key: cord-0062605-ka46me0i
authors: Bongiorno, Dafne; Musso, Nicolò; Caruso, Giuseppe; Lazzaro, Lorenzo Mattia; Caraci, Filippo; Stefani, Stefania; Campanile, Floriana
title: Staphylococcus aureus ST228 and ST239 as models for expression studies of diverse markers during osteoblast infection and persistence
date: 2021-05-01
journal: Microbiologyopen
DOI: 10.1002/mbo3.1178
sha: d6d1d5c4cf02f24fc55648702e78bb6ea03d1900
doc_id: 62605
cord_uid: ka46me0i

The ability of S. aureus to infect bone and osteoblasts is correlated with its incredible virulence armamentarium that can mediate the invasion/internalization process, cytotoxicity, membrane damage, and intracellular persistence. We comparatively analyzed the interaction, persistence, and modulation of expression of selected genes and cell viability in an ex vivo model using human MG‐63 osteoblasts of two previously studied and well‐characterized S. aureus clinical strains belonging to the ST239‐SCCmecIII‐t037 and ST228‐SCCmecI‐t041 clones at 3 h and 24 h post‐infection (p.i). S. aureus ATCC12598 ST30‐t076 was used as a control strain. Using imaging flow cytometry (IFC), we found that these strains invaded and persisted in MG‐63 osteoblasts to different extents. The invasion was evaluated at 3 h p.i and persistence at 24 h p.i., in particular: ATCC12598 internalized in 70% and persisted in 50% of MG‐63 cells; ST239‐SCCmecIII internalized in 50% and persisted in 45% of MG‐63 cells; and ST228‐SCCmecI internalized in 30% and persisted in 20% of MG‐63 cells. During the infection period, ST239‐III exerted significant cytotoxic activity resulting from overexpression of hla and psmA and increased expression of the genes involved in adhesion, probably due to the release and re‐entry of bacteria inside MG‐63 cells at 24 h p.i. The lower invasiveness of ST228‐I was also associated with non‐cytotoxic activity inside osteoblasts. This clone was unable to activate sufficient cellular reaction and succumbed inside MG‐63 cells. Our findings support the idea of considering new strategies, based on a translational approach—eukaryotic host–pathogen interaction (EHPI)—and to be applied on a large scale, to predict S. aureus /osteoblast interaction and treat bone infections. Such strategies rely on the study of the genetic and biochemical basis of both pathogen and host.

The human pathogen Staphylococcus aureus can adapt to the host/ environment, infect any organ, and damage tissues, causing severe infections, and can resist antibiotics, in particular beta-lactams and methicillin (Tong et al., 2015) . It is one of the main pathogens responsible for recurrent osteomyelitis (OM), accounting for 50% to 70% of cases, and prosthetic joint infections (PJIs) (Mruk & Record, 2012; Wu et al., 2019) . PJIs can have a dramatic impact on a patient's quality of life, often requiring surgical intervention and prosthesis removal, as well as prolonged antibiotics treatment (Moore et al., 2017; Purrello et al., 2016; Stefani et al., 2012) . These infections are often due to healthcare-associated methicillin-resistant S. aureus (HA-MRSA) belonging to clonal complex 5 (Muñoz-Gallego et al., 2017; Peng et al., 2019; Pérez-Montarelo et al., 2018) and associated with the staphylococcal chromosomal cassette (SCCmec) I and III (Hussain et al., 2009 ). Among them, ST239-III is probably the oldest pandemic MRSA clone, first discovered in 1970, isolated in most countries worldwide, and the most widespread in Europe (Campanile et al., 2015; Monecke et al., 2018; Szymanek-Majchrzak et al., 2018) . ST228-SCCmecI is one of the most widespread clones in Italy, associated with nosocomial infections (Bongiorno et al., 2018; .

