Interaction with eukaryotic cells enhances the
circularization and transcription of integrative and
conjugative elements in Mycoplasma hominis.

Bachir Boureima Abdou, Alicia Silvant, Chloé Le Roy, Jennifer Guiraud, Léa

Bientz, Laure Beven, Véronique Dubois, Eric Baranowski, Cécile Bébéar,
Sabine Pereyre

► To cite this version:

Bachir Boureima Abdou, Alicia Silvant, Chloé Le Roy, Jennifer Guiraud, Léa Bientz, et al. Interaction with eukaryotic cells enhances the circularization and transcription of integrative and conjugative elements in Mycoplasma hominis. Mobile DNA, 2025, 16 (1), pp.26. 10.1186/s13100-025-00364-x. hal-05167347

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# Interaction with eukaryotic cells enhances the circularization and transcription of integrative and conjugative elements in *Mycoplasma hominis*

Bachir Boureima Abdou¹ ID, Alicia Silvant¹,<fnref n="2" />, Chloé Le Roy¹, Jennifer Guiraud<fnref n="3" />, Léa Bientz¹,<fnref n="2" />, Laure Béven<fnref n="4" />, Véronique Dubois¹,<fnref n="2" />, Eric Baranowski<fnref n="5" /> ID, Cécile Bébéar¹,<fnref n="2" />,<fnref n="3" /> ID and Sabine Pereyre¹,<fnref n="2" />,<fnref n="3" />* ID

## Abstract

Mobile genetic elements drive bacterial evolution by promoting genetic plasticity and adaptation; among them, mycoplasma Integrative and Conjugative Elements (ICEs) challenge the notion that mycoplasmas evolved solely through genome reduction. However, they remain poorly characterized, particularly in *Mycoplasma hominis*, which is a human genital pathogen. In this study, we investigated the circularization, transcription, and protein expression of *M. hominis* 4788 ICE (ICEHo4788) under the following four environmental conditions: axenic growth, coculture with HeLa cells, mitomycin C exposure, and thermal stress. During axenic growth, ICEHo4788 circularization peaked at 12 h with a 9.4-fold increase in the number of circular forms. Mitomycin C and cold shock induced only a moderate increase in circularization (approximately 3-fold), whereas heat shock significantly reduced the number of circular forms. In coculture with HeLa cells, a strong increase in the number of circular forms was observed at 72 h and 7 days postinfection with 10- and 23-fold increases, respectively. Coculture also led to a 5- to 23-fold increase in transcription at 7 days, whereas axenic growth, mitomycin C exposure, and thermal stress did not differ. Proteomic analysis revealed that MhoH(a), CDS18, CDS17, and CDS11 were significantly overexpressed at 12 h. Neither mitomycin C nor cold shock induced changes in ICE-related proteins, but heat shock upregulated two *M. hominis* molecular chaperones and ICEHo4788 MhoH(a). In conclusion, environmental conditions modulate *M. hominis* ICE activity with strong stimulation in the presence of eukaryotic cells. These findings offer new insights into the regulation and environmental responses of *M. hominis* ICEs.

**Keywords** *Mycoplasma hominis*, Integrative and conjugative element, Circularization, Transcription, Protein expression, HeLa cells

<footnote>*Correspondence:
Sabine Pereyre
sabine.pereyre@u-bordeaux.fr
¹Univ. Bordeaux, CNRS, UMR 5234, MFP, ARMYNE team, Bâtiment BBS, 2
Rue Hoffmann Martinot, Bordeaux 33076, France</footnote>

<footnote n="2">²Bacteriology Department, Bordeaux University Hospital, Bordeaux, France</footnote>

<footnote n="3">³Bordeaux University Hospital, National Reference Centre for Bacterial
Sexually Transmitted Infections, Bordeaux, France</footnote>

<footnote n="4">⁴Univ. Bordeaux, INRAE, UMR 1332, BFP, Villenave d'Ornon, France</footnote>

<footnote n="5">⁵IHAP, Université de Toulouse, INRAE, ENVT, Toulouse, France</footnote>
**Importance**

Integrative and Conjugative Elements (ICEs) play a key role in bacterial evolution by mediating horizontal gene transfer. However, their regulation remains poorly understood in *Mycoplasma hominis*, which is a human genital pathogen. This study provides the first comprehensive analysis of ICE circularization, transcription, and protein expression under diverse environmental conditions including axenic growth, coculture with human cells, DNA damage, and thermal stress. Our findings reveal that ICE circularization is highly dynamic, peaks at specific time points, and responds differently to environmental cues. Notably, coculture with HeLa cells significantly enhances ICE circularization and transcription in *M. hominis*, which suggests a potential link between host interactions and ICE activation. By elucidating the regulatory mechanisms governing ICEs in *M. hominis*, this study expands our understanding of mobile genetic elements in minimal genomes and their potential role in adaptation, persistence, and gene transfer within the human microbiome.

**Introduction**

Integrative and Conjugative Elements (ICEs) are self-transmissible mobile genetic elements that play a fundamental role in bacterial genome evolution by facilitating horizontal gene transfer (HGT) [1]. Unlike plasmids, ICEs integrate into the host genome, excise to form circular intermediates, and then transfer to recipient cells via conjugation before reintegrating into the chromosome. These elements contribute significantly to bacterial adaptability and mediate the acquisition of antibiotic resistance, virulence factors, and metabolic traits [2]. A novel family of integrative elements that are capable of self-transmission, named MICE (*Mycoplasma Integrative and Conjugative Elements*), was discovered in the genomes of several Mollicutes, which are a class of wall-less bacteria that is derived from a low-GC% gram-positive ancestor [3] and includes several pathogenic species [4–9]. While extensively characterized in various bacterial phyla, ICEs remain poorly understood in *Mycoplasma*. Historically, the streamlined genomes of *Mycoplasma* species were thought to have a limited capacity for horizontal gene exchange, which reinforced the paradigm of reductive evolution driven solely by gene loss [10]. However, the discovery of putative ICEs in multiple *Mycoplasma* species, along with in silico analyses indicating significant chromosomal DNA exchange between phylogenetically distant groups [11, 12], has provided strong evidence for horizontal gene transfer in these minimal bacteria [10].

The distribution of ICEs within *Mycoplasma* species is highly variable. Some species, such as *Mycoplasma pneumoniae* and *Mycoplasma genitalium*, appear devoid of

ICEs, whereas others, including *Mycoplasma agalactiae*, *Mycoplasma bovis*, and *Mycoplasma fermentans*, harbor multiple ICE-related elements [4, 9]. Genomic analyses revealed the presence of MICE in *Mycoplasma hominis*, which is an opportunistic pathogen of the human urogenital tract that is responsible for genital infections in women and extragenital infections in newborns and immunocompromised patients. Among the 12 studied strains in one study, seven strains were found to carry ICEs, which ranged from 27 to 30 kb in size and were present in one or more copies in approximately 45% of strains, which indicates a highly conserved structure [13]. Among these strains, ICEHo4788 from the *M. hominis* 4788 strain has been previously characterized for its ability to form extrachromosomal circular forms as demonstrated by Meygret et al. (13).

Excision and circularization are essential steps for conjugative transfer but factors influencing these processes remain unclear. Several environmental conditions have been implicated in regulating ICE activation in other bacterial species. The stationary phase significantly increases ICE circularization and transcription in several bacterial species, including *Streptococcus thermophilus* and *Mesorhizobium loti*, and also upregulates integrase expression in *Pseudomonas knackmussii* [14–16]. Exposure to DNA-damaging agents, such as mitomycin C (MMC) at half of the minimum inhibitory concentration (MIC), increased ICE excision, circularization, core gene transcription, and overall ICE abundance in *S. thermophilus* [15, 17]. MMC also induced the expression of genes required for the transfer of SXT, which is a well-characterized ICE of *Vibrio cholerae* [18]. Cold stress in combination with a low-nutrient medium has been associated with the detection of circular ICE forms in *Mycoplasma mycoides* subsp. *capri* [8]. Recently, the cell culture environment was shown to dramatically increase ICE transfer frequency in *M. agalactiae* [19].

However, the impact of environmental conditions on *M. hominis* ICE activity has not been assessed. To fill this gap, we analyzed the circularization dynamics of ICEHo4788 under various environmental conditions including axenic culture, coculture with HeLa cells, thermal stress, and MMC exposure. The transcription and protein expression of ICEs coding sequence (CDSs) were also studied under the same environmental conditions. In addition, we investigated the transcriptional organization of ICEHo4788 to gain further insights into its regulatory mechanisms.

**Materials and methods**

**Bacterial strains, cell lines, and culture conditions**

The *M. hominis* 4788 strain, which was isolated from the peritoneal fluid of a patient with salpingitis [13], was cultured at 37 °C in Hayflick modified medium
supplemented with arginine [20] (HA medium). Bacterial concentrations were determined by measuring colony-forming units per mL (CFU/mL) on solid agar plates as previously described [20, 21].

*Escherichia coli* NEB 5α competent cells (New England Biolabs, Ipswich, MA, USA) were used for plasmid propagation and cloning experiments. Plasmid-transformed *E. coli* cells were grown at 37 °C in Lysogeny broth (LB) or on LB agar supplemented with ampicillin at 100 µg/ mL.

The human cervical carcinoma cell line HeLa (ATCC CCL-2) was cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (v/v) heat-inactivated fetal calf serum, 50 mg/mL vancomycin, 4% amphotericin B, and 10 mg/mL gentamicin. For coculture of HeLa cells with *M. hominis*, HeLa cells were resuspended in DMEM without gentamicin and seeded at a density of 3 × 10⁵ cells per well. Cells were coincubated with *M. hominis* strain 4788 at a multiplicity of infection (MOI) of 10 before incubation at 37 °C with 5% CO₂. At different post-inoculation time points, HeLa cells were lysed by three freeze-thaw cycles (-80 °C/37°C) [22].

## Stress conditions and mitomycin C treatment

*M. hominis* 4788 cultures were subjected to thermal stress after 8–15 h of incubation, which corresponds to the early and mid-exponential phases of growth. For heat stress, bacteria were exposed to 42 °C for 1 h, followed by a 3-h incubation at 37 °C under standard growth conditions. For cold stress, bacteria were exposed to -80 °C–4 °C for 1 h, followed by a 3-h incubation at 37 °C. The recovery period was applied to allow sufficient time for ICEHo4788 activation processes (circularization, transcription, and protein synthesis), as previously described for ICE induction in *S. thermophilus* [23]. The untreated culture control was maintained at 37 °C throughout the experiment.

*M. hominis* 4788 cultures were exposed to MMC at concentrations corresponding to a quarter and half MIC, which was determined at 0.062 µg/mL via the Clinical and Laboratory Standards Institute (CLSI) reference method [24]. Then, the mixture was incubated for 1 h at 37 °C, followed by centrifugation at 10,000 × g for 10 min at room temperature. Bacterial pellets were subsequently resuspended in HA liquid medium and incubated for an additional 3 h at 37 °C. The untreated culture control was centrifuged simultaneously with the treated cultures.

## Quantification of ICEHo4788 circular forms

DNA was isolated from axenic and cell culture conditions via the NucleoSpin DNA kit (Macherey-Nagel, Düren, Germany). Oligonucleotides used in quantitative PCRs (qPCR) reactions were designed via amplifix 2.1.1 (https://inp.univ-amu.fr/en/amplifx-manage-test-and-des

ign-your-primers-for-pcr). All primers used are listed in Table S1.

A plasmid calibrator, pGEMT-circu4788, was constructed to evaluate the PCR efficiency for the detection of ICEHo4788 circular forms. This plasmid was generated by cloning the 281-bp circularized fragment, which was amplified via primers 4788 Circu-F and 4788 Circu-R, into the pGEM-T vector (Promega, Madison, WI, USA). The bacterial load was normalized by amplification of the *tuf* gene [25, 26], which encodes the elongation factor Tu. The PCR efficiency for the *tuf* gene and ICEHo4788 circular form was determined via tenfold serial dilutions (from 10⁻¹ to 10⁻⁹) of *M. hominis* 4788 genomic DNA and pGEMT-circu4788, respectively. The PCR efficiency (E) was calculated via the Formula E = 10⁻¹/slope.

qPCRs were conducted in a final volume of 25 µL containing 1X SYBR® Green (Absolute blue qPCR Sybr green, Thermo Fisher Scientific, San Jose, CA, USA), 0.5 µM of each primer (Table S1), and 5 µL of the DNA template. Reactions were performed on a LightCycler 480® instrument (Roche Diagnostics, Basel, Switzerland) with the following cycling conditions: initial denaturation at 95 °C for 15 min followed by 45 cycles of 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s. The specificity of the amplification and absence of primer dimers were confirmed through melt curve analysis. The relative quantification of the ICEHo4788 circular form was determined via the 2⁻⁰⁻⁶CT method with the PCR efficiency correction available in the LC480 software [27, 28]. qPCR analyses were conducted on three biological replicates, each analyzed in two technical replicates. Statistical comparisons were made via a t-test (p-values less than 0.05 were considered significant).

