Our data show that INPs do not inhibit the entry of Chlamydia into host cells. The efficiency of bacterial invasion has been investigated with two Chlamydia species, C. trachomatis L2 and C. caviae GPIC, and it was not modified in the presence of the drug. The normal recruitment of Rac, Cdc42 and Arf6 to C. caviae GPIC entry sites in the presence of INPs further indicates that INPs do not interfere with the mechanism of Chlamydia invasion.
Previously, we had reported a partial effect on Chlamydia trachomatis L2 entry in the presence of INP0400 . This was based on the observation that treatment of the cells with 40 μM INP0400, for the first 3 hours of infection, resulted in a 40% reduction in the percentage of infected cells, compared to non-treated cells. We interpreted these data as a partial effect of the drug on bacterial entry. However, since we demonstrate here that Chlamydia invasion is not impaired by treatment with INPs, a more likely explanation is that other early events, following Chlamydia entry, are required for the onset of infection and are susceptible to the drugs. Indeed, Chlamydia genes expressed early in infection are needed to create a permissive environment for successful bacterial replication . In particular, some of the Inc proteins, which are T3S substrates, are transcribed very early during infection and can be detected in the inclusion as early as 2–4 h p.i. .
In support of our results, Wolf et al. and Slepenkin et al. had reported that they were unable to inhibit C. trachomatis L2 entry in presence of INPs [18, 19]. In the study of Wolf et al. the effect of drug on the EB translocated protein TARP, which probably plays a central role in the internalization process of C. trachomatis was examined. Upon host cell attachment, TARP is secreted in a type III dependent manner by Chlamydia trachomatis and becomes rapidly phosphorylated. Wolf et al., were unable to inhibit this early tyrosine phosphorylation of TARP in cells treated with another compound of the same family of INPs . The lack of effect of INPs, which have been identified and described as type III secretion inhibitors, on Chlamydia entry is therefore surprising. Recent reports on the mode of action of INPs which we would like to discuss here, raise the question whether these drugs interfere with the actual translocation process of T3S substrates or rather inhibit at the level of transcription of T3S associated genes or assembly of the T3S machinery.
Earlier studies suggested that INPs might affect the translocation of type III substrates per se, and indeed, in Yersinia, careful analyses suggest that it is the case . So far, the efficiency of INPs at blocking T3S in Chlamydia has been shown only for substrates secreted by RBs, and their target might be missing in EBs. In favour of this hypothesis is the observation that Chlamydiae genomes encode two homologues for the Yersinia lcrH chaperone for T3S system structural components, lcrH-1 and lcrH-2 . These genes are in clusters that are differentially expressed during the developmental cycle. It was recently shown that transcription of lcrH-1, which is expressed late in the cycle, when EBs are forming, was inhibited by INP0341, while transcription of lcrH-2, which is expressed earlier in the cycle, was not . Functional differences in the T3S apparatuses of EBs and RBs might therefore explain a difference in sensitivity to the type III secretion inhibitors. This would be consistent with our results and could explain the lack of effect of INPs on Chlamydia entry.
As an alternative, it is possible that INPs have a different mode of action on Chlamydia development than they have on Yersinia, and do not block the translocation of effectors per se. Importantly, the effect of INPs on chlamydial development is fully reversed by the addition of iron , while their inhibitory effect on Yersinia T3S is not (personal communication from Innate Pharmaceuticals AB). In this case, INPs might affect one of two requirements for effector protein secretion: (a) the assembly of functional secretion apparatuses or (b) the synthesis of the substrates recognized by the secretion machinery.
By acting on the formation of type III secretion apparatuses, INPs would only be effective when introduced while the apparatuses are being made, i.e. in the intracellular multiplication phase of Chlamydia development. In support of this hypothesis, recent data strongly suggest that, in the case of Shigella, INPs block assembly of the type III secreton . In Shigella, INPs were only effective at inhibiting host cell invasion when added during growth, rather than during the infection step.
If, on the other hand, INPs inhibited the synthesis of type III secretion substrates, they would not affect entry either, because the effectors needed for this step are not newly synthesized during entry. INP0400 has been shown to inhibit the secretion of IncA and IncG proteins, which are produced during RB proliferation, and are rapidly translocated upon synthesis, as they are only weakly detected in RBs [25, 26]. In contrast, Tarp and other potential T3S effectors participating in the entry event are at least partially stored in the RBs to be released by the EB form upon infection. Recent data show that the expression of some of the T3S genes (including genes coding for the secretion apparatus) is down-regulated by INP0341 . Similarly, the Yersinia T3S system is down-regulated upon bacterial exposure to INPs  and it has also been shown in Salmonella that these compounds cause transcriptional silencing of the Salmonella pathogenicity island 1 . It should be noted that if INPs act at a transcriptional level in Chlamydia, they might not affect the secretion of all effectors to the same extent. Therefore, at this stage INPs should only be used cautiously to assess the mechanism of secretion of a given chlamydial protein.
Down-regulation of transcription could perhaps also be due to feedback inhibition resulting from blocking T3S activity . If, in Chlamydia, either the transcription of T3S associated genes or the assembly of the T3S machinery are inhibited, addition of the drugs at the end of one cycle of infection is expected to affect the next round of infection. This is exactly what was observed when looking at the progeny of C. trachomatis infected cells treated with INP0341 24 hours post infection . In this experiment, although the inclusions formed upon late INP0341 treatment were as abundant as in control cells, there was a decrease in the infectious progeny, suggesting that EBs formed in the presence of INPs might be defective in their ability to secrete type III effectors. However, due to the asynchronicity of the Chlamydia developmental cycle, we can not definitively rule out that the decrease in the formation of infectious EBs when the drug is added late in the cycle is not due to the now well documented reduction of RB multiplication upon INP treatment.