Identification of the agr Peptide of Listeria monocytogenes

Abstract

Listeria monocytogenes (Lm) is an important food-borne human pathogen that is able to strive under a wide range of environmental conditions. Its accessory gene regulator (agr) 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 Lm agr operon is homologous to the Staphylococcus aureus system, which includes an agrD-encoded autoinducing peptide that stimulates expression of the agr 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 Lm using a luciferase reporter system in wildtype and agrD deficient strains, rational design of synthetic peptides and mass spectrometry. Upon deletion of agrD, luciferase reporter activity driven by the PII promoter of the agr operon was completely abolished and this defect was restored by co-cultivation of the agrD-negative reporter strain with a producer strain. Based on the sequence and structures of known AIPs of other organisms, a set of potential Lm AIPs was designed and tested for PII-activation. This led to the identification of a cyclic pentapeptide that was able to induce PII-driven luciferase reporter activity and restore defective invasion of the agrD deletion mutant into Caco-2 cells. Analysis of supernatants of a recombinant Escherichia coli strain expressing AgrBD identified a peptide identical in mass and charge to the cyclic pentapeptide. The Lm agr system is specific for this pentapeptide since the AIP of Lactobacillus plantarum, which also is a pentapeptide yet with different amino acid sequence, did not induce PII activity. In summary, the presented results provide further evidence for the hypothesis that the agrD gene of Lm encodes a secreted AIP responsible for autoregulation of the agr system of Lm. Additionally, the structure of the native Lm AIP was identified.

Signal Biosynthesis Inhibition with Ambuic Acid as a Strategy To Target Antibiotic-Resistant Infections

“…We report for the first time a structure of the S. saphrophyticus AIP and verify a number of reported AIP structures for other bacteria. The one case in which our AIP measurements do not agree with the literature is for the L. monocytogenes AIP, which was proposed by the Zetzmann et al (28) to be a cyclic pentapeptide, c(CysPhe-Met-Phe-Val), but detected in our studies to be a hexapeptide with an alanine N-terminal extension from the same cyclic macrocycle Ala-c(Cys-Phe-Met-Phe-Val). It should be noted that the identical hexapeptide that we propose as the L. monocytogenes AIP was detected in spent medium from the related species Listeria innocua (19).…”

Control of Staphylococcus aureus Quorum Sensing by a Membrane-Embedded Peptidase

“…Together, these findings expand the view of peptide-based signaling in S. aureus and highlight a newly uncovered regulatory input into virulence factor gene expression that dramatically affects virulence outcomes. Gram-positive pathogens of urgent clinical importance, including Clostridioides difficile and Enterococcus faecalis, as well as significant threats to food safety, such as Listeria monocytogenes, each harbor Agr systems that contribute to disease (73)(74)(75)(76)(77)(78)(79). Thus, this work provides insights into alternative pathways of regulation of the Agr system that may extend to other Gram-positive microbes of clinical importance.…”

VLP-based vaccine induces immune control of Staphylococcus aureus virulence regulation

“…For example, the food-borne pathogen Listeria monocytogenes uses a variety of communication systems to regulate virulence 70, 71 , including an agr locus and recently identified secreted AIP 72–76 . In L. monocytogenes , the agr system regulates over 650 genes contributing to virulence including ones involved in biofilm formation and host cell invasion 73 .…”