Investigating Lactococcus lactis MG1363 Response to Phage p2 Infection at the Proteome Level

Abstract

The cellular response of Lactococcus lactis MG1363 to infection by the virulent phage p2 was characterized through labelfree quantitative proteomics. Combined with a targeted approach, we identified the highest proteome coverage for phage p2, a model for the most predominant group of lactococcal phages in the dairy industry worldwide. Four bacterial gene candidates possibly involved in phage infection were disrupted through specific knockouts to investigate their importance. Deletion of gene llmg_0219 (unknown function) resulted in a resistance to phage p2. Graphical Abstract Highlights• The proteomes of L. lactis MG1363 and phage p2 at different stages of infection were characterized.• 16% (226/1412) of the bacterial proteins detected were unique to infected cultures.• A targeted approach using synthetic peptides improved the coverage of phage p2 proteome.• By means of proteogenomics, we uncovered a conserved phage protein coded by a previously unannotated gene.• Deletion of the bacterial gene llmg_0219 (unknown function) impedes phage p2 infection.

A Lactococcal Phage Protein Promotes Viral Propagation and Alters the Host Proteomic Response During Infection

“…For phage infection, growth medium was supplemented with 10 mM CaCl 2 (GM17+Ca). Phages were amplified from frozen glycerol stocks as previously described [10]. Phage titers were usually determined by double layer plaque assays [21] with plates containing a bottom layer of solid GM17+Ca and a top layer of GM17+Ca supplemented with 0.75% (w/v) agar.…”

“…The phage efficiency of plaquing (EOP) was calculated by dividing the phage titer obtained on the resistant strain by the phage titer obtained on the sensitive strain. To determine if a phage resistance phenotype was due to a deficiency in adsorption, phage adsorption assays were performed as previously described [10,22]. For phage growth curves, L. lactis MG1363 cultures were followed for 50 min post-infection with a starting MOI of 0.05, as described elsewhere [23].…”

“…The proteomic analyses of phage infections were conducted as previously described [10]. Briefly, time-course infections of L. lactis MG1363 by phage p2∆47 were performed in biological triplicate.…”

“…It infects the well-studied laboratory strain L. lactis MG1363, a plasmid-free derivative of the dairy starter strain NCDO712 [8,9]. Phage p2 dsDNA genome consists of 27,595 bp and 50 annotated open reading frames (ORFs) [10]. The ORFs are organized into three large gene clusters, chronologically transcribed upon infection ( Figure 1).…”

“…We investigated the type of resistance mechanisms in BIMs obtained after being challenged with phage p2∆47 or p2. Furthermore, we used label-free quantitative proteomics to compare the proteotypes of L. lactis MG1363 infected by either phage p2∆47 or p2 [10]. We showed that the presence of ORF47 prevents L. lactis MG1363 to rapidly adapt and become resistant to phage p2.…”