If you don't remember your password, you can reset it by entering your email address and clicking the Reset Password button. You will then receive an email that contains a secure link for resetting your password
If the address matches a valid account an email will be sent to __email__ with instructions for resetting your password
Correspondence to: Niels Odum, , LEO Foundation Skin Immunology Research Center, Department of Immunology and Microbiology; The Maersk Tower 07-12-62, University of Copenhagen, Blegdamsvej 3, DK2200 N, Copenhagen, Denmark.
Staphylococcus aureus (S. aureus) is suspected to fuel disease activity in cutaneous T cell lymphomas (CTCL). Here we investigate the effect of a recombinant, anti-bacterial protein, endolysin, XZ.700, on S. aureus skin colonization and malignant T cell activation. We show that endolysin strongly inhibits proliferation of S. aureus isolated from CTCL skin and significantly decreases S. aureus bacterial cell counts in a dose-dependent manner. Likewise, ex vivo colonization of both healthy and lesional skin by S. aureus is profoundly inhibited by endolysin. Moreover, endolysin inhibits the patient-derived S. aureus induction of Interferon-gamma (IFNγ) and IFNγ-inducible chemokine CXCL10 in healthy skin. Whereas patient-derived S. aureus stimulates activation and proliferation of malignant T cells in vitro through an indirect mechanism involving non-malignant T cells, endolysin strongly inhibits the effects of S. aureus on activation (reduced CD25 and STAT5 phosphorylation) and proliferation (reduced Ki67) of malignant T cells and cell lines in the presence of non-malignant T cells. Taken together, we provide evidence that endolysin XZ.700 inhibits skin colonization, chemokine expression, and proliferation of pathogenic S. aureus, and blocks their potential tumor-promoting effects on malignant T cells.
Cutaneous T-cell lymphomas (CTCL) are a heterogeneous group of non-Hodgkin lymphomas, with mycosis fungoides (MF) and Sézary syndrome (SS) being the most prevalent and most severe subtypes, respectively (
). Multiple cancer cell-intrinsic genetic events and epigenetic regulators seem to play an important role in CTCL pathogenesis, however, a complete understanding of disease etiology and pathogenesis remains elusive (
). Recent focus has turned to the interplay between malignant T cells, benign T cells, and stromal cells, and how these interactions shape the tumor microenvironment (TME) in ways that promote disease progression and weaken the anti-tumor and anti-microbial immune defense (
). The cellular interactions are among other affected by soluble factors in the TME, such as cytokines and chemokines. Chemokines are believed to have an important pathogenetic role in CTCL, as chemokines and their receptors are crucial for homing of malignant T cells and immune cells to the skin (
). Even without overt bacterial infection, the skin microbiota may play a role in CTCL severity. This is exemplified by the reduced disease severity and tumor burden following systemic antibiotics therapy used either prophylactic or in relation to non-cutaneous infections (
). Here S. aureus and staphylococcal enterotoxins (SE) have been suspected to fuel disease activity and cancer progression in CTCL through a direct stimulation of the malignant T cell clone and/or through modulation of the TME (
). Importantly, S. aureus toxins trigger signaling pathways associated with CTCL pathogenesis in peripheral blood mononuclear cells (PBMC) isolated from Sézary syndrome patients with both malignant and non-malignant T cells: (i) expression of oncogenic microRNA-155 and regulatory proteins (PD1, FOXP3, and IL-10), (ii) STAT3 and STAT5 activation, (iii) inhibition of anti-tumor cytotoxicity, and (iv) proliferation of primary malignant T cells (
). Nevertheless, re-colonization of skin lesions by S. aureus is seen in most CTCL patients after termination of antibiotic treatment indicative of the need for a lasting regulation of disease promoting bacteria, such as S. aureus on the skin (
). Long-term usage of antibiotics to persue this goal comes with the risk of developing drug resistance which is not warranted in a patient group already susceptible to infections thereby making it even more difficult to treat infections requiring medical attention. Thus, there is a medical need for new, specific anti-staphylococcal alternatives to the currently used antibiotics.
