4 are chemical structures capable of reducing the pathogenicity of P. aeruginosa and with the potential for the development of anti-virulence combination therapies.

A Structure-Function-Inhibition Analysis of the Pseudomonas aeruginosa Type III Secretion Needle Protein PscF.

The Pseudomonas aeruginosa type III secretion system (T3SS) needle comprised of multiple PscF subunits is essential for the translocation of effector toxins into human cells, facilitating the establishment and dissemination of infection. Mutations in the pscF gene provide resistance to the phenoxyacetamide (PhA) series of T3SS inhibitory chemical probes. To better understand PscF functions and interactions with PhA, alleles of pscF with 71 single mutations altering 49 of the 85 residues of the encoded protein were evaluated for their effects on T3SS phenotypes. Of these, 37% eliminated and 63% maintained secretion, with representatives of both evenly distributed across the entire protein. Mutations in 14 codons conferred a degree of PhA resistance without eliminating secretion, and all but one were in the alpha-helical C-terminal 25% of PscF. PhA-resistant mutants exhibited no cross-resistance to two T3SS inhibitors with different chemical scaffolds. Two mutations caused constitutive T3SS secretion. The pscF allele at its native locus, whether wild type (WT), constitutive, or PhA resistant, was dominant over other pscF alleles expressed from nonnative loci and promoters, but mixed phenotypes were observed in chromosomal ΔpscF strains with both WT and mutant alleles at nonnative loci. Some PhA-resistant mutants exhibited reduced translocation efficiency that was improved in a PhA dose-dependent manner, suggesting that PhA can bind to those resistant needles. In summary, these results are consistent with a direct interaction between PhA inhibitors and the T3SS needle, suggest a mechanism of blocking conformational changes, and demonstrate that PscF affects T3SS regulation, as well as carrying out secretion and translocation.IMPORTANCEP. aeruginosa effector toxin translocation into host innate immune cells is critical for the establishment and dissemination of P. aeruginosa infections. The medical need for new anti-P. aeruginosa agents is evident by the fact that P. aeruginosa ventilator-associated pneumonia is associated with a high mortality rate (40 to 69%) and recurs in >30% of patients, even with standard-of-care antibiotic therapy. The results described here confirm roles for the PscF needle in T3SS secretion and translocation and suggest that it affects regulation, possibly by interaction with T3SS regulatory proteins. The results also support a model of direct interaction of the needle with PhA and suggest that, with further development, members of the PhA series may prove useful as drugs for P. aeruginosa infection.

Novel Noncompetitive Type Three Secretion System ATPase Inhibitors Shut Down Shigella Effector Secretion.

Shigella is the causative agent of bacillary dysentery and is responsible for an estimated 165 million infections and 600,000 deaths annually. Like many Gram-negative pathogens, Shigella relies on a type three secretion system (T3SS) to initiate and sustain infection by directly injecting effector proteins into host cells. Protein secretion through the needle-like injectisome and overall Shigella virulence rely on the T3SS ATPase Spa47, making it a likely means for T3SS regulation and an attractive target for therapeutic small molecule inhibitors. Here, we utilize a recently solved 2.15 Å crystal structure of Spa47 to computationally screen 7.6 million drug-like compounds for candidates which avoid the highly conserved active site by targeting a distal, but critical, interface between adjacent protomers of the Spa47 homohexamer. Ten of the top inhibitor candidates were characterized, identifying novel Spa47 inhibitors that reduce in vitro ATPase activity by as much as 87.9 ± 10.5% with IC50's as low as 25 ± 20 µM and reduce in vivo Shigella T3SS protein secretion by as much as 94.7 ± 3.0%. Kinetic analyses show that the inhibitors operate through a noncompetitive mechanism that likely supports the inhibitors' low cytotoxicity, as they avoid off-target ATPases involved in either Shigella or mammalian cell metabolism. Interestingly, the inhibitors display nearly identical inhibition profiles for Spa47 and the T3SS ATPases EscN from E. coli and FliI from Salmonella. Together, the results of this study provide much-needed insight into T3SS ATPase inhibition mechanisms and a strong platform for developing broadly effective cross-pathogen T3SS ATPase inhibitors.

