Benzylidene Acylhydrazides Inhibit Chlamydial Growth in a Type III Secretion- and Iron Chelation-Independent Manner

Abstract

e Chlamydiae are widespread Gram-negative pathogens of humans and animals. Salicylidene acylhydrazides, developed as inhibitors of type III secretion system (T3SS) in Yersinia spp., have an inhibitory effect on chlamydial infection. However, these inhibitors also have the capacity to chelate iron, and it is possible that their antichlamydial effects are caused by iron starvation. Therefore, we have explored the modification of salicylidene acylhydrazides with the goal to uncouple the antichlamydial effect from iron starvation. We discovered that benzylidene acylhydrazides, which cannot chelate iron, inhibit chlamydial growth. Biochemical and genetic analyses suggest that the derivative compounds inhibit chlamydiae through a T3SS-independent mechanism. Four single nucleotide polymorphisms were identified in a Chlamydia muridarum variant resistant to benzylidene acylhydrazides, but it may be necessary to segregate the mutations to differentiate their roles in the resistance phenotype. Benzylidene acylhydrazides are well tolerated by host cells and probiotic vaginal Lactobacillus species and are therefore of potential therapeutic value.

In vitro Antichlamydial Activity of 1,2,3,5-Tetrasubstituted Pyrrole Derivatives

“…HeLa and Hep2 cells purchased from ATCC were routinely cultured as described [12]. HeLa cells were used for C. trachomatis L2 and C. muridarum , and Hep2 cells were used for C. pneumoniae .…”

“…The antichlamydial effect of the compounds was evaluated as previously described and quantified by IC 50 between 1 and 32 μM [12, 15]. Cells directly seeded in 48-well plates were infected with Chlamydia at a multiplicity of infection of 0.2 inclusion-forming units (IFUs) per cell, and the individual compound was added simultaneously unless noted.…”

“…HeLa cells were used for C. trachomatis L2 and C. muridarum , and Hep2 cells were used for C. pneumoniae . EB stocks were prepared in SPG (sucrose-phosphate-glutamate) and stored at –80°C [12]. …”

“…However, incomplete antibiotic therapy can lead to Chlamydia persistence and long-term infection [10]. The lack of approved effective vaccines for human use and the occurrence of antibiotic-resistant Chlamydia [8, 11] necessitate the selection of novel antichlamydials from synthesized or natural chemicals [12-17]. …”

GrgA as a potential target of selective antichlamydials

“…For the above-mentioned reasons, it is important to identify new antichlamydial leads, particularly selective antichlamydial leads without adverse effects on either the host or other microbes, and identify their antichlamydial mechanisms. We have reported benzal-N-acylhydrazones (BAH) as novel antichlamydial leads capable of inhibiting all three Chlamydia species tested, C. trachomatis, C. pneumoniae and C. muridarum [25]. Significantly, at concentrations above minimal inhibition concentrations, BAH have no adverse effects on animal cells or vaginal lactobacilli [25].…”