# Timely excision of prophage Φ13 is essential for the Staphylococcus aureus infectious process

Olivier Poupel, Gérald Kenanian, Lhousseine Touqui, Charlotte Abrial, Tarek Msadek, Sarah Dubrac

► To cite this version:

Olivier Poupel, Gérald Kenanian, Lhousseine Touqui, Charlotte Abrial, Tarek Msadek, et al. Timely excision of prophage Φ13 is essential for the Staphylococcus aureus infectious process. Infection and Immunity, 2025, 93 (10), 10.1128/iai.00314-25. pasteur-05446243

HAL Id: pasteur-05446243

https://pasteur.hal.science/pasteur-05446243v1

Submitted on 7 Jan 2026

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons CC BY 4.0 - Attribution - International License
# Timely excision of prophage Φ13 is essential for the Staphylococcus aureus infectious process

Olivier Poupel,¹ Gérald Kenanian,¹ Lhousseine Touqui,² Charlotte Abrial,² Tarek Msadek,¹ Sarah Dubrac¹

**AUTHOR AFFILIATIONS** See affiliation list on p. 13.

**ABSTRACT** Mobile genetic elements play an essential part in the infectious process of major pathogens, yet the role of prophage dynamics in *Staphylococcus aureus* pathogenesis is still not well understood. Here, we studied the impact of the Φ13 *hlb*-convert-ing prophage, whose integration inactivates the *hlb* β-toxin gene, on staphylococcal pathogenesis. We showed that prophage Φ13 is lost in approximately half the bacterial population during the course of infection. Inactivation of the Φ13 *int* recombinase gene, essential for insertion/excision, locked the prophage in the bacterial chromosome, leading to a significant loss of virulence in a murine systemic infection model. In contrast, the non-lysogen strain (ΔΦ13), where the *hlb* beta-hemolysin gene is reconstituted, displayed strongly increased virulence. Accordingly, histopathological analyses revealed more severe nephritis in mice infected with bacteria lacking prophage Φ13 (ΔΦ13), compared to infection with the parental strain. Infection with the *Δint* mutant, where beta-hemolysin production is abolished, led to the least severe renal lesions. Cytokine induction in a human neutrophil model showed significantly increased IL-6 expression following infection with the beta-hemolysin producing strain (ΔΦ13). Our results indicate that timely *in vivo* excision of the Φ13 prophage is essential for progression of the *S. aureus* infectious process: early excision leads to rapid host death, whereas the inability to excise the prophage significantly reduces staphylococcal virulence.

**IMPORTANCE** This study highlights prophage Φ13 excision as a critical factor in *Staphylococcus aureus* pathogenesis, influencing infection outcomes by balancing rapid host killing with reduced bacterial virulence. This mechanism may represent a bet-hedging strategy in genetic regulation, resulting in a mixed bacterial population capable of rapidly switching between two processes: bacterial colonization and host damage. Unraveling this dynamic opens new possibilities for developing targeted therapies to disrupt or modulate prophage activity, offering a novel approach to mitigating *S. aureus* infections.

**KEYWORDS** *Staphylococcus aureus*, prophage excision, virulence, host-pathogen interactions, infectious process, beta-hemolysis, timing device

Staphylococcus aureus, a major human pathogen, causes severe invasive infectious diseases targeting every type of organ, such as pneumonia, endocarditis, osteomyelitis, or septicemia. Yet it is also a commensal, asymptomatically colonizing up to 30% of the human population, with the transition between the two lifestyles poorly understood (1). Mobile genetic elements make up approximately 20% of its genome, leading to variations in gene combinations that can strongly influence *S. aureus* virulence (2). Among these, temperate bacteriophages play an important role in genetic variability. Φ13, a member of the Sa3int Siphoviridae *S. aureus* bacteriophage family, is responsible for a dual phenotypic conversion (3, 4) and is by far the most widespread among human clinical isolates (5). Its chromosomal attachment site (*attB*) lies within the coding

**Editor** Victor J. Torres, St Jude Children's Research Hospital, Memphis, Tennessee, USA

Address correspondence to Tarek Msadek, tmsadek@pasteur.fr, or Sarah Dubrac, sarah.dubrac@pasteur.fr.

Tarek Msadek and Sarah Dubrac contributed equally to this article.

The authors declare no conflict of interest.

See the funding table on p. 13.

Received 16 June 2025

Accepted 15 July 2025

Published 3 September 2025

Copyright © 2025 Poupel et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
sequence of the hemolysin B gene, *hlb*, encoding the neutral sphingomyelinase C β-toxin, such that it acts as a phage regulatory switch (6). Φ13 lysogeny leads to disruption of *hlb* and loss of β-hemolytic activity, with excision reconstituting an intact *hlb* gene (5, 7, 8).

Phage Φ13 also carries the *iec* immune evasion cluster including several genes involved in escape from the host innate immune response: *scn*, *chp*, and *sak* (9, 10). Natural mobility has been shown *in vivo* for Φ13-related phages during murine skin colonization and rabbit invasive infection as well as in isolates from cystic fibrosis patients and healthy individuals (5, 11–13). Additionally, among *S. aureus* isolates from recurrent furunculosis, 45% produced β-hemolysin compared to only 19% of those from nasal colonization (14).

Given the importance of phage Φ13 excision on β-hemolytic activity, we wished to study the effect of the presence or absence of phage Φ13 on *S. aureus* virulence during systemic infection in three different configurations that had not been previously studied: (i) preventing phage excision and consequently β-hemolytic activity, (ii) absence of phage Φ13 leading to continuous β-hemolytic activity, and (iii) simultaneous presence of phage Φ13 and a functional *hlb* gene.

We show that timely excision of the Φ13 prophage is essential for pathogenesis, with early β-hemolysin production leading to a significant increase in virulence and elevated levels of the pro-inflammatory cytokine IL-6. Conversely, preventing phage Φ13 excision significantly lowers staphylococcal virulence.

# RESULTS

## Mobility dynamics of *S. aureus* prophages

*S. aureus* strain HG001, a derivative of sepsis isolate NCTC 8325 (15), carries prophages Φ11, Φ12, and Φ13 (7, 16). Specific oligonucleotide primer pairs were designed to detect the presence or absence of each prophage (Table S2). Using chromosomal DNA from a closely related strain cured of all phages, SH1000, an amplicon corresponding to the native bacterial locus without Φ13 (oligonucleotide pair OMA7/OMA9, hybridizing on either side of the *attB* site, Fig. S1A) was detected, but no amplification occurred with oligonucleotides specific for Φ13 insertion (OMA7/OMA8; Fig. S1B). With the HG001 Φ13 lysogen strain, both types of amplicon were generated by PCR, suggesting spontaneous Φ13 excision within the bacterial population (Fig. S1B). We quantified relative prophage levels within the bacterial population by qRT-PCR during growth under optimal laboratory conditions (TSB at 37°C with aeration). Our results indicate that prophage Φ11 is highly mobile with approximately 10% of cells lacking the phage, whereas Φ12 was much more stable with less than 0.01% of non-lysogen cells (Fig. 1A). Interestingly, the Φ13 prophage was lost in about 0.5% of the population under these growth conditions (Fig. 1A).

## Excision of prophage Φ13 is increased *in vivo*

As shown above, prophage Φ13 is spontaneously excised during growth under laboratory conditions. Since this phage carries genes encoding the SAK staphylokinase virulence factor, as well as SCIN and CHIP, involved in escape from the innate immune response (17), we examined Φ13 excision frequency during the infectious process.

We generated a strain carrying the *aad* (9) spectinomycin resistance gene inserted within the Φ13 genome of the HG001 strain (see Materials and Methods, and Table S1 strain ST1387). We verified that the *aad* (9) cassette did not interfere with Φ13 stability during growth *in vitro*. In the ST1387 strain (HG001Φ13-*aad* [9]), the prophage excision frequency was 0.58 ± 0.05%, which is comparable to the excision rate observed in the wild-type (WT) HG001 strain (0.59 ± 0.05%), as determined by qRT-PCR. Four days following sub-lethal (2 × 10⁷ c.f.u.) intravenous inoculation with *S. aureus* strain ST1387, mice were sacrificed and kidneys collected for c.f.u. counts. Out of 25 harvested kidneys, 18 were colonized by a mixed bacterial population with respect to Φ13 lysogeny (Fig. 1B).
<figure><img src="image_2.png" /><figcaption>FIG 1 Prophage Φ13 excision *in vitro* and *in vivo*. (A) Prophage excision frequencies in strain HG001. qRT-PCR quantification of Φ11, Φ12, and Φ13 prophage content in *S. aureus* strain HG001 grown in TSB at 37°C with aeration. Specific primer pairs are described in Table S2. The results are the mean of four technical replicates and are representative of three independent biological replicates. (B) Quantification of Φ13 lysogens in bacterial populations from infected kidneys. Mice were infected from the caudal lateral vein with a sub-lethal dose (2 × 10⁷ c.f.u.) of *S. aureus* strain HG001 Φ13::aad(9). Mice were sacrificed 4 days post-infection, and kidneys harvested for c.f.u. counts on TSA and TSA with spectinomycin. Twenty-five infected kidneys were examined, and the proportion of homogeneous lysogen population (less than 0.5% non-lysogen) and heterogeneous bacterial population with a mix of lysogen and non-lysogen bacteria is shown. (C) Proportion of non-lysogen bacteria per kidney in organs infected with a mixed population of lysogenic and non-lysogenic bacteria.</figcaption></figure>
In some mice, a single kidney was colonized with a mixed population with the other only by Φ13 lysogens, suggesting that loss of Φ13 is a *post facto* event to kidney colonization. The average loss of Φ13 was around 50% in the 18 kidneys colonized with a mixed bacterial population (Fig. 1C), a significant increase over the spontaneous excision rate of 0.5% observed during growth *in vitro*. The specific *in vivo* dynamics of prophage content suggest either an induction of Φ13 excision in response to conditions encountered during infection or a selective advantage for the non-lysogenic strain, potentially due to enhanced fitness during kidney colonization through loss of the prophage.

## Excision of prophage Φ13 is essential for the *S. aureus* infectious process

Increased prophage Φ13 excision during infection suggests it may impact virulence. The Φ13 SAOUHSC-02293 gene, referred to henceforth as *int*, encodes a protein highly similar to tyrosine recombinase integrases such as Int of phage lambda (16, 18). We deleted the entire coding sequence of the Φ13 *int* gene in *S. aureus* strain HG001 in order to prevent excision, locking the prophage within the bacterial chromosome. We also constructed a strain where the entire Φ13 genome is deleted, restoring an intact and functional *hlb* gene (see Materials and Methods). These mutations had no effect on bacterial growth *in vitro* (Fig. S2). β-hemolytic activity following Φ13 excision was detected on sheep blood agar plates as a distal cold-inducible halo of partial hemolysis (indicated by the white arrow in Fig. 2A).

As shown in Fig. 2B, the frequency of Φ13 excision in the *Δint* mutant is reduced more than 1000-fold compared to the WT strain (0.6%), confirming the essential role of Int in Φ13 excision. Full complementation of the *Δint* mutant was obtained by expressing the *int* gene from the Pprot constitutive promoter in the pMK4Pprot-*int* plasmid (Fig. 2B).

We next examined the effect on *S. aureus* virulence of either the absence of prophage Φ13 (HG001ΔΦ13 strain, intact *hlb* gene) or the lack of Φ13 excision (HG001Δ*int* strain, *hlb* gene disrupted). As shown in Fig. 3A, using a systemic model of murine infection (5 × 10⁷ c.f.u. intravenous injection), the absence of Φ13 with a resulting intact *hlb* gene led to acute virulence with the rapid death of infected animals (50% mortality 24 h post-infection for the ΔΦ13 strain vs 6 days for the WT strain), and more than 90% mortality at the end of the assay (10 days post-infection). Conversely, when Φ13 excision is abolished (*Δint* strain), *S. aureus* virulence is strongly reduced compared to the WT strain (31% mortality at the end of the assay, vs 63%). These results indicate that Φ13 excision and its timing are crucial parameters for the *S. aureus* infectious process.

## Φ13 dynamics are involved in organ damage during infection

To understand the effect of Φ13 dynamics on *S. aureus* virulence, we measured bacterial colonization and evaluated the kidney lesions of infected mice by histopathological analysis. Mice were inoculated intravenously with sub-lethal concentrations of *S. aureus* cells (approximately 2 × 10⁷ c.f.u.). At 6 days post-infection, mice were sacrificed, and one kidney was fixed for histopathological analysis, and the other was homogenized for c.f.u. counts. Kidney bacterial loads for the parental HG001 strain and the HG001ΔΦ13 and HG001Δ*int* mutants were not significantly different (approximately 5 × 10⁷ c.f.u. per kidney; Fig. S3). However, histopathological analysis revealed nephritis (very probably embolic nephritis) with significantly varying degrees of severity between mice infected with the parental HG001 strain or the ΔΦ13 and *Δint* mutants. At 6 days post-infection, mice inoculated with the WT strain generally displayed mild to moderate nephritis (5/6 mice) or multifocal inflammatory infiltrates (macrophages and other mononucleated cells) in the renal cortex (1/6 mouse) (Fig. 3B1 and B2). In contrast, infection with the ΔΦ13 strain leads to more severe nephritis (5/6 mice), with neutrophilic inflammation forming multifocal abscesses, extension to tubules and interstitium, and ischemic necrosis of the papillae (Fig. 3B3 and B4). Mice infected with the *Δint* strain displayed less severe lesions, with mononuclear cell infiltrates mostly in the cortex (4/6 mice) or mild to moderate (1/6 mouse) or marked (1/6 mouse) nephritis (Fig. 3B5 and B6).
A.

<figure><img src="image_3.png" /></figure>

B.

<figure><img src="image_4.png" /><figcaption>**FIG 2** Characterization of the *S. aureus* Δ*int* and ΔΦ13 mutants. Strains were grown overnight at 37°C with aeration. (A) 3 µL of the bacterial cultures was spotted on sheep blood agar plates and incubated consecutively for 24 h at 37°C and 24 h at 4°C to detect hemolytic activities. The white arrow indicates the β-hemolytic halo characteristic of bacteria lacking prophage Φ13. (B) Prophage Φ13 excision frequency detected by qRT-PCR using oligonucleotide pairs OMA7-OMA8 and OMA7-OMA9, specific for the presence or absence of Φ13, respectively. A Φ13 lysogen strain was included as a control to confirm the specificity of the amplicons used to quantify Φ13 lysogeny. Results are presented as means ± SEM of three replicates, and a t-test was used to compare WT and Δ*int* results (***, *P* < 0,0001).</figcaption></figure>

These results show that Φ13 dynamics and correlated β-hemolysin production are not essential for host colonization but instead are key factors in host organ damage during infection.
<figure><img src="image_5.png" /><figcaption>FIG 3 Impact of prophage Φ13 and *hlb* on *S. aureus* virulence. (A) Kaplan-Meier survival curves of *RjOrl:SWISS* mice infected intravenously with *S. aureus* HG001 wild-type (WT) strain and the *Δint* and *ΔΦ13* mutants (5 × 10⁷ c.f.u./injection). A total of 14 mice were used in each group in two independent experiments. For each strain, the genotype is indicated followed by the relevant genetic context (presence or absence of prophage Φ13 and a functional *hlb* gene). Statistical analysis of data using the Log-rank (Mantel-Cox) test was performed to compare each mutant strain to the HG001 parental strain. WT/Δint: *P* = 0.0077; WT/ΔΦ13: *P* = 0.0021. (B) Histopathological kidney analyses 6 days post-infection by intravenous inoculation with sub-lethal bacterial loads (2 × 10⁷ c.f.u./injection) of the *S. aureus* HG001 WT (1 and 2), *Δint* (3 and 4) and *ΔΦ13* (5 and 6) strains. Most (5/6) mice infected with the WT strain displayed mild to moderate nephritis, with neutrophilic infiltrates/abscesses (black arrowheads) of varying sizes in the cortex (1), medulla, and papilla (2). Infection with strain *ΔΦ13* led to more severe nephritis (5/6 mice), with multifocal abscesses (3 and 4, black arrowheads), extension to tubules and interstitium, and ischemic necrosis of the papilla (4, black arrow). Most mice (4/6) infected with the *Δint* strain displayed less severe lesions, with mononuclear cell infiltrates most often in the cortex (5, not seen at a low magnification) but also in the papillae (6, arrowhead).</figcaption></figure>

## Φ13 excision triggers host inflammatory response in infected THP-1 cells

Given the major impact of Φ13 excision on virulence and inflammation, we tested its effect on the induction of the cellular inflammatory response using human monocytic
THP-1 cells. We compared the bacterial internalization capacity of THP-1 cells, the intracellular viability of bacterial cells, and the induction of inflammatory markers following infection with the parental HG001 strain and the ΔΦ13 and Δ*int* mutants. THP-1 differentiated macrophages were very efficient for *S. aureus* phagocytosis, regardless of prophage content, with nearly 100% of bacteria internalized with an m.o.i. (multiplicity of infection) of 10 after 2 h (Fig. S4A). Intracellular survival was not significantly different for the three strains with approximately 10% survival after 24 h post-infection and less than 5% after 48 h (Fig. S4B). To measure induction of the inflammatory response, duplicate aliquots of infected THP-1 cells were frozen 24 h post-infection, treated to recover cellular RNA and induction of inflammatory cytokine genes measured by qRT-PCR. We measured induction of three pro-inflammatory cytokines previously shown to be induced by *S. aureus* infection, TNFα, IL-1β, and IL-6 (19). Although there was no significant difference in induction levels of TNFα and Pro-IL-1β for the three strains, IL-6 gene expression was strongly increased when THP-1 cells were infected with the ΔΦ13 strain as compared to the HG001 strain and the Δ*int* mutant (Fig. 4). Thus, lysogeny by Φ13 and the concomitant loss of β-hemolytic activity appear to downregulate the inflammatory response through lower induction of the pro-inflammatory cytokine IL-6.

## Impact of *hlb* on *S. aureus* virulence

Taken together, the results shown above suggest a major role of β-hemolysin production in the increased virulence of the ΔΦ13 strain. To test this hypothesis, we compared the virulence of the ΔΦ13 strain, which produces β-hemolysin, with the virulence of the ΔΦ13Δ*hlb* strain in which the *hlb* gene has also been deleted. As shown in Fig. 5A, 44% of mice infected by the ΔΦ13Δ*hlb* strain survived at 9 days post-infection, whereas the survival of mice infected by the strain producing β-hemolysin (ΔΦ13) was only 14%, indicating that it is the major factor responsible for the effect of Φ13 excision on *S. aureus* virulence. To test the contribution of Φ13 in virulence, we constructed a strain where both Φ13 and an intact *hlb* gene are present (Δ*int* *hlb* complemented strain; see Materials and Methods, and Table S1). As shown in Fig. 5B, mouse mortality caused by the ΔΦ13 strain producing β-hemolysin but lacking the Φ13 prophage was essentially the same as that following infection by the Δ*int* *hlb* complemented strain (with both the Φ13 prophage and a functional *hlb* gene). This result confirms the major role of Φ13 dynamics

<figure><img src="image_6.png" /><figcaption>**FIG 4** Impact of Φ13 on cytokine induction by THP-1 neutrophils. PMA-activated THP-1 cells were infected with the HG001 wild-type (WT) strain or the ΔΦ13 and Δ*int* mutants with an m.o.i. = 10. After 24 h of infection, cytokine gene expression was determined by qRT-PCR performed with cDNA synthesized from RNA of THP-1 cells infected by the indicated strains (dark gray) or non-infected control (light gray). Expression levels are given in arbitrary units normalized against that of HPRT. Each experiment was carried out six times in two independent series (means ± SD). ***, *P* < 0.005 (t-test).</figcaption></figure>
<figure><img src="image_7.png" /><figcaption>FIG 5 Impact of *hlb* on *S. aureus* virulence. Kaplan-Meier survival curves of *RjOrl:SWISS* mice infected intravenously with *S. aureus* HG001 wild-type (WT) strain and the *Δint hlbcomp*, *ΔΦ13*, and *ΔΦ13Δhlb* mutants (5 × 10⁷ c.f.u./injection). A total of 14 mice were used in each group in two independent experiments. *, *P* < 0.05 and **, *P* < 0.01 (Wilcoxon test). For each strain, the genotype is indicated followed by the relevant genetic context (presence or absence of prophage Φ13 and a functional *hlb* gene). (A) Role of *hlb* in virulence by comparing infection with the HG001 strain and the *ΔΦ13* and *ΔΦ13Δhlb* derivatives. Statistical analysis of data using Log-rank (Mantel-Cox) test was performed to compare results from each mutant strain to the HG001 parental strain. WT/*ΔΦ13Δhlb*: *P* = 0.6157 (ns); WT/*ΔΦ13*: *P* = 0.0039. (B) Role of Φ13 in virulence by comparing infection with the HG001 strain and the *Δint hlbcomp* (dashed line) and *ΔΦ13* derivatives. Statistical analysis of data using the Log-rank (Mantel-Cox) test was performed to compare data for mutant strains to the HG001 parental strain. WT/*Δint hlbcomp*; *P* = 0.0147; WT/*ΔΦ13*; *P* = 0.0066.</figcaption></figure>

in *S. aureus* virulence by controlling the timing of β-hemolysin production during the infectious process.
DISCUSSION

