Limits...
Comparative proteome analysis reveals conserved and specific adaptation patterns of Staphylococcus aureus after internalization by different types of human non-professional phagocytic host cells.

Surmann K, Michalik S, Hildebrandt P, Gierok P, Depke M, Brinkmann L, Bernhardt J, Salazar MG, Sun Z, Shteynberg D, Kusebauch U, Moritz RL, Wollscheid B, Lalk M, Völker U, Schmidt F - Front Microbiol (2014)

Bottom Line: Most of the bacterial adaptation reactions, such as decreased levels of ribosomal proteins and metabolic enzymes or increased amounts of proteins involved in arginine and lysine biosynthesis, enzymes coding for terminal oxidases and stress responsive proteins or activation of the sigma factor SigB were observed after internalization into any of the three cell lines studied.This revealed similar levels of intracellular glucose but host cell specific differences in the amounts of amino acids such as glycine, threonine or glutamate.With this comparative study we provide an impression of the common and specific features of the adaptation of S. aureus HG001 to specific host cell environments as a starting point for follow-up studies with different strain isolates and regulatory mutants.

View Article: PubMed Central - PubMed

Affiliation: Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald Greifswald, Germany.

ABSTRACT
Staphylococcus aureus is a human pathogen that can cause a wide range of diseases. Although formerly regarded as extracellular pathogen, it has been shown that S. aureus can also be internalized by host cells and persist within these cells. In the present study, we comparatively analyzed survival and physiological adaptation of S. aureus HG001 after internalization by two human lung epithelial cell lines (S9 and A549), and human embryonic kidney cells (HEK 293). Combining enrichment of bacteria from host-pathogen assays by cell sorting and quantitation of the pathogen's proteome by mass spectrometry we characterized S. aureus adaptation during the initial phase between 2.5 h and 6.5 h post-infection. Starting with about 2 × 10(6) bacteria, roughly 1450 S. aureus proteins, including virulence factors and metabolic enzymes were identified by spectral comparison and classical database searches. Most of the bacterial adaptation reactions, such as decreased levels of ribosomal proteins and metabolic enzymes or increased amounts of proteins involved in arginine and lysine biosynthesis, enzymes coding for terminal oxidases and stress responsive proteins or activation of the sigma factor SigB were observed after internalization into any of the three cell lines studied. However, differences were noted in central carbon metabolism including regulation of fermentation and threonine degradation. Since these differences coincided with different intracellular growth behavior, complementary profiling of the metabolome of the different non-infected host cell types was performed. This revealed similar levels of intracellular glucose but host cell specific differences in the amounts of amino acids such as glycine, threonine or glutamate. With this comparative study we provide an impression of the common and specific features of the adaptation of S. aureus HG001 to specific host cell environments as a starting point for follow-up studies with different strain isolates and regulatory mutants.

No MeSH data available.


Related in: MedlinePlus

Activation of SigB following internalization. Average values of log2 intensities from three biological replicates each for non-adherent bacteria as well as 2.5 h and 6.5 h p.i. are depicted for six proteins encoded by members of the SigB regulon. Blue spots indicate lower levels in the internalized bacteria compared to the non-adherent control; red colors represent higher levels of proteins in response to internalization compared to the non-adherent control. (A) Asp23, (B) SpoVG, (C) ClfA, (D) SAOUHSC_02665, (E) ClpL, (F) YfkM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4117987&req=5

Figure 7: Activation of SigB following internalization. Average values of log2 intensities from three biological replicates each for non-adherent bacteria as well as 2.5 h and 6.5 h p.i. are depicted for six proteins encoded by members of the SigB regulon. Blue spots indicate lower levels in the internalized bacteria compared to the non-adherent control; red colors represent higher levels of proteins in response to internalization compared to the non-adherent control. (A) Asp23, (B) SpoVG, (C) ClfA, (D) SAOUHSC_02665, (E) ClpL, (F) YfkM.

