Isolation and characterisation of ΦcrAss002, a crAss-like phage from the human gut that infects Bacteroides xylanisolvens

Abstract

Background The gut phageome comprises a complex phage community of thousands of individual strains, with a few highly abundant bacteriophages. CrAss-like phages, which infect bacteria of the order Bacteroidales, are the most abundant bacteriophage family in the human gut and make an important contribution to an individual’s core virome. Based on metagenomic data, crAss-like phages form a family, with four sub-families and ten candidate genera. To date, only three representatives isolated in pure culture have been reported: ΦcrAss001 and two closely related phages DAC15 and DAC17; all are members of the less abundant candidate genus VI. The persistence at high levels of both crAss-like phage and their Bacteroidales hosts in the human gut has not been explained mechanistically, and this phage-host relationship can only be properly studied with isolated phage-host pairs from as many genera as possible. Results Faeces from a healthy donor with high levels of crAss-like phage was used to initiate a faecal fermentation in a chemostat, with selected antibiotics chosen to inhibit rapidly growing bacteria and selectively enrich for Gram-negative Bacteroidales. This had the objective of promoting the simultaneous expansion of crAss-like phages on their native hosts. The levels of seven different crAss-like phages expanded during the fermentation, indicating that their hosts were also present in the fermenter. The enriched supernatant was then tested against individual Bacteroidales strains isolated from the same faecal sample. This resulted in the isolation of a previously uncharacterised crAss-like phage of candidate genus IV of the proposed Alphacrassvirinae sub-family, ΦcrAss002, that infects the gut commensal Bacteroides xylanisolvens. ΦcrAss002 does not form plaques or spots on lawns of sensitive cells, nor does it lyse liquid cultures, even at high titres. In keeping with the co-abundance of phage and host in the human gut, ΦcrAss002 and Bacteroides xylanisolvens can also co-exist at high levels when co-cultured in laboratory media. Conclusions We report the isolation and characterisation of ΦcrAss002, the first representative of the proposed Alphacrassvirinae sub-family of crAss-like phages. ΦcrAss002 cannot form plaques or spots on bacterial lawns but can co-exist with its host, Bacteroides xylanisolvens, at very high levels in liquid culture without impacting on bacterial numbers.

Discovery, diversity and functional associations of crAss-like phages in human gut metagenomes from four Dutch cohorts

“…Despite the 4-year period separating the two timepoints, we observed a degree of stability both at the level of individual genus-level clusters (on average, 41% of individuals positive for a frequently detected genus-level cluster were positive at both timepoints) and at the level of the overall composition of the crAss-like fraction of the virome (on average, dissimilarity between samples collected from the same individual at the two timepoints was lower than the dissimilarity between pairs of samples collected from different individuals at the same timepoint). These observations are in line with previous reports of temporal stability of crAss-like phages (Edwards et al , 2019; Shkoporov et al , 2019; Siranosian et al , 2020), which may be explained by a relatively peaceful co-existence of phage and host (Guerin et al , 2021; Shkoporov et al , 2018; Shkoporov et al , 2021).…”

“…In this study, both CRISPR-based and co-abundance analyses strongly pointed to Prevotella copri as a host of the crAss-like phages belonging to the genus-level cluster delta27. The fact that the correlation between the relative abundance of the delta27 phages and their potential host is positive suggests that the dynamics in this phage–host pair may be similar to the co-existence in equilibrium reported for the four crAss-like phages isolated in cultures of their hosts (Guerin et al , 2021; Hryckowian et al , 2020; Shkoporov et al , 2018; Shkoporov et al , 2021).…”

“…Numerous crAss-like phages have been predicted to infect bacteria of the phylum Bacteroidetes (Dutilh et al , 2014; Yutin et al , 2021), and accordingly, several crAss-like phages have been isolated in cultures of Bacteroidetes hosts or enriched in the presence of Bacteroidetes hosts (Fitzgerald et al, 2021; Guerin et al, 2021; Hryckowian et al, 2020; Shkoporov et al , 2018), although there is also evidence that some crAss-like phages may infect hosts from different phyla (Yutin et al , 2021). Apparently, crAss-like phages may coexist with their hosts relatively peacefully, as the same crAss-like phages were often recurrently detected in metagenomic samples of longitudinal study participants (Edwards et al, 2019; Shkoporov et al, 2019; Siranosian et al, 2020) and four crAss-like phages were shown to persist in the cultures of their hosts for a long time without a significant reduction in the host population (Guerin et al , 2021; Shkoporov et al , 2018; Shkoporov et al , 2021). Interestingly, hosts were shown to develop resistance to crAss-like phage infection via the phase variation mechanism: inversions in the bacterial genome loci responsible for cell surface coating biosynthesis were linked to altered cell surface architecture and a reduced ability of the bacteria to absorb phage (Shkoporov et al , 2021).…”

Long-term persistence of crAss-like phage crAss001 is associated with phase variation in Bacteroides intestinalis

“…Other members of the family belonging to sub-families Alpha- and Betacrassvirinae , moderately abundant in the human gut, appear to be cultivable. The exemplar strains were isolated from human feces (ΦcrAss001 and ΦcrAss002), wastewater effluent (DAC15 and DAC17), and seawater (Φ14:2), infecting Bacteroides intestinalis [ 11 ], Bacteroides xylanisolvens [ 12 ], Bacteroides thetaiotaomicron [ 13 ], and Cellulophaga baltica , respectively [ 14 ]. The genus Bacteroides , predicted or demonstrated to serve as host to many crAss-like phages [ 6 , 11 , 12 ], comprises strictly anaerobic Gram-negative non-spore-forming bacteria, many of which are predominant members of the human gut microbiome and are highly adapted to the gut environment [ 15 ].…”

“…The exemplar strains were isolated from human feces (ΦcrAss001 and ΦcrAss002), wastewater effluent (DAC15 and DAC17), and seawater (Φ14:2), infecting Bacteroides intestinalis [ 11 ], Bacteroides xylanisolvens [ 12 ], Bacteroides thetaiotaomicron [ 13 ], and Cellulophaga baltica , respectively [ 14 ]. The genus Bacteroides , predicted or demonstrated to serve as host to many crAss-like phages [ 6 , 11 , 12 ], comprises strictly anaerobic Gram-negative non-spore-forming bacteria, many of which are predominant members of the human gut microbiome and are highly adapted to the gut environment [ 15 ]. Genome plasticity, extensive regulation of gene expression, and high adaptability to changes in their environment are characteristic features of this bacterial group [ 16 ].…”