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An Efficient, Counter-Selection-Based Method for Prophage Curing in Pseudomonas aeruginosa Strains.

ABSTRACT: Prophages are bacteriophages in the lysogenic state, where the viral genome is inserted within the bacterial chromosome. They contribute to strain genetic variability and can influence bacterial phenotypes. Prophages are highly abundant among the strains of the opportunistic pathogen Pseudomonas aeruginosa and were shown to confer specific traits that can promote strain pathogenicity. The main difficulty of studying those regions is the lack of a simple prophage-curing method for P. aeruginosa strains. In this study, we developed a novel, targeted-curing approach for prophages in P. aeruginosa. In the first step, we tagged the prophage for curing with an ampicillin resistance cassette (ampR) and further used this strain for the sacB counter-selection marker's temporal insertion into the prophage region. The sucrose counter-selection resulted in different variants when the prophage-cured mutant is the sole variant that lost the ampR cassette. Next, we validated the targeted-curing with local PCR amplification and Whole Genome Sequencing. The application of the strategy resulted in high efficiency both for curing the Pf4 prophage of the laboratory wild-type (WT) strain PAO1 and for PR2 prophage from the clinical, hard to genetically manipulate, 39016 strain. We believe this method can support the research and growing interest in prophage biology in P. aeruginosa as well as additional Gram-negative bacteria.

SUBMITTER: Shmidov E

PROVIDER: S-EPMC7926659 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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An Efficient, Counter-Selection-Based Method for Prophage Curing in Pseudomonas aeruginosa Strains.

Shmidov Esther E Zander Itzhak I Lebenthal-Loinger Ilana I Karako-Lampert Sarit S Shoshani Sivan S Banin Ehud E

Viruses 20210221 2

Prophages are bacteriophages in the lysogenic state, where the viral genome is inserted within the bacterial chromosome. They contribute to strain genetic variability and can influence bacterial phenotypes. Prophages are highly abundant among the strains of the opportunistic pathogen Pseudomonas aeruginosa and were shown to confer specific traits that can promote strain pathogenicity. The main difficulty of studying those regions is the lack of a simple prophage-curing method for P. ae ...[more]

PMID: 33670076

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Project description:Prophages are bacteriophages integrated into the bacterial host's chromosome. This research aims to analyze and characterize the existing prophages within a collection of 53 Pseudomonas aeruginosa strains from intensive care units (ICUs) in Portugal and Spain. A total of 113 prophages were localized in the collection, with 18 of them being present in more than one strain simultaneously. After annotation, five of them were discarded as incomplete, and the 13 remaining prophages were characterized. Of 13, 10 belonged to the siphovirus tail morphology group, 2 to the podovirus tail morphology group, and 1 to the myovirus tail morphology group. All prophages had a length ranging from 20,199 to 63,401 bp and a GC% between 56.2% and 63.6%. The number of open reading frames (ORFs) oscillated between 32 and 88, and in 3/13 prophages, more than 50% of the ORFs had an unknown function. With our findings, we show that prophages are present in the majority of the P. aeruginosa strains isolated from Portuguese and Spanish critically ill patients, many of them found in more than one circulating strain at the same time and following a similar clonal distribution pattern. Although a great sum of ORFs had an unknown function, number of proteins in relation to viral defense (anti-CRISPR proteins, toxin/antitoxin modules, proteins against restriction-modification systems) as well as to prophage interference into their host's quorum sensing system and regulatory cascades were found. This supports the idea that prophages have an influence in bacterial pathogenesis and anti-phage defense. IMPORTANCE Despite being known for decades, prophages remain understudied when compared to the lytic phages employed in phage therapy. This research aims to shed some light into the nature, composition, and role of prophages found within a set of circulating strains of Pseudomas aeruginosa, with special attention to high-risk clones. Given the fact that prophages can effectively influence bacterial pathogenesis, prophage basic research constitutes a topic of growing interest. Furthermore, the abundance of viral defense and regulatory proteins within prophage genomes detected in this study evidences the importance of characterizing the most frequent prophages in circulating clinical strains and in high-risk clones if phage therapy is to be used.

