Putative pseudolysogeny-dependent phage gene implicated in the superinfection resistance of Cutibacterium acnes - PubMed (original) (raw)
Putative pseudolysogeny-dependent phage gene implicated in the superinfection resistance of Cutibacterium acnes
Stephanie Wottrich et al. Microbiome Res Rep. 2024.
Abstract
Objectives: Cutibacterium acnes, formerly Propionibacterium acnes, is a bacterial species characterized by tenacious acne-contributing pathogenic strains. Therefore, bacteriophage therapy has become an attractive treatment route to circumvent issues such as evolved bacterial antibiotic resistance. However, medical and commercial use of phage therapy for C. acnes has been elusive, necessitating ongoing exploration of phage characteristics that confer bactericidal capacity. Methods: A novel phage (Aquarius) was isolated and analyzed. Testing included genomic sequencing and annotation, electron microscopy, patch testing, reinfection assays, and qPCR to confirm pseudolysogeny and putative superinfection exclusion (SIE) protein expression. Results: Given a superinfection-resistant phenotype was observed, reinfection assays and patch tests were performed, which confirmed the re-cultured bacteria were resistant to superinfection. Subsequent qPCR indicated pseudolysogeny was a concomitantly present phenomenon. Phage genomic analysis identified the presence of a conserved gene (gp41) with a product containing Ltp family-like protein signatures which may contribute to phage-mediated bacterial superinfection resistance (SIR) in a pseudolysogeny-dependent manner. qPCR was performed to analyze and roughly quantify gp41 activity, and mRNA expression was high during infection, implicating a role for the protein during the phage life cycle. Conclusions: This study confirms that C. acnes bacteria are capable of harboring phage pseudolysogens and suggests that this phenomenon plays a role in bacterial SIR. This mechanism may be conferred by the expression of phage proteins while the phage persists within the host in the pseudolysogenic state. This parameter must be considered in future endeavors for efficacious application of C. acnes phage-based therapeutics.
Keywords: Cutibacterium acnes; Propionibacterium acnes; antibiotic resistance; bacteriophage; phage therapy; pseudolysogeny; superinfection exclusion; superinfection resistance.
© The Author(s) 2024.
Conflict of interest statement
All authors declared that there are no conflicts of interest.
Figures
Figure 1
Phage Aquarius Characterization. (A) Plaque morphology of Aquarius was of variable size with clear to turbid plaques. The plate image was enhanced by 30% in brightness and contrast to show detail; (B) Transmission electron microscopy at 52000X magnification. The length of the phage tail and the diameter of the phage head were measured using the software program ImageJ[52] and were found to be 150.3 and 58.8 nm, respectively. The presence of the long non-contractile tail and the icosahedral head are characteristic of Caudoviricetes phages; (C) SIR phenotype characterized by the growth of bacteria in the center of an area of clearing following spot inoculation of phage Aquarius on cultures of C. acnes ATCC 6919 (second row streak) and two C. acnes clinical isolates (third and fourth row streaks) during an initial host range assay. No bacterial lysis was observed for the negative control spot inoculation on E. coli (first row streak). SIR: superinfection resistance.
Figure 2
Phamerator Comparative Genomics Maps. Comparative genomic maps generated using Phamerator.org for the eight C. acnes phages used in this study. Each genome is arranged along a kilobase ruler with forward transcribed genes marked above the ruler and reverse transcribed genes marked below the ruler. Each gene product is color coded by related protein “phamilies” or “phams” determined by BLASTP and ClustalW as described by Cresawn et al. (2011)[34]. Phams with known functions are labeled along the Aquarius genome map. Nucleotide sequence similarity based on BLASTN is shown by the shaded regions between genomes, and is colored based on its E value, with violet representing the best matches (lowest E values) and red the worst matches (highest E values). White areas indicate that there is no nucleotide similarity in those regions. As reported for previously studied C. acnes phages, the phages used in this study have genomes with a high degree of synteny and nucleotide conservation, as demonstrated by the mostly violet shading between genomes[3,6,11].
