AUTHOR=Zetzmann Marion , Sánchez-Kopper Andrés , Waidmann Mark S. , Blombach Bastian , Riedel Christian U. 

TITLE=Identification of the agr Peptide of Listeria monocytogenes

JOURNAL=Frontiers in Microbiology

VOLUME=7

YEAR=2016

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2016.00989

DOI=10.3389/fmicb.2016.00989

ISSN=1664-302X

ABSTRACT=<p><italic>Listeria monocytogenes</italic> (<italic>Lm</italic>) is an important food-borne human pathogen that is able to strive under a wide range of environmental conditions. Its accessory gene regulator (<italic>agr</italic>) system was shown to impact on biofilm formation and virulence and has been proposed as one of the regulatory mechanisms involved in adaptation to these changing environments. The <italic>Lm agr</italic> operon is homologous to the <italic>Staphylococcus aureus</italic> system, which includes an <italic>agrD</italic>-encoded autoinducing peptide that stimulates expression of the <italic>agr</italic> genes via the AgrCA two-component system and is required for regulation of target genes. The aim of the present study was to identify the native autoinducing peptide (AIP) of <italic>Lm</italic> using a luciferase reporter system in wildtype and <italic>agrD</italic> deficient strains, rational design of synthetic peptides and mass spectrometry. Upon deletion of <italic>agrD</italic>, luciferase reporter activity driven by the P<sub>II</sub> promoter of the <italic>agr</italic> operon was completely abolished and this defect was restored by co-cultivation of the <italic>agrD</italic>-negative reporter strain with a producer strain. Based on the sequence and structures of known AIPs of other organisms, a set of potential <italic>Lm</italic> AIPs was designed and tested for P<sub>II</sub>-activation. This led to the identification of a cyclic pentapeptide that was able to induce P<sub>II</sub>-driven luciferase reporter activity and restore defective invasion of the <italic>agrD</italic> deletion mutant into Caco-2 cells. Analysis of supernatants of a recombinant <italic>Escherichia coli</italic> strain expressing AgrBD identified a peptide identical in mass and charge to the cyclic pentapeptide. The <italic>Lm agr</italic> system is specific for this pentapeptide since the AIP of <italic>Lactobacillus plantarum</italic>, which also is a pentapeptide yet with different amino acid sequence, did not induce P<sub>II</sub> activity. In summary, the presented results provide further evidence for the hypothesis that the <italic>agrD</italic> gene of <italic>Lm</italic> encodes a secreted AIP responsible for autoregulation of the <italic>agr</italic> system of <italic>Lm</italic>. Additionally, the structure of the native <italic>Lm</italic> AIP was identified.</p>