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

Abstract

Phages are viruses that specifically infect and eventually kill their bacterial hosts. Bacterial fermentation and biotechnology industries see them as enemies, however, they are also investigated as antibacterial agents for the treatment or prevention of bacterial infections in various sectors. They also play key ecological roles in all ecosystems. Despite decades of research some aspects of phage biology are still poorly understood. In this study, we used label-free quantitative proteomics to reveal the proteotypes of Lactococcus lactis MG1363 during infection by the virulent phage p2, a model for studying the biology of phages infecting Gram-positive bacteria. Our approach resulted in the high-confidence detection and quantification of 59% of the theoretical bacterial proteome, including 226 bacterial proteins detected only during phage infection and 6 proteins unique to uninfected bacteria. We also identified many bacterial proteins of differing abundance during the infection. Using this high-throughput proteomic datasets, we selected specific bacterial genes for inactivation using CRISPR-Cas9 to investigate their involvement in phage replication. One knockout mutant lacking gene llmg_0219 showed resistance to phage p2 because of a deficiency in phage adsorption. Furthermore, we detected and quantified 78% of the theoretical phage proteome and identified many proteins of phage p2 that had not been previously detected. Among others, we uncovered a conserved small phage protein (pORFN1) coded by an unannotated gene. We also applied a targeted approach to achieve greater sensitivity and identify undetected phage proteins that were expected to be present. This allowed us to follow the fate of pORF46, a small phage protein of low abundance. In summary, this work offers a unique view of the virulent phages' takeover of bacterial cells and provides novel information on phage-host interactions.

Keywords: Bacteria; Genome editing; Label-free quantification; Lactococcus lactis; Mass Spectrometry; Phage infection; Proteogenomics; Viruses.

Graphical abstract

Fig. 1.

Proteins detected in this study. A, Bar graph comparing the number of proteins detected with the theoretical annotated proteome for both L. lactis MG1363 and phage p2. B, Venn diagram depicting the overlaps of identified bacterial proteins among samples. C, Pie chart illustrating the proportion and the classification according to metabolic pathways of bacterial proteins detected only in uninfected cultures or D, detected strictly in cultures infected with phage p2. E, The blue pie chart illustrates the proportion and the classification according to the expression modules of phage proteins detected. In (C), (D) and (E), the number of proteins are indicated after their respective classification.

Fig. 2.

Viral proteins detected during time-course infections. The genome of phage p2 now contains 50 annotated genes represented by arrows and numbers (top). Early-expressed genes are in green, middle-expressed in yellow and late-expressed in dark red. Colored boxes (bottom) indicate that the corresponding phage proteins were detected at the time-points indicated on the y axis. The newly annotated orfN1 is located between orf22 and orf23. The data for the orf46-encoded protein (boxes colored in lighter yellow) was obtained separately, by SRM (see Fig. 3 for more details). MCP: Major capsid protein, MTP: Major tail protein, TMP: Tail tape measure protein, Dit: Distal tail protein, Tal: Tail associated lysozyme, RBP: Receptor binding protein, Hol: Holin, Lys: Lysin, SaV: Sensitivity to AbiV protein, SSB: Single stranded binding protein.

Fig. 3.

Expression profile of the phage p2 protein encoded by orf46. The orf46-encoded protein was first detected 10 min post-infection and its abundance peaked 20 to 30 min post-infection.

Fig. 4.

Voronoi treemaps depicting the proteotypes of L. lactis cells during phage p2 infection. Each cell represents a single gene locus grouped with other functionally related elements. Metabolic pathways assigned to gene locus are based on TIGR roles (left panel) and TIGR subroles (right panel). Protein abundances were determined at 10 (T10), 20 (T20) and 40 (T40) minutes post-infection and compared with uninfected cultures (T0). For high-resolution images, see supplemental Figs. S1, S2, and S3.

Fig. 5.

Growth curves of phage-sensitive L. lactis pKO0219 pL2Cas9 (control) and derivative L. lactis MGΔ0219 in presence or absence of phage p2. The knockout strain (orange and gray) takes longer to grow than the control strain (yellow and blue) and it is resistant to phage p2 infection (gray).