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Coli4Lan - Better systems to produce two ­peptide lantibiotics with diverse structures completely in vivo using Escherichia coli and establishment of their mode of action.
Coordinator - Tânia Caetano
Programme - EXPL/BBB-BEP/0496/2012
Execution dates - 2013-05-01 - 2014-10-31 (18 Months)
Funding Entity - Fundação para a Ciência e Tecnologia
Funding for CESAM - 44.856 €
Total Funding - 44.856 €
Proponent Institution - Universidade de Aveiro
Participating Institutions
Tecnhical University of Berlin
University of Bonn

The progressive worldwide increase and spread of drug­resistant pathogens like the methicillin resistant Staphylococcus aureus (MRSA) and vancomycin intermediate Staphylococcus aureus (VISA) makes the identification of novel antibiotics directed against new targets a high priority. Lantibiotics are a diverse and promising class of antimicrobial peptides produced by a variety of bacteria. They display a broad spectrum of activity to a number of pathogenic bacteria including MRSA, VISA and vancomycin resistant Enterococcus (VRE) among others. Therefore lantibiotics are promising candidates to develop therapeutic agents against these microorganisms. However, to fully realize their potential, it is first necessary to understand their biology and, in particular, to elucidate their structure­function relationships, production, immunity, regulation and mode of action. Initial in vivo trials with animal models have demonstrated the success of lantibiotics in treating infections caused by S. pneumoniae, and MRSA and in preventing tooth decay and gingivitis. One of the most studied lantibiotics is nisin, a class I lantibiotic produced by certain Lactococcus lactis strains with broad activity spectrum against Gram+ pathogens, including S. pneumonia. It has a long record of safe industrial use as a food preservative. The development of methods to produce new lantibiotic variants has enabled the investigation of the structure­activity relationships of these compounds and hence an evaluation of this hitherto underexploited class of natural products as a source of potential therapeutic drug candidates. The study of two­ peptide lantibiotics represents a relatively new field of science and consequently less exploited. Bacillus licheniformis I89, a bacteria isolated from a hot spring in the Azores islands, produces the two ­peptide lantibiotic lichenicidin. In this class of lantibiotics, two peptides are produced (α and a β)that act synergistically to exert full antibacterial activity. The lichenicidin spectrum of activity includes several pathogens such as S. aureus (including MRSA), Enterococcus faecium, Haemophilus influenza and Listeria monocytogenes. Lichenicidin was the first lantibiotic of its class to be produced totally in vivo using the Gram negative­ E. coli. This was a major breakthrough, since the system allows the manipulation of lantibiotic peptides using simpler and less time consuming procedures. For instance, the system enabled the production in vivo of some a­variants harboring non­canonical amino acids. Coli4Lan will explore the aspects necessary to understand the structure­-activity function relationship of two peptide lantibiotics and also explore their bioengineering, using lichenicidin as a model. To that end, several E. coli expression systems will be tested for the output of lichenicidin peptides. The best system will be used to generate new molecules using random mutagenesis. The variants showing increased or abolished bioactivity will be selected for nucleotide sequencing and LC­-ESI-MS/MS analysis. Also, the diversity of lichenicidin will be enriched by the introduction of non­proteinogenic amino acids in their structure by the methodology optimized in the present proposal. Bioactivity of the engineered peptides will always be accessed. The impact of the substitutions in the peptide structure will be investigated also by LC­-ESI-­MS/MS analysis. Secondly, the mechanism of action of lichenicidin will be investigated. It is expected that lichenicidin has the same mode of action of the two­ component lantibiotic lacticin 3147. It seems that the α peptide forms a complex with lipid II, inhibiting cell wall synthesis and the β peptide forms a pore in the bacterial membrane. However lichenicidin and lacticin 3147 structures are quite different. Moreover, the lichenicidin α peptide is closely related to mersacidin lantibiotic. However, it is necessary 13 times more of α peptide (without the β peptide) compared to mersacidin to inhibit the growth of M. luteus indicator strain. Therefore, we are also interested to understand the biological explanation behind these facts. The project will bring together a synergistic team with the necessary know-­how that guarantees the successful development of the proposed tasks: microbiology, molecular biotechnology and biology (T. Caetano and S. Mendo, UA and G. Bierbaum, University of Bonn), and analytical chemistry and biochemistry (R. Suessmuth, TUBerlin). 

Members on this project
Sónia Mendo
Tânia Caetano
Principal Investigator

CESAM Funding: