Discussion
Internalization of S. aureus in non-phagocytic cells is an attractive field of study relying on the particular ability of the microorganism to generate chronic diseases.
The process of internalization includes different phases: in the early phase, microbial surface proteins (MSCRAMMs) are implicated in host-cell adhesion and invasion, fighting against the immune system and entering the deep tissue structure (Painter et al., 2014); subsequently, the pathogenicity and virulence of S. aureus is entrusted to the capacity to produce several virulence factors including enterotoxins (SEs) and toxic shock syndrome toxin-1 (TSST), pore-forming toxins (hemolysins; Panton Valentine Leukocidin; phenol-soluble modulins), exfoliative toxins (ETs), protein A, and several enzymes able to damage host cells by virtue of their cytotoxic/cytolytic activity and by modulating immune responses.
In a previous study, we analysed, by a highly performing IFC assay, the differential proclivity of the main staphylococcal clones to invade, internalize, and persist within human cells. 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, Musso et al ., 2020).
Based on the above observations, by using the same model of infections, we examined the differential expression of a set of genes responsible for the adhesion, invasion and internalization of two S. aureusclones (ST228-I and ST239-III) compared with the control ATCC12598 invasive isolate.
The pathotype gene profile of the two strains included in the study showed only few differences.
S. aureus ST228 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 hospital stay. It exhibited two more enterotoxins (SEA and SEO), suggesting a potential augmented role in eliciting and deregulating the immune system of the host (Hussain et al., 2009).
Conversely, ST239 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.
To further investigate these differences, we evaluated the expression of a selected set of genes grouped into three different general categories, i.e. regulators, adhesion factors (the MSCRAMMs), and toxins (table 5). Moreover, to evaluate the metabolic activity of internalized bacteria, we evaluated a hexose phosphate antiporter for G6P. The expression of the regulator global network SigB, SarA, SarS, agr locus and Rot controls the expression of a set of virulence factors involved in adhesion and cytotoxic/cytolytic activities.
Genes responsible for invasiveness and persistence were analyzed comparing the ability to infect and internalize into MG-63 osteoblasts, according to their cytotoxic potential (by means of cellular metabolic status of infected MG-63 osteoblasts) and the expression levels of surface and secreted toxins, at 3h and 24h p.i.
As was preliminary observed and now confirmed (Bongiorno, Musso et al ., 2020), the ability of MSSA ATCC12598-ST30 and MRSA ST239-III to internalized and persist in MG-63 is very similar, while MRSA ST228-I behaves differently (20.7±1.80%). These differences among clones were evaluated also by measuring the MG-63 metabolic status in terms of cytotoxicity, demonstrating that ST239 was immediately able to decrease the cell metabolic activity at 3h p.i. maintaining it at 24h p.i. The ATCC12598 clone was able to interfere with the cells at 24h p.i., while ST228 only slightly affected the metabolic status of the cells.
These results are in agreement with other studies (Botelho et al ., 2019), demonstrating that the ST239 clone is able to adapt to diverse pathological conditions (diverse syndromes), undergoes genomic adaptations, developing a distinct pattern of virulence-associated genes.
Strain ATCC12598 was included as an invasive model. It displayed, at 3h p.i., a higher ability to infect and internalize into MG-63 human osteoblasts (70%), associated with a significative increment of the cellular metabolic status compared to the uninfected cell-line. At 24h p.i., despite the fact that intracellular persistence decreased to 50%, ATCC12598 bacteria exerted a high intracellular cytotoxic activity against osteoblasts, as demonstrated by a decrease in the cellular metabolic status. The regulation of gene expression in ATCC12598, during internalization and, especially, during persistence steps, followed an expected scheme: a significant increase of the expression of the genes involved in the regulation to environmental stress (in particularsar A, sig B), resulting in the up-regulation of the expression of the genes involved both in adherence (in particular tofnb A and sdr E) and virulence (psm A and hla ) at 3h p.i, although rot , the toxin repressor gene, did not undergo statistically significant changes, this is still in line with the expression of toxins.