Identification of the natural product paeonol derived from peony bark as an inhibitor of the Salmonella enterica serovar Typhimurium type III secretion system.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important zoonotic pathogen in public health and food safety. The type III secretion system (T3SS) encoded by Salmonella pathogenicity island (SPI) is a sophisticated molecular machine that facilitates active invasion, intracellular replication, and host inflammation. Due to increasing antibiotic resistance, new therapeutic strategies that target the Salmonella T3SS have received considerable attention. In this study, paeonol was identified as an inhibitor of the S. Typhimurium T3SS. Paeonol significantly blocked the translocation of SipA into host cells and suppressed the expression of effector proteins without affecting bacterial growth in the effective concentration range. Additionally, S. Typhimurium-mediated cell injury and invasion levels were significantly reduced after treatment with paeonol, without cytotoxicity. Most importantly, the comprehensive protective effect of paeonol was confirmed in an S. Typhimurium mouse infection model. Preliminary mechanistic studies suggest that paeonol inhibits the expression of effector proteins by reducing the transcription level of the SPI-1 regulatory pathway gene hilA. This work provides proof that paeonol could be used as a potential drug to treat infections caused by Salmonella.

Inhibition of the type III secretion system by syringaldehyde protects mice from Salmonella enterica serovar Typhimurium.

The invasiveness of Salmonella enterica serovar Typhimurium (S. Typhimurium) is closely associated with the Salmonella pathogenicity island (SPI)-encoded type Ⅲ secretion system (T3SS), which can directly inject a series of effector proteins into eukaryotic cells to enable bacterial infection. In this study, syringaldehyde was identified as an effective inhibitor of the S. Typhimurium T3SS using an effector protein-lactamase fusion reporter system. Syringaldehyde treatment could inhibit the expression of important effector proteins (SipA, SipB and SipC) at a concentration of 0.18 mM without affecting bacterial growth. Additionally, significant inhibition of bacterial invasion and cellular injury was observed following the syringaldehyde treatment in the co-infection system of HeLa cells and S. Typhimurium. Furthermore, treatment with syringaldehyde provided systemic protection to mice infected with S. Typhimurium, reducing mortality (40.00%) and bacterial loads and relieving caecal damage and systemic inflammation. The results presented in this study indicate that syringaldehyde significantly affects T3SS activity and is a potential leading compound for treating S. Typhimurium infections.

Characterization of the Mode of Action of Aurodox, a Type III Secretion System Inhibitor from Streptomyces goldiniensis.

Recent work has demonstrated that the polyketide natural product Aurodox from Streptomyces goldiniensis is able to block the pathogenesis of the murine pathogen Citrobacter rodentium In this work, we aimed to gain a better understanding of the mechanism of action of the compound. We show that Aurodox downregulates the expression of the type III secretion systems of enteropathogenic and enterohemorrhagic Escherichia coli Furthermore, we have used transcriptomic analysis to show that Aurodox inhibits the expression at the transcriptional level by repressing the master regulator, ler Our data support a model in which Aurodox acts upstream of ler and not directly on the secretion system itself. Finally, we have shown that Aurodox, unlike some traditional antibiotics, does not induce expression of RecA, which is essential for the production of Shiga toxin. We propose that these properties nominate Aurodox as a promising antivirulence therapy for the treatment of these infections.

Transcriptional responses of Xanthomonas oryzae pv. oryzae to type III secretion system inhibitor ortho-coumaric acid.