S. aureus is a highly successful human pathogen with a sophisticated arsenal of virulence factors whose production is tightly controlled by multiple regulatory networks, such as two-component systems (20). Further fine-tuning is provided by mobile genetic elements such as pathogenicity islands, plasmids, transposons, staphylococcal cassette chromosomes and bacteriophages conferring additional virulence factors or antibiotic resistance genes (21). Prophages are widely distributed amongst S. aureus strains. Among these, the Φ13 S. aureus prophage is inserted within the *hlb* gene encoding β-hemolysin and carries at least three genes involved in host interactions and escape from the immune response, known as the immune evasion cluster (IEC): *sak*, encoding staphylo-kinase, involved in converting host plasminogen to active plasmin, *scn*, encoding a staphylococcal complement inhibitor, and *chp*, encoding a neutrophil and monocyte chemotaxis inhibitory protein (9). The Φ13 *hlb*-converting prophage family is present in approximately 90% of S. aureus clinical isolates (9).

β-hemolysin is a sphingomyelinase that can lyse proliferating human lymphocytes, contributing to the host immune evasion (22). Independently of its pore forming activity, β-hemolysin has a biofilm ligase effect by forming a nucleoprotein matrix both *in vitro* and during infectious endocarditis (23, 24). It has also been shown that Hlb favors mouse skin colonization (12). We noted that S. aureus cells isolated from infected mice frequently displayed β-hemolytic activity due to *in vivo* excision of the Φ13 prophage. This prompted us to examine the effect of phage mobility on host-pathogen interactions. Our results indicate that mobility of this prophage is required for full virulence of S. aureus as part of the time-course of the infectious process. We identified and inactivated the potential integrase gene of this prophage, effectively locking the prophage within the S. aureus genome by preventing its excision, and showed that this leads to strongly decreased virulence, with 70% of the mice still surviving after 10 days. Conversely, a strain lacking the Φ13 prophage exhibited greater virulence than the WT strain, with 50% of infected mice succumbing within the first day of infection, compared to 6 days for the WT strain. These Φ13-associated virulence traits are not correlated to differences in the strain's ability to colonize the host, as bacterial loads in the infected kidneys were similar regardless of the strain's lysogenic status. However, kidney damage following infection was strongly associated with the presence of Φ13, showing a clear gradient where a lower proportion of lysogenic bacteria resulted in more severe tissue lesions. In a human monocyte (THP-1) infection model used to assess inflammatory response, we found that the non-lysogenic strain significantly increased the production of the pro-inflammatory cytokine IL-6. IL-6 triggers the release of acute-phase response proteins into the bloodstream, leading to a systemic inflammatory response (25). It is also considered a key biomarker for predicting poor outcomes in sepsis patients and is a promising target for inhibitors to help mitigate the adverse effects of inflammation (19, 26). The induction of IL-6 by the non-lysogenic strain may be linked to its heightened virulence and the significant neutrophil recruitment observed at the inflammation site, as shown by the histopathological analysis of infected kidneys.

The exact trigger causing excision of Φ13 during infection remains to be established. Our observations show that after intravenous infection, the kidneys of the same mouse can be colonized differently, one kidney by a lysogenic population, while the other harbors a mixed population. This suggests that Φ13 excision is likely induced in the kidneys following colonization rather than during the initial spread of bacteria through the bloodstream. A previous study using a rabbit infection model found that bacteria producing β-hemolysin emerged during organ colonization, but this was not observed in the bacterial population recovered from the bloodstream (11). Stress conditions activating the SOS response are known to induce prophage excision. However, despite many attempts, we were not able to induce Φ13 excision from the bacterial chromosome through treatment with Mitomycin C as an SOS-inducing agent. Similar results were previously described with a Newman strain carrying the closely related *hlb*-converting prophage, ΦNM3 (27). More recently, it has been demonstrated that exposure to
oxidative stress (such as hydrogen peroxide) and biofilm growth conditions promote the excision of ΦSA3mw (28). This may correlate with our *in vivo* observations, that is, that phage induction occurs after organ colonization, under biofilm development conditions.

Epidemiological studies have shown that *hlb*-converting prophages are more commonly found in strains colonizing humans (from nasal swabs) than in invasive strains (isolated from blood) (3). Additionally, Φ13-derivative carriers are significantly more frequent in human *S. aureus* isolates than in animal strains (29). These *hlb*-con- verting phages not only inactivate *hlb* but also carry virulence genes, including an immune evasion cluster (IEC) (9). The Sak, SCIN, and CHIPS determinants encoded by this cluster play crucial roles in modulating neutrophil chemotaxis, bacterial phagocytosis, and killing (17). However, their activity is highly specific, primarily targeting the human immune system (30–32). This specificity of the Φ13-derivative IEC in counteracting the human immune response likely contributes to the greater prevalence of *hlb*-converting prophages in human *S. aureus* strains.

Interestingly, we demonstrated using a murine systemic infection model, that the *hlb* gene is the major determinant responsible for the increased virulence observed in the absence of the Φ13 phage. When Hlb is produced, no effect of Φ13 alone was observed (Fig. 5B), likely because the strain is already highly virulent due to constant Hlb production. However, in the absence of *hlb*, the presence of Φ13 led to lowered mouse mortality (Fig. 3A and 5A). While the immune evasion factors encoded by Φ13 have been described as human-specific, it cannot be excluded that they may also exhibit some activity during mouse colonization. Alternatively, Φ13 may encode uncharacter- ized proteins that could modulate *S. aureus* virulence.

Excision of Φ13 and the chromosomal rearrangement reconstituting an intact *hlb* gene can be viewed as a timing device, ensuring that the bacteria first colonize the host, benefiting from the immune evasion cluster proteins encoded by the prophage, with production of β-hemolysin and host tissue damage occurring once the bacteria have reached a threshold level, in agreement with the temporal control of *S. aureus* virulence factors (33). One of the first examples of prophage excision as a timing device for a developmental process is the *skin* element whose excision leads to a chromosomal rearrangement reconstituting the gene encoding the Sigma K sigma factor, essential for *Bacillus subtilis* sporulation (34, 35).

