A protocol to study bacteriophage adaptation to new hosts

Luzon-Hidalgo, Raquel; Risso, Valeria A.; Delgado, Asuncion; Ibarra‐Molero, Beatriz; Sanchez-Ruiz, José M.

doi:10.1016/j.xpro.2021.100784

Cited by 3 publications

(3 citation statements)

References 7 publications

(14 reference statements)

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“…Lysis experiments were performed using a simplified adaptation of a previously employed protocol (Luzon-Hidalgo et al, 2021). Briefly, overnight preinocula were incubated in LB medium at 37 ºC, which, for transformed cells, had been supplemented with kanamycin (50 mg/mL).…”

Section: Methodsmentioning

confidence: 99%

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Virus propagation linked to exceedingly rare gene-expression errors

Luzón-Hidalgo,

d’Agostino,

Risso

et al. 2024

Preprint

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Viruses are obligate parasites that establish extensive interactions with proteins and other biomolecules of their hosts. About 20% of protein molecules bear phenotypic mutations due to errors during gene expression. Phenotypic mutations are not inherited and are not purged/amplified by natural selection. Therefore, protein variants harboring phenotypic mutations remain at very low levels. Here, we show that proteins at exceedingly low levels may enable virus propagation. Bacteriophage T7 recruits the host thioredoxin as an essential processivity factor for its replisome. Thioredoxin constitutive expression yields 10000-20000 molecules per E. coli cell. We inserted early stop codons in the thioredoxin gene and appended to its end the sequence encoding for a photoconvertible fluorescent protein. Virus propagation was not abolished, indicating that some thioredoxin molecules were produced through mistranscription or mistranslation. Single-molecule localization microscopy detected 12+-5 molecules per cell when an ochre codon was inserted. This work demonstrates that crucial virus-host biomolecular interactions may need occur only a few times to trigger virus propagation and supports that viruses may exploit the wide diversity of host and viral protein variants arising from gene-expression errors to establish such interactions. Immediate implications of this notion for the mechanisms of cross-species transmission and antibody evasion are discussed.

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Section: Methodsmentioning

confidence: 99%

“…Except for the knockout Trx - , the strains are identified by the gene used in the transformation. Values of PFU/mL for the phage solutions were calculated from the numbers of plaques upon serial dilutions, as described in Luzon-Hidalgo et al (2021). Average values and standard deviations from three determinations are given.…”

Section: Extended Data Figmentioning

confidence: 99%

Virus propagation linked to exceedingly rare gene-expression errors

Luzón-Hidalgo,

d’Agostino,

Risso

et al. 2024

Preprint

Self Cite

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“…The researchers concluded that although the role of phage adaptation in the efficacy of in vivo therapy has yet to be demonstrated, the ability of trained phages to coevolve with and counter host defenses may improve treatment outcomes [ 175 ]. Similarly, Luzon-Hidalgo and colleagues recently published an analytical protocol for the study of phage adaptation to new hosts [ 176 ]. One of the biggest problems of phage therapy is that during treatment, phage-resistant isolates (“resisters”) arise.…”

Section: Broad-range Phages Should Be Either Made By Expansion or By ...mentioning

confidence: 99%

Does Phage Therapy Need a Pan-Phage?

Bozidis,

Markou,

Gouni

et al. 2024

Pathogens

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The emergence of multidrug-resistant bacteria is undoubtedly one of the most serious global health threats. One response to this threat that has been gaining momentum over the past decade is ‘phage therapy’. According to this, lytic bacteriophages are used for the treatment of bacterial infections, either alone or in combination with antimicrobial agents. However, to ensure the efficacy and broad applicability of phage therapy, several challenges must be overcome. These challenges encompass the development of methods and strategies for the host range manipulation and bypass of the resistance mechanisms developed by pathogenic bacteria, as has been the case since the advent of antibiotics. As our knowledge and understanding of the interactions between phages and their hosts evolves, the key issue is to define the host range for each application. In this article, we discuss the factors that affect host range and how this determines the classification of phages into different categories of action. For each host range group, recent representative examples are provided, together with suggestions on how the different groups can be used to combat certain types of bacterial infections. The available methodologies for host range expansion, either through sequential adaptation to a new pathogen or through genetic engineering techniques, are also reviewed.

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A sensitive one-pot ROA assay for rapid miRNA detection

Hou,

Deng,

et al. 2024

aBIOTECH

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MicroRNAs (miRNAs) and short RNA fragments (18–25 nt) are crucial biomarkers in biological research and disease diagnostics. However, their accurate and rapid detection remains a challenge, largely due to their low abundance, short length, and sequence similarities. In this study, we report on a highly sensitive, one-step RNA O-circle amplification (ROA) assay for rapid and accurate miRNA detection. The ROA assay commences with the hybridization of a circular probe with the test RNA, followed by a linear rolling circle amplification (RCA) using dUTP. This amplification process is facilitated by U-nick reactions, which lead to an exponential amplification for readout. Under optimized conditions, assays can be completed within an hour, producing an amplification yield up to the microgram level, with a detection limit as low as 0.15 fmol (6 pM). Notably, the ROA assay requires only one step, and the results can be easily read visually, making it user-friendly. This ROA assay has proven effective in detecting various miRNAs and phage ssRNA. Overall, the ROA assay offers a user-friendly, rapid, and accurate solution for miRNA detection.

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A protocol to study bacteriophage adaptation to new hosts

Cited by 3 publications

References 7 publications

Virus propagation linked to exceedingly rare gene-expression errors

Virus propagation linked to exceedingly rare gene-expression errors

Does Phage Therapy Need a Pan-Phage?

A sensitive one-pot ROA assay for rapid miRNA detection

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