BACKGROUND: We previously identified a plant-derived phenolic compound ortho-coumaric acid (OCA) as an inhibitor of type III secretion system (T3SS) of Xanthomonas oryzae pv. oryzae (Xoo), the pathogen causing bacterial leaf blight of rice, one of the most devastating bacterial diseases of this staple crop worldwide. However, the molecular mechanisms by which OCA suppresses T3SS and the transcriptional responses to the OCA treatments in Xoo remains unclear. RESULTS: The present study conducted the RNA-seq-based transcriptomic analysis to reveal changes in gene expression in Xoo in response to 30 min, 1 h, 3 h, and 6 h of OCA treatment. Results showed that OCA significantly inhibited the expression of T3SS genes after 30 min, and the inhibition also existed after 1 h, 3 h, and 6 h. After treatment for 30 min, membrane proteins in the functional category of cellular process was the predominant group affected, indicating that Xoo was in the early stress stage. Over time, more differentially-expressed genes (DEGs) gathered in the functional category of biological process. Analysis of common DEGs at all four of time points revealed the core elements of Xoo during the response to OCA treatment. Notable, a multidrug transporter cluster that consisted of a MarR-family protein (PXO_RS13760), a multidrug RND transporter (PXO_RS13755), a multidrug transporter (PXO_RS13750), and an MFS transporter (PXO_RS13745) were significantly up-regulated at all four of the time points. Although these three transporter genes were not upregulated by OCA in the PXO_RS13760 deletion mutant, the deficiency of PXO_RS13760 in Xoo did not affect T3SS transcript, and OCA still had the ability to inhibit the expression of T3SS in the mutant, suggesting that the MarR-family protein was involved in bacterial responses to OCA, but not direct OCA inhibition of T3SS in Xoo. CONCLUSIONS: We analyzed the transcriptome of Xoo during OCA treatment at both early and late stages, which revealed the landscape of Xoo responses to OCA at the whole-genome transcription level. A multidrug transporter cluster was identified to be involved in the response process, but had no direct relation to T3SS in Xoo.

Synthesis and bioactivity of 1,3-thiazolidine-2-thione derivatives against type III secretion system of Xanthomonas oryzae.

Targeting virulence factors of bacterial without affecting their growth and survival, has been an initiative strategy for the development of novel anti-microbial agents. The type III secretion system (T3SS), one of essential and highly conserved virulence factors in most Gram-negative pathogenic bacteria, has been regarded as an effective target that developed new anti-microbial drugs. Xanthomonas oryzae pv. oryzae (Xoo) is one of the most important bacterial pathogens on rice, which causes leaf blight disease. To discover potential anti-virulence agents against the pathogens, a new series of 1,3-thiazolidine-2-thione derivatives containing 5-phenyl-2-furan were designed and synthesized. Their structures were characterized by 1H NMR, 13C NMR, MS, and elemental analysis. All the title compounds inhibited the promoter activity of a harpin gene hpa1, significantly, that were further checked for the impact on bacterial growth. The results indicated that treatment of Xoo with the title compound III-7 did not affect bacterial growth or survival. Moreover, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that the expression of the Xoo T3SS was suppressed by treatment with the inhibitor. The mRNA levels of representative genes in the hrp (hypersensitive response and pathogenicity) cluster, as well as the regulatory genes hrpG and hrpX, were reduced. Finally, the in vivo test demonstrated that the compounds could reduce the disease symptoms of Xoo on the rice cultivar (Oryza sativa) IR24.

Shutting Down Shigella Secretion: Characterizing Small Molecule Type Three Secretion System ATPase Inhibitors.

