020; Fig. 2), confirming the transcriptome data. Strain JH3009 harbouring a gfp+ fusion to the T3SS-2 gene ssaG exhibited
a threefold decrease in fluorescence in the presence of INP0403 (P=0.023; Fig. 2), supporting the microarray data, although ssaG did not meet the stringent filtering criteria (Table S1). A control strain JH3016 containing a gfp+ fusion to the rpsM promoter showed equivalent levels of fluorescence when treated with DMSO or INP0403 (Fig. 2). Reverse transcriptase-PCR analysis of the same RNA samples used for microarray analysis did not detect prgH transcripts in the INP0403-treated sample, but they were detected in Sotrastaurin cost the DMSO-treated sample, while the housekeeping gene, rpoD, was transcribed in equivalent amounts in both INP0403- and DMSO-treated samples (data not shown). Comparison of the INP0403-sensitive transcriptome to the HilA regulon (De Keersmaecker et al., 2005; Thijs
et al., 2007) indicated that only one gene (prgH) in the HilA regulon was significantly (at least twofold) repressed, suggesting that inhibition of T3SS-1 by INP0403 may occur in a HilA-independent manner. A large number of positive and negative regulators of Salmonella T3SS-1 exist (reviewed in Altier, 2005; Ellermeier & Slauch, 2007); thus, we sought to determine whether transcription of any of these was affected by INP0403. Only four previously characterized positive regulators Talazoparib of SPI-1 were significantly (P≤0.05) repressed at least twofold in the presence of INP0403 (Table S3). These included RtsA (11-fold), HilC (5.4-fold) and HilD (9.7-fold), all of which are AraC-like transcriptional activators that constitute a feed forward loop that controls hilA expression in S. Typhimurium (Ellermeier et al., 2005). RtsA, HilC and HilD each independently activate the transcription
of hilA, as well as each other (Ellermeier et al., 2005). HilD also activates the SPI-2 regulon in a medium- and growth phase-dependent manner (Bustamante et al., 2008). FliZ was also repressed by INP0403 (2.2-fold), and is an FlhD4C2-dependent activator of flagellar Pclass2/middle gene expression (Saini et al., 2008) and a positive regulator of SPI-1 gene expression (Lucas et al., 2000; Iyoda et al., 2001), via post-transcriptional control of HilD (Kage et al., 2008). Although Methocarbamol the effect of INP0403 on hilA expression was not statistically significant, it remains feasible that it produces a biologically significant effect on T3S even though transcription of few genes under the control of HilA was significantly modulated. No other flagellar genes were significantly affected by INP0403 in the filtered dataset (Table S2), but fliA and fliY, which are in an operon with fliZ, were repressed approximately twofold, and most other flagellar genes showed a similar pattern of 1.5–2-fold downregulation (Table S1).