Membrane proteins and virulence pathways: new approaches to design antimicrobial peptides

Abstract

In the last two decades many studies have shown that lipid composition of biologic membranes as well as their structure have a primary role in cellular signaling. In particular, the research group in which I have worked for my PhD thesis, has demonstrated that the primary sensor of temperature variations and, in general, of other kinds of stress is localized in the membrane. The imbalance in the membrane lipid/protein ratio of the pathogen Salmonella Typhimurium, due to over-expression either of the protein 12-desaturase of the cyanobacterium Synechocistis or individually expressed membrane regions of the enzyme, caused major changes in the MPS (Membrane Physical State) and a significant impairment of the heat shock response (Porta et al. 2010). These results highlighted the possibility to identify molecules that, interacting with specific membrane lipids, can modify the functionality of membrane itself and consequently the biochemical/genetical properties of the whole cell. About thirty years ago, antimicrobial peptides (AMPs), molecules that are part of an ancient mechanism of the innate immunity of all Metazoa and plants, have been identified. However, from the over 1,000 identified peptides, no consensus sequence has been recognized and thus, as to-day, the possibility to predict and design an active AMP is still not feasible. Starting from these assertions, through the study of membrane proteins and virulence pathways, my project has focused on the rational design of new AMPs and identification of key rules that allow the correct insertion of short peptides inside the phospholipid bilayer. The amino acid sequence of the first trans-membrane region (p200) of the 12-desaturase, that was shown to have antimicrobial activity (AMA), has been reduced to identify the minimal active sequence. Thus, three peptides, called A2/I2, A3/I3 and A4/I4, have been designed and their corresponding oligos inserted into the vector pBAD. The plasmids were used to transform S. Typhimurium LT2 strain. Transformants were then tested in terms of growth and survival outside and inside host cells (murine macrophages, ϕ). The results have shown that trimming of p200 determined a reduction of its AMA. Further, it is well acknowledged that almost all, if not all, membrane proteins have more lysine and arginine residues on the sides of trans-membrane protein domains, and these residues are necessary for the insertion of proteins into the membrane bilayer. Thus, to verify the importance of this rule, called “positive inside rule”, a scrambled sequence of p200 has been designed, maintaining unchanged the five aminoacids preceding the trans-membrane region. From the growth curve and in vitro infection it was observed that p200 scrambled conserved the same activity of the original sequence, as expected. Studying the virulence mechanisms of Salmonella survival inside host cells, the protein PhoQ of the two components system PhoP/PhoQ has been identified as the protein mostly involved in sensing and responding to ϕ environment and as a likely target to develop a new AMP. Based on our model, an AMP has AMA if it can interfere with lipids/proteins interaction of a specific domain and if this domain has a fundamental role in the pathogen’s survival and/or growth. Thus, the original sequence of the first trans-membrane region of PhoQ has been modified, obtaining two potential AMP candidates. The two resulting sequences, called NUF and STA, have been cloned and expressed in Salmonella. In particular, the peptide NUF has been very promising in determining the decrease of Salmonella persistence inside ϕ. [edited by Author]