Recombinantly produced endolysins represent an interesting new class of antibacterial agents. Endolysins are peptidoglycan (PG) hydrolases expressed by bacteriophages. As endolysins cleave the bonds within the PG, they can target both metabolically active and inactive bacteria, such as bacterial biofilms (
). Bacterial species show unique PG compositions that can be specifically targeted by endolysins, and thereby enabling species-specificity of antimicrobial action, such as S. aureus including MRSA, while sparing the remaining commensal microbiota (
). Several endolysins with specificity toward S. aureus have been recombinantly produced including endolysin XZ.700. In vitro experiments using this endolysin in sublethal dosages did not lead to the development of resistance (
). By specifically targeting S. aureus and thereby maintaining the commensal microbiota, the hope is that re-colonization by S. aureus will be hampered.
The present study was undertaken to investigate the effect of the chimeric endolysin XZ.700 on skin colonization, chemokine expression, and possible pro-tumorigenic effects of CTCL patient-derived enterotoxin-producing S. aureus.
We first determined the bactericidal effect of the recombinant endolysin XZ.700 on a S. aureus laboratorium strain (S. aureus LAB) and S. aureus isolated from lesional skin from CTCL patients (S. aureus from patient skin (S. aureus Pt)) (Lindahl et al., 2019, Willerslev-Olsen et al., 2021). Endolysin effectively inhibited planktonic growth of S. aureus LAB (Fig. 1a) as well as a S. aureus Pt (Fig. 1b) as measured by optical density (OD). Moreover, endolysin killed both S. aureus LAB and S. aureus Pt in a dosage responsive manner with a reduction by more than four log-units at and above 1 μg/mL as determined by colony forming unit (CFU) (Fig. 1c and d). Two additional patient-derived S. aureus isolates showed similar results (Fig. 1e). As expected, the killing was abrogated by prior heat-inactivation of the endolysin (Fig. 1f) supporting that the effect is mediated by the enzymatic activity of endolysin.
We setup a human ex vivo skin model to determine the effect of endolysin on S. aureus Pt colonization. Healthy skin was colonized by S. aureus Pt as indicated by arrows on the H&E and FISH stainings while no bacteria was observed in the skin without S. aureus Pt added (Fig. 2a). Importantly, endolysin profoundly inhibited colonization by S. aureus Pt (Fig. 2a). This observations was quantified by CFU measurement (Fig. 2b). When 1 μg/mL of XZ.700 was added simultaneously with S. aureus Pt, almost no skin colonization was observed (Fig. 2b). In parallel, we investigated whether endolysin could also reduce already established S. aureus Pt on skin. A significant, dose-dependent reduction of bacteria was observed for all concentrations with an almost complete eradication of S. aureus Pt using 10μg/mL (Fig. 2b). Taken together, these data provide evidence that endolysin has a potent inhibitory effect on S. aureus Pt growing on human skin.
Lesional skin from CTCL patients differs from healthy skin due to changes in skin architecture, barrier proteins, and levels of endogenous antimicrobial peptides, all of which could change S. aureus colonization and accessibility for S. aureus killing by endolysin. Accordingly, we examined the effect of endolysin on S. aureus Pt colonization of lesional skin from CTCL patients (Fig. 3). S. aureus Pt colonized the lesional CTCL skin while co-addition of endolysin significantly reduced colonization (Fig. 3). These findings suggest that the lesional skin architecture and other CTCL-associated conditions did not affect the mode of action and efficacy of endolysin on pathogenetic S. aureus.
As chemokines are believed to play an important pathogenetic role in CTCL, we examined the effect of S. aureus Pt on the expression of CTCL-associated chemokines in human skin. Accordingly, healthy skin specimens were incubated with S. aureus Pt supernatant for 18 hours prior to analysis of chemokine mRNA expression. As shown in figure 4a, S. aureus Pt stimulation triggered a pronounced expression of the IFNγ-inducible chemokine CXCL10, whereas the other tested CTCL-associated chemokines were not significantly upregulated (Fig. 4a). Importantly, pretreatment with endolysin partly inhibited induction of CXCL10 expression by S. aureus Pt supernatant. To validate this finding, the expression of additional S. aureus associated cytokines including IFNγ and IL-6 were analyzed. We also measured expression of IL-37, which has been inversely linked to infection-associated inflammation (
). While IL-6 and IL-37 remained stable, incubation with S. aureus Pt supernatant induced IFNγ expression, which was strongly inhibited by endolysin (Fig. 4b). These findings suggest a novel mechanism for S. aureus to accelerate pathogenic events in CTCL, which is partially blocked by endolysin.
