Prophage Activation in the Intestine: Insights Into Functions and Possible Applications

Prophage activation in intestinal environments has been frequently reported to affect host adaptability, pathogen virulence, gut bacterial community composition, and intestinal health. Prophage activation is mostly caused by various stimulators, such as diet, antibiotics, some bacterial metabolites, gastrointestinal transit, inflammatory environment, oxidative stress, and quorum sensing. Moreover, with advancements in biotechnology and the deepening cognition of prophages, prophage activation regulation therapy is currently applied to the treatment of some bacterial intestinal diseases such as Shiga toxin-producing Escherichia coli infection. This review aims to make headway on prophage induction in the intestine, in order to make a better understanding of dynamic changes of prophages, effects of prophage activation on physiological characteristics of bacteria and intestinal health, and subsequently provide guidance on prophage activation regulation therapy.

The hokW-sokW Locus Encodes a Type I Toxin–Antitoxin System That Facilitates the Release of Lysogenic Sp5 Phage in Enterohemorrhagic Escherichia coli O157

The toxin-antitoxin (TA) genetic modules control various bacterial events, such as plasmid maintenance, persister cell formation, and phage defense. They also exist in mobile genetic elements, including prophages; however, their physiological roles remain poorly understood. Here, we demonstrate that hokW-sokW, a putative TA locus encoded in Sakai prophage 5 (Sp5) in enterohemorrhagic Escherichia coli O157: H7 Sakai strain, functions as a type I TA system. Bacterial growth assays showed that the antitoxic activity of sokW RNA against HokW toxin partially requires an endoribonuclease, RNase III, and an RNA chaperone, Hfq. We also demonstrated that hokW-sokW assists Sp5-mediated lysis of E. coli cells when prophage induction is promoted by the DNA-damaging agent mitomycin C (MMC). We found that MMC treatment diminished sokW RNA and increased both the expression level and inner membrane localization of HokW in a RecA-dependent manner. Remarkably, the number of released Sp5 phages decreased by half in the absence of hokW-sokW. These results suggest that hokW-sokW plays a novel role as a TA system that facilitates the release of Sp5 phage progeny through E. coli lysis.

Characterization of a Novel Phage ΦAb1656-2 and Its Endolysin with Higher Antimicrobial Activity against Multidrug-Resistant Acinetobacter baumannii

Acinetobacter baumannii is a nosocomial pathogen, which is a problem worldwide due to the emergence of a difficult-to-treat multidrug-resistant A. baumannii (MDRAB). Endolysins are hydrolytic enzymes produced by a bacteriophage that can be used as a potential therapeutic agent for multidrug-resistant bacterial infection in replacing antibiotics. Here, we isolated a novel bacteriophage through prophage induction using mitomycin C from clinical A. baumannii 1656-2. Morphologically, ΦAb1656-2 was identified as a Siphoviridae family bacteriophage, which can infect MDRAB. The whole genome of ΦAb1656-2 was sequenced, and it showed that it is 50.9 kb with a G + C content of 38.6% and 68 putative open reading frames (ORFs). A novel endolysin named AbEndolysin with an N-acetylmuramidase-containing catalytic domain was identified, expressed, and purified from ΦAb1656-2. Recombinant AbEndolysin showed significant antibacterial activity against MDRAB clinical strains without any outer membrane permeabilizer. These results suggest that AbEndolysin could represent a potential antimicrobial agent for treating MDRAB clinical isolates.

Confocal microscopy reveals alterations of thylakoids in Limnospira fusiformis during prophage induction

The alkaliphilic cyanobacterium Limnospira fusiformis is an integral part in food webs of tropical soda lakes. Recently, sudden breakdowns of Limnospira sp. blooms in their natural environment have been linked to cyanophage infections. We studied ultrastructural details and prophage components in the laboratory by means of confocal laser scanning microscopy (CLSM) and transmission electron microscopy (TEM). For a comparison at the subcellular level, we included transmission electron microscopy (TEM) material of infected cells collected during a field survey. Compared to TEM, CLSM has the advantage to rapidly providing results for whole, intact cells. Moreover, many cells can be studied at once. We chemically induced lysogenic cyanophages by means of mitomycin C (MMC) treatments and studied the ultrastructural alterations of host cells. In parallel, the number of cyanophages was obtained by flow cytometry. After treatment of the culture with MMC, flow cytometry showed a strong increase in viral counts, i.e., prophage induction. CLSM reflected the re-organization of L. fusiformis with remarkable alterations of thylakoid arrangements after prophage induction. Our study provides a first step towards 3D visualization of ultrastructure of cyanobacteria and showed the high potential of CLSM to investigate viral-mediated modifications in these groups.

Common Oral Medications Lead to Prophage Induction in Bacterial Isolates from the Human Gut

Many bacteria carry bacteriophages (bacterial viruses) integrated in their genomes in the form of prophages, which replicate passively alongside their bacterial host. Environmental conditions can lead to prophage induction; the switching from prophage replication to lytic replication, that results in new bacteriophage progeny and the lysis of the bacterial host. Despite their abundance in the gut, little is known about what could be inducing these prophages. We show that several medications, at concentrations predicted in the gut, lead to prophage induction of bacterial isolates from the human gut. We tested five medication classes (non-steroidal anti-inflammatory, chemotherapy, mild analgesic, cardiac, and antibiotic) for antimicrobial activity against eight prophage-carrying human gut bacterial representative isolates in vitro. Seven out of eight bacteria showed signs of growth inhibition in response to at least one medication. All medications led to growth inhibition of at least one bacterial isolate. Prophage induction was confirmed in half of the treatments showing antimicrobial activity. Unlike antibiotics, host-targeted medications led to a species-specific induction of Clostridium beijerinckii, Bacteroides caccae, and to a lesser extent Bacteroides eggerthii. These results show how common medication consumption can lead to phage-mediated effects, which in turn would alter the human gut microbiome through increased prophage induction.

Instability of CII is needed for efficient switching between lytic and lysogenic development in bacteriophage 186

The CII protein of temperate coliphage 186, like the unrelated CII protein of phage λ, is a transcriptional activator that primes expression of the CI immunity repressor and is critical for efficient establishment of lysogeny. 186-CII is also highly unstable, and we show that in vivo degradation is mediated by both FtsH and RseP. We investigated the role of CII instability by constructing a 186 phage encoding a protease resistant CII. The stabilised-CII phage was defective in the lysis-lysogeny decision: choosing lysogeny with close to 100% frequency after infection, and forming prophages that were defective in entering lytic development after UV treatment. While lysogenic CI concentration was unaffected by CII stabilisation, lysogenic transcription and CI expression was elevated after UV. A stochastic model of the 186 network after infection indicated that an unstable CII allowed a rapid increase in CI expression without a large overshoot of the lysogenic level, suggesting that instability enables a decisive commitment to lysogeny with a rapid attainment of sensitivity to prophage induction.