## RNA isolation and quantitative reverse transcription PCR

Total RNA was extracted from *M. hominis* 4788 cultures under axenic and cell culture conditions via the NucleoSpin® RNA Plus kit (Macherey-Nagel). The RNA concentration was determined by measuring the absorbance at 260 nm via a Qubit RNA HS Assay Kit (Thermo Fisher Scientific). The integrity and quality of RNA samples were assessed via the Qubit RNA IQ Assay (Thermo Fisher Scientific) with RNA IQ values equal to or greater than 9.7, which indicated sufficient quality for downstream applications.

cDNA was synthesized via the RT² First Strand Kit (Qiagen, Hilden, Germany). Reverse transcription was performed at 42 °C for 15 min followed by enzyme inactivation at 95 °C for 5 min. A no-RT control was included and showed no amplification, confirming the absence of genomic DNA contamination.

PCRs were conducted in a final volume of 25 µL containing 1 U HotStar Taq DNA Polymerase (Qiagen), 1X PCR buffer, 3 mM MgCl₂, 200 µM dNTPs, 0.5 µM of each
primer (Table S1), and 1.25 µL of cDNA. The following
amplification conditions were used: initial denaturation
at 95 °C for 15 min; 35 cycles of 94 °C for 1 min, 52 °C for
1 min, and 72 °C for 2 min; and a final extension at 72 °C
for 10 min.

Quantitative RT-PCR (RT-qPCR) was performed in a
final volume of 25 µL, as described in the previous sec-
tion, with 2.5 µL of cDNA template using three biological
replicates, each analyzed in two technical replicates.

In Silico transcription terminator prediction

The prediction of Rho-independent transcription termi-
nators was performed on ICEHo4788 via the web-based
ARNold tool, which is available at http://rna.igmors.u-p
sud.fr/toolbox/arnold/ (Table S2). ARNold utilizes two
distinct algorithms, Erpin [29] and RNAmotif [30], which
are based on terminator sequences from *E. coli* and
*Bacillus subtilis*. These algorithms predict terminators
independently of the gene context in which they occur.

Proteomic analysis by LC-MS/MS

M. hominis 4788 was cultured in 50 mL of HA media under different conditions that were selected based on qPCR results. Cells were harvested via centrifugation at 12,000 × g for 30 min, washed five times with Dulbecco's phosphate-buffered saline (Eurobio Scientific, Les Ulis, France) and resuspended in the same buffer. The protein concentration was determined via a DC Protein Assay kit (Bio-Rad Laboratories, Hercules, CA, USA) to standardize the input concentrations.

Protein samples were solubilized in Laemmli buffer, and 5 µg of protein was separated via SDS-PAGE gel. After colloidal blue staining, each lane was cut from the gel and subsequently cut into 1 mm × 1 mm gel pieces. These gel pieces were destained in 25 mM ammonium bicarbonate 50% acetonitrile (ACN), rinsed twice in ultrapure water, and shrunk in ACN for 10 min. After ACN removal, gel pieces were dried at room temperature, covered with a trypsin solution (10 ng/µL in 50 mM NH₄HCO₃), rehydrated at 4 °C for 10 min, and finally incubated overnight at 37 °C. Then, spots were incubated for 15 min in 50 mM NH₄HCO₃ at room temperature with rotary shaking. The supernatant was collected, and an H₂O/ACN/HCOOH (47.5:47.5:5) extraction mixture was added to the gel slices for 15 min. This extraction step was repeated twice and then supernatants were pooled and dried in a vacuum centrifuge. Finally, digests were solubilized in 0.1% HCOOH.

The peptide mixture was analyzed on an Ultimate 3000
nanoLC system (Dionex, Amsterdam, The Netherlands)
coupled to an Electrospray Orbitrap Fusion™ Lumos™
Tribrid™ Mass Spectrometer (Thermo Fisher Scientific,
San Jose, CA, USA). Peptide digests were loaded onto
a 300 µm-inner diameter × 5 mm C₁₈ PepMap™ trap

column (LC Packings) at a flow rate of 10 µL/min and eluted from the trap column onto an analytical 75 mm id × 60 cm C18 column (IonOpticks). This column had a 4–25% linear gradient of solvent B for 87 min (solvent A was 0.1% formic acid, and solvent B was 0.1% formic acid in 80% ACN), followed by a 30-min gradient from 25 to 40% solvent B. The separation flow rate was set at 200 nL/min. The mass spectrometer was operated in the positive ion mode at a 2-kV needle voltage. Data were acquired via Xcalibur 4.4 software in data-independent mode. A duty cycle alternated one full scan (resolution 60,000, AGC target 4E5, maximum injection time 50 ms, 350–1400 m/z) with 35 DIA MS/MS scans (resolution 30,000, AGC target 5e5, maximum injection time 54 ms) covering the mass range from 380–980 with a variable width isolation window. The Higher-energy collisional dissociation (HCD) collision energy was set to 25% [31].

Data-independent acquisition (DIA) data were searched using Chimerys through Proteome Discoverer 3.1 (Thermo Fisher Scientific, Inc.) against an *M. hominis* 4788 protein database generated via PROKKA (589 entries) from the complete genome sequence [13]. Spectra from peptides greater than 5000 Da or less than 350 Da in size were excluded. The maximum fragment tolerance was set at 20 ppm, and two missed trypsin cleavages were allowed. The oxidation of methionines (+16 Da) was considered a variable modification, whereas the carbamidomethylation of cysteines (+57 Da) was considered a fixed modification. Only “high confidence” peptides corresponding to a 1% false-positive rate at the peptide level were retained. Peptide quantitation was achieved via the Fragment Ion Quantifier node. Normalization was performed based on the total *M. hominis* peptide amount. Protein ratios were calculated as the median of all possible pairwise peptide ratios. A t-test was performed based on the background population of peptides or proteins and was adjusted via a Benjamini-Hochberg correction. Quantitative data were considered for proteins quantified by a minimum of two peptides and with a statistical p-value lower than 0.05 based on three independent biological replicates.