Toxins including SE secreted from S. aureus are suspected to change the TME (
). Accordingly, we examined the effect of endolysin on SE production. A qualitative screening confirmed that S. aureus Pt produced staphylococcal enterotoxin A (SEA) (Suppl Fig. S1). Four hours after setting up the S. aureus Pt cultures, SEA was detected in the supernatant of S. aureus Pt at concentrations above 15 ng/mL (Fig. 5a). In contrast, the concentration of SEA in the S. aureus Pt cultures with endolysin remained below 1 ng/mL (Fig. 4a). It has previously been shown that recombinant SEA can activate non-malignant Myla1850 T cells (
), and thus we next addressed the effect of endolysin treated S. aureus Pt supernatants on activation of malignant and non-malignant T cells. As expected, SEA activated the non-malignant MyLa1850 T cells (CD25 expression) (Fig. 5b). Similarly, culturing Myla1850 with supernatants harvested from S. aureus Pt, resulted in T cell activation (CD25 expression) at all dilutions used (Fig. 5b). In comparison, supernatant from S. aureus Pt with 1μg/mL XZ.700 induced only weak T cell activation (Fig. 5b).
Malignant SeAx T cells do not respond directly to SEA but become activated via an indirect, IL-2-dependent pathway, when exposed to SEA in co-culture with non-malignant T cells (
). Thus, we setup co-cultures of non-malignant Myla1850 and malignant SeAx cells and assesed T cell activation (CD25 expression and STAT5 phosphorylation). SEA and S. aureus Pt supernatant induced CD25 expression and STAT5 phosphorylation in the malignant SeAx cells (Fig. 5c), whereas pre-treatment of S. aureus Pt with endolysin inhibited supernatant-mediated T cell activation (Fig. 5c).
We next examined the effect of S. aureus Pt supernatant and endolysin in primary malignant T cells from Sézary Syndrome patients. We used peripheral blood mononuclear cells (PBMCs), because SE-induced activation of malignant T cells usually requires crosstalk between malignant and non-malignant T cells (
). As expected, S. aureus Pt supernatant induced CD25 expression and STAT5 phosphorylation in primary malignant T cells (Fig. 5d, brown graph). Importantly, activation of primary malignant T cells was abolished when pre-treating S. aureus Pt with endolysin (Fig. 5d, green versus brown graph).
Next, we examined the effect of endolysin on S. aureus-mediated proliferation of malignant T cell lines from Sézary syndrome (SeAx) and mycosis fungoides (Myla3675) in co-culture with the non-malignant T cell line Myla1850. As shown in Fig. 6a, S. aureus Pt increased proliferation (Ki67 expression) of SeAx cells when cultured with MyLa1850 cells (Fig. 6a, upper row, third column) but not in SeAx cultures without MyLa1850 (Fig. 6a, upper row, first column) indicating that S. aureus Pt induced enhanced proliferation of malignant cells in co-cultures with non-malignant T cells. S. aureus Pt also increased Ki67 expression in MyLa3675 cells co-cultured with MyLa1850 cells (Fig. 6a, bottom row, third column) when compared to cultures without MyLa1850 (Fig. 6a, bottom row, first column). Of note, the high basic level of Ki67 expression in unstimulated MyLa3675 cells reflect the high level of spontaneous proliferation of this and other malignant T cell lines from this patient (
). Importantly, endolysin almost completely blocked the effect of S. aureus Pt on Ki67 expression in both SeAx (fig. 6a, upper row, right) and MyLa3675 cells (Fig. 6a, bottom row, right) indicating that endolysin prevented S. aureus Pt enhanced proliferation of malignant T cells. This promted us to test if endolysin could prevent S. aureus-induced proliferation of primary malignant T cells in Sézary syndrome PBMC cultures. Indeed, supernatant from S. aureus Pt - and recombinant SE induced comparable levels of mitotic (Ki67) and activated (CD25) primary malignant T cells (Fig. 6b). Importantly, endolysin treatment of S. aureus Pt supernatant prevented this effect (Fig. 6b). Similar results were obtained with PBMCs isolated at a later time-point from the same Sézary syndrome patient (Supl. fig. S2a). As endolysin strongly inhibited activation of non-malignant T cells (c.f. above), we examined whether endolysin-treatment also inhibited the effect of S. aureus Pt supernatant on proliferation of non-malignant T cells (Suppl. Fig. S2b and c). As expected, endolysin inhibited Ki67 expression in S. aureus Pt- treated patient-derived primary non-malignant CD4+ T cells (suppl. Fig. S2b) and non-malignant T cell lines (MyLa1850 cells - suppl Fig. S2c) supporting the hypothesis that endolysin inhibits S. aureus Pt mediated crosstalk between malignant and non-malignant T cells.
