Recent efforts in the field of high-throughput sequencing contributed to the public release of numerous bacterial genomes of medical importance. This information allowed the development of microarrays able to monitor gene expression changes at the genome scale. We report here the utilization of a microarray covering the whole genome of Staphylococcus aureus to evaluate the behavior of the bacterium upon cellular internalization, a phenomenon potentially contributing to bacterial protection against host defenses, and important in the context of chronic infections. Our overall findings are in agreement with previous studies utilizing experimental cellular models of bacterial internalization, using either intracellular or facultative intracellular pathogens. If the ability of S. aureus to persist in different in vitro models is widely accepted, the capacity of S. aureus to escape from host-defenses and persist intracellularly in vivo is still debated . In this respect, the call to document "the presence of staphylococci within non-professional phagocytes at sites of infection in clinicopathological tissues"  has been recently demonstrated by Clément and colleagues, who identified S. aureus residing in human nasal epithelial cells in vivo, a probable cause of recurrent rhinosinusitis infection . This in vivo survival capacity was further documented in the same population and shown to constitute a significant risk factor for recurrent episodes of rhinosinusitis refractory to antimicrobial and surgical therapy . Similar behavior was also observed in an experimental model of mastitis .
In our model, the presence of cells or the attachment of bacteria to A549 cells (in the presence of cytochalasin) showed limited impact on bacterial transcriptome. This observation is in accordance with previous reports from our group showing that no active bacterial process is require for internalization . With respect to bacterial pathogenesis, this observation suggests that S. aureus internalization is probably not rare as it requires only a minimal number of bacterial factors . In contrast to other microorganisms known to invade eukaryotic cells and rapidly divert cellular compounds and energy to actively multiply , our study indicates that upon cellular internalization, S. aureus transiently shuts down most of its metabolic functions and adopts an apparent "dormant state" without extensively initiating classic starvation or stress process. These profound changes in the bacterial transcriptome affect approximately 40% of all putative ORFs identified in the genomic sequence of the bacterium and appear related to an extensive adaptation to the new environment constituted by the intracellular compartment. The majority of genes involved in energy production (citrate and ATP cycles, pyruvate metabolism, and oxidative phosphorylation) were drastically reduced after 2 h internalization. Consequently, > 75% of S. aureus ribosomal proteins showed a significant down-regulation, which constitutes a common trait with streptococcal exposition to endothelial cell , another Gram positive pathogen able to invade non-phagocytic cells before inducing apoptosis . For these 2 pathogens, metabolic and cellular division machineries appear strongly down-regulated rapidly after internalization . This situation is markedly different from that observed in invasive intracellular pathogens such as Salmonella , Shigella [66, 70], or Pasteurella , which show up-regulation of most cellular processes upon internalization and are able to multiply actively before disseminating to other cells [69, 70]. In our model, a significant increase in colony forming unit counts was observed between 2 and 6 h of intracellular persistence. This cellular multiplication parallels up-regulation of lytM, as well as that of numerous amidases involved in cell-wall processing . Concurrently, a slight up-regulation of cell-wall and techoic acid synthesis genes was observed between these time points, suggesting evidence of intracellular replication or completion of cell division, as observed in the context of S. aureus phagocytosis by neutrophils . This metabolic evolution was paralleled by a massive up-regulation of numerous transporters. In our study, more than 40 transporters, involved in inorganic ions, nucleotides or peptides, or urea were found significantly up-regulated. Transporters involved in metabolic pathways contribute to the direct rerouting of cellular resources by the internalized bacterium. Illustrative examples are the up-regulation of uhpT (observed at 2 and 6 h internalization), allowing glucose-6-phosphate acquisition, as observed during internalization of other bacterial species [66, 70] or during in vitro  or intracellular exposition of S. aureus to acidic environments . S. aureus genome contains 7 putative iron transporters  that are important mediators of bacterial virulence. Iron transporters are typically up-regulated in various internalized invasive pathogens [67, 74, 75]. In our study, isdF, SA0567 and particularly bitC, were drastically up-regulated in S. aureus, thus providing probably sufficient amounts of iron from the intracellular medium, whereas all genes composing the fhu operon, encoding ferric hydroxamate assimilation proteins were down-regulated, in accordance with their function of iron transporters under specific environmental conditions .
