STIs remain a worldwide problem especially in light of their association with increased HIV acquisition [1
]. Vaginal microbicides have been proposed as an approach to reduce the spread of STIs [7
], an attractive concept since it would afford women the ability to protect themselves [21
]. A limited number of formulations of vaginal microbicides have been investigated in clinical trials, but in general results have been disappointing [22
]. Some of this is due to the fact that they were shown to damage the vaginal epithelium resulting in inflammation and ulceration that ultimately led to an increase in STIs. The work presented here lays the foundation for the development of a vaginal microbicide based on local iron withholding as a strategy to selectively attenuate pathogenic bacterial growth. The ability of pathogenic organisms to acquire iron is essential to their survival [24
]. However, the human host keeps iron tightly regulated and bound to limit the free supply available. We have explored the concept of introducing iron-sequestering compounds into this ‘tug-a-war’ between host and pathogen in order to favour the host and thus limit the infectivity of the pathogen.
The INPs used in this report were originally described to interfere with the type three secretion system (T3S) of several virulent Gram-negative bacilli including Yersinia
]. In an attempt to dissect the mechanism for this apparent effect on the Chlamydia
T3S, we explored their role as a chelating agent. Using different metal ions to reverse the inhibition of the genital pathogen C. trachomatis
serovar D, we found this effect to be specifically reversed by iron [13
]. Any effect these compounds had on host cell proliferation was also reversed by iron, leading us to hypothesise that these compounds were able to indirectly limit Chlamydia
growth by restricting available iron within the host. Chlamydia
, like most intracellular pathogens, is dependent upon host cell iron supplies [28
With the ultimate goal of developing a vaginal microbicide, we wanted to expand our findings to determine whether other genital bacterial pathogens would be inhibited by these compounds. LGV serovars of C. trachomatis
are more aggressive and invasive than the other genital serovars of C. trachomatis
. LGV strains are prevalent in underdeveloped nations, many of which also have a high prevalence of HIV. However, over the last decade there have been several reports of increasing numbers of LGV from countries in Western Europe and North America [30
]. The results obtained with the INPs for C. trachomatis
serovar L2 mirrored those that we had previously reported for serovar D. We also wanted to determine whether lactoferrin could compete with INPs since it is in high concentration in the vaginal tract and a key protein in maintaining iron homeostasis. Apo-lactoferrin did not interfere or compete with the inhibitory properties of the INP; only when it was saturated with iron in its holo form did it reverse the INP effect.
was also inhibited by the INPs and in general those compounds that were effective against C. trachomatis
also had activity toward N. gonorrhoeae
. This pathogen also requires iron for growth and has multiple mechanisms for acquiring iron, including high-affinity receptors for both transferrin and lactoferrin [14
]. We attempted to determine whether natural and laboratory strains lacking lactoferrin or transferrin receptors were inhibited by the INPs and, if so, could the inhibition be reversed by the iron-rich form of either lactoferrin or transferrin. Our results showed that INP inhibition could be reversed by an iron source with strains possessing the full complement of lactoferrin and transferrin receptor genes, but was not reversed with strains lacking one or both set of genes coding for these receptors. The reason for this is not clear, but resolving this may add to our understanding of the complex iron acquisition systems in N. gonorrhoeae
that have both unique features but also share some common pathways within the bacterial membrane. Results with N. gonorrhoeae
corroborated those obtained with Chlamydia
, suggesting that iron withholding is one, if not the main, mechanism by which the INPs attenuate the growth of these STI agents.
When developing a vaginal microbicide, candidate compounds should have minimal activity toward organisms that comprise the normal vaginal flora [34
]. The hydrogen peroxide-producing lactobacilli have been shown to be part of the innate vaginal defence, being essential in maintaining homeostasis of the vaginal tract [36
]. Since lactobacilli are somewhat unique in that they do not require iron for growth but instead rely on manganese, we reasoned that INPs should not have appreciable activity against these organisms [37
]. To test this hypothesis, both L. crispatus
and L. jensenii
, two of the lactobacilli most frequently recovered from the normal human vaginal tract [34
], were employed. None of the compounds tested had bactericidal activity towards L. crispatus
and L. jensenii
. Therefore, whilst the compounds were able to kill the genital pathogens, they had minimal to no effect on organisms considered essential for the maintenance of pH and bacterial balance in the vagina.
In the vaginal milieu many factors may compromise the activity of the INPs. Among them are the vaginal secretions, pH, lactoferrin and blood cells. In addition, a microbicide needs to be carefully formulated for ease of delivery and retention of activity. To begin to address the in vivo activity of the INP, a genital mouse model of a Chlamydia infection was employed. Here it was possible to demonstrate that the group of mice that were INP-treated had a significantly lower rate of infection compared with the control untreated group. These experiments demonstrate the proof of principle that INP 0341 can attenuate C. trachomatis infection in vivo.
In conclusion, the in vitro results presented here demonstrate that select INPs, in particular 0029 and 0341, inhibited both C. trachomatis and N. gonorrhoeae, had minimal to no effect towards organisms considered to be part of the normal vaginal flora, were not cytotoxic to HeLa cells and were active in vivo. These findings suggest that they are good candidates to consider further for formulation and continued testing as a vaginal microbicide.