In conclusion, these findings indicate that endolysin treatment profoundly reduced bacterial counts and SE-release to levels below the threshold required for induction of malignant T cell proliferation in co-culture with non-malignant cells.
Here, we present evidence that recombinant endolysin XZ.700 has the potential to reduce S. aureus on the skin of CTCL patients. Thus, endolysin profoundly inhibited skin colonization by S. aureus of both healthy skin and lesional CTCL skin. Importantly, endolysin killed S. aureus after the S. aureus colonization had been established. This suggests that endolysin has potential in both a treatment-setting and as a prophylactic treatment. Although treatment with endolysin strongly inhibited S. aureus-mediated release of SE, the blockage was not complete. Importantly, only a low number of SE molecules per cell is needed to trigger a T cell response (
). Yet, endolysin-treated S. aureus Pt culture supernatants induced little or no T cell activation and proliferation indicating that endolysin-mediated reduction in S. aureus (and SE-secretion) was sufficient to prevent the mitogenic effect. Thus, our data show that endolysin profoundly inhibited both skin colonization by live patient-derived S. aureus and their ability to stimulate activation and proliferation of primary malignant T cells and T cell lines. In addition, we show that S. aureus Pt culture supernatants induced selective expression of IFNγ and IFNγ-inducible CXCL10 in healthy skin. Since CXCL10 is known to play a key role in CD4 T cell epidermotropism (
), we hypothesize that this could be a novel mechanism by which S. aureus modulates the TME and potentially promotes disease progresion and, consequently, another potential benefit from endolysin-mediated removal of S. aureus from lesional skin.
Similar to antibiotics, resistance against bacteriophages exists. However, resistance against recombinant endolysins is less likely due to (i) their highly conserved targets in the PG and (ii) their action on the cell envelope, without having to enter the cell (when applied from the outside), thereby avoiding a majority of known resistance mechanisms (
). As recombinant endolysins have only been around for a few years, it is imposible to know whether S. aureus may eventually develop resistance to recombinant endolysin in a real life setting, but studies suggest that S. aureus do not develop resistance to endolysin-mediated killing in long-term in vitro cultures (
). Whether the targeted killing of S. aureus by endolysin will be sufficient to reinforce beneficial commensal bacteria or enable other potential pathogenic bacteria to increase in presence will remain to be seen.
Thus, studies are warranted to address whether recombinant endolysins are useful as a non-antibiotic alternative for eradication of S. aureus colonization from lesional skin and/or as prophylactic measures against skin colonization by S. aureus in CTCL patients. Since S. aureus also plays an important role in other dermatological diseases, we speculate that recombinant endolysin may also be of interest for anti-S. aureus treatment and prophylaxis against S. aureus-mediated flare-ups in diseases, such as, atopic dermatitis.
In conclusion, we provide evidence that a recombinant endolysin, XZ.700, potently inhibits proliferation of pathogenic S. aureus, their skin colonization, and their tumor-promoting effects on malignant T cells. These findings suggest that recombinant endolysin comprises a non-antibiotic treatment strategy to control S. aureus colonization of lesional skin in CTCL.