Figure 3
Genome-BLAST Distance Phylogeny tree for C. acnes and S. thermophilus phages. The numbers above the branches are GBDP pseudo-bootstrap support values from 100 replications. The branch lengths of the resulting trees are scaled in terms of the recommended VICTOR formula (D0)[40]. GBDP: Genome-BLAST Distance Phylogeny.
Figure 4
High Stability/Low Stability Patch Test Samples, derived from passage 4, on ATCC 6919. The above picture illustrates the capacity of the passaged lysogenic bacteria taken from the fourth pass of the patch test to produce an area of clearing surrounding the lysogen streak (high stability group, on the right). Also shown is a prior putative pseudolysogen that possessed the ability to cause lysis but lost its lysing capacity after the fourth passage (low stability group, on the left). Positive and negative controls were also prepared (lawn of pure ATCC 6919, and putative pseudolysogens with no bacterial lawn, respectively), and demonstrated successful bacterial growth (not shown).
Figure 5
Protein signatures of Phage TP-J34 Ltp and Phage Aquarius gp41. (A) Phage TP-J34 Ltp. Output from InterPro (blue) indicated the presence of several protein signatures, including the two conserved domains that comprise the active site region of Ltp (residues 49-92 and 96-141). Other notable signatures included a prokaryotic lipoprotein (residues 1-20), regions of disorder (residues 21-50 and 21-37), signal peptide H-region (residues 4-15), signal peptide C-region (residues 16-20), signal peptide N-region (residues 1-3), signal peptide (residues 1-20), transmembrane signal peptide (residues 1-28), and a non-cytoplasmic domain (residues 21-142). Output from MEME (red) indicated the presence of two conserved motifs as well, spanning residues 46-77 and 91-141. Output from RaptorX (green; depicted on ruler) also identified a generally high region of disorder spanning from the first residue to roughly residue 50, and a small region at the very end of the peptide spanning roughly one to two residues; (B) Protein signatures of Phage Aquarius gp41. Output from InterPro (blue) indicated the presence of several notable protein signatures, including regions of disorder (residues 23-89 and 34-51), signal peptide H-region (residues 8-19), signal peptide C-region (residues 20-24), signal peptide N-region (residues 1-7), signal peptide (residues 1-24), transmembrane signal peptide (residues 1-25), transmembrane helix (residues 7-26), and a non-cytoplasmic domain (residues 25-179). Output from MEME (red) indicated the presence of two conserved motifs as well, spanning residues 84-133 and 137-177. Output from RaptorX (green; depicted on ruler) also identified a generally high region of disorder spanning from the first residue to roughly residue 91, and a small region at the very end of the peptide spanning roughly three to five residues. MEME: Multiple Em for Motif Elicitation.
Figure 6
The predicted structure of Aquarius gp41 resembles factors that facilitate protein-protein interactions. (A) Aquarius gp41structure predicted by AlphaFold2. Structure is colored by confidence and functional motifs are labeled; (B) Closest structural homologs to Aquarius gp41 as determined by the DALI structural homology server - Vir8B (Green: pdbid is 6IQT) and mouse Cystatin (orange:pdbid 6UIO); (C) Structural alignment between gp41 95-179 (blue) and mouse cystatin (orange); (D) Electrostic potential was solved using the APBS for Aquarius gp41 (left) and TP-J34 Ltp (right). Electrostatic potential scale is given in kT/e where negative (red) and positive (blue) surface potentials are shown. APBS: Adaptive Poisson-Boltzmann Solver.
Figure 7
Gp41 expression relative to the uninfected control. The average fold increase in phage Aquarius gp41 mRNA expression averaged over three PCR trials, normalized to the bacterial housekeeping gene RecA. A one-way ANOVA with post hoc Tukey HSD comparison indicated a significant difference between the active infection and pseudolysogen groups (*P < 0.05). Error bars the Standard Error.
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