Bacteriophage protein PEIP is a potent Bacillus subtilis enolase inhibitor

Zhang, Kaining; Li, Shanshan; Wang, Yawen; Wang, Wenwen; Mulvenna, Nancy; Yang, Hang; Zhang, Peipei; Chen, Huan; Li, Yan; Wang, Hongliang; Gao, Yongxiang; Wigneshweraraj, Sivaramesh; Matthews, Steve; Zhang, Kaiming; Liu, Bing

doi:10.1016/j.celrep.2022.111026

Cited by 6 publications

(5 citation statements)

References 63 publications

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“…There is evidence of protein–protein interactions between coliphage T1 and E. coli enolase and direct inhibition of enolase by Bacillus subtilis phage SPO1, so it stands to reason that there may be interactions between T7 and enolase. ,, Because rne inhibition is part of the T7 lifecycle, we expected rne knockdown to have a neutral to positive effect on EOP. However, as with eno, rne knockdown lowered EOP.…”

Section: Resultsmentioning

confidence: 99%

Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield

Brooks,

Morici,

Sandoval

2023

ACS Synth. Biol.

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Phage therapy to treat life-threatening drugresistant infections has been hampered by technical challenges in phage production. Cell-free bacteriophage synthesis (CFBS) can overcome the limitations of standard phage production methods by manufacturing phage virions in vitro. CFBS mimics intracellular phage assembly using transcription/translation machinery (TXTL) harvested from bacterial lysates and combined with reagents to synthesize proteins encoded by a phage genomic DNA template. These systems may enable rapid phage production and engineering to accelerate phages from bench-to-bedside. TXTL harvested from wild type or commonly used bacterial strains was not optimized for bacteriophage production. Here, we demonstrate that TXTL from genetically modified E. coli BL21 can be used to enhance phage T7 yields in vitro by CFBS. Expression of 18 E. coli BL21 genes was manipulated by inducible CRISPR interference (CRISPRi) mediated by nuclease deficient Cas12a from F. novicida (dFnCas12a) to identify genes implicated in T7 propagation as positive or negative effectors. Genes shown to have a significant effect were overexpressed (positive effectors) or repressed (negative effectors) to modify the genetic background of TXTL harvested for CFBS. Phage T7 CFBS yields were improved by up to 10-fold in vitro through overexpression of translation initiation factor IF-3 (inf C) and small RNAs OxyS and CyaR and by repression of RecC subunit exonuclease RecBCD. Continued improvement of CFBS will mitigate phage manufacturing bottlenecks and lower hurdles to widespread adoption of phage therapy.

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Section: Resultsmentioning

confidence: 99%

Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield

Brooks,

Morici,

Sandoval

2023

ACS Synth. Biol.

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“…While the overexpressed Gp49 clearly did not affect the growth of B. subtilis during the early lag and exponential phases, it does not have a statistically significant attenuation effect on the growth of the bacteria during the stationary phase ( Figure 2 A). Inspired by another gene product—PEIP of the HTM—whose overexpression defies the rule of Gram staining by making B. subtilis stains resemble a Gram-negative bacterium [ 8 ], we then used the Gram stain for phenotyping the effect of overexpressing Gp49 during the stationary phase at OD 600 = 1.4 ( Figure 2 B). Interestingly, the Gp49 overexpressing B. subtilis exhibited safranin color that characterizes Gram-negative bacteria, while bacteria in the pHT01 (control) groups showed the typical crystal violet for Gram-positive bacteria under the stain.…”

Section: Resultsmentioning

confidence: 99%

“…Gp46 is a cross-species HU inhibitor that causes filamentous morphology and nucleoid deformation in B. subtilis [ 7 ]. PEIP (also known as Gp60) has been shown to be a potent glycolysis inhibitor that dissembles the octameric enolase that results in growth attenuation and disruption of the cell wall [ 8 ]. Other studies also suggest that Gp53 is a host ClpCP modifier [ 9 ] and Gp56 inhibits cell division by interacting with FtsL [ 10 ].…”

Section: Introductionmentioning

confidence: 99%

Phage SPO1 Protein Gp49 Is a Novel RNA Binding Protein That Is Involved in Host Iron Metabolism

Yang,

Hu,

Kang

et al. 2023

IJMS

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Bacillus subtilis is a model organism for studying Gram-positive bacteria and serves as a cell factory in the industry for enzyme and chemical production. Additionally, it functions as a probiotic in the gastrointestinal tract, modulating the gut microbiota. Its lytic phage SPO1 is also the most studied phage among the genus Okubovrius, including Bacillus phage SPO1 and Camphawk. One of the notable features of SPO1 is the existence of a “host-takeover module”, a cluster of 24 genes which occupies most of the terminal redundancy. Some of the gene products from the module have been characterized, revealing their ability to disrupt host metabolism by inhibiting DNA replication, RNA transcription, cell division, and glycolysis. However, many of the gene products which share limited similarity to known proteins remain under researched. In this study, we highlight the involvement of Gp49, a gene product from the module, in host RNA binding and heme metabolism—no observation has been reported in other phages. Gp49 folds into a structure that does not resemble any protein in the database and has a new putative RNA binding motif. The transcriptome study reveals that Gp49 primarily upregulates host heme synthesis which captures cytosolic iron to facilitate phage development.

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“…A small protein like Gp11 targets the essential proteins MurG and DivIC to control its host. This implies the strong evolutionary pressure on phages to develop strategies to control host function by targeting two proteins simultaneously ( 19 , 20 , 47 ). DivIC is a key component of cell wall dynamics during division, which is essential for proper septum formation ( 48 ).…”

Section: Discussionmentioning

confidence: 99%

“…Studies have shown that phage proteins inhibit bacterial DNA biosynthesis by targeting host DNA polymerase and DNA gyrase ( 9 , 10 ), and affect cell division by inhibiting the essential cell division proteins FtsZ and FtsL or the cytoskeletal protein MreB ( 11 – 14 ), or modifying or inhibiting bacterial RNA polymerase ( 15 – 18 ). In addition, other key host processes, such as quorum sensing and glycolysis, are also targeted by phages ( 19 , 20 ).…”

Section: Introductionmentioning

confidence: 99%

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

Xu,

Tang,

Liu

et al. 2024

mBio

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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.

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Bacteriophage protein PEIP is a potent Bacillus subtilis enolase inhibitor

Cited by 6 publications

References 63 publications

Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield

Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield

Phage SPO1 Protein Gp49 Is a Novel RNA Binding Protein That Is Involved in Host Iron Metabolism

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

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