Some of the first work done to characterize the T3SS involved analyzing regulatory mechanisms of expression of the LEE.60,289,379 Expression of the T3SS-related genes has since been linked to major global regulators of E. coli as well as other adaptive virulence mechanisms, such as quorum sensing.379 Quorum sensing is a mechanism of regulation of gene expression in response to environmental chemical signals. These signaling molecules are indicative of cell density and increases in their concentrations result in alterations in expression patterns in genes related to antibiotic resistance, horizontal gene transfer, biofilm formation, and secretion systems. Quorum sensing is imperative for successful propagation of infection and evasion of antibiotic treatment. The links between quorum sensing and the T3SS, while understood, have not been explored in terms of identification of potential therapeutic targets. We chose several modulators of quorum sensing to analyze their effects on T3SS activity. These include analogs of trans- cinnamaldehyde (36-39), ellagic acid (40), tannic acid (41), secreted quorum sensing signaling molecules L-homoserine lactone analogs (42-46), bergamottin (47), naringenin (48), penicillic acid (49), and patulin (50).
Tannic acid (41) is a well-known inhibitor of quorum sensing and it has been used as a positive control in screens for quorum sensing inhibitiors.380 Tannic acid and trans- cinnamaldehyde (36) were identified as inhibitors of the AHL synthase production in 2014.23 In one study, we analyzed para-substituted analogs of trans-cinnamaldehyde. We were initially interested in examining the inhibitory activity of 36 in our CPG2 reporter assay. Our initial results
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indicated that 36 does not inhibit CPG2 secretion. We screened analogs of 36 and found that para substitution with a dimethylamino group (39) results in 76.5% inhibition of type III secretion. Since these analogs have not been analyzed, it is unclear if there is a parallel pattern of inhibition in quorum sensing.
Figure 30. Of the trans-cinnamaldehyde analogs studied, only 4-
(dimethylamino)cinnamaldehyde (39) shows inhibition in the CPG2 reporter assay. Compounds were screened at 50 μM.
We next analyzed ellagic acid (40) and tannic acid (41), which have the same mechanism of quorum sensing inhibition as 36, to see if we would observe comparable results. One previously published study compared the AHL-inhibitory abilities of tannic acid, EGCG, and ellagic acid, all polyphenolic compounds containing a gallic acid moiety.24 All three compounds indicated an ability to inhibit AHL signaling. We were interested in comparing their T3SS inhibitory activity. We initially screened 40 and 41 at 50 μM in our CPG2 reporter assay (Figure 31). We found that 41 is a more potent inhibitor than 40 and decided to characterize this inhibition further. 41 shows concentration-dependent inhibition of T3SS-mediated CPG2 translocation with an IC50 of <1 μM.
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Our results indicate that these polyphenolic compounds may inhibit the T3SS in a mechanism separate from their quorum sensing targets, since trans-cinnamaldehyde does not appear to inhibit the T3SS.
Figure 31. A) Ellagic acid (40) and tannic acid (41) are polyphenols containing gallate. B) 40 has an IC50 of approximately 50 μM. 41 inhibits T3SS activity almost entirely at that concentration. C) 41 inhibits the secretion of CPG2 in a concentration-dependent manner.
The best-characterized quorum sensing pathway involves the N-acylhomoserine lactones (AHL) autoinducers.381 AHLs are synthesized by AHL synthases, and consist of a homoserine lactone ring that is N-acylated. These molecules induce the expression of the biosynthetic mechanisms that result in their production, making them autoinducers. We analyzed a collection
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of N-acylhomoserine lactones for their ability to overcome tannic acid inhibition of the T3SS (Figure 32). An initial screen was run to identify the effects of the AHLs independently (20 μM, Figure 32B). The compounds were then rescreened in the presence of tannic acid (Figure 32C). The C. rodentium cells harboring the pBAD-EspF-CPG2 plasmid were incubated in the presence of 1 μM tannic acid in addition to the listed AHL at 20 μM. DMSO was normalized and used as the vehicle control.
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C
Figure 32. N-acylhomoserine lactones (AHLs) and their ability to overcome tannic acid inhibition. A) Structures of the AHLs analyzed. B) AHLs were studied in the CPG2 reporter assay for their effect on the T3SS. 42 and 43 appear to inhibit T3SS activity, so they were not carried forward to test their ability to overcome 41 inhibition. C) AHLs were analyzed in the CPG2 reporter assay in the presence of 1 μM 41. While 44 and 46 appear to overcome tannic acid inhibition to the extent of regaining control activity, 45 appears to enhance T3SS activity.
From the initial screen, it appeared that 42 and 43 inhibited T3SS activity. We then rescreened 44-46 for their effect on tannic acid’s inhibitory activity. We observed that 44 and 46 were able to overcome the inhibition to the same level as the DMSO control, while 45 appeared to increase T3SS activity significantly. These results indicate that tannic acid’s inhibition of the T3SS may be related to AHL signaling mechanisms in C. rodentium. The ability to overcome tannic acid inhibition indicates a competitive relationship between AHLs and the T3SS target of tannic acid that should be further analyzed.
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Bergamottin (47) and naringenin (48) are cytochrome p450 inhibitors derived from grapefruit juice (Figure 33). 47 has been indicated as an inhibitor of biofilm formation in EHEC O157:H7.382 48 is a flavonoid compound shown to inhibit the production of AHLs and decrease the expression of some quorum sensing-related genes in P. aeruginosa. Our screening of these compounds indicated that neither bergamottin nor naringenin act as inhibitors of the C. rodentium T3SS (Figure 33).
Figure 33. Bergamottin and naringenin do not inhibit the T3SS at 50 μM. A) Structures of bergamottin (47) and naringenin (48). B) When 47 was analyzed in the CPG2 reporter assay at 50 μM, no inhibition was observed. C) 50 μM 48 did not inhibit T3SS-mediated secretion of CPG2.
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Penicillic acid (49) and patulin (50) are secondary metabolites produced by Penicillium spp. of fungi. Both compounds were found to downregulate the expression of QS-related genes in
P. aeruginosa.383 Our results indicate that 49 and 50 are not inhibitors of the T3SS at 50 μM.
Figure 34. Penicillic acid and patulin do not inhibit the T3SS at 50 μM. A) Structures of penicillic acid (49) and patulin (50). B) Compared to the DMSO vehicle control, 50 μM of 49 or 50 does not inhibit T3SS-mediated secretion of CPG2.
Our screen of quorum sensing modulators revealed a correlation between polyphenolic compounds with gallic acid moieties as a structural class of T3SS inhibitors. Tannic acid (41) has a known mechanism of quorum sensing inhibition that it shares with trans-cinnamaldehyde (36). 41 most likely has a separate mechanism of action as a T3SS inhibitor, since 36 does not share the same efficacy. It is possible that 41, EGCG (9), and ellagic acid (40) act through related
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mechanisms given their structural similarities, but much more investigation is required to corroborate those claims.