Many important human pathogens rely on one or more type three secretion systems (T3SSs) to inject bacterial effector proteins directly into the host cell cytoplasm. Secretion of protein through the needlelike type three secretion apparatus (T3SA) is essential for pathogen virulence and relies on a highly conserved ATPase at the base of the apparatus, making it an attractive target for anti-infective therapeutics. Here, we leveraged the ability to purify an active oligomeric Shigella T3SS ATPase to provide kinetic analyses of three T3SS ATPase inhibitors of Spa47. In agreement with in silico docking simulations, the inhibitors displayed noncompetitive inhibition profiles and efficiently reduced Spa47 ATPase activity with IC50s as low as 52 ± 3 µM. Two of the inhibitors functioned well in vivo, nearly abolishing effector protein secretion without significantly affecting the Shigella growth phenotype or HeLa cell viability. Furthermore, characterization of Spa47 complexes in vitro and Shigella T3SA formation in vivo showed that the inhibitors do not function through disruption of Spa47 oligomers or by preventing T3SA formation. Together, these findings suggest that inhibitors targeting Spa47 may be an effective means of combating Shigella infection by shutting down type three secretion without preventing presentation of the highly antigenic T3SA tip proteins that aid in clearing the infection and developing pan- Shigella immunological memory. In summary, this is the first report of Shigella T3SS ATPase inhibitors and one of only a small number of studies characterizing T3SS ATPase inhibition in general. The work presented here provides much-needed insight into T3SS ATPase inhibition mechanisms and provides a strong platform for developing and evaluating non-antibiotic therapeutics targeting Spa47 and other T3SS ATPases.

Novel compounds targeting the enterohemorrhagic Escherichia coli type three secretion system reveal insights into mechanisms of secretion inhibition.

Anti-virulence (AV) compounds are a promising alternative to traditional antibiotics for fighting bacterial infections. The Type Three Secretion System (T3SS) is a well-studied and attractive AV target, given that it is widespread in more than 25 species of Gram-negative bacteria, including enterohemorrhagic E. coli (EHEC), and as it is essential for host colonization by many pathogens. In this work, we designed, synthesized and tested a new series of compounds that block the functionality of the T3SS of EHEC. Affinity chromatography experiments identified the primary target of the compounds as the T3SS needle pore protein EspD, which is essential for effector protein translocation into host cells. These data were supported by mechanistic studies that determined the coiled-coil domain 1 of EspD as a key compound-binding site, thereby preventing correct assembly of the T3SS complex on the cell surface. However, binding of inhibitors to EspD or deletion of EspD itself did not result in transcriptional down-regulation of effector proteins. Instead, we found the compounds to exhibit dual-functionality by also down-regulating transcription of the entire chromosomal locus encoding the T3SS, further demonstrating their desirability and effectiveness.

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.

Inhibition of Pseudomonas aeruginosa ExsA DNA-Binding Activity by N-Hydroxybenzimidazoles.

The Pseudomonas aeruginosa type III secretion system (T3SS) is a primary virulence determinant and a potential target for antivirulence drugs. One candidate target is ExsA, a member of the AraC family of DNA-binding proteins required for expression of the T3SS. A previous study identified small molecules based on an N-hydroxybenzimidazole scaffold that inhibit the DNA-binding activity of several AraC proteins, including ExsA. In this study, we further characterized a panel of N-hydroxybenzimidazoles. The half-maximal inhibitory concentrations (IC50s) for the tested N-hydroxybenzimidazoles ranged from 8 to 45 µM in DNA-binding assays. Each of the N-hydroxybenzimidazoles protected mammalian cells from T3SS-dependent cytotoxicity, and protection correlated with reduced T3SS gene expression in a coculture infection model. Binding studies with the purified ExsA DNA-binding domain (i.e., lacking the amino-terminal self-association domain) confirmed that the activity of N-hydroxybenzimidazoles results from interactions with the DNA-binding domain. The interaction is specific, as an unrelated DNA-binding protein (Vfr) was unaffected by N-hydroxybenzimidazoles. ExsA homologs that control T3SS gene expression in Yersinia pestis, Aeromonas hydrophila, and Vibrio parahaemolyticus were also sensitive to N-hydroxybenzimidazoles. Although ExsA and Y. pestis LcrF share 79% sequence identity in the DNA-binding domain, differential sensitivities to several of the N-hydroxybenzimidazoles were observed. Site-directed mutagenesis based on in silico docking of inhibitors to the DNA-binding domain, and on amino acid differences between ExsA and LcrF, resulted in the identification of several substitutions that altered the sensitivity of ExsA to N-hydroxybenzimidazoles. Development of second-generation compounds targeted to the same binding pocket could lead to drugs with improved pharmacological properties.

