Macrophages suppress Escherichia coli LF82 Gally prophage lytic cycle

By Pooja Toshniwal PahariaFeb 8 2023Reviewed by Benedette Cuffari, M.Sc.

In a recent study published in the journal PLoS Pathogens, researchers analyze prophages of the LF82 strain of adherent-invasive Escherichia coli (AIEC) to better understand their behavior in vitro and within macrophages.

Study: Phage production is blocked in the adherent invasive Escherichia coli LF82 upon macrophage infection. Image Credit: fusebulb / Shutterstock.com

What are prophages?

Prophages like lysogens can either be directly integrated into the genome of their host bacteria or exist as freely replicating organisms. The expression of prophage genes, otherwise known as ‘morons,’ provide advantageous properties to the bacteria ranging from protection against other pathogenic species or the ability to adapt to certain environments, to name a few.

Conversely, the induction of prophages into bacteria may induce stress, which can ultimately lead to lysis of the prophage. Thus, the wide range of prophage behaviors must be further understood, particularly when considering their potential impact on bacterial pathogens.

AIEC genomes, for example, host many different prophages, with the LF82 strain encoding for five functional prophages. The LF82 prophage 1 has been reported in E. coli strains obtained from patients with Crohn’s disease.

Lambda prophage induction increases by 26-fold when present in a macrophage-rich environment compared to its activity in laboratory conditions. This high induction level leads to the lysis of about 90% of E. coli bacteria, which may be directly attributed to macrophage activity.

About the study

Five LF82 prophages were named Gally (prophage 1), Perceval (prophage 2), Tritos (prophage 3), Cartapus (prophage 4), and Cyrano (pLF82). These prophages were uploaded to the European Nucleotide Archive database with re-annotations.

Genes differing from the master regulator or typical bacteriophage genes that transcribed five-fold greater than the local transcription rates in the in vitro LF82 transcriptome analysis were identified as morons. Homology between Escherichia coli LF82 prophages and reference phages from NCBI, as of March 2020, was assessed.

Ribonucleic acid (RNA) sequencing data was used for transcriptomic and differential gene expression analyses. In addition, to detect spontaneous virus production among prophages, encapsidated virome deoxyribonucleic acid (DNA) was subjected to whole-genome sequencing analysis.

Further, virion genomes were measured by quantitative polymerase chain reaction (qPCR) to verify Gally prevalence in vitro. Finally, the bacteriophages were propagated as plaques and subsequently examined by transmission electron microscopy (TEM).

The researchers also assessed whether antibiotics such as trimethoprim, gentamycin, ciprofloxacin, and cefotaxim could induce LF82 prophages beyond the spontaneous induction level using epifluorescence microscopy.

Gally-Perceval hybrid genome assembly was assessed, and the minimal inhibitory concentrations of antibiotics for LF82 were determined. Survival of E. coli LF82 in the presence of ciprofloxacin and a macrophage-rich environment was assessed.  

Transcription profiles of Gally within macrophages after six hours of infection and those cultured in vitro were comparatively assessed. MAC2225, a ΔGally LF82 strain, was obtained by curating prophages with ciprofloxacin, with MAC2459, MAC2774, MAC2222, and OEC2481 strains were by recombineering.

Study findings

All AIEC prophages formed virions in vitro. Gally showed the most significant activity by spontaneously producing over 10⁸ virus particles/milliliter (mL) of culture supernatant, which was over 100-fold greater than other prophages. Over-induction of Gally occurred under genotoxic stressful conditions created by trimethoprim and ciprofloxacin but not within macrophages.

LF82 inhibited prophage awakening within macrophages, thereby enabling their survival. Gally deletion did not affect LF82 viability in the genotoxic environment.

Any fitness advantage conferred by LF82 prophages occurs in environments differing from macrophages. The noteworthy survival of LF82 within macrophages could partially be due to the ability of LF82 to control Gally prophage induction.

All five LF82 prophages were spontaneously induced in vitro. In addition, LF82 prophages, except Gally, encoded morons, and Gally-mediated lateral transduction events were observed. A minimum of two prophages were induced by trimethoprim and ciprofloxacin.

LF82 survival within macrophages was unaffected by Gally’s presence, with Gally partially transcribed within macrophages. Gally prophage induction was inhibited within macrophages, and the lytic stage of the life cycle was repressed at the excision/replication stage within macrophages.

The Cyrano prophage showed the greatest moron abundance of 13 genes, followed by Tritos and Perceval, with seven genes each, and Cartapus with two genes, whereas Gally lacked morons. About 95% of reads were mapped on Gally, thus indicating that the bacteriophage was highly produced from LF82 strain cultures, with qPCR showing elevated concentrations of 4.30 x 10⁸ Gally genomes/mL.

Perceval and Tritos were successfully isolated and visualized by TEM. Gally showed no plaque production under any condition. Gally was ultimately found to be infectious; however, it formed invisible plaques, or ‘chose’ lysogeny at high frequencies on infection.

Two hybrids of Gally and Perceval, Galper1 and Galper2, were isolated during Gally plaque isolation attempts. In addition, TEM showed a podovirus abundance in overnight LF82 cultures that were inferred to correspond to the Gally prophage.

In silico analysis of reconstructed bacterial lodgement sites showed no open-reading frame (ORF) restoration post-excision of prophage or pre-existing ORF modification in the lysogenic strain genome.

The bacterial genetic content was found to be unaffected by bacterial prophages post-excision. However, Gally was located between the torS protein (pointing towards the left) and the torT protein (pointing towards the right), the latter of which is the site targeted for prophage insertion among 5.0% of Escherichia coli strains.

Genotoxic stress comparable to that induced by ciprofloxacin exposure partially induced the LF82 phageome. Gally induction frequency was 0.04% in LF82-infecting macrophages, 10.0-fold lesser than that in the in vitro unstressed environments.

Overall, the study findings showed the repression of the LF82 strain Gally prophage lytic cycle within macrophages.