Differential transcription profiling of the phage LUZ19 infection process in different growth media

Brandão, Ana Catarina; Pires, Diana Priscila Penso; Coppens, Lucas; Voet, Marleen; Lavigne, Rob; Azeredo, Joana

doi:10.1080/15476286.2020.1870844

Cited by 20 publications

(24 citation statements)

References 52 publications

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“…In the present study, we intended to further expand this technique for real-time assessment of phage-bacteria interactions as a reliable and fast method to assess host susceptibility to phages, overcoming the time-consuming plating Regarding phage PE3, the levels of gp3 increased significantly during the first 3 min of infection (p < 0.05). This was expected, as this gene is referred to as an early gene of phage replication cycle [16] (Figure 3a). The levels of expression of this gene were stable during 9 min of infection and decreased after 15 min of phage-host interaction (p < 0.05).…”

Section: Discussionmentioning

confidence: 58%

“…Regarding phage PE3, the levels of gp3 increased significantly during the first 3 min of infection ( p < 0.05). This was expected, as this gene is referred to as an early gene of phage replication cycle [ 16 ] ( Figure 3 a). The levels of expression of this gene were stable during 9 min of infection and decreased after 15 min of phage–host interaction ( p < 0.05).…”

Section: Resultsmentioning

confidence: 68%

“…To guarantee that all cells are infected at the same time, synchronous infections were used. These infections have been used, for example, to study the molecular profiling of infection through qPCR [ 20 ] or RNA-Seq [ 16 , 21 ], and even suggested to study one-step growth curves [ 22 ]. Here, to guarantee a synchronous infection of P. aeruginosa PAO1 with phages PE3 and DP1, a MOI of 50 was used.…”

Section: Discussionmentioning

confidence: 99%

“…Synchronized infection experiments were performed with phages DP1 and PE3 according to a procedure published elsewhere [ 16 ]. Briefly, P. aeruginosa cells were grown overnight, diluted 1:100 in 20 mL of fresh LB media, and incubated at 37 °C and 120 rpm, until reaching an optical density (OD 600 nm ) of 0.3, which corresponds to approximately 2 × 10 8 CFU/mL.…”

Section: Methodsmentioning

confidence: 99%

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Phage-Host Interaction Analysis by Flow Cytometry Allows for Rapid and Efficient Screening of Phages

et al. 2022

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Recently, phages have become popular as an alternative to antibiotics. This increased demand for phage therapy needs rapid and efficient methods to screen phages infecting specific hosts. Existing methods are time-consuming, and for clinical purposes, novel, quick, and reliable screening methods are highly needed. Flow cytometry (FC) allows a quick differentiation and enumeration of bacterial cell populations and has been used to assess in vitro the activity of antimicrobial compounds. In this work, we propose FC as a rapid and reliable method to assess the susceptibility of a bacterial population to phage infection. For that, the interaction of phages vB_PaeM_CEB_DP1 and vB_PaeP_PE3 with Pseudomonas aeruginosa PAO1 was characterized by FC. Synchronous infection assays were performed, and samples were collected at different time points and stained with SYTO BC and PI before analysis. Part of the collected samples was used to characterize the expression of early, middle, and late genes by qPCR. Both FC and qPCR results were correlated with phage propagation assays. Results showed that SYTO BC median fluorescence intensity (MFI) values increased in the first 25 min of PE3 and DP1 infection. The increase of fluorescence is due to the expression of phage genes observed by qPCR. Since SYTO BC MFI values increase with gene expression, it allows the determination of host susceptibility to a phage in a short period of time, avoiding false positives caused by lysis from without. In conclusion, this method may allow for a quick and high-throughput real-time screening of different phages to a specific host, which can be crucial for a quick phage selection in clinical practice.

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Section: Discussionmentioning

confidence: 58%

Section: Resultsmentioning

confidence: 68%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Phage-Host Interaction Analysis by Flow Cytometry Allows for Rapid and Efficient Screening of Phages

et al. 2022

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“…We postulate that there are likely additional constraints affecting optimal phage infectivity, and probably we might have missed uncovering these additional factors in our model strain because of highly fit phage receptor mutants. Considering differences in phage infectivity in a panel of strains with highly conserved genetic determinants, we posit that systematic study of differences in transcriptional and translation processes in these strains might provide more insights as illustrated in a few recent phage-host interaction studies [152,153]. Future studies could also employ recently developed methods [49,154,155] to provide higher resolution into phage-host interactions and may aid in filling the knowledge gaps on phage host-range.…”

Section: Discussionmentioning

confidence: 99%

The genetic basis of phage susceptibility, cross-resistance and host-range in Salmonella

et al. 2021

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Though bacteriophages (phages) are known to play a crucial role in bacterial fitness and virulence, our knowledge about the genetic basis of their interaction, cross-resistance and host-range is sparse. Here, we employed genome-wide screens in Salmonella enterica serovar Typhimurium to discover host determinants involved in resistance to eleven diverse lytic phages including four new phages isolated from a therapeutic phage cocktail. We uncovered 301 diverse host factors essential in phage infection, many of which are shared between multiple phages demonstrating potential cross-resistance mechanisms. We validate many of these novel findings and uncover the intricate interplay between RpoS, the virulence-associated general stress response sigma factor and RpoN, the nitrogen starvation sigma factor in phage cross-resistance. Finally, the infectivity pattern of eleven phages across a panel of 23 genome sequenced Salmonella strains indicates that additional constraints and interactions beyond the host factors uncovered here define the phage host range.

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Obtaining Detailed Phage Transcriptomes Using ONT-Cappable-Seq

Putzeys,

Intizar,

Lavigne

et al. 2024

Methods in Molecular Biology

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Differential transcription profiling of the phage LUZ19 infection process in different growth media

Cited by 20 publications

References 52 publications

Phage-Host Interaction Analysis by Flow Cytometry Allows for Rapid and Efficient Screening of Phages

Phage-Host Interaction Analysis by Flow Cytometry Allows for Rapid and Efficient Screening of Phages

The genetic basis of phage susceptibility, cross-resistance and host-range in Salmonella

Obtaining Detailed Phage Transcriptomes Using ONT-Cappable-Seq

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