As demonstrated in this study and supported by the literature, the heterogeneity within *S. aureus* populations with respect to *hlb*-converting prophages appears to be a common trait. This could be seen as a bet-hedging strategy, allowing the bacteria to retain the ability to produce both immune evasion proteins (in the lysogenic popula- tion) and the potent Hlb cytotoxin (in the non-lysogenic cells). Additionally, since both β-hemolysin and the immune evasion cluster proteins encoded by the prophage are secreted, it is plausible that functional complementation may occur within a mixed population of lysogenic and non-lysogenic bacteria. Similar dual strategies have been observed before, where bistability leads to cell-to-cell variation in gene expression (36, 37). This mixed population with distinct genetic content would be well-equipped to resist host defenses during colonization as well as causing cellular damage.

# MATERIALS AND METHODS

## Bacterial strains and growth media

*Escherichia coli* K12 strain DH5α (Invitrogen) was used for cloning experiments. *S. aureus* strain HG001, derived from strain NCTC 8325 by repairing the mutated *rsbU* gene (15), was used for genetic and functional studies. Plasmids were first transformed into the restriction-deficient *S. aureus* strain RN4220 before introduction into the HG001 or derivative strains. *E. coli* strains were grown in LB medium with ampicillin (100 µg/mL) added when required. *S. aureus* strains and plasmids used in this study are listed in Table S1. *S. aureus* strains were grown in trypticase soy broth (TSB; Difco) supplemented with chloramphenicol (10 µg/mL) or erythromycin (1 µg/mL) when required. *E. coli* and *S. aureus* were grown in trypticase soy broth (TSB; Difco) supplemented with chloramphenicol (10 µg/mL) or erythromycin (1 µg/mL) when required.
*aureus* strains were transformed by electroporation using standard protocols (38), and transformants were selected on LB or trypticase soy agar (TSA; Difco) plates, respectively, with the appropriate antibiotics.

## DNA manipulations

Oligonucleotides used in this study were synthesized by Eurofins Genomics and are listed in Table S2. *S. aureus* chromosomal DNA was isolated using the MasterPure Gram-Positive DNA Purification Kit (Epicenter Biotechnologies). Plasmid DNA was isolated using a QIAprep Spin Miniprep Kit (Qiagen) and PCR fragments were purified using the Qiaquick PCR Purification Kit (Qiagen). T4 DNA ligase and restriction enzymes, PCR reagents, and Q5 high-fidelity DNA polymerase (New England Biolabs) were used according to the manufacturer's recommendations. Nucleotide sequencing of plasmid constructs was carried out by Eurofins Genomics.

## Plasmids and mutant strain construction

Construction of the ΔΦ13 mutant strain was carried out by cloning an 1,800 bp DNA fragment centered around the *attB* site, generated by PCR using oligonucleotides OSA418 and OSA419 and chromosomal DNA from the SH1000 strain (a NCTC8325-4 derivative cured for prophages), between the *Bam*HI and *Eco*RI sites of the pMAD vector (39), to give plasmid pMAD-Φ13. For deletion of the Φ13 integrase (*int*; *saouhsc-02239*), DNA fragments corresponding to the *int* upstream and downstream regions were generated using oligonucleotides OSA411/OSA412 (900 bp) and OSA413/OSA414 (820 bp) cloned between the pMAD *Bam*HI and *Ncol* sites. The *aad* (9) spectinomycin resistance gene (40) was inserted in the center of the 23 nt intergenic region between the convergent Φ13 *saouhsc-02232* and *saouhsc-02233* genes, to avoid any polar effects on transcription. DNA fragments corresponding to the regions upstream and downstream from the insertion site were generated by PCR using primers OSA508/OSA509 and OSA510/OSA511, respectively, and primers OSA299/OSA470 for the *aad* (9) cassette with plasmid pLC333 (41) as the DNA matrix. The DNA fragments were cloned into pMAD using the *Bam*HI/*Sall* sites for the upstream fragment, *Sall/Ncol* for the *aad* (9) cassette, and the *Ncol/Bgl*I sites for the downstream fragment, generating plasmid pMAD-Φ13*aad* (9). For deletion of *hlb*, we generated upstream and downstream fragments by PCR using oligonucleotides OSA442/OSA443 (884 bp) and OSA444/OSA445 (889 bp), respectively. These fragments were introduced into pMAD between the *Bam*HI and *Eco*RI sites, thus generating the pMAD-*hlb* plasmid. To introduce a functional *hlb* allele into the Φ13 lysogenic strain, a pMAD-*hlb*comp plasmid was constructed to place the intact *hlb* gene upstream from the Φ13 genome. A PCR fragment including the *hlb* upstream region and the *hlb* intact gene (1,760 bp) was generated using the OSA418 and OSA573 primers and the SH1000 genomic DNA as matrix and cloned into pMAD using the *Bam*HI and *Sall* restriction sites. A second PCR fragment corresponding to the part of Φ13 adjacent to *attL* was generated with the OSA574-575 primers and cloned between the *Sall* and *Bgl*I restriction sites of pMAD.

Plasmids were introduced by electroporation into *S. aureus* strain RN4220, and transformants selected at 30°C on TSA plates containing erythromycin and 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) (100 µg/mL). Plasmids were then introduced into strain HG001 and integration and excision performed as previously described (39), yielding strains HG001ΔΦ13, HG001Δ*int*, and HG001Φ13-*aad* (9) and into strain HG001ΔΦ13 for pMAD-*hlb* and HG001Δ*int* for pMAD-*hlb*comp to give strains HG001Δ*int* *hlb*comp and HG001ΔΦ13Δ*hlb*.

Plasmid pMK4Pprot-*int* was used for complementation of the *Δint* mutant and constructed by cloning the entire *int* coding sequence (amplicon OSA446/OSA447) under the control of the constitutive Pprot promoter (42).
## Prophage content measurement by quantitative real-time PCRs (qRT-PCRs)

Strains were grown overnight in TSB at 37°C, with aeration. The cells were pelleted by centrifugation (2 min, 20,800 × g) from 2 mL culture samples and genomic DNA extracted using the MasterPure Gram-Positive DNA Purification Kit (Tebu-Bio).

Oligonucleotides were designed with the BEACON Designer 4.02 software (Premier Biosoft International, Palo Alto, CA, USA) to synthesize 100–200 bp amplicons (see Table S2). qRT-PCRs and critical threshold (CT) cycles were performed and determined using the SsoFast EvaGreen Supermix (Bio-Rad, Hercules, CA, USA). All assays were performed using quadruplicate technical replicates and repeated with three independent biological samples, with one representative experiment shown. Prophage loss frequency was calculated using the formula: $1/(1 + 2^{\Delta Ct})$, $\Delta Ct = Ct_{phage negative} - Ct_{phage positive}$ derived from the $\Delta Ct$ method (43).

## Hemolysis detection

Bacteria were grown overnight in TSB at 37°C with aeration. Culture aliquots (3 µL) were spotted on Columbia plates containing 5% sheep blood (COS, Biomerieux Craponne). Plates were incubated for 24 h at 37°C and transferred to 4°C for an additional 24 h for detection of β-hemolysis (44).

