Development of new antimicrobial peptides and peptidomimetics and mechanism of resistance to peptide antibiotics

Abstract

Nowadays in the world there is a very big problem associated with two factors related to each other. The first factor is the increase in the resistant of certain bacteria, especially the bacteria from the ESKPAE group. The second factor is the dramatically decrease of new antibiotics approved by the FDA. These two problems show that there is an urgent need to find new antibiotics active against these resistant bacteria.

In this thesis, we have tackled two different topics closely related in the race to find new antimicrobials. The first topic tackled was the knowledge of the mechanism of resistance of Gram-negative (A. nosocomialis) and Gram-positive (S. mitis) bacteria. The two antibiotics studied were peptides, colistin and daptomicin, these two peptides are resistant to A. nosocomialis and S. mitis, respectively. Both peptides had a similar mechanism of action related to the membrane of bacteria, therefore we are going to focus just in the modifications in the membrane of the strains resistant to the antibiotic peptides. In S. mitis it was observed, using proteomic techniques, that two proteins related with the membrane were observed. These two proteins has some homologue domains to several proteins involved in daptomycin resistant in S. aureus and Enterococci. In A. nosocomialis, the bacteria showed a high tolerance to colistin, and at 8 mg/L an inflexion point is observed. In this inflexion point, the MIC of colistin, against bacteria increase from <0.1 mg/L to 128 mg/L. These bacteria with high resistance to colistin showed no production of LPS due to the fact that mutations and a stop codon in lpxD gene were observed. This gene is involved in the synthetic pathway of the LPS.

Apart from the understanding of the mechanism of action of peptide antibiotics, we have proposed several peptides and peptidomimetics against Acinetobacter species. We have used two different approaches.

The first approach is the normal approach, testing several peptides or peptidomimetics against the desired bacteria. The first peptides tested were commercially available, and we found mastoparan that was active against both colistin-susceptible and colistin-resistant A. baumannii. This peptide was optimized specially in terms of stability in human serum. After several in vivo trials we did not observe any activity of the peptides tested, however we found a very strong bindoing with some proteins present in the human serum. Frog skin secretions peptides were also tested against colistin-susceptible and colistin-resistant Acinetobacter species, the results obtained were really interesting specially in two peptides. The last peptides tested were peptidomimetics. These peptidomimetics act as an antimicrobial peptide, with two different faces, one face with a cation charge and the other very amphipathic. These peptidomimetics are analogues from the original structure of cholic acid, the structure was modified in order to have antibacterial activity that was found in colistin-susceptible and colistin-resistant A. baumannii, K. pneumonia and P. aeruginosa.

The second approach was completely different, in this case the idea was to block the virulence of bacteria caused by OmpA. This protein is involved in the adherence between bacteria and host cells, therefore several hexacylcic peptides were synthesized in order to inhibit the action of this protein. The results obtained were satisfactory, obtaining good activity in both in vitro and in vivo.

Actualment al mon hi ha un greu problema derivat de dos factors relacionats, el primer factor es el increment de la resistència, especialment del bacteris del grup ESKAPE. El segon factor es la disminució dràstica en el nombre d’antibiòtics aprovats per la FDA. Aquests dos problemes fan que hi hagi una urgència per trobar nous antimicrobians efectius en front d’aquestes soques resistents.

En aquesta tesi hem abordat dos temes diferents però que estan relacionats a la hora de trobar nous antibiòtics. El primer tema abordat es el de conèixer a fons els mecanismes de resistència de certs antibiòtics, en aquest cas peptídics, en front diferents tipus de soques tant Gram-positives (S. mitis) com Gram-negatives (A. nosocomialis). Els dos antibiòtics peptídics pels que s’ha estudiat la resistència son daptomicina i colistina, en front de S. mitis i A. nosocomialis respectivament. Ambdós pèptids actuen a nivell de membrana, per tant ens centrarem en veure les modificacions produïdes en els soques resistents. Per part de S. mitis resistent a daptomicina, es pot veure una sobreexpressió de dues proteïnes que tenen dominis homòlegs amb altres proteïnes involucrades en la resistència a daptomicina en altres bacteris. En la resistència a colistina es pot apreciar com les soques resistents d’A. nosocomialis presenten una deficiència del LPS.

També hem proposat diferents alternatives com a nous antibiòtics, en aquest cas en front de soques A. baumannii. Dos tipus d’aproximacions van ser utilitzades, la primera, i mes clàssica es la de trobar nous antimicrobians, vàrem trobar mastoparan i va diferents paràmetres van ser optimitzat però sense obtindré bons resultats in vivo. També es van provar diferents pèptids provinents de les secrecions de les granotes, presentant bona activitat en front soques d’Acinetobacter, i per últim, les ceragenines, anàlegs del àcid cólic, que tenen bona activitat en front de totes les soques tant colistina sensibles com colistina resistents en Gram-negatius.

La segona aproximació es buscant pèptids capaços d’inhibir l’adherència entre el bacteri i la cèl•lula del hoste bloquejant l’acció de la proteïna OmpA. S’ha trobat un pèptid amb bona activitat fins i tot in vivo.