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Wottrich et al. Microbiome Res Rep 2024;3:27 https://dx.doi.org/10.20517/mrr.2023.42 Page 11 of 17

Table 5. BLASTp results for Ltp-like proteins
Ltp Ltp Ltp Ltp Ltp Ltp
Phage Host TP-J34 TP-J34 TP-J34 TP-778L TP-778L TP-778L
score E-value identities (%) score E-value identities (%)
Lauchelly C. acnes 30 1.1 26/124 (20) 31 0.83 26/121 (21)
PFR1 P. freudenreichii 110 6e-25 65/138 (47) 108 4e-24 66/149 (44)
PFR2 P. freudenreichii 110 6e-25 63/138 (47) 108 4e-24 66/149 (44)

No escape mutants found
Since bioinformatics suggested the presence of a conserved Ltp-like protein in Aquarius’s genome, three
attempts were made to isolate escape mutant phages capable of infecting pseudolysogens. It was
hypothesized that after several reinfection attempts, pseudolysogens conferring SIR may eventually acquire
SIR-compromising mutations mapping to either gp41 or the tape measure protein of Aquarius, which
would support an SIE mechanism characteristic of that described by Bebeacua et al. (2013) . However, all
[22]
attempts at plating Aquarius on pseudolysogens were unsuccessful in producing plaques, indicating a tight
immunity mechanism at play.

qPCR of Aquarius Ltp-like gp41
To find evidence linking gp41 to SIR, qPCR was performed to assess gene expression in a phage-free control
compared to actively infected bacteria and Aquarius pseudolysogens. The gp41 expression was normalized
to the bacterial housekeeping gene RecA [Table 2]. The average results of three qPCR trials indicated a fold
increase of approximately 333,000 times more expression of Aquarius gp41 in the active infection group and
40,000 times more expression in the pseudolysogen group compared to the phage-free control [Figure 7].
This represents an expression ratio of 8.3:1 for the active phage infection to the pseudolysogen. A one-way
ANOVA with post hoc Tukey HSD comparison of the qPCR results yielded a significant result (P < 0.05),
suggesting an important role for gp41 in early phage infection. Thus, it may be that gp41 is highly expressed
during initial infection, the period in which many individual phages have recently entered their bacterial
hosts and are undergoing replication, whereas expression may be maintained at a lower level during the
pseudolysogenic life cycle.

DISCUSSION
The results of this research support the previous findings that Aquarius and other C. acnes phages can
undergo pseudolysogeny and support the hypothesis of a SIR mechanism conferred via expression of an
Lpt-like gene (gp41) from a semi-stable phage episome within the bacterial host. Gene mapping has
demonstrated that C. acnes phages have little genomic diversity, which may account for the effectiveness of
the SIR mechanism against other C. acnes phages in cross spot testing [3,11] . Bioinformatics demonstrated the
presence of gp41, a conserved protein in all C. acnes phage genomes tested, which bears structural
resemblance to the known phage SIR protein Ltp. Gp41 was also expressed at high levels during active
infection, as well as in established pseudolysogen strains as demonstrated by qPCR. The lower gp41
expression in latent pseudolysogen samples relative to the sharp increase in the newly infected samples may
suggest the breaching of a saturation point or feedback mechanism that results in waning gp41 expression
over time. However, without further data, definitive conclusions cannot be drawn about the precise
mechanism, nor the evolutionary implications of the SIE phenomenon in C. acnes phages as described here.
A future study assessing the expression of gp41 in other C. acnes phages, as well as the expression of Ltp in
TP-J34 and/or TP-778L, may provide further support for a similar mechanism if a similar expression
pattern were to be found. Additionally, there are various proposals as to the evolutionary etiology of SIR in
general which may also be applicable with regard to the pattern of waning expression over time throughout
the phage life cycle. Recent studies have demonstrated that in some cases of SIE, superinfecting phages are