LENGUAJE C# 41

In this work, we have presented the structural and functional analysis performed on MOMP and Omp50 from C. jejuni and Omp35 and Omp36 from E. aerogenes. Both bacteria are pathogenic for humans, causing gastrointestinal and respiratory infections, respectively.

MOMP was expressed in the native C. jejuni and its x-ray structure was solved. The structure showed that MOMP was a trimeric porin of 18 β-strand monomers. The obtained structure was compared to the structure of the recombinantly expressed MOMP, and despite differences in the LPS composition between E. coli and C. jejuni, we found the two structures to be identical. We then focused our attention on the calcium binding site found at the constriction zone. Electrophysiology analysis of MOMP behavior in the presence and absence of calcium led us to conclude that the metal ion is important for the stability of the structure, as it holds together a number of key loops (L3, L4 and L6) involved in its binding. Furthermore, in silico data obtained in collaboration with the University of Cagliari gave us some insight on the functional role of the calcium, specifically, highlighting the effect of the calcium on the ciprofloxacin permeation.

It is well established that the constriction zone of porins plays a crucial role in substrates permeation and thus any alterations at this level can lead to impaired diffusions [243]. In MOMP, the presence of the calcium neutralizes the negative charges of the residues D155, D120 and E288 in the eyelet, weakening the electric

observations, it would be useful to test several antibiotics, using for example liposome swelling assay, and to analyze their permeation rate with and without calcium. This would provide us with more data on how the calcium effects the translocation of different classes of antibiotics and thus improve the design of new ones.

Omp50, also a porin from C. jejuni, was also the object of study in this work.

Omp50 purification was first attempted in the native Campylobacter, but due to the low expression level and low purity we decided to use a recombinant system. Omp50 was successfully expressed in E. coli and the purified porin showed pore activity, when inserted in a lipid bilayer comparable to the protein natively expressed. Crystallization of Omp50 was, however, hard to achieve. Omp50 was also tested in order to find evidences of its proposed kinase activity. None of the experiments we performed, with either the recombinant or the native protein, suggested that Omp50 is able to auto-phosphorylize or is able to hydrolyze ATP. Hence, our data are in disagreement with that proposed by Corcionivoschi et al [207].

Lastly, we determined the X-ray structures of Omp35 and Omp36 from E. aerogenes, orthologues of the E. coli OmpF and OmpC, respectively. The structures we obtained showed that both Omp35 and Omp36 were homotrimer of 16 β- strands monomers. Omp35 and Omp36 structures also showed high levels of homology with OmpC and OmpF from E. coli, OmpE35/36 from E. cloacae and Ompk35/36 from K. pneumonie.

We analyzed the permeation of clinically relevant antibiotics through the OmpC- like (OmpC, Omp36, OmpE36, OmpK36) and the OmpF-like (OmpF, Omp35, OmpE35, OmpK36) porins using liposome swelling assay. We detected difference in the rate of permeation among the different antibiotics. Specifically, in both the OmpC-like and OmpF-like porins, we observed that the zwitterionic antibiotics were the most permeable and the negatively charged one the least permeable. Interestingly, the antibiotic Ertapenem, despite having a net negative charge, showed a rate of permeation over 80%. Further analysis, (in collaboration with the University of Cagliari), led us to conclude that it is not crucial to have a net positive charge to improve the rate of permeation but it is the ability of the molecule to align its dipole moment to the electric field at the constriction zone, instead, correlated with increased permeation.

E. aerogenes strains isolated from hospitalized patients have been shown to be resistant to a wide range of antibiotics [220]. Omp35 and Omp36, E. aerogenes major porins, play an important role in the multi-drug resistance phenomenon [235]. Variation in their expression of these two porins and mutation at the level of the constriction zone are the main mechanisms that lead to antibiotic resistance in E. aerogenes [235], [236]. The structural and functional analysis we presented in this work represent, a good starting point to better understand translocation of antibiotics through porins of pathogenic bacteria. In order to investigate how (and to what extent) these mutations impair the permeation of different classes of antibiotics when compared to the wild-type, such analysis could also be extended to Omp35 and Omp36 (and also their orthologues) that carry mutations at the level of the constriction zone.

Antibiotics resistance in Gram-negative bacteria has become a threat for public health worldwide. As already discussed in the general introduction of this work, porins play an important role in the translocation of antibiotics and thus in bacteria resistance. It is for this reason that we structurally and functionally characterized the major porins of two pathogenic bacteria. We believe that the data collected in this study will give a better understanding of drug translocation through porins and hence improve the rational design of high permeable antibiotics.