Results

Growth of *M. hominis* 4788 under axenic and cell culture conditions

Given that mycoplasma coincubation with eukaryotic
cells shows enhanced conjugation in *M. agalactiae* [19],
we assessed the ability of *M. hominis* 4788 to grow in
coculture with HeLa cells and compared growth curves
under axenic and cell culture conditions. Under axenic
culture conditions, bacterial growth peaked at approxi-
mately 10⁹ CFU/mL after 22 h of incubation (Fig. 1A).
This was followed by a rapid decline in bacterial count
with no stationary phase and a complete loss of viable
<figure><img src="image_3.png" /><figcaption>**Fig. 1** Growth curve of *M. hominis* 4788 under axenic and cell culture conditions. (A) Axenic conditions: *M. hominis* 4788 was cultured in Hayflick medium supplemented with arginine, and the *M. hominis* titer was determined at various time points. (B) Cell culture conditions: a monolayer of HeLa cells was infected with *M. hominis* 4788 at an MOI of 10. Samples were collected at various time points post-inoculation, and CFU counts were determined via agar cultures. Data points represent the means ± standard deviations from three independent experiments</figcaption></figure>

bacteria after 40 h. In coculture with HeLa cells, there was an initial significant decrease in the bacterial count to 10² CFU/mL, which occurred at 24 h, and this was then followed by an exponential increase, up to approximately 10⁷ CFU/mL, after 168 h (7 days) of incubation (Fig. 1B). Although overall titers were lower than those under axenic conditions, *M. hominis* 4788 remained viable for up to 7 days in coculture with HeLa cells. Furthermore, *M. hominis* 4788 was unable to grow in DMEM without HeLa cells despite the presence of fetal calf serum in the cell culture medium (data not shown). These differences in *M. hominis* 4788 proliferation between axenic and cell culture conditions highlight the potential influence of the replicative environment on the biology of this *Mycoplasma* species.

## Circularization of ICEHo4788 under different environmental conditions

The excision of ICE leads to the formation of transient circular intermediates, which are essential for ICE transfer. To quantify the circular extrachromosomal forms of ICEHo4788, we developed a qPCR assay using divergent primers, 4788 Circu-F and 4788 Circu-R, which are located at both ends of the integrated ICEHo4788 (Fig. S1). The bacterial load was normalized via amplification of the *tuf* gene. The circularization of ICEHo4788 was assessed under the following four different environmental conditions: axenic growth, coculture with HeLa cells, exposure to subinhibitory concentrations of MMC, and thermal stress.

Under axenic conditions, ICEHo4788 circularization peaked at 12 h of culture, which corresponded to the mid-exponential phase with a 9.4-fold increase in the number of circular forms compared with the 6-h reference point (Fig. 2A). At 19 h, 22 h, and 26 h, the number of circular forms was significantly lower than that at 6 h. Under coculture with HeLa cells, there was no significant increase in the number of circular forms until 48 h (Fig. 2B). However, high levels of circularization were observed at 72 h and 7 days with fold changes of 10 and 23, respectively, compared with the 0-h baseline. These findings suggest that the presence of HeLa cells stimulates ICEHo4788 circularization after 72 h of coculture.

The effect of MMC at subinhibitory concentrations (0.25 and 0.5 MIC) on circularization was also assessed at two time points (12 h and 19 h) (Fig. 3A). At 12 h, only 0.25 MIC resulted in a significant increase in circularization with a 3.3-fold change compared with the untreated condition. No significant change was observed at 19 h or with 0.5 MIC. The impact of thermal stress was evaluated by applying cold shock at -80 °C-4 °C and heat shock at 42 °C (Fig. 3B). At 12 h, cold shocks at 4 °C and -80 °C resulted in a slight but significant increase in circularization with 2.4-fold and 3-fold increases, respectively, compared with untreated conditions. At 19 h, no significant variation was observed. Heat shock at 42 °C led to a decrease in circular forms at both time points but the decrease was significant only at 19 h.
<figure><img src="image_4.png" /><figcaption>**Fig. 2** Effect of growth under axenic and cell culture conditions on ICEHo4788 circularization. qPCR experiments were conducted using DNA extracts from *M. hominis* 4788 collected at different growth stages under both axenic and cell culture conditions. **A)** Under axenic conditions, samples were collected at 6 h (early exponential phase), 12 h (mid-exponential phase), 15 h, 19 h (late exponential phase), 22 h, and 26 h (death phase). **B)** In coculture with HeLa cells, samples were collected at 0 h, 4 h, 24 h, 48 h, 72 h, and 168 h. The fold change in ICE circularization (RQ) was calculated via the 2^(-ΔΔCT) method with corrections for PCR efficiency of the circular target gene and the *tuf* reference gene. For the axenic condition, time points were compared to the 6-h time point used as a reference, and for the cell culture condition, time points were compared to 0-h. Data are presented as the mean ± standard deviation from three (cell culture) or four (axenic culture) independent biological replicates. Statistical significance (t-test) is indicated as follows: \*p < 0.05; \*\*p < 0.01; \*\*\*p < 0.001</figcaption></figure>

## Transcription of ICEHo4788 CDSs under different environmental conditions

To further understand the potential impact of environmental conditions on ICEHo4788 activity, we also explored the transcriptional response of ICE-related CDSs under the same conditions. RT-PCR analysis first revealed that all ICEHo4788 CDSs were transcribed under axenic culture conditions at 12 h and 19 h (Fig. S2). A total of 10 ICEHo4788 CDSs that were evenly distributed on the ICE were selected (Fig. 4A) to assess the potential impact of environmental conditions on ICEHo4788 transcription via RT-qPCR and the 2^(-ΔΔCT) method (the *tuf* gene was used as a reference gene). Axenic growth, exposure to MMC at 0.25 MIC, cold thermal stress at -80 °C and heat shock at 42 °C had no significant effect on the transcription of these ICE CDSs (Fig. S3 and Fig. S4). In coculture with HeLa cells, no significant variation in CDS transcription was detected at 72 h (Fig. 4B). In contrast, a marked increase in the transcription of the 10 ICE CDSs was observed after 7 days of

incubation with changes ranging from 5-fold for MhoK to 23-fold for CDS21(b).

Overall, compared with exposure to thermal stresses and a DNA-damaging agent, *M. hominis* coincubation with HeLa cells was identified as a major trigger of ICEHo4788 activation, which only moderately affected ICEHo4788 circularization and transcription.

