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Published on: October 14, 2011
Investigating Lactococcus lactis MG1363 Response to Phage p2 Infection at the Proteome Level
Marie-Laurence Lemay1, Andreas Otto2, Sandra Maaß2
1From the ‡Département de biochimie, de microbiologie, et de bio-informatique, Faculté des sciences et de génie, Université Laval, Québec City, QC, G1V 0A6, Canada;; §Groupe de recherche en écologie buccale, Faculté de médecine dentaire, Université Laval, Québec City, QC, G1V 0A6, Canada;; Félix d'Hérelle Reference Center for Bacterial Viruses, Faculté de médecine dentaire, Université Laval, Québec City, QC, G1V 0A6, Canada.
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.
Insights
Bacteriophages (phages) are viruses that infect bacteria. This study used proteomics to analyze phage p2 infecting Lactococcus lactis, revealing new phage proteins and host interactions, including a gene essential for phage adsorption.
Area of Science:
- Microbiology
- Virology
- Proteomics
Background:
- Bacteriophages (phages) are bacterial viruses with ecological and industrial significance.
- Phage-host interactions are crucial but not fully understood, particularly during virulent infections.
- Lactococcus lactis is a key bacterium in food fermentation, and phage p2 is a model for Gram-positive phages.
Purpose of the Study:
- To comprehensively analyze the proteome of Lactococcus lactis during infection by virulent phage p2 using label-free quantitative proteomics.
- To identify bacterial and phage proteins involved in the infection process and uncover novel phage-encoded proteins.
- To investigate the role of specific bacterial genes in phage replication and host resistance.
Main Methods:
- Label-free quantitative proteomics was employed to profile the proteomes of infected and uninfected Lactococcus lactis MG1363.
- CRISPR-Cas9 gene editing was used to create knockout mutants for functional analysis of bacterial genes.
- Targeted proteomics approaches were applied to detect low-abundance phage proteins.
Main Results:
- High-confidence detection of 59% of the bacterial proteome and 78% of the phage proteome during infection.
- Identification of 226 bacterial proteins upregulated during phage infection and 6 proteins unique to uninfected bacteria.
- Discovery of novel phage proteins, including a conserved small protein (pORFN1) from an unannotated gene, and identification of pORF46.
- A knockout mutant for gene llmg_0219 exhibited resistance to phage p2 due to impaired phage adsorption.
Conclusions:
- This study provides an in-depth proteomic view of virulent phage takeover of bacterial cells.
- Novel insights into phage-host interactions and the identification of previously undetected phage proteins were achieved.
- The findings highlight the importance of bacterial factors, like llmg_0219, in controlling phage infection and offer a foundation for phage-based antibacterial strategies.
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