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.