## THP-1 infection experiments

THP-1 monocytes were maintained as previously described (45). Differentiation and adherence were obtained by incubation with Phorbol 12-Myristate 13-Acetate (PMA, 50 ng/mL) during 4 days at 37°C in a 5% CO₂ atmosphere. Bacterial uptake experiments and survival assays were carried out as previously described (46). Briefly, bacteria were grown in TSB until OD₆₀₀ nm = 1. Cultures were washed in PBS and adjusted to the desired inoculum in RPMI 1640 medium (Gibco), and c.f.u. counts were determined by plating serial dilutions on TSA plates. Differentiated THP-1 macrophages were incubated with bacteria (m.o.i. = 10) in RPMI 1640 at 37°C with 5% CO₂ for 1 h to allow bacterial phagocytosis. They were then washed with RPMI and incubated in RPMI-10% fetal calf serum with streptomycin (100 µg/mL), penicillin (100 U/mL), and gentamycin (40 µg/mL) to eliminate extracellular bacteria. At the indicated times, infected macrophages were washed once with RPMI and lysed by incubation in ice-cold water for 15 min, and c.f.u. counts were determined by plating serial dilutions on TSA plates. For cytokine gene expression studies by qRT-PCR, THP-1 cells were frozen at -20°C 24 h post-infection. Total RNA was extracted using an RNeasy Mini Kit (Qiagen, CA, USA). Cytokine gene expression was analyzed by qRT-PCR using the StepOnePlus real-time PCR system (Applied Biosystems). Primers used for qRT-PCR were TNFα, IL6, pro-IL1beta, and HPRT as the reference gene. The qRT-PCRs were performed by Taqman, and results were presented as relative quantification of gene expression using the 2⁻⁰⁻ΔΔCT method.

## Animal experiments

Seven-week-old female *RjOrl:SWISS* mice (Janvier Labs) were inoculated intravenously with the specified *S. aureus* strains. To analyze Φ13 excision during the infectious process, mice were infected with a sublethal dose (2 × 10⁷ c.f.u.) of the Φ13-*aad* (9) strain. Mice were sacrificed 4 days post-infection, and the kidneys were harvested and homogenized for c.f.u. determination following dilution and plating on TSA and TSA with spectinomycin (100 µg/mL). The rate of prophage excision was calculated as [(c.f.u. on TSA) – (c.f.u. on TSA + spectinomycin)]/(c.f.u. on TSA).

Strain HG001 and the *Δint*, *ΔΦ13*, *ΔΦ13Δhlb*, and *Δint hlbcomp* mutants were used for survival studies. Groups of seven mice were injected in the caudal lateral vein with 5 × 10⁷ c.f.u. per mouse in 0.2 mL. Survival was monitored daily for 9 days following infection and three independent experiments were carried out. To measure kidney colonization and histopathological analysis, mice were infected intravenously with a sublethal dose (1 × 10⁷ c.f.u.) of the HG001 parental and *Δint* and *ΔΦ13* mutant strains (six mice for each
strain). At 6 days post-infection, the kidneys were collected and for each mouse, bacterial c.f.u. counts were carried out using one kidney, and histopathological observations were done on the other kidney. For histopathology, the kidneys were removed and immediately fixed in 10% neutral buffered formalin. After 48 h of fixation, the kidneys were longitudinally sectioned through the hilus in order to obtain the renal cortex, medulla, crest, and pelvis on the same section. These samples were embedded in paraffin, and 4 µm sections were cut and stained with hematoxylin and eosin (lesion description) and Gram (bacterial detection).

## Statistical analysis

Statistical significance in survival experiments was determined using the log-rank, Mantel-Cox test, and significance across means was carried out using t-test (Graph-Pad Prism Software).

## ACKNOWLEDGMENTS

We are grateful to Grégory Jouvion for the histopathological analysis, Patrick Trieu-Cuot, in whose lab this work was carried out, and Shaynoor Dramsi for helpful discussion. All authors have read and agreed to the submitted version of the manuscript.

This work was supported by funds from Institut Pasteur, CNRS, and Vaincre La Mucoviscidose Association (Grant "Phage induction, a novel essential factor for *Staphylococcus aureus* infections?").

Conceptualization: S.D., L.T., and T.M. Data curation: O.P., S.D., L.T., and T.M. Formal analysis: S.D., L.T., and T.M. Funding acquisition: S.D., L.T., and T.M. Investigation: O.P., G.K., C.A., and S.D. Methodology: O.P. and S.D. Project administration: T.M. and S.D. Resources: T.M. and S.D. Supervision: T.M. and S.D. Validation: T.M. and S.D. Writing – original draft: T.M. and S.D. Writing – review & editing: T.M. and S.D.

## AUTHOR AFFILIATIONS

## PRESENT ADDRESS

Sarah Dubrac, Department of Microbiology, Stress Adaptation and Metabolism in Enterobacteria Unit, Institut Pasteur, Université Paris Cité, CNRS UMR6047, Paris, France

## AUTHOR ORCIDs

Tarek Msadek http://orcid.org/0000-0002-9841-8223

Sarah Dubrac http://orcid.org/0000-0002-9810-5390

## FUNDING

<table><thead><tr><th>Funder</th><th>Grant(s)</th><th>Author(s)</th></tr></thead><tbody><tr><td rowspan="6">Institut Pasteur</td><td></td><td>Olivier Poupel</td></tr><tr><td></td><td>Gérald Kenanian</td></tr><tr><td></td><td>Lhousseine Touqui</td></tr><tr><td></td><td>Tarek Msadek</td></tr><tr><td></td><td>Sarah Dubrac</td></tr><tr><td></td><td>Olivier Poupel</td></tr><tr><td></td><td>Gérald Kenanian</td></tr></tbody></table>

<footnote n="1">¹Department of Microbiology, Biology of Gram-Positive Pathogens Unit, Institut Pasteur, Université Paris Cité, CNRS UMR2001, Paris, France</footnote>

<footnote n="2">²Department of Global Health, Cystic Fibrosis and Bronchial Diseases Unit, Institut Pasteur, Université Paris Cité, Paris, France</footnote>
<table><thead><tr><th>Funder</th><th>Grant(s)</th><th>Author(s)</th></tr></thead><tbody><tr><td></td><td></td><td>Tarek Msadek</td></tr><tr><td></td><td></td><td>Sarah Dubrac</td></tr><tr><td>Association Vaincre la Mucoviscidose</td><td>Lhousseine Touqui</td><td>Tarek Msadek</td></tr><tr><td></td><td></td><td>Sarah Dubrac</td></tr></tbody></table>

## AUTHOR CONTRIBUTIONS

Olivier Poupel, Data curation, Investigation, Methodology | Gérald Kenanian, Investigation | Lhousseine Touqui, Conceptualization, Data curation, Formal analysis, Funding acquisition | Charlotte Abrial, Investigation | Tarek Msadek, Conceptualization, Data curation, Formal analysis, Funding acquisition, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review and editing | Sarah Dubrac, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Project administration, Resources, Supervision, Validation, Writing – original draft, Writing – review and editing

## ETHICS APPROVAL

Animal experiments were carried out at the Institut Pasteur according to European Union guidelines for handling of laboratory animals (http://ec.europa.eu/environment/chemicals/lab_animals/index_en.htm). Animals were monitored daily with a particular attention for any signs of distress. Protocols used were approved by the Institut Pasteur ethics committee (dap160057).