MATERIALS AND METHODS
Planktonic S. aureus culture and endolysin activity assay
S. aureus NCTC 8325, designated S. aureus LAB, and the clinical CTCL patient isolates Pt126.96.36.199 (S. aureus Pt), Pt188.8.131.52 (S. aureus Pt 1), and Pt184.108.40.206 (S. aureus Pt 2) were used (
). The clinical S. aureus isolates were identified by detection of coagulase and catalase activity and by MALDI-TOF MS. In addition, the isolates were characterized by multilocus sequence typing and characterized in relation to toxin genes by NGS as previously described (
). S. aureus was prepared from an overnight culture diluted to an OD600nm of 0.01 (Shimadzu UV spectrophotometer, UV-1800) in tryptic soy broth (TSB) and regrown till early log phase (OD600nm of 0.2-0.7) at 37°C under shaking conditions (180 rpm). Colony forming units (CFU) was determined by serial dilution, plating on LB agar plates, overnight incubation at 37 °C, and counting the number of colonies. The growth inhibitory effect of XZ.700 (Micreos Pharma B.V., Netherlands) in liquid culture was determined by measuring the OD595m (Victor X4 2030 multiplate reader, PerkinElmer). XZ.700 was heat-inactivating at 70°C for 10 min.
Patients skin specimens
Biopsies from CTCL patients were obtained from donors at Bispebjerg Hospital and Gentofte Hospital, Denmark. In accordance with the Declaration of Helsinki, all biopsies were obtained with written, informed consent after approval by the Committee on Health Research Ethics (H-16025331). Healthy adult human skin designated “healthy skin” were obtained from donors below the age of 70 undergoing blepharoplasty at ophthalmologist Neel Gerner, Lyngby, Denmark. The use of anonymized leftover skin from cosmetic surgeries was obtained with written, informed consent and approved by the Danish Data Protection Agency. Healthy skin biopsies and CTCL patient biopsies were maintained in DMEM (Thermo, #11965) and Ham’s F12 (Thermo, #11765) supplemented with 2% heat-inactivated FBS (Biological Industries, #04-007-1A), EGF (Sigma, #SRP3027), insulin (Merck, #I9278), and penicillin/streptomycin (Sigma-Aldrich, #P7539). Biopsies were incubated in a humidified 37 °C chamber with 5% CO2.
Ex vivo skin experiments
S. aureus (OD600nm 0.2-0.7) was resuspended to about 1·106 CFU/10 μL PBS based on previous correlation between OD600nm and CFU. Actual CFU seeded was determined for each seeding. S. aureus was seeded at a density of about 5·106 CFU/cm2 (1-10·106 CFU/cm2). Following incubation, biopsies were washed in 0.1 M citrate buffer (pH 4.5) and S. aureus was released by swabbing with a cotton bud 10 times, transferred to 0.1 M citrate buffer (pH 4.5) by sonication (5 min degassing, 5 min sonication (Branson 2510)) and 10 sec vortex before determining CFU.
Immunofluorescence labeling and imaging
Skin biopsies were fixed in fresh 4% paraformaldehyde at 4 °C and paraffin embedded. 3-5μm sections were used for hematoxylin and eosin staining (Histolab, Department of Biomedical Sciences, University of Copenhagen) or fluorescence in situ hybridization (FISH) after deparaffinization. FISH was performed using fluorescently labeled PNA-probes for S. aureus (AdvanDx, #Sta16S03-TXR, OpGen) according to manufacturer’s protocol, stained with DAPI (Thermo, #62248) and mounted with ProLong Diamont Antifade Reagent (Thermo Fisher, #P36961). Images were collected using Zeiss Axio Scan.Z1 with a 405 nm (Dapi) and a 561 nm (Texas red) laser lines and a 10x/0.45 objective.
S. aureus supernatants and S. aureus enterotoxin (SE) determination
S. aureus was prepared from an overnight culture diluted to an OD600nm of 0.01 (Shimadzu UV spectrophotometer, UV-1800) in TSB) and regrown for 4 hours with or without 1μg/mL XZ.700. S. aureus Pt supernatant was prepared by centrifugation (10.000xg for 10 min) followed by sterile filtration (0.22μm filter). SE in S. aureus supernatants were detected by ELISA according to manufacturer’s protocol; SEA (Chrondrex, #6029), SE (R-Biopharm, RIDASCREEN SET A/B/C/D/E kit). Signal was detected by UV plate reader (Thermoscientific, Multiscan FC).
