ACCIÓN ANTAGÓNICA DE Bacillus subtilis PARA CONTROLAR Rhizoctonia sp IN VITRO E IN VIVO

7.1 Conclusion

The aim of this project was to develop pro-HDPs for CF with two main goals: the first was to demonstrate the utility of the NE-targeted oligoglutamic acid model for HDPs in CF, the second was to bring one of these pro-HDP therapeutics closer to the clinic by evaluating its use in vitro and in vivo. The sum of the four results chapter has achieved these goals.

Modification of five HDPs: Bac8c, HB43, P18, WMR, and WR12 has produced pro-HDPs with NE-dependent activity. In addition, reduction in cytotoxicity has been shown in four of these. The application of CF BAL fluid underlines the potential of the pro-HDPs to work in the CF lung and

reinforces the potential for targeting prodrugs to NE. Some of the other enzymes relevant to CF such as PE and CG have been tested against the AAAG linker but the lack of proteolysis coupled with the concentrations several times lower than NE (which represents approximately 90% of the protease load) would render them inferior targets, should an alternative linker be used. The fact that the low levels of mNE found in the mouse BAL fluids were insufficient for cleavage emphasises this; the highest concentration

found (0.393µg/ml) might be considered a high for another enzyme. While

the pro-HDP model is versatile, and could be applied to other disease models such as cancer with a suitable linker, the requirement for an extracellular protease of sufficiently high concentration must be carefully considered. An appropriate combination of infectious disease/cellular dysfunction and co-localised high levels of protease would be an attractive target for HDPs. With this combination also comes the consideration of what the fate of the active sequence in the same conditions will be: it is shown that

for CF, D-amino acids can provide stability in hostile proteolytic conditions

where L-amino acid peptides would be quickly degraded and that this is more

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The development of pro-WMR not only represents a proof-of-concept for pro- HDPs, it has the potential to be used as an antibacterial therapeutic in its own right. CF provides the required high levels of protease but also an uncomplicated means of delivering the peptide. It is demonstrated here that the nebulised delivery of pro-WMR is facile and that the additional glutamic acid residues are not antagonist to the generation of a respirable aerosol. The % deliverable to a model human lung compares well to other CF therapeutics. In other disease models this delivery may not be as

straightforward. For example, injection of pro-HDPs for cancer introduces the complications of circulatory proteases and the exposure of off-target sites to the HDPs. In addition, the local drug concentrations with intravenous delivery are not as high as with inhalation.

The results of the in vivo tests complement the in vitro data and demonstrate the reduction of host toxicity afforded by the pro-HDP modification. This was evident in the significant differential in outcome between AAG-P18 and the pro-HDP and also in the reduction in weight loss and cytokine release afforded by pro-WMR compared to AAG-WMR treatment. The trend is also for the less-cytotoxic AAG-WMR to reduce infection with P. aeruginosa in contrast to AAG-P18, which may facilitate bacterial infection by inducing lung damage, despite the fact that it has a lower MIC against the bacterium. This illustrates how the evaluation of in vitro antimicrobial activity and cytotoxicity may be used to predict which HDPs will perform better in vivo.

In many respects, while pro-HDPs show great promise in treating CF

infections, CF itself is ideally suited for pro-HDPs. The predominance of one particular pathogen, P. aeruginosa, a high concentration of co-localised enzyme, NE, and availability for local delivery simplifies the design and delivery of the peptides. We demonstrate with pro-WMR the potential for therapy of CF, killing the pathogen and targeting the enzyme. In other biological systems the difficulties in delivery and enzyme-targeting may be compounded, but the progress made with these peptides in CF should

advise design. More-labile linkers can be developed for enzymes that appear in lower concentrations and modern drug-delivery systems such as

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these considerations are thankfully not necessary and the present work validates that pro-HDPs should form part a potential strategy for developing new therapeutics for this disease.