Project description:Streptococcus pyogenes is a human commensal and a bacterial pathogen responsible for a wide variety of human diseases differing in symptoms, severity, and tissue tropism. The completed genome sequences of >37 strains of S. pyogenes, representing diverse disease-causing serotypes, have been published. The greatest genetic variation among these strains is attributed to numerous integrated prophage and prophage-like elements, encoding several virulence factors. A comparison of isogenic strains, differing in prophage content, would reveal the effects of these elements on streptococcal pathogenesis. However, curing strains of prophage is often difficult and sometimes unattainable. We have applied a novel counter-selection approach to identify rare S. pyogenes mutants spontaneously cured of select prophage. To accomplish this, we first inserted a two-gene cassette containing a gene for kanamycin resistance (KanR) and the rpsL wild-type gene, responsible for dominant streptomycin sensitivity (SmS), into a targeted prophage on the chromosome of a streptomycin resistant (SmR) mutant of S. pyogenes strain SF370. We then applied antibiotic counter-selection for the re-establishment of the KanS/SmR phenotype to select for isolates cured of targeted prophage. This methodology allowed for the precise selection of spontaneous phage loss and restoration of the natural phage attB attachment sites for all four prophage-like elements in this S. pyogenes chromosome. Overall, 15 mutants were constructed that encompassed every permutation of phage knockout as well as a mutant strain, named CEM1ΔΦ, completely cured of all bacteriophage elements (a ~10% loss of the genome); the only reported S. pyogenes strain free of prophage-like elements. We compared CEM1ΔΦ to the WT strain by analyzing differences in secreted DNase activity, as well as lytic and lysogenic potential. These mutant strains should allow for the direct examination of bacteriophage relationships within S. pyogenes and further elucidate how the presence of prophage may affect overall streptococcal survival, pathogenicity, and evolution.

Project description:Pseudomonas aeruginosa is a notorious pathogen that causes various nosocomial infections. Several prophage genes located on the chromosomes of P. aeruginosa have been reported to contribute to bacterial pathogenesis via host phenotype transformations, such as serotype conversion and antibiotic resistance. However, our understanding of the molecular mechanism behind host phenotype shifts induced by prophage genes remains largely unknown. Here, we report a systematic study around a hypothetical recombinase, Pg54 (RecT), located on a 48-kb putative prophage (designated PP9W) of a clinical P. aeruginosa strain P9W. Using a ΔrecT mutant (designated P9D), we found that RecT promoted prophage PP9W excision and gene transcription via the inhibition of the gene expression level of pg40, which encodes a CI-like repressor protein. Further transcriptomic profiling and various phenotypic tests showed that RecT modulated like a suppressor to some transcription factors and vital genes of diverse cellular processes, providing multiple advantages for the host, including cell growth, biofilm formation, and virulence. The versatile functions of RecT hint at a strong impact of phage proteins on host P. aeruginosa phenotypic flexibility. IMPORTANCE Multidrug-resistant and metabolically versatile P. aeruginosa are difficult to eradicate by anti-infective therapy and frequently lead to significant morbidity and mortality. This study characterizes a putative recombinase (RecT) encoded by a prophage of a clinical P. aeruginosa strain isolated from severely burned patients, altering prophage lifestyle and host core cellular processes. It implies the potential role of RecT in the coevolution arm race between bacteria and phage. The excised free phages from the chromosome of host bacteria can be used as weapons against other sensitive competitors in diverse environments, which may increase the lysogeny frequency of different P. aeruginosa subgroups. Subsequent analyses revealed that RecT both positively and negatively affects different phenotypic traits of the host. These findings concerning RecT functions of host phenotypic flexibility improve our understanding of the association between phage recombinases and clinical P. aeruginosa, providing new insight into mitigating the pathogen infection.

Dataset Information

An Efficient, Counter-Selection-Based Method for Prophage Curing in Pseudomonas aeruginosa Strains.

Publications

An Efficient, Counter-Selection-Based Method for Prophage Curing in <i>Pseudomonas aeruginosa</i> Strains.

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