Assessing the interaction between S. aureus isolates and osteoblasts during PJIs and OM includes three crucial events: adhesion, invasion, and post-invasion, where S. aureus controls the expression of adhesion and virulence determinants with its large armamentarium of regulatory genes.

Many studies have been carried out in this field. Different authors have shown the role of many regulators involved in the invasion and adaptation to host tissue. The role of sigB in persistence and stress response, together with its link to sarA and, consequently, to its action on the agr locus was studied in osteoblasts, in an in vivo model, using two different strains of S. aureus; in particular, the authors used the wild-type and the defective strain for agr, sigB, and sarA (Tuchscherr et al., 2015) . The global regulatory systemagr locus-cell density-dependent, controls virulence factor expression. Through its effector molecule RNAIII, the agr locus controls the post-transcriptional regulation of proteins involved in cell-surface interaction and virulence cytotoxic factors (Painter et al., 2014) .

Two other genes belonging to the SARA protein family are involved in the regulation of virulence genes: sarS (SarH1), whose expression is repressed by sarA and agr, is a repressor of hla and etb and is a positive regulator of spa and rot, the "repressor of toxin," which is a repressor of enterotoxin B (seb) and alpha-toxin (hla) and is repressed by the agr effector RNAIII and SarA (Jenul & Horswill, 2018) . (Otsuka et al., 2006; Pérez-Montarelo et al., 2018) ; and SdrE, a serine-aspartate (SD) protein that anchors the cell wall interacting with complement factor H and facilitates colonization through adherence to the cell surface or extracellular matrix (ECM) components (Herr & Thorman, 2017) .

S. aureus can invade endothelial cells and osteoblasts using the cell surface integrin α5β1, binding Fn on the surface of human cells.

In particular, as already demonstrated, FnBPA and FnBPB are involved not only in adhesion but also in internalization (Shinji et al., 2011) . Pore-forming proteins, such as Panton-Valentine leukocidin (PVL) and α-and δ-hemolysin (Hla and Hld), together with phenolsoluble modulins (PSMs), were able to induce local complications such as bone deformation, systemic complications as severe sepsis in rabbit osteomyelitis, or neutrophil and osteoblast cytotoxicity in an ex vivo model (Davido et al., 2016) . The hla gene was frequently present in strains associated with osteoarticular bacteremia (Pérez-Montarelo et al., 2018) . PSMs are small peptides with amphipathic properties that destabilize the lipid bilayer of the host cell; this activity is related to receptor-independent cytotoxicity to osteoblasts and specialized cells such as the neutrophils. PSMs are also implicated in biofilm formation, in bacterial interference, and in cell-cycle disruptions (Davido et al., 2016) .

During the infection process, S. aureus can use alternative carbon sources and, in particular, glucose-6-phosphate (G6P); the uptake of these alternative carbon sources occurs through the hexose phosphate antiporter UhpT (Yang et al., 2016) .

After observing that interaction, internalization, and persistence during an ex vivo osteoblast infection are a strain-dependent process, we selected two strains belonging to different genetic backgrounds showing a preliminary difference in their ability to internalize as a model of infection to study how differently they adapt their strategies to react to changing environmental conditions and how they adjust their virulence factor expression at different times of infection inside the MG-63 cell line.

The study sample consisted of 2 invasive MRSA isolates, 2SA ST239-SCCmecIII-t036 and 10SA ST228-SCCmecI-t041, already molecularly characterized using internationally recognized standard genotyping methods to determine MRSA clones. These strains were selected from a large collection of 15 MRSA strains phenotypically and molecularly (SCCmec-spa type) characterized as previously reported (Bongiorno et al., 2018; Campanile et al., 2015) and tested for their ability to internalize and persist in MG-63 human osteoblasts (Bongiorno et al., 2020) .

The invasive ATCC12598 isolate (Cowan ST30-t076) (ATCC ® Standards Development Organization, LGC Standards S.r.l.) was used, as previously described, as a control strain for invasion and persistence assays and the statistical analysis of the results obtained at imaging flow cytometry (IFC), as previously reported (Bongiorno et al., 2020) .