## Prediction of ICEHo4788 terminators and experimental validation

We explored whether ICEHo4788 CDSs are transcribed as individual units or organized into operons. This analysis was motivated by two key observations. First, our RT-qPCR data revealed that CDSs located close to each other within ICEHo4788 presented similar Ct values (Fig. S2), which suggested potential cotranscription. Second, the genomic organization of ICEHo4788 revealed minimal intergenic regions between adjacent CDSs [13], which suggests the hypothesis of polycistronic transcription. The ARNOLD web server was used to predict Rho-independent transcription terminators within ICEHo4788.
<figure><img src="image_5.png" /><figcaption>**Fig. 3** Effect of MMC treatment and thermal stress on ICEHo4788 circularization. qPCR amplification was performed on DNA extracts from *M. hominis* 4788 cultured in Hayflick medium under different conditions. A) For the MMC treatment, bacteria were exposed for 1 h to 0.03 µg/ml MMC (corresponding to 0.5 MIC) or 0.015 µg/ml MMC (MMC 0.25 MIC) after 8–15 h of culture. Both treated and untreated (MMC-) cultures were then centrifuged and pellets were resuspended in fresh medium for an additional 3 h of incubation at 37 °C. B) For thermal stress, cold shocks (4 °C and –80 °C) and heat shock (42 °C) were applied for 1 h, which was followed by a 3-h incubation at 37 °C. The fold change in ICE circularization (RQ) was calculated via the 2^(-ΔΔCt) method with corrections for the PCR efficiency of the circular target and the *tuf* reference gene. These data are presented as means ± standard deviations from three independent biological replicates. \*p-value < 0.05 according to a t-test</figcaption></figure>

Four transcription terminators were predicted along the entire length of ICEHo4788 (Fig. 5A). The first terminator was located upstream of the first CDS and between the inverted repeat and the initiation codon; this showed a very low stem-loop free energy of -10.6 kcal/mol, which reflects a high degree of stability. The remaining three terminators were predicted to be located toward the end of ICEHo4788, between CDS19 and MhoK and between MhoA(b) and CDS21(a), with free energies of -8.2 kcal/mol for both, and between CDS21(b) and CDS22 for which the free energy could not be determined (ND) by the ARNold web server.

To experimentally validate the functionality of these predicted terminators, we conducted RT-PCR assays targeting each intergenic region of ICEHo4788. Positive cDNA amplification was obtained for all intergenic regions within the ICE, which suggests potential cotranscription of adjacent CDSs (Fig. 5B). However, no cDNA amplification of the region encompassing the first predicted transcription terminator was obtained (Fig. 5B). This indicates that this terminator is effective

in halting transcription between the host chromosomal DNA encoding the N-terminal region of the DUF4011 domain-containing protein and into which ICEHo4788 is inserted with the ICEHo4788 CDS1. In contrast, positive cDNA amplification was observed at the 3' end of ICEHo4788 between CDS22 and the downstream CDS fragment of the disrupted DUF4011 domain-containing protein. Unexpectedly, amplification was also observed between this latter disrupted CDS fragment and the adjacent *ribF* gene, which is oriented on the reverse strand (Fig. 5). Collectively, these results suggest that ICEHo4788 exhibits predominantly polycistronic transcriptional organization with putative internal terminators that do not interrupt transcription. This efficient upstream terminator highlights the strict separation between the host and ICEHo4788 transcription.

ICEHo4788 protein expression profile under different environmental conditions

Because the correlation of transcription levels varies with protein abundance due to posttranscriptional and
<figure><img src="image_6.png" /><figcaption>**Fig. 4** Effect of HeLa cell culture conditions on the transcription of 10 ICEHo4788 CDSs. (A) Schematic representation of ICEHo4788 CDSs. The 10 CDSs selected for mRNA quantification are highlighted in dark gray. (B) RT-qPCR experiments were performed on a monolayer of HeLa cells infected with *M. hominis* 4788 at an MOI of 10 at different time points (0 h, 72 h, and 168 h). At each time point, the mRNA levels of 10 ICEHo4788 CDSs were quantified. The fold change was determined via the 2^−ΔΔCt^ method with the *tuf* gene as the reference gene and the 0 h time point used as the reference condition. These data are presented as means ± standard deviations from three independent biological replicates. Statistical significance (t-test) is indicated as follows: ***p < 0.001; ****p < 0.0001</figcaption></figure>

<figure><img src="image_7.png" /><figcaption>**Fig. 5** RT-PCR analysis of ICEHo4788 intergenic regions. (A) Schematic representation of the primer design used for RT-PCR analysis. Reverse transcription followed by end-point PCR experiments were conducted on intergenic regions to assess the cotranscription of ICEHo4788 CDSs. Arrows indicate CDSs and their orientations with the CDS name above. The predicted terminators, which were identified by the ARNOLD Web server, are marked with a hairpin symbol with the associated free energy values displayed above or marked as ND (not determined) when the prediction could not yield a reliable value due to sequence or structural limitations. The DUF4011 domain-containing protein-encoding gene disrupted by the ICE insertion is shown with hatched markings at both ends of the ICE. The expected transcript sizes are indicated and primers used are presented in Table S1. A "+" indicates successful amplification of the intergenic region, whereas a "-" indicates no amplification. (B) Agarose gel electrophoresis of RT-PCR products from corresponding intergenic regions. C, cDNA; D, DNA amplification used as a positive control</figcaption></figure>

posttranslational regulation, we performed a proteomic analysis of ICEHo4788 via LC-MS/MS under the same previous environmental axenic conditions. The proteomic analysis identified 10 ICEHo4788 CDSs, which were consistently expressed regardless of the condition

tested, out of 520 total *M. hominis* proteins detected with at least two unique peptides (Fig. 6). These included CDS11, MhoH(a), CDS14, CDS17, CDS18, CDS12, CDS19, MhoK, CDS21a and CDS21b. The remaining CDSs were detected below the detection threshold (only
<figure><img src="image_8.png" /><figcaption>**Fig. 6** Schematic representation of proteins expressed from ICEHo4788 CDSs under six tested axenic conditions as follows: 12 h, 19 h, 19 h -80 °C, 19 h 42 °C, 19 h with and without 0.25 MIC MMC. Each arrow represents a CDS. The gray-filled arrows indicate CDSs with detected protein expression, whereas the hatched arrows represent CDSs detected below the threshold and with only a single unique peptide identified. The white-filled arrows correspond to CDSs that were not detected. This analysis is based on LC-MS/MS data obtained from three biological replicates for each of the six tested conditions for a total of 18 samples</figcaption></figure>

<table><caption>**Table 1** A list of proteins differentially expressed between 12 h and 19 h of culture. The values correspond to the 12 h/19 h abundance ratio and the associated p values. A ratio ≥ 2 or ≤ 0.5, combined with a p value < 0.05, was used as the significance threshold, which was based on data from three biological replicates. ICEHo4788 CDSs are shown in bold. The putative protein functions were determined based on the *M. hominis* 4788 genome annotation via the Prokka annotation pipeline</caption><thead><tr><th>Accession number (CP035542)</th><th>Putative Protein Function</th><th>Abundance ratio (12 h / 19 h)</th><th>P-value (12 h / 19 h)</th></tr></thead><tbody><tr><td colspan="4">Downexpressed at 12 h</td></tr><tr><td>LANHJHLF_00357</td><td>30 S ribosomal protein S14 type Z</td><td>0.13</td><td>1.46E-04</td></tr><tr><td>LANHJHLF_00433</td><td>Hypothetical protein</td><td>0.15</td><td>8.58E-04</td></tr><tr><td>LANHJHLF_00435</td><td>High affinity transport system protein p37</td><td>0.17</td><td>1.63E-03</td></tr><tr><td>LANHJHLF_00308</td><td>Hypothetical protein</td><td>0.18</td><td>2.97E-03</td></tr><tr><td>LANHJHLF_00549</td><td>Hypothetical protein</td><td>0.19</td><td>4.50E-03</td></tr><tr><td>LANHJHLF_00586</td><td>Putative lipid kinase YtIR</td><td>0.20</td><td>7.38E-03</td></tr><tr><td>LANHJHLF_00532</td><td>Hypothetical protein</td><td>0.21</td><td>1.26E-02</td></tr><tr><td>LANHJHLF_00104</td><td>Hypothetical protein</td><td>0.22</td><td>1.49E-02</td></tr><tr><td>LANHJHLF_00062</td><td>Hypothetical protein</td><td>0.25</td><td>4.05E-02</td></tr><tr><td>LANHJHLF_00421</td><td>Purine nucleoside phosphoramidase</td><td>0.25</td><td>4.64E-02</td></tr><tr><td>LANHJHLF_00360</td><td>50 S ribosomal protein L14</td><td>0.26</td><td>4.93E-02</td></tr><tr><td colspan="4">Overexpressed at 12 h</td></tr><tr><td>LANHJHLF_00300</td><td>ICE hypothetical protein (CDS11)</td><td>2.14</td><td>1.79E-04</td></tr><tr><td>LANHJHLF_00420</td><td>Chromosome partition protein Smc</td><td>2.30</td><td>8.43E-05</td></tr><tr><td>LANHJHLF_00020</td><td>Hypothetical protein</td><td>2.31</td><td>8.01E-05</td></tr><tr><td>LANHJHLF_00111</td><td>30 S ribosomal protein S20</td><td>2.42</td><td>4.77E-05</td></tr><tr><td>LANHJHLF_00288</td><td>ICE hypothetical protein (CDS17)</td><td>2.43</td><td>4.50E-05</td></tr><tr><td>LANHJHLF_00609</td><td>Hypothetical protein</td><td>2.46</td><td>3.89E-05</td></tr><tr><td>LANHJHLF_00480</td><td>Hypothetical protein</td><td>2.54</td><td>2.61E-05</td></tr><tr><td>LANHJHLF_00287</td><td>ICE hypothetical protein (CDS18)</td><td>2.80</td><td>8.19E-06</td></tr><tr><td>LANHJHLF_00210</td><td>Holliday junction ATP-dependent DNA helicase RuvA</td><td>2.81</td><td>7.53E-06</td></tr><tr><td>LANHJHLF_00103</td><td>Putative mutator protein MutT4</td><td>2.91</td><td>4.98E-06</td></tr><tr><td>LANHJHLF_00397</td><td>Hypothetical protein</td><td>2.92</td><td>4.75E-06</td></tr><tr><td>LANHJHLF_00587</td><td>Hypothetical protein</td><td>4.07</td><td>4.45E-08</td></tr><tr><td>LANHJHLF_00434</td><td>High affinity transport system protein p37</td><td>4.20</td><td>2.78E-08</td></tr><tr><td>LANHJHLF_00628</td><td>Hypothetical protein</td><td>5.21</td><td>8.46E-10</td></tr><tr><td>LANHJHLF_00180</td><td>Meromycolate extension acyl carrier protein</td><td>6.20</td><td>3.97E-11</td></tr><tr><td>LANHJHLF_00190</td><td>Hypothetical protein</td><td>6.52</td><td>1.58E-11</td></tr><tr><td>LANHJHLF_00418</td><td>Chromosome partition protein Smc</td><td>6.97</td><td>4.53E-12</td></tr><tr><td>LANHJHLF_00237</td><td>Hypothetical protein</td><td>8.86</td><td>3.86E-14</td></tr><tr><td>LANHJHLF_00537</td><td>Hypothetical protein</td><td>11.15</td><td>2.22E-16</td></tr><tr><td>LANHJHLF_00223</td><td>Hypothetical protein</td><td>20.77</td><td>1.00E-17</td></tr><tr><td>LANHJHLF_00299</td><td>ICE hypothetical protein (MhoH(a))</td><td>62.33</td><td>1.00E-17</td></tr></tbody></table>

one unique peptide was identified for CDS3, MhoA(b), and CDS22) or not detected.

Results of the quantitative analysis of protein expression between 12 h and 19 h under axenic conditions

revealed significant differences. At 12 h, 21 proteins were significantly overexpressed compared with those at 19 h (Table 1; Fig. 7), including four ICEHo4788-associated proteins as follows: MhoH(a) (62.3-fold), CDS18
<figure><img src="image_9.png" /><figcaption>**Fig. 7** Effect of growth phase on *M. hominis* 4788 protein expression. The volcano plot illustrates differential protein expression between the 12-h and 19-h cultures. Each point represents a protein with the x-axis showing the log2-fold change in protein abundance and the y-axis showing the -log10 of the unadjusted p-value derived from the statistical t-test. Proteins significantly overexpressed at 12 h are shown on the right, whereas those down-expressed are plotted to the left. The dashed lines indicate the statistical significance threshold (p < 0.05) and log2-fold change threshold (± 1)</figcaption></figure>

(2.8-fold), CDS17 (2.4-fold), and CDS11 (2.1-fold). A total of 11 proteins were significantly downregulated at 12 h but none were located within ICEHo4788 (Table 1). The expression levels of other detected ICEHo4788-associated proteins remained unchanged between the two time points.

Compared with no treatment, exposure to MMC at 0.25 MIC and cold stress at -80 °C resulted in minimal changes in protein expression with no modification of ICEHo4788 protein expression (Fig. S5 and Fig. S6). Heat shock at 42 °C induced the overexpression of five proteins including the molecular chaperones ClpB and DnaK and the MhoH(a) ICEHo4788 protein whose expression increased 2.1-fold (Fig. S7).

Overall, these findings demonstrate that nearly half of ICEHo4788 CDSs are expressed under axenic conditions with several CDSs showing increased expression during

the mid-exponential phase. This pattern aligns with the peak of ICEHo4788 circularization observed at 12 h.

## Discussion

In this study, we investigated the dynamics of *M. hominis* ICEHo4788 under various environmental conditions including axenic and cell culture growth, exposure to a DNA-damaging agent, and thermal stress.

## Growth in axenic culture

The excision of ICEs results in the formation of circular intermediates that are necessary for ICE transfer. These circular forms have been previously identified in *M. hominis* [13]. Under axenic conditions, the highest number of ICEHo4788 circular forms was observed at 12 h, which corresponded with the mid-exponential growth phase of the strain. This peak suggests a potential link
between active bacterial replication and ICE mobiliza-
tion. The subsequent decrease in circular forms during
the decline and death phases aligns with reduced meta-
bolic activity and indicates that ICE circularization in
*M. hominis* may be tightly coordinated with cellular
processes. In contrast, ICEs from other bacterial spe-
cies, such as *S. thermophilus*, *M. loti*, and *P. knackmussii*,
exhibit circularization peaks during the stationary phase
[14–16]. Interestingly, *M. hominis* 4788 lacks a stationary
phase (Fig. 1), which likely explains the earlier timing of
ICEHo4788 circularization. In addition to circularization,
some ICEs, such as ICEBs1 of *Bacillus subtilis*, replicate
autonomously once excised [32]. Whether ICEHo4788
possesses a similar capacity remains unknown but inves-
tigating its potential for autonomous replication could
further clarify its maintenance and dissemination within
*M. hominis* populations. Future studies using primers
flanking the ICE insertion site could enable more precise
quantification of excision events by PCR, as performed
for other ICEs [15].

Unlike circularization, variation in the transcription of ICEHo4788 CDSs was not significant between 12 h and 19 h of growth. However, proteomic analysis revealed discrepancies between transcriptional activity and protein abundance because ICE-associated proteins, such as MhoH(a), CDS18, CDS17, and CDS11, were overexpressed during the mid-exponential phase at 12 h, which coincided with the circularization peak. These findings suggest that while ICEHo4788 circularization and transcription do not seem to be correlated in *M. hominis*, discrepancies between transcriptional activity and protein abundance may have several causes, such as a lack of sensitivity of the LC-MS/MS method used for proteomic analysis, posttranscriptional regulation, differences in mRNA and protein stability, or delayed translation [33–36].

Moreover, our results suggest that ICEHo4788 CDSs
might be continuously transcribed regardless of the
bacterial growth phase in axenic culture. The absence
of active transcription terminators within ICEHo4788
could contribute to this sustained transcription. Notably,
based on our RT-PCR data, only the terminator located
upstream of the first ICE CDS appears to effectively halt
transcription. Interestingly, the ARNOLD web server
analysis predicted the presence of an identical terminator
located upstream of the first CDS in all reported *M. hom-
inis* ICEs [13] (data not shown), which suggests that it
may serve as a regulatory element controlling transcrip-
tion initiation in *M. hominis* ICEs. Given the random
integration of *M. hominis* ICEs within the genome [13],
this transcription terminator likely plays a role in pre-
venting ICE gene transcription from being influenced by
upstream genomic promoters. By effectively isolating ICE
gene transcription from adjacent genes this mechanism

may ensure proper ICE regulation and maintain its func-
tional autonomy.

RT-PCR data revealed positive amplification for all
intergenic regions within ICEHo4788, which suggests
that the element may be transcribed as a single operon.
In support of this, intergenic regions within ICEHo4788
are generally short, with the longest predicted inter-
genic distance being 426 bp between MhoC and CDS14.
Similarly, large transcriptional units have been identified
in other ICEs, such as ICESt1 and ICESt3 from S. ther-
mophilus, where sixteen conjugation–recombination
genes are cotranscribed as a single polycistronic mRNA
of approximately 14.6 kb [15]. However, the hypothesis
of a single operon should be advanced with caution due
to the inherent limitations of RT-PCR. One possibility is
that the observed inter-CDS amplifications result from
promoter leakage where transcription from one CDS
unintentionally extends into the next. Additionally, the
presence of cryptic promoters within ICEHo4788 can-
not be ruled out. By cryptic promoters, we refer to tran-
scriptional start sites that escape standard bioinformatic
prediction, either because of non-canonical motifs or
atypical locations such as within coding regions. Such
atypical promoters have been reported in the genomes of
*M. agalactiae* [37] and *M. gallisepticum* [38]. Indeed, pre-
dicting promoters in ICEHo4788 remained challenging
due to its low GC content (27.31%) [13] and the absence
of a typical –35 region [33, 39, 40], which made accu-
rate identification difficult. Thus, multiple potentially
undetected promoters may contribute to the observed
transcriptional activity. It should also be noted that the
end-point PCR used in this study may only identify
strong terminators while weaker terminators may lead
to read-through amplification during long PCR cycles.
Further studies, including Northern blot or RNA-seq
analyses, are necessary to better understand the operon
structure of ICEHo4788. Surprisingly, we also detected
positive amplification between the 3' end of the disrupted
DUF-containing domain protein and the adjacent *ribF*
gene, which is oriented in the reverse direction. Anti-
sense transcription has been previously reported in *M.
genitalium* via novel promoterless reporter vectors [41]
and in *M. pneumoniae* [34] via RNA-seq analyses, which
suggests that such regulatory mechanisms may be wide-
spread among Mollicutes; this highlights the potential
for complex posttranscriptional regulation in *M. homi-
nis* and possibly compensates for the paucity of classical
transcriptional regulators [42].

MMC exposure

Exposure to sublethal concentrations of MMC had a lim-
ited influence on the circularization of ICEHo4788 with a
slight effect observed only when one quarter of the MIC
was applied at 12 h of culture. Higher concentrations
failed to elicit similar responses, which was likely due to DNA damage overwhelming the bacterial repair mechanisms if present and disrupting ICE activation [43, 44]. This result contrasts sharply with other ICEs, such as ICESt1 and ICESt3 from S. thermophilus, where MMC exposure at half the MIC led to significant excision increases of 38-fold and 18-fold, respectively [17]. These effects were attributed to the presence of regulatory modules encoding cl or ImmR repressors within ICE. In such systems, MMC-induced DNA damage generates single-stranded DNA, which activates the RecA protein. RecA subsequently triggers the autoproteolysis of cl repressors, which reduces repression and initiates ICE activation. In the case of ICEHo4788, the absence of similar repressors likely accounts for the minimal response to MMC. Notably, the timing of the MMC application in this study (12 h) coincided with mid-exponential growth, a phase that is characterized by active replication and metabolic activity. These findings suggest that MMC-induced effects, if present, might be modulated by the metabolic state of the bacteria. The lack of significant circularization at 19 h of culture, i.e., at the end of the exponential growth phase, further supports the idea that ICEHo4788 circularization potentially has little reliance on DNA damage signaling. Additionally, transcriptional analysis revealed no significant changes following MMC exposure, and proteomic analysis revealed minimal changes in ICEHo4788-associated protein expression under MMC exposure. Notably, no stress-related proteins were overexpressed, which suggests that MMC at one-quarter MIC did not induce a detectable stress response in M. hominis. The lack of upregulation of RecA and other repair proteins after MMC exposure suggests that M. hominis may not mount a strong DNA damage response under these conditions. Collectively, these data suggest that ICEHo4788 activity may be somewhat dependent on MMC-induced DNA damage signaling.

