and were grown, unless otherwise specified, in 2xYT (yeast extract-tryptone) at 26C shaking overnight. III secretion at different points in T3SS assembly and function. For example, our data suggests that Compound 3, a malic diamide, blocks either activity of the assembled T3SS or alters the structure of the T3SS in a way that blocks T3SS cargo secretion but not antibody recognition of the T3SS needle. In contrast, our data predicts that Compound 4, a haloid-containing sulfonamidobenzamide, disrupts T3SS needle subunit secretion or assembly. Furthermore, we suggest that misregulation of copy number control of the pYV virulence plasmid, which encodes the T3SS, should be considered as a possible mode of action for compounds with T3SS inhibitory activity against species pathogenic to mammals, the fully assembled Ysc T3SS needle is composed of ~140 SctF subunits, is usually 65 nm in length, and harbors a tip complex PD-1-IN-18 composed of a pentamer of the hydrophilic LcrV translocator protein (Broz et al., 2007). Upon host cell contact, two additional hydrophobic translocator proteins, YopD and YopB, are secreted through the Ysc needle to form a translocon complex that leads to pore formation in the host membrane, facilitating the translocation of effector proteins to the host cytoplasm (Bttner and Bonas, 2002). The Ysc T3SS is usually highly regulated at the transcriptional, translational, and post-translational levels (Francis et al., 2002; Heroven et al., 2012). The transcription factor LcrF directs transcription of genes encoding PD-1-IN-18 the T3SS structural, regulatory, and effector proteins, all of which are encoded around the 70 kb pYV virulence plasmid (Schwiesow et al., 2015). Several factors govern regulation of LcrF expression, including temperature and the transcription factor IscR (Schwiesow et al., 2015). Importantly, pYV copy EN-7 number increases during active type III secretion, and this is usually important for virulence (Wang et al., 2016). In addition, the T3SS functions on a positive feedback loop in which active secretion leads to upregulated transcription of T3SS genes (Cornelis et al., 1987; Francis et al., 2002), although the mechanism behind this remains unclear. A number of PD-1-IN-18 pathogens require one or more T3SSs for virulence, as genetic ablation causes attenuation in animal models and clinical isolates harbor plasmids or pathogenicity islands that encode T3SS genes (Coburn et al., 2007). An antibody against the T3SS needle tip protein PcrV is usually part of a current Phase II clinical trial to treat nosocomial ventilator-associated pneumonia (“type”:”clinical-trial”,”attrs”:”text”:”NCT02696902″,”term_id”:”NCT02696902″NCT02696902), indicating that antibodies targeting the T3SS may be used as therapeutics. However, antibodies have low oral bioavailability and must be administered by injection; small molecules with high oral bioavailability are more attractive as therapeutic brokers. A number of putative small molecule T3SS inhibitors have been identified in the past 15 years (Duncan et al., 2012; Marshall and Finlay, 2014; Anantharajah et al., 2017), yet only one class of compounds can be considered validated. Many published T3SS inhibitors have off target effects that may underlie their T3SS disruption. For example, the best studied class of T3SS inhibitors, the salicylidene acylhydrazides, are thought to cause deregulation of T3SS genes through an unknown mechanism, yet the activity of some salicylidene acylhydrazides is dependent on iron chelation (Beckham and Roe, 2014). The phenoxyacetamides represent the only class of compounds that inhibit the T3SS in a physiologically relevant cellular context, protect against a bacterial infection (abscess formation in mice), and have a validated molecular target, the SctF needle subunit (Bowlin et al., 2014; Berube et al., 2017). We have developed an experimental pipeline that can be employed to determine initial mode of action for compounds with T3SS inhibitory activity. We chose to use the enteropathogens and as the workhorses for this assay pipeline because is usually susceptible to the majority of T3SS inhibitors described and because of the wealth of genetic and biochemical tools available. In addition, are extracellular pathogens that use their T3SS to prevent phagocytosis, negating the need for a.