Novel Moraxella catarrhalis prophages display hyperconserved non-structural genes despite their genomic diversity

Abstract

Background: Moraxella catarrhalis is an important pathogen that often causes otitis media in children, a disease that is not currently vaccine preventable. Asymptomatic colonisation of the human upper respiratory tract is common and lack of clearance by the immune system is likely due to the emergence of seroresistant genetic lineages. No active bacteriophages or prophages have been described in this species. This study was undertaken to identify and categorise prophages in M. catarrhalis, their genetic diversity and the relationship of such diversity with the host-species phylogeny.

Results: This study presents a comparative analysis of 32 putative prophages identified in 95 phylogenetically variable, newly sequenced M. catarrhalis genomes. The prophages were genotypically classified into four diverse clades. The genetic synteny of each clade is similar to the group 1 phage family Siphoviridae, however, they form genotypic clusters that are distinct from other members of this family. No core genetic sequences exist across the 32 prophages despite clades 2, 3, and 4 sharing the most sequence identity. The analysis of non-structural prophage genes (coding the integrase, and terminase), and portal gene showed that the respective genes were identical for clades 2, 3, and 4, but unique for clade 1. Empirical analysis calculated that these genes are unexpectedly hyperconserved, under purifying selection, suggesting a tightly regulated functional role. As such, it is improbable that the prophages are decaying remnants but stable components of a fluctuating, flexible and unpredictable system ultimately maintained by functional constraints on non-structural and packaging genes. Additionally, the plate encoding genes were well conserved across all four prophage clades, and the tail fibre genes, commonly responsible for receptor recognition, were clustered into three major groups distributed across the prophage clades. A pan-genome of 283,622 bp was identified, and the prophages were mapped onto the diverse M. catarrhalis multi-locus sequence type (MLST) backbone.

Conclusion: This study has provided the first evidence of putatively mobile prophages in M. catarrhalis, identifying a diverse and fluctuating system dependent on the hyperconservation of a few key, non-structural genes. Some prophages harbour virulence-related genes, and potentially influence the physiology and virulence of M. catarrhalis. Importantly our data will provide supporting information on the identification of novel prophages in other species by adding greater weight to the identification of non-structural genes.

Fig. 1

Prophages of Moraxella catarrhalis are divided into four clades, grouped by nucleotide similarity. The 32 prophages identified in 95 sequences of M. catarrhalis were aligned using progressiveMauve and compared to five reference Siphoviridae genera: λ-like, L5-like, N15-like, ϕC31-like, and Tuna-like viruses (purple) and their similarities represented by the Mauve output guide tree. The M. catarrhalis prophages are grouped into four clades (red = clade 1, blue = clade 2, orange = clade 3, green = clade 4)

Fig. 2

Alignment of M. catarrhalis prophages in clade 1. Phage-related genes are shown as arrows to indicate their direction relative to the prophage sequence, and are colour coded as follows: integrase (red), terminase (light blue), coat (dark blue), portal (dark orange), tail shaft (light green), tail fibre (dark green), plate (purple), and protease (yellow). Vertical blue lines between prophages show conserved areas without inversion, and red lines between prophages show conserved areas with inversion; the intensities of these lines correspond to percent identity between adjacent prophages as indicated by the scale (lower right)

Fig. 3

Phage-related gene analysis of M. catarrhalis prophages. Distance trees displaying the diversity of phage-related genes in M. catarrhalis prophages. a Large terminase subunit. b Small terminase subunit. c Integrase. d Portal. e Plate. f Tail Fibre. The M. catarrhalis prophage names are coloured according to clade assignment in Fig. 1; red = clade 1, blue = clade 2, orange = clade 3, green = clade 4. Reference phage genes, as detailed in the methods section, are coloured purple. Scale at lower left of each panel corresponds to 0.2 nucleotide substitutions per site. The image of a Siphoviridae phage presented in the centre with 100 nm scale [66] is labelled according to the analysed genes. The a terminase large subunit, b terminase small subunit, and c integrase proteins are found inside the capsid, whereas the d portal, e plate, and f tail fibre are structural

Fig. 4

Codon-based measure of evolutionary pressure on M. catarrhalis prophage genes. The d_N/d_S value for each codon (represented by each blue +) is plotted against the length of each prophage gene encoding large terminase subunit, integrase, and portal. Similarly, the d_N/d_S value for each codon is plotted for the concatenation of 7 M. catarrhalis MLST housekeeping genes. An average value for four subsequent codons is represented by the red line. Values of d_N/d_S < 1 indicates purifying selection, d_N/d_S > 1 indicates diversifying selection, and d_N/d_S = 1 indicates neutrality

Fig. 5

Similarity of antitoxins found in M. catarrhalis prophages. Eleven antitoxin components were identified in 9 M. catarrhalis prophages with their putative evolutionary relationship depicted above. ¹The antitoxins are found in prophages contained in clades 1 (red), 2 (green), and 4 (blue). ²The frequency at which the respective antitoxins or analogues can be found throughout the studied 95 M. catarrhalis genomes. The numbers above each branch in the distance tree represent the bootstrap percentage, and the scale in the lower left corner corresponds to 0.1 nucleotide substitutions per site

Fig. 6

Pan-genome analysis of M. catarrhalis prophages. The pan-genome of 32 M. catarrhalis prophages is 283,622 bp in length, and is represented in the BRIG image above as the central black ring. Each concentric circle outwards from the reference pan-genome represents an individual prophage colour coded according to the assigned clades (red = clade 1, blue = clade 2, orange and yellow = clade 3, green = clade 4). A percentage identity cut-off above 90 % was used, which is represented by different hues in each ring (minimum of 90 % = grey, and up to maximum of 100 % = colour assigned to specific ring and prophage)

Fig. 7

Distribution of prophages and prophage hosts on M. catarrhalis MLST phylogenetic tree. The phylogenetic tree based on M. catarrhalis MLST data is shown on the left. The two putative sero-groups based on MLST data are shown on the right where green = seroresistant and red = serosensitive. Each M. catarrhalis strain that hosts a complete prophage as described in this study is labelled according to colour of the prophage clade, red = prophage from clade 1, blue = clade 2, orange = clade 3, and green = clade 4. The scale on the lower left of image is equal to 0.05 nucleotide substitutions per site