The genomic DNA used as a template for PCR amplification was extracted with QIAamp ® DNA Mini Kit (cat. No. 51306, Qiagen) following the manufacturer's instructions with some modifications.

Briefly, a bacterial suspension was centrifuged and the pellet was resuspended in 200 μl of physiological saline solution 0.9% and subjected to freezing and thawing twice. After centrifugation, the bacterial pellet was resuspended in 180 µl of enzyme solution: 20 mg/ ml lysozyme (cat. No. 10837059001, Sigma-Aldrich, Merck KGaA) and 100 μg/ml lysostaphin (cat. No. L7386-15MG, Sigma-Aldrich, Merck KGaA) in Tris-EDTA (TE) buffer, pH 8.0 (cat. No. AM9849, Ambion, Invitrogen). Apart from these differences, the indications provided by the manufacturer were followed.

The toxin and MSCRAMM genes included in the study and listed in Table A1 were tested as previously described (Stefani et al., 2009) . The agr locus was typed using a multiplex PCR assay (Gilot et al., 2002) . The cna gene was PCR-tested using the following primers in 5ʹ-3ʹ: F-GGAAAACGACCAACTGAAATCAAAG, R-TCTGGCGTATATTTATTCGTCACAATC. PCR was performed at 57°C, the product size was 239 bp, and the MW2 strain was used as an internal control.

PCR amplification was carried out in a Veriti Thermal Cycler (Applied Biosystems, Thermo Fisher) in a total volume of 25 µl containing 2× Multiplex PCR Master Mix (cat. No. BR0200804, biotechrabbit GmbH) and 10 ng template DNA.

δ-Hemolysis production was evaluated by cross-streaking perpendicularly our sample to S. aureus RN4220, using 5% sheep blood agar Columbia base with 6 mg/L vancomycin, as previously described. The TA B L E 1 Regulators, MSCRAMMs, toxins, and other genes used to evaluate gene expression in real-time PCR 

Infection experiments were performed on the human osteosarcoma cell line MG-63 (ATCC ® CRL-1427™, Standards Development

Organization, LGC Standards S.r.l.), as previously described (Bongiorno et al., 2020) .

A single 6-well plate was used for the imaging flow cytometry (IFC), a single 6-well plate was used for RNA extraction, and a 96well plate was used for the evaluation of eukaryotic cell metabolism.

All experiments were performed twice in triplicate.

The frequency of internalization was evaluated in a cell culture model of infection in MG-63 osteoblasts at a multiplicity of infection (MOI) of 100:1, as previously reported (Bongiorno et al., 2020) . In this work, we evaluated, by IFC, bacterial internalization at 3 h p.i., and persistence at 24 h p.i, as previously reported (Bongiorno et al., 2020) . Briefly, bacterial isolates were grown in We acquired 10,000 events per sample, and as quality control, we used the stained and unstained bacterial suspension to exclude autofluorescence; a negative control of infected, but not permeabilized, cells was also acquired to guarantee the exclusively intracellular localization of green spots. Acquisition analysis was performed using the powerful INSPIRE ® and IDEAS ® software (Amnis, EMD Millipore).

To evaluate the metabolic status of MG-63 cells following infection with S. aureus for 3 h p.i. and 24 h p.i., we used the MTT ([3-(4,5-di methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]) assay (Fresta et al., 2018; Pedotti et al., 2017) . The formazan crystals obtained at the end of the procedure were dissolved by dimethyl sulfoxide, and the multiplate reader Synergy™ H1 (BioTek) was used to determine the colorimetric differences, detected as absorbance at 569 nm, between samples. The values obtained for the infected cells were expressed as percent variation from the metabolic status detected in not infected (control) cells (as 100%). 