## ADDITIONAL FILES

The following material is available online.

## Supplemental Material

Supplemental material (IAI00314-25-s0001.pdf). Fig. S1 to S4; Tables S1 and S2.

## REFERENCES

1. Piewngam P, Otto M. 2024. *Staphylococcus aureus* colonisation and strategies for decolonisation. Lancet Microbe 5:e606–e618. https://doi.org/10.1016/S2666-5247(24)00040-5

2. Fitzgerald JR, Sturdevant DE, Mackie SM, Gill SR, Musser JM. 2001. Evolutionary genomics of *Staphylococcus aureus*: insights into the origin of methicillin-resistant strains and the toxic shock syndrome epidemic. Proc Natl Acad Sci USA 98:8821–8826. https://doi.org/10.1073/pnas.161098098

3. Goerke C, Pantucek R, Holtfreter S, Schulte B, Zink M, Grumann D, Bröker BM, Doskar J, Wolz C. 2009. Diversity of prophages in dominant *Staphylococcus aureus* clonal lineages. J Bacteriol 191:3462–3468. https://doi.org/10.1128/JB.01804-08

4. Xia G, Wolz C. 2014. Phages of *Staphylococcus aureus* and their impact on host evolution. Infect Genet Evol 21:593–601. https://doi.org/10.1016/j.meegid.2013.04.022

5. Goerke C, Wirtz C, Flückiger U, Wolz C. 2006. Extensive phage dynamics in *Staphylococcus aureus* contributes to adaptation to the human host during infection. Mol Microbiol 61:1673–1685. https://doi.org/10.1111/j.1365-2958.2006.05354.x

6. Feiner R, Argov T, Rabinovich L, Sigal N, Borovok I, Herskovits AA. 2015. A new perspective on lysogeny: prophages as active regulatory switches of bacteria. Nat Rev Microbiol 13:641–650. https://doi.org/10.1038/nrmicro03527

7. Iandolo JJ, Worrell V, Groicher KH, Qian Y, Tian R, Kenton S, Dorman A, Ji H, Lin S, Loh P, Qi S, Zhu H, Roe BA. 2002. Comparative analysis of the genomes of the temperate bacteriophages φ11, φ12 and φ13 of *Staphylococcus aureus* 8325. Gene 289:109–118. https://doi.org/10.1016/S0378-1119(02)00481-x

8. Coleman DC, Sullivan DJ, Russell RJ, Arbuthnott JP, Carey BF, Pomeroy HM. 1989. *Staphylococcus aureus* bacteriophages mediating the simultaneous lysogenic conversion of β-lysin, staphylokinase and enterotoxin A: molecular mechanism of triple conversion. J Gen Microbiol 135:1679–1697. https://doi.org/10.1099/00221287-135-6-1679

9. van Wamel WJB, Rooijakkers SHM, Ruyken M, van Kessel KPM, van Strijp JAG. 2006. The innate immune modulators staphylococcal complement inhibitor and chemotaxis inhibitory protein of *Staphylococcus aureus* are located on β-hemolysin-converting bacteriophages. J Bacteriol 188:1310–1315. https://doi.org/10.1128/JB.188.4.1310-1315.2006

10. Rohmer C, Wolz C. 2021. The role of hlb-converting bacteriophages in *Staphylococcus aureus* host adaption. Microb Physiol 31:109–122. https://doi.org/10.1159/000516645

11. Salgado-Pabón W, Herrera A, Vu BG, Stach CS, Merriman JA, Spaulding AR, Schlievert PM. 2014. *Staphylococcus aureus* β-toxin production is common in strains with the β-toxin gene inactivated by bacteriophage. J Infect Dis 210:784–792. https://doi.org/10.1093/infdis/jiu146

12. Katayama Y, Baba T, Sekine M, Fukuda M, Hiramatsu K. 2013. Beta-hemolysin promotes skin colonization by *Staphylococcus aureus*. J Bacteriol 195:1194–1203. https://doi.org/10.1128/JB.01786-12

13. Goerke C, Matias y Papenberg S, Dasbach S, Dietz K, Ziebach R, Kahl BC, Wolz C. 2004. Increased frequency of genomic alterations in *Staphylococcus aureus* during chronic infection is in part due to phage mobilization. J Infect Dis 189:724–734. https://doi.org/10.1086/381502

14. Hedström SA, Malmqvist T. 1982. Sphingomyelinase activity of *Staphylococcus aureus* strains from recurrent furunculosis and other infections. Acta Pathol Microbiol Immunol Scand B 90:217–220. https://doi.org/10.1111/j.1699-0463.1982.tb00108.x
15. Herbert S, Ziebandt A-K, Ohlsen K, Schäfer T, Hecker M, Albrecht D, Novick R, Götz F. 2010. Repair of global regulators in *Staphylococcus aureus* 8325 and comparative analysis with other clinical isolates. Infect Immun 78:2877-2889. https://doi.org/10.1128/IAI.00088-10

16. Gillaspy AF, Worrell V, Orvis J, Roe BA, Dyer DW, landolo JJ. 2006. The *Staphylococcus aureus* NCTC 8325 genome, p 381-412. In Fischetti VA, Novick RP, Ferretti JJ, Portnoy DA, Rood JI (ed), Gram-positive pathogens, 2nd ed. ASM Press, Washington, DC.

17. Rooijakkers SHM, van Kessel KPM, van Strijp JAG. 2005. Staphylococcal innate immune evasion. Trends Microbiol 13:596-601. https://doi.org/10.1016/j.tim.2005.10.002

18. Nunes-Düby SE, Kwon HJ, Tirumalai RS, Ellenberger T, Landy A. 1998. Similarities and differences among 105 members of the Int family of site-specific recombinases. Nucleic Acids Res 26:391-406. https://doi.org/10.1093/nar/26.2.391

19. van den Berg S, Laman JD, Boon L, ten Kate MT, de Knegt GJ, Verdijk RM, Verbrugh HA, Nouwen JL, Bakker-Woudenberg IAJM. 2013. Distinctive cytokines as biomarkers predicting fatal outcome of severe *Staphylococcus aureus* bacteremia in mice. PLoS One 8:e59107. https://doi.org/10.1371/journal.pone.0059107

20. Haag AF, Bagnoli F. 2017. The role of two-component signal transduction systems in *Staphylococcus aureus* virulence regulation. Curr Top Microbiol Immunol 409:145-198. https://doi.org/10.1007/82_2015_5019

21. Lindsay JA. 2010. Genomic variation and evolution of *Staphylococcus aureus*. Int J Med Microbiol 300:98-103. https://doi.org/10.1016/j.ijmm.2009.08.013

