Reversal of the Antichlamydial Activity of Putative Type III Secretion Inhibitors by Iron

Abstract

INPs, which are chemically synthesized compounds belonging to a class of acylated hydrazones of salicylaldehydes, can inhibit the growth of Chlamydiaceae. Evidence has been presented that in Yersinia and Chlamydia INPs may affect the type III secretion (T3S) system. In the present study 25 INPs were screened for antichlamydial activity at a concentration of 50 M, and 14 were able to completely inhibit the growth of Chlamydia trachomatis serovar D in McCoy and HeLa 229 cells. The antichlamydial activities of two of these INPs, INPs 0341 and 0400, were further characterized due to their low cytotoxicity. These compounds were found to inhibit C. trachomatis in a dose-dependent manner; were not toxic to elementary bodies; were cidal at a concentration of >20 M; inhibited all Chlamydiaceae tested; and could inhibit the development of C. trachomatis as determined by the yield of progeny when they were added up to 24 h postinfection. INP 0341 was able to affect the expression of several T3S genes. Compared to the expression in control cultures, lcrH-1, copB, and incA, all middle-to late-expressed T3S genes, were not expressed in the INP 0341-treated cultures 24 to 36 h postinfection. Iron, supplied as ferrous sulfate, as ferric chloride, or as holo-transferrin, was able to negate the antichlamydial properties of the INPs. In contrast, apo-transferrin and other divalent metal ions tested were not able to reverse the inhibitory effect of the INPs. In conclusion, the potent antichlamydial activity of INPs is directly or indirectly linked with iron, and this inhibition of Chlamydia has an effect on the T3S system of this intracellular pathogen.The type III secretion (T3S) system is known to be a potent virulence mechanism shared by several pathogenic bacteria, including the Chlamydiaceae (10). All T3S systems share common structural components, while their effector proteins and methods of gene regulation vary widely. Targeting and inactivating common T3S components has been proposed as a strategy to fight infections caused by pathogens that require a T3S system for virulence (13). In an attempt to identify such compounds, Kauppi et al. (13) used a chemical genetics approach to screen a large number of synthetic compounds for the ability to inhibit Yersinia T3S gene expression. They identified compounds with the general structure of an acylated hydrazone of salicylaldehydes that were able to inhibit the pathogenic Yersinia T3S system, neutralizing the virulence while not affecting the growth of the organism (13, 17).We have previously reported that INP 0400 was able to inhibit the growth of Chlamydophila pneumoniae (27a). We reported that this compound inhibited C. pneumoniae development in a dose-dependent manner, was not cytotoxic, was not directly toxic to elementary bodies (EBs), and was effective at inhibiting the growth of Chlamydia trachomatis and Chlamydia muridarum. The appearance of inclusions at lower concentrations (Ͻ20 M) of INP 0400 resembled the appearance of inclusions seen in persistent infections resulting...

Mutations in hemG Mediate Resistance to Salicylidene Acylhydrazides, Demonstrating a Novel Link between Protoporphyrinogen Oxidase (HemG) and Chlamydia trachomatis Infectivity

“…Because the growth inhibition of Chlamydia by SAHs is reversed by exogenous iron, it has been postulated that iron chelation by SAHs may be responsible for their antichlamydial properties (27,28). However, the SAH INP0406, which does not inhibit T3S, retains iron-chelating properties, yet it cannot inhibit Chlamydia growth (28). In this study, we have investigated how SAHs affect Chlamydia trachomatis development and secretion, in both the presence and absence of exogenously added iron.…”

“…Subsequent studies indicate that secretion or localization of predicted T3S effectors is altered by SAHs (19,(23)(24)(25)(26). Because the growth inhibition of Chlamydia by SAHs is reversed by exogenous iron, it has been postulated that iron chelation by SAHs may be responsible for their antichlamydial properties (27,28). However, the SAH INP0406, which does not inhibit T3S, retains iron-chelating properties, yet it cannot inhibit Chlamydia growth (28).…”

Anti-virulence Strategies to Target Bacterial Infections

“…The two main virulence factors of V. cholerae are cholera toxin (CT) and toxin co- Hudson et al 2007;Negrea et al 2007), Y. pseudotuberculosis (Nordfelth et al 2005), Chlamydia (Bailey et al 2007;Muschiol et al 2006;Slepenkin et al 2007;Wolf et al 2006), E. coli (Tree et al 2009) and Shigella (Veenendaal et al 2009). The mode of action seems to be via the inhibition of needle subunit secretion or assembly, but recent analysis further demonstrated that SAHs significantly repressed the expression of main regulators of the T3SS machinery and modulate the function or activity of several protein targets (Layton et al 2010;Tree et al 2009;Wang et al 2011).…”

“…The mode of action seems to be via the inhibition of needle subunit secretion or assembly, but recent analysis further demonstrated that SAHs significantly repressed the expression of main regulators of the T3SS machinery and modulate the function or activity of several protein targets (Layton et al 2010;Tree et al 2009;Wang et al 2011). An involvement in iron chelation (Layton et al 2010;Slepenkin et al 2007) and a role for the metabolism (Wang et al 2011) The Brucella abortus protein VirB8 is an essential component of the T4SS and indispensible for its assembly. B8I-2 was identified to inhibit the dimerization of VirB8 as well as the interaction with other VirB proteins (Paschos et al 2011).…”

Solving the Supply of Resveratrol Tetramers from Papua New Guinean Rainforest Anisoptera Species That Inhibit Bacterial Type III Secretion Systems

“…15,16 It has been shown that the salicylidene acylhydrazide class of inhibitors can attenuate virulence without affecting bacterial growth in a number of bacterial species. [17][18][19][20][21][22][23] Several studies suggest that one of the key advantages of using virulence systems as targets for novel anti-infectives is a low probability for development of resistance. 4,24 Virulence blockers target extracellular processes and are therefore less likely to be impacted by drug efflux mechanisms which have transmembrane and cytoplasmic domains.…”