Clone characterization through: ST-sequence type; SCCmec-Staphylococcal Cassette Chromosome mec; spa type-staphylococcal protein A; agr type-locus agr; delta-hemolysis production; fnbA-fibronectin-binding protein A; icaA-intracellular adhesion; sdrE-platelet aggregation; clfA/Bclumping factor A/B; cna-adhesin binding to collagen; luk-PV-Panton-Valentine leukocidin; eta/b-exfoliative toxin A/B; hlb/d/g-hemolysins beta/ delta/gamma; tst-toxic shock syndrome toxin; seA-P-Staphylococcal enterotoxin from A to P; spa-protein A; agr-accessory gene regulated. 

+ + − − − − − − + − + − − − + − − − + + − − − − − − − − − − − − − − − − + + − + − − − − + − + − − − + − + − F I G U R E 1

The statistical analysis and related graphs were obtained using GraphPad Prism 6 (GraphPad Software Inc.).

The statistical significance of cytofluorimetric analyses was assessed using Student's t test, and the number of single-cell events analyzed was never below 9980 cells out of 10,000 events at the outset (Bongiorno et al., 2020) . The statistical significance of the MTT assay was evaluated using ANOVA and Sidak's multiple comparison test (post hoc test). The statistical significance of the expression analysis was assessed using ordinary one-way ANOVA and Bonferroni's multiple comparison test. The significance threshold was set to p-value ≤0.05 (*), p-value ≤0.01 (**), p-value ≤0.001 (***), and p-value ≤0.0001(****).

The molecular features of the strains in the study are reported in Table 2 . Up-regulated. Down-regulated. Up-Regulated But No Significant difference. Down-regulated but no significant difference.

= same expression level as a basal condition.

S. aureus ATCC12598 was associated with agr type III, while ST239-III was associated with agr type I and ST228-I with agr type II.

δ-Hemolysis production was observed in ATCC12598 and ST239-III but not in ST228-I. 

We used imaging flow cytometry (IFC) to evaluate the number of bacteria that internalized and persisted in MG-63 cells. We previously demonstrated the power of IFC in precisely estimating the percentage of osteoblasts infected with S. aureus strains in a sample of 10,000 MG-63 cells. Here, we report the internalization rate, expressed as a percentage of internalization at 3 h p.i. and the persistence rate at 24 h p.i (Figure 1 and Table 3) .

At 3 h p.i., ATCC12598 internalized in 70 ± 17.04% of MG-63 cells, a slightly lower ability to internalize was found for the ST239-III clone (50.24 ± 2.26%), while ST228-I showed the lowest internalization rate, at 29.8 ± 2.31% (p = 0.015). The lower ability to internalize the ST228-I strain was also evident when compared to ST239-III (p = 0.0004) (Figure 1a and Table 3A ). Persistence inside cells was measured at 24 h p.i.: 49 ± 1.96% of the ATCC12598 bacterial cells persisted inside MG-63 cells; the same ability to persist was found in the ST239-III clone (45.2 ± 6.2%), while ST228-I showed a lower rate of persistence, 20.7 ± 1.80% (p < 0.0001). The lower ability to persist of ST228-I was also evident when compared to ST239-III (p = 0.0028) (Figure 1b and Table 3B ).

This technique allowed us to estimate the percentage of MG-63 cells that were internalized (full vs. empty cells) and the number of bacteria that internalized in a sample of 10,000 MG-63 cells; moreover, we were able to establish bacterial persistence at 24 h p.i.

Alterations in the metabolic status of human osteoblasts induced during internalization and persistence were evaluated in the same experimental condition by analyzing the MG-63 cell viability at 3 h p.i. and 24 h p.i. (Table 4) . 

We decided to study the expression of different staphylococcal These findings support the idea that variations in the regulator, MSCRAMM, toxins, and metabolic gene transcription were related to the degree of invasiveness and persistence in MG-63 cells.

