Phage protein Gp11 blocks Staphylococcus aureus cell division by inhibiting peptidoglycan biosynthesis

Abstract

Phages and bacteria have a long history of co-evolution. However, these dynamics of phage-host interactions are still largely unknown; identification of phage inhibitors that remodel host metabolism will provide valuable information for target development for antimicrobials. Here, we perform a comprehensive screen for early-gene products of ΦNM1 that inhibit cell growth in Staphylococcus aureus. A small membrane protein, Gp11, with inhibitory effects on S. aureus cell division was identified. A bacterial two-hybrid library containing 345 essential S. aureus genes was constructed to screen for targets of Gp11, and Gp11 was found to interact with MurG and DivIC. Defects in cell growth and division caused by Gp11 were dependent on MurG and DivIC, which was further confirmed using CRISPRi hypersensitivity assay. Gp11 interacts with MurG, the protein essential for cell wall formation, by inhibiting the production of lipid II to regulate peptidoglycan (PG) biosynthesis on the cell membrane. Gp11 also interacts with cell division protein DivIC, an essential part of the division machinery necessary for septal cell wall assembly, to disrupt the recruitment of division protein FtsW. Mutations in Gp11 result in loss of its ability to cause growth defects, whereas infection with phage in which the gp11 gene has been deleted showed a significant increase in lipid II production in S. aureus. Together, our findings reveal that a phage early-gene product interacts with essential host proteins to disrupt PG biosynthesis and block S. aureus cell division, suggesting a potential pathway for the development of therapeutic approaches to treat pathogenic bacterial infections.

Importance: Understanding the interplay between phages and their hosts is important for the development of novel therapies against pathogenic bacteria. Although phages have been used to control methicillin-resistant Staphylococcus aureus infections, our knowledge related to the processes in the early stages of phage infection is still limited. Owing to the fact that most of the phage early proteins have been classified as hypothetical proteins with uncertain functions, we screened phage early-gene products that inhibit cell growth in S. aureus, and one protein, Gp11, selectively targets essential host genes to block the synthesis of the peptidoglycan component lipid II, ultimately leading to cell growth arrest in S. aureus. Our study provides a novel insight into the strategy by which Gp11 blocks essential host cellular metabolism to influence phage-host interaction. Importantly, dissecting the interactions between phages and host cells will contribute to the development of new and effective therapies to treat bacterial infections.

Keywords: Staphylococcus aureus; essential gene; lipid II; peptidoglycan; phage.

Fig 1

Overexpression of Gp11 in S. aureus alters cell morphology. (A) Effect of Gp11 overexpression on S. aureus cell growth. (B) Effect of Gp11 overexpression on S. aureus cell shape as visualized by phase-contrast and fluorescence microscopy. Arrows indicate septal defects as multiple. Cells were stained with membrane dye Nile Red. Scale bar, 2.5 µm. (C) Cell volume was measured using MicrobeJ. Cells were harvested after 2 h incubation with 10 µm NaAsO₂. (D) Quantitation of cells with septal defects as multiple septa (abnormal), and nascent or complete septum (normal) after treatment with 10 µm NaAsO₂ for 2 h. In this plot, n =  198 (Vec) and 91 (Gp11) cells; P values were determined by unpaired Student’s t-test. **P < 0.01 and *P < 0.05.