Intracellular survival can potentially expose the bacterium to cellular defense mechanisms such as oxygen radicals. In our study, the number of up-regulated genes involved in stress protection or bacterial defense revealed surprisingly low. Except for sodA and cysM, whose products protect bacterial cell against oxidative stress , a majority of genes involved in oxidative stress response appeared down-regulated. This finding is markedly different than the behavior of Shigella , but consistent with previous reports about S. pneumoniae . Note however that experiments performed in A549 cells failed to detect production of reactive oxygen species upon bacterial uptake (not shown). Internalized S. aureus appeared to solicit protective functions but in a different context than that requiring preservation of the cellular machinery integrity as described by Voyish in the context of resistance to neutrophil phagocytosis . In non-phagocytic cells, the expression of capsule operon, Clp proteases family and chaperones, as well as their known regulators ctsR and sigB , potentially conferring survival against toxic cellular metabolites was drastically down-regulated.
The expression of numerous bacterial virulence factors is modulated through complex networks of regulatory molecules including two-component systems and global regulators such as agr or sar. Multiple regulatory pathways contributed not only to the rapid adaptation of the bacteria to environmental changes but also to the regulation of toxins or cell-surface components expression during bacterial growth-phases [79, 80]. In our system, upon contact with cell monolayers, strain 6850 was in early exponential phase of growth where agr activity is limited. Following internalization and metabolic restart, general metabolic functions appeared up-regulated in the absence of agr induction. In addition, internalized bacteria showed an increase in expression of sarS and svrA simultaneously to a marked down-regulation of saeRS genes, which are important compounds known to modulate agr expression [35, 63]. In parallel to a moderately increased expression of RNAIII, a compound known to induce agr expression in vitro, this regulation pattern results in the reduced expression of hla that potentially mediates apoptosis induction in some systems [25, 27, 30]. This uncoupling between agr and RNAIII expression has been previously observed in vivo in an experimental model of endocarditis . The restricted level of hla expression suggests a different regulation behavior under these experimental conditions or the action of another unknown regulator. The latter hypothesis is further supported by the results of Goerke , who showed that mutation in agr and sar had no consequence for hla expression in vivo. This unknown target is probably not sigB, a regulator found drastically down-regulated upon internalization as well as the totality of related rsb partners in our model. SigB has been recently suggested as a potential partner involved in the persistence of SCVs recovered from cystic fibrosis . In our study, we failed to detect such SCVs as S. aureus recovered after 2 weeks of internalization yielded significant hemolysis when plated on blood agar. However, this observation does not rule out the emergence of SCVs in our model as this phenotype appears able to revert spontaneously . These observations are directly related to the absence of cytotoxicity on A549 cells even after two weeks of infection, an observation common between our study and previous work using SCVs .
In contrast to the behavior of internalized SCVs, Bantel and colleagues reported 6850-evoked induction of apoptosis using Jurkat T cells , which is consistent with our control assays confirming that strain 6850 was able of provoking cytotoxic effects. In addition, Qazi and colleagues reported the induction of agr and bacterial escape from endosomal vesicles rapidly after internalization using S. aureus RN6390 and MAC-T . On the contrary, Kahl reported that i) rifampicin-treated bacteria failed to induce apoptosis and that ii) Cowan I -a strain that failed to multiply in epithelial cells- does not show appreciable cytotoxic effects . These observations are important concerning S. aureus pathogenicity and suggest that certain combinations of host cells and bacteria can result in different biological outcomes. Altogether, results obtained with different models strongly suggest that the internalization of S. aureus is a key factor in chronic infections, as intracellular survival occurs in cells showing a long lifespan such as fibroblasts or epithelial cells. S. aureus intracellular survival appears related to its capability to adopt a discrete behavior instead of actively duplicating. S. aureus then benefits from natural or programmed cell death to re-emerge and trigger another episode of infection, leading to chronicity.