Type III Secretion-Dependent Sensitivity of Escherichia coli O157 to Specific Ketolides.

A subset of Gram-negative bacterial pathogens uses a type III secretion system (T3SS) to open up a conduit into eukaryotic cells in order to inject effector proteins. These modulate pathways to enhance bacterial colonization. In this study, we screened established bioactive compounds for any that could repress T3SS expression in enterohemorrhagic Escherichia coli (EHEC) O157. The ketolides telithromycin and, subsequently, solithromycin both demonstrated repressive effects on expression of the bacterial T3SS at sub-MICs, leading to significant reductions in bacterial binding and actin-rich pedestal formation on epithelial cells. Preincubation of epithelial cells with solithromycin resulted in significantly less attachment of E. coli O157. Moreover, bacteria expressing the T3SS were more susceptible to solithromycin, and there was significant preferential killing of E. coli O157 bacteria when they were added to epithelial cells that had been preexposed to the ketolide. This killing was dependent on expression of the T3SS. Taken together, this research indicates that the ketolide that has accumulated in epithelial cells may traffic back into the bacteria via the T3SS. Considering that neither ketolide induces the SOS response, nontoxic members of this class of antibiotics, such as solithromycin, should be considered for future testing and trials evaluating their use for treatment of EHEC infections. These antibiotics may also have broader significance for treating infections caused by other pathogenic bacteria, including intracellular bacteria, that express a T3SS.

Licoflavonol is an inhibitor of the type three secretion system of Salmonella enterica serovar Typhimurium.

As an important food-borne human pathogen, Salmonella enterica serovar Typhimurium depends on its type III secretion system (T3SS) as a major virulence factor to cause disease all over the world. The T3SS secretes effector proteins to facilitate invasion into host cells. In this study, twenty prenylated flavonoids (1-20) were screened for their anti-T3SS activity, revealing that several analogs exhibited strong inhibitory effects on the secretion of Salmonella pathogenicity island 1 (SPI-1)-associated effector proteins without affecting the growth of bacteria and the secretion of the flagellar protein FliC. Among the flavonoids 1-20, licoflavonol (20) exhibited a strong inhibitory effect on the secretion of the SPI-1 effector proteins via regulating the transcription of the SicA/InvF genes, and the transportation of the effector protein SipC. In summary, licoflavonol, a novel natural inhibitor of Salmonella T3SS, could be a promising candidate for novel type of anti-virulence drugs.

Small-Molecule Inhibitors of the Type III Secretion System.

Drug-resistant pathogens have presented increasing challenges to the discovery and development of new antibacterial agents. The type III secretion system (T3SS), existing in bacterial chromosomes or plasmids, is one of the most complicated protein secretion systems. T3SSs of animal and plant pathogens possess many highly conserved main structural components comprised of about 20 proteins. Many Gram-negative bacteria carry T3SS as a major virulence determinant, and using the T3SS, the bacteria secrete and inject effector proteins into target host cells, triggering disease symptoms. Therefore, T3SS has emerged as an attractive target for antimicrobial therapeutics. In recent years, many T3SS-targeting small-molecule inhibitors have been discovered; these inhibitors prevent the bacteria from injecting effector proteins and from causing pathophysiology in host cells. Targeting the virulence of Gram-negative pathogens, rather than their survival, is an innovative and promising approach that may greatly reduce selection pressures on pathogens to develop drug-resistant mutations. This article summarizes recent progress in the search for promising small-molecule T3SS inhibitors that target the secretion and translocation of bacterial effector proteins.

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.

Identification of Type III Secretion Inhibitors for Plant Disease Management.