Fig 2

Validation of the targets of Gp11 in S. aureus. (A) Flowchart for screening Gp11-interacting proteins from S. aureus essential-gene BACTH libraries. With libraries being collected in an essential-gene plasmid pool and equimolar mix, the plasmid library was then transformed into Escherichia coli BTH101 reporter cells, which contain the phage gene, and plated on MacConkey agar plates. (B) In a bacterial two-hybrid assay of Gp11 interaction with MurG and DivIC, the known interaction pair Gp104 and DnaI was used as a positive control, while Gp11 and MraY as a negative control. (C) CRISPRi-based assay revealed MurG as the target of Gp11. The growth of S. aureus by knockdown of murG after expression of Gp11 (Gp11 + sgRNA^murG) relative to no sgRNA control (Vec₁ + Vec₂). (D) The growth of S. aureus by knockdown of divIC after expression of Gp11 (Gp11 + sgRNA^divIC) relative to no sgRNA control (Vec₁ + Vec₂). Each data point represents three independent replicates. P values were determined by unpaired Student’s t-test. **P < 0.01, *P < 0.05, and n.s.: no significant difference. (E) The growth of S. aureus by knockdown of mraY after expression of Gp11 (Gp11 + sgRNA^mraY) relative to no sgRNA control (Vec₁ + Vec₂). (F) Growth assay of cells expressing Gp11, MurG, DivIC, or MraY on solid medium. (G) Microscopic images of S. aureus expressing Gp11, MurG, and DivIC. Cells were stained with membrane dye Nile Red. Scale bars, 2.5 µm. (H) Cell volume was measured using MicrobeJ. Cells were collected after 2 h incubation with 10 µM NaAsO₂ and 100 µM IPTG. From left to right, n =  467, 418, 118, and 510 cells. P values were determined by unpaired Student’s t-test. **P < 0.01. (I) Quantitation of cells with septal defects (abnormal), and nascent or complete septum (normal) after cells were treated with 10 µM NaAsO₂ and 100 µM IPTG for 2 h.

Fig 3

Gp11 acts to block cell division. (A) Summary of the identified mutants, all of which map to a hypothetical protein encoded by the gene gp11 in S. aureus ΦNM1. The structural model of Gp11 predicted by AlphaFold (30), and the fraction of each mutant of Gp11 is indicated. (B) Analysis of the interactions between MurG and the mutants of Gp11 through the bacterial adenylate cyclase-based two-hybrid (BACTH) system. (C) Analysis of the interactions between DivIC and the mutants of Gp11 through BACTH. (D) The localization of MurG, DivIB, FtsW, and EzrA were monitored in the presence of Gp11 or its mutants. The cells were collected after 2 h incubation with 10 µM NaAsO₂ and 100 µM IPTG. Scale bars, 2.5 µm.

Fig 4

Gp11 targets peptidoglycan biosynthesis by inhibiting the activity of MurG. (A) S. aureus cells expressing Gp11 or its mutants were stained with the fluorescent cell wall marker D-amino acid 7-hydroxycoumarin carbonyl amino-D-alanine (HADA) and analyzed by phase-contrast and fluorescence microscopy. Scale bars, 2.5 µm. (B) Images of individual cells expressing Gp11 or its mutants were used to calculate the fluorescence ratio of the septal versus cell peripheral fluorescence signal. n ≥ 30. P values were determined by unpaired Student’s t-test. **P < 0.01 and n.s.: no significant difference. (C) The terminal D-Ala residue of the lipid II stem peptide was labeled with the biotin-D-lysine probe BDL using S. aureus PBP4 (34). (D) Western blot of lipid II after overexpression of Gp11 and its point mutants in S. aureus strain RN4220. (E) Western blot of lipid II after cells were infected with ΦNM1 and ΦNM1^Δgp11.

Fig 5

ΦNM1 gp11 is contributed to phage infection. (A) The scheme for sgRNA-guided, CRISPR/Cas 9-induced homologous recombination in the gp11 gene. (B) Growth curves of S. aureus RN4220 treated with ΦNM1 or ΦNM1^Δgp11 at a multiplicity of infection (MOI) of 0.01 or 1. (C) Serial, 10-fold dilutions of the phages ФNM1 or ΦNM1^Δgp11 spotted on lawns of cells harboring plasmid expressing Gp11 with 2.5 µM NaAsO₂ or an empty vector (Vec). (D) Plaque morphologies of S. aureus RN4220 after infected with ФNM1, ФNM1^Δgp11 (delete the entire gene gp11), or its complementary phage ФNM1^Δgp11::gp11. For complementation assay, ФNM1^Δgp11 was used to infect S. aureus RN4220, which contained the plasmid expressing gp11 with 2.5 µM NaAsO₂. The experiments were repeated at least three times.