In particular, psm A and hla over-expression could be related to the 20% decrease in persistence; moreover, the over-expression of α-hemolysin could enhance internalization and survival by modulating osteoblast expression of β1 integrin, as previously reported (Goldmann et al ., 2016), and also could be involved in pore formation and cellular lysis, which could explain the cellular metabolism decrease associated with enhanced cytotoxic activity. This is in line with previous studies in which Hla expression was required for the pathogenesis of invasive disease (Oliveira et al., 2018). At 24h p.i there was an over-decrease in the expression of regulatory genes, with the exception of sig B, probably due to cellular stimulation, which results in a decrease of the expression of genes involved in adhesion (fnb A and sdr E) and virulence (psm A and hla ). We can conclude that for the ATCC12598 strain, all the events involving both adhesion/invasion and damage of host cells, occurred within 3h p.i. Furthermore, its metabolism, adapted to intracellular conditions, showed an up-regulation of the alternative antiporter of carbon source (UhpT) at 3h p.i., confirmed by the 12% increase in the cellular metabolic status; while there was a significant decrease in metabolism at 24h p.i. vs 3h, confirmed by the loss of -7.11% of the cell metabolic status. The down-regulation ofhld gene expression in ATCC12598 leads us to conclude that it was not involved in cellular adaptation or invasion.
The strain belonging to ST239-III, which did not show any toxin gene (Botelho et al ., 2019) except for sdr E presence, was able to infect (3h p.i.) 50% of MG-63 human osteoblasts, and induced a cellular metabolic slowdown activity, that could be due to the production of hemolysins Hlb, Hla, and PsmA. The ST239-III strain displayed, at 3h p.i., a significant increase of bbp that is necessary for adhesion and invasion in MG-63 cells, and the increase ofagr A expression, resulting in an up-regulation of hla andpsm A, associated with a good ability to internalize and infect MG-63 cells. At 24h p.i., ST239-III bacteria stably persisted intracellularly (45%) and exerted an extremely significative cytotoxic activity against osteoblasts, due to the over-expression of hla,as demonstrated by a remarkable decrease in the cellular metabolic status, also confirmed by the expression of the uhp T gene. Some studies have demonstrated the importance of this toxin for S. aureus pathogenicity, phagosomal escape and induction of biofilm formation (Katayama et al., 2013 ; Tajima et al., 2009). At 24h p.i there was an increased expression of the genes involved in adhesion. A possible explanation could be that the bacterial cells exit and enter MG-63 through the membrane, using the integrins located in the cell wall.
The significatively higher rates of intracellular invasion of ST239 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 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 differences in the expression of the genes involved in the regulation to environmental stress (sar A, sig B, sarS,agr ) and in those involved in adherence (fnb A and fnb B) with respect to the basal condition; this phenomenon could be probably due to the fact that these genes are normally over-expressed at the basal condition; while in the same condition, genes involved in toxicity and phagosomal escape (psm A and hla ) were down-regulated.
On the contrary, the strain belonging to ST228-I was found to be less able to internalize (30%) after 3h p.i.; correlation analysis between regulatory genes and virulence factors showed that the ST228-I strain displayed at 3h p.i. a basal level of agr A expression and an up-regulation of sig B and sar A, leading to an up-regulation of the surface-proteins and up-regulation of secreted toxins, such as PSMα, Hla and Hld, associated with a significative non-cytotoxic activity inside osteoblasts, and a lower ability to internalize 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 thepls gene, carried by SCCmec I, known to be involved in the failure to adhere to the cell surface (Hussain et al. , 2009).
After 24h of intracellular persistence, all the regulatory genes were down-expressed, in particular rot and consequently the genes involved in adhesion and virulence (except for an increased expression of hla at 3h p.i.): 20% of MG-63 cells were infected and the cellular metabolic status did not show any variation with respect to the uninfected cell-line; the expression of uhp T supported these results.
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, rather it seemed to “succumb” inside the MG-63 cells. This variation in the expression of agr , sig B,hla and fnb A was previously associated with the passage from extracellular to intracellular behavior, due to changes in the expression of the fibronectin-binding protein and adhesion binding protein, important for host/cell invasion but difficult for intracellular persistence (Tuchscherr and Löffler, 2016).
Our results, arising from the qualitative and quantitative assays of virulence and toxin factors, are in line with recently published studies in which the genetic and phenotypic different characteristics of staphylococcal strains favor the infection process, invasiveness and persistence inside host cells (Davis and Isberg., 2018; Recker et al., 2017; Tuchscherr et al. , 2019). In our study, we analyzed two different genetic backgrounds to elucidate how bacteria can differentially adapt strategies with the interplay among regulatory and adhesion/toxin genes, and rapidly react to changing environmental conditions and dynamically adjust their virulence factor expression at different times of infection. Recent data from our group considering pro-inflammatory and pro-oxidant response in an ST239-III osteoblast infection, demonstrated a significant increase of gene expression of both interleukin-6 and TNF-α (paper submitted). This ability was identified in this clone and not in others. The idea to consider new strategies, including the clonal approach, to treat S. aureusbone infection is being studied.