22. Huseby M, Shi K, Brown CK, Digre J, Mengistu F, Seo KS, Bohach GA, Schlievert PM, Ohlendorf DH, Earhart CA. 2007. Structure and biological activities of beta toxin from *Staphylococcus aureus*. J Bacteriol 189:8719-8726. https://doi.org/10.1128/JB.00741-07

23. Herrera A, Vu BG, Stach CS, Merriman JA, Horswill AR, Salgado-Pabón W, Schlievert PM. 2016. *Staphylococcus aureus* β-toxin mutants are defective in biofilm ligase and sphingomyelinase activity, and causation of infective endocarditis and sepsis. Biochemistry 55:2510-2517. https://doi.org/10.1021/acs.biochem.6b00083

24. Huseby MJ, Kruse AC, Digre J, Kohler PL, Vocke JA, Mann EE, Baykes KW, Bohach GA, Schlievert PM, Ohlendorf DH, Earhart CA. 2010. Beta toxin catalyzes formation of nucleoprotein matrix in staphylococcal biofilms. Proc Natl Acad Sci USA 107:14407-14412. https://doi.org/10.1073/pnas.0911032107

25. Tanaka T, Narazaki M, Kishimoto T. 2014. IL-6 in inflammation, immunity, and disease. Cold Spring Harb Perspect Biol 6:a016295. https://doi.org/10.1101/cshperspect.a016295

26. Cao YZ, Tu YY, Chen X, Wang BL, Zhong YX, Liu MH. 2012. Protective effect of ulinastatin against murine models of sepsis: inhibition of TNF-α and IL-6 and augmentation of IL-10 and IL-13. Exp Toxicol Pathol 64:543-547. https://doi.org/10.1016/j.etp.2010.11.011

27. Bae T, Baba T, Hiramatsu K, Schneewind O. 2006. Prophages of *Staphylococcus aureus* Newman and their contribution to virulence. Mol Microbiol 62:1035-1047. https://doi.org/10.1111/j.1365-2958.2006.05441.x

28. Tran PM, Feiss M, Kinney KJ, Salgado-Pabón W. 2019. ØSa3mw prophage as a molecular regulatory switch of *Staphylococcus aureus* β-toxin production. J Bacteriol 201:e00766-18. https://doi.org/10.1128/JB.00766-18

29. Verkaik NJ, Benard M, Boelens HA, de Vogel CP, Nouwen JL, Verbrugh HA, Melles DC, van Belkum A, van Wamel WJB. 2011. Immune evasion cluster-positive bacteriophages are highly prevalent among human *Staphylococcus aureus* strains, but they are not essential in the first stages of nasal colonization. Clin Microbiol Infect 17:343-348. https://doi.org/10.1111/j.1469-0691.2010.03227.x

30. de Haas CJC, Veldkamp KE, Peschel A, Weerkamp F, Van Wamel WJB, Heezius ECJM, Poppelier MJG, Van Kessel KPM, van Strijp JAG. 2004. Chemotaxis inhibitory protein of *Staphylococcus aureus*, a bacterial antiinflammatory agent. J Exp Med 199:687-695. https://doi.org/10.1084/jem.20031636

31. Kwieciński J, Josefsson E, Mitchell J, Higgins J, Magnusson M, Foster T, Jin T, Bokarewa M. 2010. Activation of plasminogen by staphylokinase reduces the severity of *Staphylococcus aureus* systemic infection. J Infect Dis 202:1041-1049. https://doi.org/10.1086/656140

32. Rooijakkers SHM, Ruyken M, Roos A, Daha MR, Presanis JS, Sim RB, van Wamel WJB, van Kessel KPM, van Strijp JAG. 2005. Immune evasion by a staphylococcal complement inhibitor that acts on C3 convertases. Nat Immunol 6:920-927. https://doi.org/10.1038/ni1235

33. Novick RP. 2003. Autoinduction and signal transduction in the regulation of staphylococcal virulence. Mol Microbiol 48:1429-1449. https://doi.org/10.1046/j.1365-2958.2003.03526.x

34. Stragier P, Kunkel B, Kroos L, Losick R. 1989. Chromosomal rearrangement generating a composite gene for a developmental transcription factor. Science 243:507-512. https://doi.org/10.1126/science.2536191

35. Kunkel B, Losick R, Stragier P. 1990. The *Bacillus subtilis* gene for the development transcription factor sigma K is generated by excision of a dispensable DNA element containing a sporulation recombinase gene. Genes Dev 4:525-535. https://doi.org/10.1101/gad.4.4.525

36. Dubnau D, Losick R. 2006. Bistability in bacteria. Mol Microbiol 61:564-572. https://doi.org/10.1111/j.1365-2958.2006.05249.x

37. Veening JW, Smits WK, Kuipers OP. 2008. Bistability, epigenetics, and bet-hedging in bacteria. Annu Rev Microbiol 62:193-210. https://doi.org/10.1146/annurev.micro.62.081307.163002

38. Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual. 2nd edition. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

39. Arnaud M, Chastanet A, Débarbouillé M. 2004. New vector for efficient allelic replacement in naturally nontransformable, low-GC-content, Gram-positive bacteria. Appl Environ Microbiol 70:6887-6891. https://doi.org/10.1128/AEM.70.11.6887-6891.2004

40. Murphy E. 1985. Nucleotide sequence of a spectinomycin adenyltransferase AAD(9) determinant from *Staphylococcus aureus* and its relationship to AAD(3") (9). Mol Gen Genet 200:33-39. https://doi.org/10.1007/BF00383309

41. Steinmetz M, Richter R. 1994. Easy cloning of mini-Tn10 insertions from the *Bacillus subtilis* chromosome. J Bacteriol 176:1761-1763. https://doi.org/10.1128/jb.176.6.1761-1763.1994

42. Archambaud C, Gouin E, Pizarro-Cerda J, Cossart P, Dussurget O. 2005. Translation elongation factor EF-Tu is a target for Stp, a serine-threonine phosphatase involved in virulence of *Listeria monocytogenes*. Mol Microbiol 56:383-396. https://doi.org/10.1111/j.1365-2958.2005.04551.x

43. Livak KJ, Schmittgen TD. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2^ΔΔCT^ method. Methods 25:402-408. https://doi.org/10.1006/meth.2001.1262

44. Haque RU, Baldwin JN. 1964. Types of hemolysins produced by *Staphylococcus aureus*, as determined by the replica plating technique. J Bacteriol 88:1442-1447. https://doi.org/10.1128/jb.88.5.1442-1447.1964

45. Stokes RW, Doxsee D. 1999. The receptor-mediated uptake, survival, replication, and drug sensitivity of *Mycobacterium tuberculosis* within the macrophage-like cell line THP-1: a comparison with human monocyte-derived macrophages. Cell Immunol 197:1-9. https://doi.org/10.1006/ciimm.1999.1554

46. Soutourina O, Dubrac S, Poupel O, Msadek T, Martin-Verstraete I. 2010. The pleiotropic CymR regulator of *Staphylococcus aureus* plays an important role in virulence and stress response. PLoS Pathog 6:e1000894. https://doi.org/10.1371/journal.ppat.1000894