A global view of Staphylococcus aureus whole genome expression upon internalization in human epithelial cells

Abstract

Background: Staphylococcus aureus, a leading cause of chronic or acute infections, is traditionally considered an extracellular pathogen despite repeated reports of S. aureus internalization by a variety of non-myeloid cells in vitro. This property potentially contributes to bacterial persistence, protection from antibiotics and evasion of immune defenses. Mechanisms contributing to internalization have been partly elucidated, but bacterial processes triggered intracellularly are largely unknown.

Results: We have developed an in vitro model using human lung epithelial cells that shows intracellular bacterial persistence for up to 2 weeks. Using an original approach we successfully collected and amplified low amounts of bacterial RNA recovered from infected eukaryotic cells. Transcriptomic analysis using an oligoarray covering the whole S. aureus genome was performed at two post-internalization times and compared to gene expression of non-internalized bacteria. No signs of cellular death were observed after prolonged internalization of Staphylococcus aureus 6850 in epithelial cells. Following internalization, extensive alterations of bacterial gene expression were observed. Whereas major metabolic pathways including cell division, nutrient transport and regulatory processes were drastically down-regulated, numerous genes involved in iron scavenging and virulence were up-regulated. This initial adaptation was followed by a transcriptional increase in several metabolic functions. However, expression of several toxin genes known to affect host cell integrity appeared strictly limited.

Conclusion: These molecular insights correlated with phenotypic observations and demonstrated that S. aureus modulates gene expression at early times post infection to promote survival. Staphylococcus aureus appears adapted to intracellular survival in non-phagocytic cells.

Figure 1

Internalization of S. aureus 6850 by A549 cells. A. Dose-response of internalization assay performed by adding increasing number of bacteria to constant number of cells. The internalization assay was performed as described in the experimental section. After 2 h, cells were detached with trypsin lyzed with dilute Triton X-100, and then internalized bacteria were assessed by count of colony forming units (cfu). B. Intracellular survival assay of S. aureus over a two-week interval. Note that at times after 6 hours, viable bacterial counts decreased, though viable bacteria were recovered from monolayers even 2 weeks after internalization. All values are mean ± standard deviation (SD) of at least three independent experiments. C. Viability tests were performed after 24 hours of internalization using bacterial m.o.i. of 100:1, or the presence of anti-CD95 (1 μg/ml) or etoposide (100 μM final concentration) as pro-apoptotic controls.

Figure 2

Schematic representation of conditions subjected to microarray analysis and number of differentially expressed genes. A. Similar expression profiles were observed for the comparisons between the reference condition and the control conditions consisting in i) mock infection (bacteria in cell culture medium) or ii) adherent bacteria (in the presence of cytochalasin D 1 μg/ml). On the opposite, an important number of genes were found differentially regulated between reference condition and internalized bacteria after 2 or 6 h of intracellular survival. See material and methods section for details. B. Numbers of genes found differentially regulated are indicated for each condition. The proportion of non-characterized genes annotated as genes with "unknown function" appears dark grey.

Figure 3

Changes in gene expression 2 h and 6 h after internalization shown by functional categories. Percent changes at 2 h (white) and 6 h (grey) was calculated by dividing the number of significantly changed genes by total genes at the given time point. Genes were assigned functional groups using annotated public database and metabolic pathways databases (COG). Categories are: E: Amino acid transport and metabolism, G: Carbohydrate transport and metabolism, D: Cell cycle control, cell division, chromosome partitioning, M: Cell wall/membrane/envelope biogenesis, H: Coenzyme transport and metabolism, V: Defense mechanisms, C: Energy production and conversion, R: General function prediction only, P: Inorganic ion transport and metabolism, U: Intracellular trafficking, secretion, and vesicular transport I: Lipid transport and metabolism, F: Nucleotide transport and metabolism, O: Posttranslational modification, protein turnover, chaperones, L: Replication, recombination and repair, Q: Secondary metabolites biosynthesis, transport and catabolism, T: Signal transduction mechanisms, K: Transcription, J: Translation, ribosomal structure and biogenesis, and VF for virulence factor.

Figure 4

Global pattern of expression. Patterns of gene expression changes are shown for each of nine possible categories. The most abundant categories (B) were analyzed in depth after grouping by gene function. Numbers of genes in these two categories are also shown.

Figure 5

Quantitative PCR validation of microarray data. Dynamic of gene expression changes is shown as fold change by real-time PCR (black bars), or by microarray determination (white bars), at 2 h (A) or 6 h (B) post-infection. Data are mean ± SD of triplicate measurements from 2 independent experiments.