7.2 Future Plans

The most important set of experiments that must be carried out to advance the results of this project is the expansion of the animal study. While many interesting conclusions could be drawn, more work beyond analysis of the current data should be ideally undertaken. Some of the treatment groups (especially AAG-P18) were too small for complete observations to be made. For example, the trend was for AAG-WMR to produce a 2 log reduction in CFU/ml, a figure which is consistent with some of the more active in vivo HDPs, but more subjects are required to bring the result to statistical significance. In addition, while it seems that the mouse model may not be suitable for testing a NE-dependent prodrug, they must first be tested in a chronic infection model before this can be fully ruled out. The literature is conflicting on whether enough free mNE can be induced, but the most optimistic model we could adopt is to infect mice with bacteria embedded in agarose beads and treat with pro-HDP several days later (256).

There are several other interesting directions that may be taken to evaluate pro-HDPs for CF:

7.2.1 Expanded clinical isolate groups

The study has focused on reference strains and four clinical isolates of P. aeruginosa. The bacterium is usually initially acquired from the environment but may sometimes be transferred from patient-to-patient, leading to

genotypically-indistinguishable and potentially-resistant strains passing between individuals. Infection with one strain for example, Liverpool Epidemic Strain, can lead to rapid loss of lung function and increased mortality while another strain resistant to colistin has been reported in a paediatric CF clinic (273). Classical phenotypic methods usually applied to

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other bacteria such as serotyping are less discriminatory for P. aeruginosa, with the correlation with genotype being less clear (274). While clonal and epidemic strains are typically more resistant, they often do not show

distinctive antibiograms which, along with other phenotypes, such as being mucoid, have been seen to vary over time (275). Despite the availability of fast and cost-effective PCR-based genotyping methods, the more laborious pulsed-field gel electrophoresis (PFGE) remains the gold standard for typing P. aeruginosa. This is useful for examining local populations but less so for cross-sectional studies (276). The demonstration of pro-HDP efficacy against well-characterised and representative P. aeruginosa clones that are

circulating in the Irish CF population would be important. Nearly 30 CF isolates have so far been collected and characterised from patients in

Beaumont Hospital. After collection and genotyping of the CF P. aeruginosa isolates a comprehensive review of the susceptibility to the HDPs could be carried out using MIC assays. Recently, an analysis of the activity of WR12 against 100 CF P. aeruginosa strains was carried out, demonstrating the lack of intrinsic bacterial resistance to the peptide in the wider population (210).

7.2.2 Quorum sensing effects

Another interesting direction would be the effects of these HDP prodrugs on P. aeruginosa virulence factors at sub-inhibitory concentrations, specifically factors associated with Quorum Sensing (QS). QS describes the ability of populations of bacteria to coordinate gene expression in response to their surroundings, relying on the transmission of small diffusible signal molecules (277). Virulence factors such as rhamnolipids, elastases, and pyocyanin have been used before as indicators of QS activity and could be the subject of investigation in this study (277-282). QS is associated with much of the pathogenicity of P. aeruginosa infections and so any reduction would be beneficial (282). It has previously been shown that structurally-unrelated compounds such as azithromycin, ceftazidime, ciprofloxacin, and certain sesquiterpene lactones have the ability to reduce QS activity, and it has been suggested that the diversity of the compounds may indicate that membrane disruption is the source of the anti-QS activity. On the other hand, other

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antibiotics do not have anti-QS activity, including tobramycin (277, 280). The effect of HDPs, which are known to disrupt membranes, on these virulence factors has not been extensively studied. The factors for investigation, elastase (LasB protease), rhamnolipid, and pyocyanin production, are associated with increased QS activity. As many reference strains can lose QS characteristics over time, clinical isolates would be used for comparison (281). The measurement of many of these factors is relatively

straightforward. Elastase can be measured by incubating filtered culture supernatants in elastin congo red and measuring absorbance at 495nm, correcting for pigments already produced by P. aeruginosa (278, 281). Rhamnolipids can be measured by culture on M8 salt-based agar and the measurement of the blue rhamnolipid halo produced by the cultures (282). Pyocyanin can be measured at 520nm after extracting it from broths with chloroform and 0.2N HCl (279). The expression of specific QS genes after exposure to the HDPs may also be investigated using reverse transcriptase PCR (277).

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