F I G U R E 5 Regulation of adhesion and virulence determinants in S. aureus by global regulatory loci. Arrows stand for activation; bars for repression. The molecules that act as activators or repressors (members of SarA protein family, SarA, SarS, and rot), that is, regulating protease expression, are represented in green ovals; the alternate sigma factor (SigB) in blue rectangles; the agr quorum-sensing system (AgrA) in red hexagons; the toxins in pink rumbles (Hla, Hld, SdrE, and PsmA); and the adhesion factors in yellow circles (Bbp and FnaA/B) (Jenul & Horswill, 2018; Romilly et al., 2014) .

Internalization of S. aureus in non-phagocytic cells is an attractive field of study owing to the particular ability of this microorganism to generate chronic diseases. In a previous study, we analyzed the different proclivity of the main staphylococcal clones to invade, internalize, and persist within human cells, using a highly performing IFC assay. In particular, we demonstrated that ST5-II, ST8-IV, and ST228-I showed a statistically significant lower aptitude to intracellular subsistence, while ST239-III and ST22-IVh isolates had intracellular frequencies equal to, or greater than, those of the invasive ATCC12598 strain, suggesting their possible role as invasive and persistent clones responsible for chronic and recurrent infections (Bongiorno et al., 2020) .

Based on the above observations, we used the same infection models to examine the differential expression of a set of genes responsible for the adhesion, invasion, and internalization of two S.

aureus clones ST239-III and ST228-I compared with the control invasive ATCC12598 isolate.

The pathotype gene profile of the two strains included in the study showed only a few differences.

S. aureus ST228-I is one of the most predominant clones associated with orthopedic infection (Jain et al., 2019) showing a high rate of virulence, drug resistance, and longer duration of hospitalization. It exhibited two more enterotoxins (SEA and SEO), suggesting a potentially augmented role in eliciting and deregulating the immune system of the host (Hussain et al., 2009 ).

Conversely, ST239-III was the only strain carrying the hlb gene coding for a pore-forming toxin involved in chronic skin infections and phagosomal escape (Katayama et al., 2013) and producing δ-hemolysis, demonstrating that the agr locus is not defective.

Moreover, this strain, belonging to the ST239 clone, did not carry the other toxin genes; therefore, its ability to damage host cells is related to hla and psmα or other unknown factors.

To further investigate these differences, we evaluated the expression of a selected set of genes grouped into three different general categories, that is, regulators, adhesion factors (MSCRAMMs), and toxins (Table 6, Figure 5) . In particular, psmA and hla overexpression may be related to the 20% decrease in persistence; moreover, the overexpression of α-hemolysin may enhance internalization and survival by modulating osteoblast expression of β1 integrin, as previously reported (Goldmann et al., 2016) , and may also be involved in pore formation and cellular lysis, which might explain the decrease in cellular metabolism associated with enhanced cytotoxic activity. This is in line with previous studies in which hla expression was a requirement for the pathogenesis of the invasive disease (Oliveira et al., 2018) . At 24 h p.i, an over decrease in the expression of regulatory genes was observed, except for sigB, probably due to cellular stimulation, which resulted in a decreased expression of genes involved in adhesion (fnbA and sdrE) and virulence (psmA and hla). We can conclude that, The significantly higher rates of intracellular invasion by ST239-III bacteria could justify their cytotoxicity despite the loss of toxin production and contribute to their survival in a higher number.

These findings suggest that ST239-III is associated with an intracellular adaptation that leads to decreased virulence and host immune escape, making this clone more prone to persistent infections.

During internalization and persistence, we did not find any difference in the expression of the genes involved in adaptation to environmental stress (sarA, sigB, sarS, agr) and of those involved in adherence (fnbA and fnbB) with respect to the baseline condition;

this phenomenon could be probably because these genes were normally overexpressed at the baseline condition, while, under the same conditions, the genes involved in toxicity and phagosomal escape (psmA and hla) were down-regulated. This regulation was in line with rot expression.