Bacterial plant pathogens are among the most devastating threats to agriculture. To date, there are no effective means to control bacterial plant diseases due to the restrictions in the use of antibiotics in agriculture. A novel strategy under study is the use of chemical compounds that inhibit the expression of key bacterial virulence determinants. The type III secretion system is essential for virulence of many Gram-negative bacteria because it injects into the plant host cells bacterial proteins that interfere with their immune system. Here, we describe the methodology to identify bacterial type III secretion inhibitors, including a series of protocols that combine in planta and in vitro experiments. We use Ralstonia solanacearum as a model because of the number of genetic tools available in this organism and because it causes bacterial wilt, one of the most threatening plant diseases worldwide. The procedures presented can be used to evaluate the effect of different chemical compounds on bacterial growth and virulence.

Adjunctive therapy for multidrug-resistant bacterial infections: Type III secretion system and efflux inhibitors.

The increasing prevalence of multidrug-resistant (MDR) bacterial infections has created a crucial need for new therapeutics that avoid or minimize existing resistance mechanisms. In this review, we describe the development of novel classes of small-molecule adjunctive agents targeting either a bacterial virulence factor, the Pseudomonas aeruginosa type III secretion system (T3SS), or an intrinsic resistance factor, resistance-nodulation-cell division superfamily (RND) efflux pumps of the Enterobacteriaceae. These agents are designed to be administered with antibacterials to improve their efficacy. T3SS inhibition rescues host innate immune system cells from injection with bacterial toxins, whereas RND efflux pump inhibition increases antibiotic susceptibility, in both cases improving the efficacy of the combined antibacterial.

Novel Disk Diffusion Assay on Magnesium Oxalate Agar To Evaluate the Susceptibility of Yersinia pestis to Type III Secretion System Inhibitors.

Current methods for screening small molecules that inhibit the plasmid pCD1-encoded Yersinia pestis type III secretion system (T3SS) include lengthy growth curves followed by multistep luminescence assays or Western blot assays to detect secretion, or lack thereof, of effector proteins. The goal of this research was to develop a novel disk diffusion assay on magnesium oxalate (MOX) agar as a simple way to evaluate the susceptibility of Y. pestis to type III secretion system inhibitors. MOX agar produces distinct Y. pestis growth characteristics based on the bacteria's ability or inability to secrete effector proteins; small, barely visible colonies are observed when secretion is activated versus larger, readily visible colonies when secretion is inhibited. Wild-type Y. pestis was diluted and spread onto a MOX agar plate. Disks containing 20 µl of various concentrations of imidocarb dipropionate, a known Y. pestis T3SS inhibitor, or distilled water (dH2O) were placed on the plate. After incubation at 37°C for 48 h, visible colonies of Y. pestis were observed surrounding the disks with imidocarb dipropionate, suggesting that T3S was inhibited. The diameter of the growth of colonies surrounding the disks increased as the concentration of the T3SS inhibitor increased. Imidocarb dipropionate was also able to inhibit Y. pestis strains lacking effector Yops and Yop chaperones, suggesting that they are not necessary for T3S inhibition. This disk diffusion assay is a feasible and useful method for testing the susceptibility of Y. pestis to type III secretion system inhibitors and has the potential to be used in a clinical setting. IMPORTANCE Disk diffusion assays have traditionally been used as a simple and effective way to screen compounds for antibacterial activity and to determine the susceptibility of pathogens to antibiotics; however, they are limited to detecting growth inhibition only. Consequently, antimicrobial agents that inhibit virulence factors, but not growth, would not be detected. Therefore, we developed a disk diffusion assay that could detect inhibition of bacterial virulence factors, specifically, type III secretion systems (T3SSs), needle-like structures used by several pathogenic bacteria to inject host cells with effector proteins and cause disease. We demonstrate that magnesium oxalate (MOX) agar can be used in a disk diffusion assay to detect inhibition of the T3SS of Yersinia pestis, the causative agent of bubonic plague, by small-molecule inhibitors. This assay may be useful for screening additional small molecules that target bacterial T3SSs or testing the susceptibility of patient-derived samples to drugs that target T3SSs.