) were cultured in RPMI1640 (Sigma, #R2405) containing penicillin/streptomycin (Sigma-Aldrich, #P7539), 10% human serum (Bloodbank, Copenhagen University Hospital, Denmark), IL-2, and for MyLa1850 also IL-4 as described (
Sézary Syndrome patient PBMC culture and Flow cytometry
Peripheral blood mononuclear cells (PBMCs) were isolated from Sézary Syndrome patient blood by lymphoprep density-gradient centrifugation (Stemcell Technologies, Vancouver, Canada). SE (50 ng/mL; SEA, SEB, SEC2, SED, SEI; Toxin Technology, Sarasota, FL, USA). Cells were stained for CellTrace, propidium iodide and Fixable Viability Stain Dye eFluor780 (ThermoFisher) and fluorochrome-conjugated CD3, CD4, CD7, CD8, CD19, CD25, CD26, Ki-67, pY-STAT5 and respective fluorochrome-conjugated isotype control antibodies (Biolegend,San Diego, CA, USA; BD Biosciences, San Jose, CA, USA). Flow cytometry analysis was performed on a LSR Fortessa flow cytometer (BD Biosciences) using FlowJo software (Tree Star, Ashland, OR). Malignant T cells were gated as CD3+ CD4+, CD7dim/-, and CD26-. Non-malignant T cells were gated as CD3+ CD4+, CD7+, and CD26+.
Quantitative reverse transcription PCR (RT-qPCR)
Biopsies were homogenized (Tissuelyser at 20000 HZ, Qiagen or Precellys, hard tissue program, Bertin) prior to RNA isolation (Qiagen #74134 or AllPrep DNA/RNA kit). cDNA was prepared (Applied biosystems, #4368814) and subjected to RT-qPCR (Lightcycler480) using β-actin as reference gene (TaqMan probe, ThermoFisher). Relative expression was calculated according to the 2ΔΔCT method.
All statistical analyses and graphs were done using GraphPad Prism® software (Prism 9.0.1 version). For statistical analysis, a 2-tailed Student’s t test or an Ordinary one-way ANOVA with Dunnett's multiple comparisons test were made. * significant at p<0.05, ** significant at p>0.01, *** significant at p<0.001, **** significant at p<0.0001. Error bars represent standard deviation.
DATA AVAILABILITY STATEMENT
No datasets were generated or analyzed during the current study.
CONFLICT OF INTERESTS
Niels Ødum has an advisory consultant honorarium from Micreos Pharma B.V and Almirall. Bob de Rooij and Johan Frieling work for Micreos Pharma B.V., Christian Röhrig, and Mathias Schmelcher work for Micreos GmbH. Lars Iversen served as a consultant and/or paid speaker for and/or participated in clinical trials sponsored by AbbVie, Almirall, Amgen, Astra Zeneca, BMS, Boehringer Ingelheim, Celgene, Centocor, Eli Lilly, Janssen Cilag, Kyowa, Leo Pharma, Micreos Human Health, MSD, Novartis, Pfizer, Regranion, Samsung, Union Therapeutics, and UCB. Koralov Laboratory (Sergei Koralov) has a sponsored research agreement with Micreos company to explore potential for recombinant endolysins as a de-colonization agent in the context of T cell lymphoma. This sponsored work is not covered by the present manuscript and the agreement did not influence interpretation of results. All other authors declare no potential conflicts of interest.
This research was funded by LEO Foundation, The Danish Cancer Society (Kræftens Bekæmpelse), the Fight Cancer Program (Knæk Cancer), Novo Nordisk Research Foundation, Novo Nordisk Foundation Tandem Program (grant number NNF21OC0066950), and The Danish Council for Independent Research (Danmarks Frie Forskningsfond, 2 project grants).
Emil M. H. Pallesen (EMHP), Maria Gluud (MG), Chella K. Vadivel (CKV), Terkild B. Buus (TBB), Bob de Rooij (BDR), Ziao Zeng (ZZ), Sana Ahmad (SA), Andreas Willerslev-Olsen (AWO), Christian Röhrig (CR), Maria R. Kamstrup (MRK), Lene Bay (LB), Lise Lindahl (LL), Thorbjørn Krejsgaard (TK), Carsten Geisler (CG), Charlotte M. Bonefeld (CMB), Lars Iversen (LI), Anders Woetmann (AW), Sergei B. Koralov (SBK), Thomas Bjarnsholt (TB), Johan Frieling (JF), Mathias Schmelcher (MS), Niels Ødum (NØ)