## Thermal stresses

The impact of thermal stress on the circularization of ICEHo4788 was also examined and revealed distinct responses to cold and heat stress. Under cold stress (-80 °C/4°C), a slight increase in circular forms was observed, whereas heat stress at 42 °C reduced the number of circular forms. Neither condition led to variations in the transcription levels of ICEHo4788 CDSs. Proteomic analysis confirmed the effectiveness of the heat treatment as ClpB and DnaK, which are two key components of a stress-induced multichaperone system involved in cellular recovery from heat-induced damage, were overexpressed. This also confirms that the bacterial response to heat stress was still active and detectable after the 3-h recovery period at 37 °C. These two proteins were previously reported to be overexpressed under heat

stress conditions in M. pneumoniae [45] and M. gallisepticum [46]. In contrast, no stress marker proteins were overexpressed under cold stress, which suggests that low temperatures do not trigger a significant stress response in M. hominis. These findings illustrate that heat stress, but not cold stress, negatively impacts ICEHo4788 dynamics, which suggests an adaptive mechanism in M. hominis to balance genetic element activation with survival priorities.

## Coculture with eukaryotic cells

To assess the influence of eukaryotic cells, we chose HeLa cells derived from the human cervix as they are more likely to be adapted to a human genital mycoplasma. Among all of the tested conditions, coculture with HeLa cells had the strongest effect on ICEHo4788 activity. First, the bacterial growth dynamics were markedly different with a significant decline during the first 24 h; this is a phenomenon also reported for the M. hominis FBG strain under similar conditions [47]. This initial decrease may reflect the adaptation period required for M. hominis to acclimate to the host cellular environment. In contrast to axenic culture, where M. hominis rapidly loses viability after the exponential phase, coculture with HeLa cells maintains bacterial survival for several days, thereby offering a potential more favorable context for ICEHo4788 activation. Interestingly, ICEHo4788 circularization strongly increased during the later stages of coculture, which suggests that host-associated factors or prolonged bacterial persistence in this environment can stimulate ICE mobilization. Additionally, coculture with HeLa cells for seven days was the only condition that led to a significant increase in CDS transcription, which suggests a correlation between ICE circularization and its transcriptional activation in this setting. The influence of eukaryotic cells on ICE activity is not exclusive to M. hominis. In M. agalactiae, the mating frequency was 200-fold greater during coincubation with T-antigen-immortalized goat milk epithelial cells (TiGMEG) than under axenic conditions [19]. Similarly, coincubation with eukaryotic cells stimulated conjugation in M. bovis and provided the first report of ICEB transfer in this species [19]. Notably, nucleotide stress might be a key trigger for mycoplasma conjugation and thus ICE activation [19]. This finding raises the possibility that M. hominis may respond to similar host-induced metabolic constraints and reinforces the idea that stress signals encountered in the host environment could drive ICE mobilization. A limitation of our study is that proteomic analysis under coculture with HeLa cells failed. In our attempts, the detection of approximately 7,300 HeLa cell proteins decreased the sensitivity of detection of M. hominis proteins with only 186 M. hominis proteins identified under coculture conditions compared with 520 under axenic
conditions. The strong ability of mycoplasmas to adhere to eukaryotic cells [48] prevents the use of differential centrifugation methods to accurately separate mycoplasma bacteria from HeLa cells in these experiments.

Together, our results indicate that coculture with HeLa cells is the most favorable condition for ICEHo4788 activity and should be used to assess the transfer capacity of *M. hominis* ICEs from a donor to a recipient cell through mating experiments. Some previously transformed strains harboring an ICE marked with a resistance gene are available as donor cells [49]. Indeed, several mating experiments in axenic conditions have failed as have the first attempts in HeLa cell coculture conditions (data not shown). Considering that *M. hominis* ICE may be transcribed as a single operon, inserting a resistance gene marker may disrupt ICE functions. The insertion of a resistance gene marker in a strain harboring two ICEs may be needed to overcome this problem to achieve ICE complementation.

In conclusion, our findings highlight a complex regulatory network governing ICEHo4788 activity and suggest that transcription, protein expression, and circularization may not always be correlated. The presence of eukaryotic cells, such as HeLa cells, strongly enhances ICEHo4788 CDS transcription, excision, and circularization, which suggests that the interaction between *M. hominis* and its host cell may serve as a key signal to activate functions that are useful for the physiology or pathogenicity of this human pathogen.

**Supplementary Information**

The online version contains supplementary material available at https://doi.org/10.1186/s13100-025-00364-x.

Supplementary Material 1

Supplementary Material 2

**Acknowledgements**

We would like to thank S. Claverol for the LC-MS/MS analysis and helpful discussions.

**Author contributions**

B.B.A. contributed to conceptualization, methodology, validation, formal analysis, investigation, data curation, and writing original draft. A.S. contributed to conceptualization and methodology. C.L.R. contributed to conceptualization and data curation. J.G. and L.Bi. contributed to methodology and data interpretation. L.Be contributed to conceptualization, methodology, and data curation. V.D. contributed to writing, review and editing. E.B. contributed to conceptualization, validation, and writing, review and editing. C.B. contributed to validation, writing, review and editing, and funding acquisition. S.P. contributed to conceptualization, methodology, validation, writing, review and editing, supervision, and funding acquisition. All authors approved the final manuscript.

**Funding**

This work was supported by internal funding.

**Data availability**

No datasets were generated or analysed during the current study.

**Declarations**

**Ethics and consent to participate**

Not applicable.

**Consent to publish**

Not applicable.

**Competing interests**

The authors declare no competing interests.

**Transparency declarations**

None to declare.

Received: 17 April 2025 / Accepted: 16 June 2025

Published online: 04 July 2025

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