On the contrary, the strain belonging to ST228-I was found to be less able to internalize (30%) at 3 h p.i.; correlation analysis between regulatory genes and virulence factors showed that at 3 h p.i., the ST228-I strain displayed a baseline level of agrA expression and an upregulation of sigB and sarA, leading to up-regulation of the surface proteins and up-regulation of secreted toxins, such as PSMα, Hla, and Hld, associated with a significant non-cytotoxic activity inside osteoblasts, and a lower ability to internalize into and infect MG-63 cells. Even if these variations were not statistically significant, their expression levels showed an expected scheme. This strain is also characterized by the presence of the pls gene, carried by SCCmecI, known to be involved in the failure to adhere to the cell surface (Hussain et al., 2009) .

After 24 h of intracellular persistence, all regulatory genes were down-expressed, in particular, rot and consequently the genes in- The impact of PSMα suggests that regardless of the lower invasiveness, ST228 bacteria exert their potential to damage osteoblasts by a cytotoxic effect. ST228 is not capable of activating a sufficient cellular reaction;

quite on the contrary, it seemed to "succumb" inside MG-63 cells. This variation in the expression of agr, sigB, rot, hla, and fnbA was previously associated with the switch from an extracellular to an intracellular behavior, due to changes in the expression of the fibronectin-binding protein and adhesion-binding protein, important for host/cell invasion but not required difficult for intracellular persistence (Tuchscherr & Löffler, 2016) .

Although rot variations were not statistically significant, except for the ST228-I strain at 24 h p.i., they were still in line with the variations found in the toxin genes. Two further considerations are necessary:

The regulation of toxin production was not dependent on rot regulation only, but also on other members of the SarA family, accessory sensor/regulators (RSP), and/or two-component systems (i.e., SaeRS) (Horn et al., 2018) ; and rot was regulated by other genes, which have not been considered in this context, such as rbf (Gimza et al., 2019 

We found that the ST239-III clone was able to infect, at 3 h p.i., 50%

of MG-63 human osteoblasts, and this rate stably persisted at 24 h p.i.; during the infection period, it exerted a highly significant cytotoxic activity against osteoblasts, due to the overexpression of hla, as demonstrated by a remarkable decrease in the cellular metabolic status. The increase in hla and, to a lesser extent, of psmA has as a consequence the increased expression of genes involved in adhesion (bbp), probably due to the release and re-entry of bacteria inside MG-63 cells at 24 h p.i. Our results lead us to conclude that the ST239-III clone is more prone to persistent infections. Recent data from our group considering pro-inflammatory and pro-oxidant response in an ST239-III osteoblast infection demonstrated a significant increase in gene expression of both interleukin-6 and TNFα (Musso et al., 2021) .

On the contrary, ST228-I was found to be less able to internalize (30%), compared with the control strain and ST239-III at 3 h p.i., and to persist (20%) at 24 h p.i., and this lower invasiveness was also correlated with the non-cytotoxic activity inside osteoblasts. This is probably due to the presence of the pls gene in SCCmecI, which is involved in the failure to adhere to the cell surface. This clone is not able to activate a sufficient cellular reaction and succumbs inside MG-63 cells.

Our idea is to consider new strategies, including a clonal and translational approach. We believe that our translational approacheukaryotic host-pathogen interaction (EHPI)-based on the study of the genetic and biochemical basis of both pathogen and host can be applied on a large scale to predict S. aureus /osteoblast interaction and treat bone infections.

None required.

Some of the results of this study were presented at the 29th ECCMID (O0927) and the 44th Italian Society of Microbiology (SIM) congress (P127). We would like to thank the BRIT laboratory at the University of Catania (Italy) for valuable technical assistance and use of their laboratories. We also wish to thank the PharmaTranslated (http://www.pharm atran slated.com/) and in particular to Silvia Montanari for language support. The manu- 

All data generated or analyzed during this study are included in this published article. 

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Additional supporting information may be found online in the Supporting Information section.How to cite this article: Bongiorno D, Musso N, Caruso G, et al