Mentions: It has recently been shown that the alternative sigma factor SigB which controls many S. aureus genes also with impact on virulence is activated following internalization of S. aureus HG001 by S9 cells (Pförtner et al., 2014). Since reports on the role of the alternative sigma factor SigB in different animal models and cell culture settings differ (Jonsson et al., 2004; Depke et al., 2012), we wanted to assess if the activation described for S9 epithelial cells is also conserved upon internalization by A549 and HEK 293 cells. The data presented in Figure 7 support this notion. Of the six proteins for which Pförtner et al. (2014) have clearly shown SigB-dependent increases in level following internalization of S. aureus HG001 by S9 cells, five [Asp23 (Figure 7A), SpoVG (Figure 7B), CflA (Figure 7C), ClpL (Figure 7D), and SAOUHSC_02665 (Figure 7F)] displayed increases in level following internalization that were maintained for ClpL even 6.5 h after internalization. The increase in protein content was observed following internalization by all three cell lines, even if the particular patterns sometimes differed. The increase in level could not be confirmed for YfkM (Figure 7F).


Comparative proteome analysis reveals conserved and specific adaptation patterns of Staphylococcus aureus after internalization by different types of human non-professional phagocytic host cells.

Surmann K, Michalik S, Hildebrandt P, Gierok P, Depke M, Brinkmann L, Bernhardt J, Salazar MG, Sun Z, Shteynberg D, Kusebauch U, Moritz RL, Wollscheid B, Lalk M, Völker U, Schmidt F - Front Microbiol (2014)

Activation of SigB following internalization. Average values of log2 intensities from three biological replicates each for non-adherent bacteria as well as 2.5 h and 6.5 h p.i. are depicted for six proteins encoded by members of the SigB regulon. Blue spots indicate lower levels in the internalized bacteria compared to the non-adherent control; red colors represent higher levels of proteins in response to internalization compared to the non-adherent control. (A) Asp23, (B) SpoVG, (C) ClfA, (D) SAOUHSC_02665, (E) ClpL, (F) YfkM.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4117987&req=5

Figure 7: Activation of SigB following internalization. Average values of log2 intensities from three biological replicates each for non-adherent bacteria as well as 2.5 h and 6.5 h p.i. are depicted for six proteins encoded by members of the SigB regulon. Blue spots indicate lower levels in the internalized bacteria compared to the non-adherent control; red colors represent higher levels of proteins in response to internalization compared to the non-adherent control. (A) Asp23, (B) SpoVG, (C) ClfA, (D) SAOUHSC_02665, (E) ClpL, (F) YfkM.
Mentions: It has recently been shown that the alternative sigma factor SigB which controls many S. aureus genes also with impact on virulence is activated following internalization of S. aureus HG001 by S9 cells (Pförtner et al., 2014). Since reports on the role of the alternative sigma factor SigB in different animal models and cell culture settings differ (Jonsson et al., 2004; Depke et al., 2012), we wanted to assess if the activation described for S9 epithelial cells is also conserved upon internalization by A549 and HEK 293 cells. The data presented in Figure 7 support this notion. Of the six proteins for which Pförtner et al. (2014) have clearly shown SigB-dependent increases in level following internalization of S. aureus HG001 by S9 cells, five [Asp23 (Figure 7A), SpoVG (Figure 7B), CflA (Figure 7C), ClpL (Figure 7D), and SAOUHSC_02665 (Figure 7F)] displayed increases in level following internalization that were maintained for ClpL even 6.5 h after internalization. The increase in protein content was observed following internalization by all three cell lines, even if the particular patterns sometimes differed. The increase in level could not be confirmed for YfkM (Figure 7F).

Bottom Line: Most of the bacterial adaptation reactions, such as decreased levels of ribosomal proteins and metabolic enzymes or increased amounts of proteins involved in arginine and lysine biosynthesis, enzymes coding for terminal oxidases and stress responsive proteins or activation of the sigma factor SigB were observed after internalization into any of the three cell lines studied.This revealed similar levels of intracellular glucose but host cell specific differences in the amounts of amino acids such as glycine, threonine or glutamate.With this comparative study we provide an impression of the common and specific features of the adaptation of S. aureus HG001 to specific host cell environments as a starting point for follow-up studies with different strain isolates and regulatory mutants.

View Article: PubMed Central - PubMed

Affiliation: Interfaculty Institute for Genetics and Functional Genomics, University Medicine Greifswald Greifswald, Germany.

ABSTRACT
Staphylococcus aureus is a human pathogen that can cause a wide range of diseases. Although formerly regarded as extracellular pathogen, it has been shown that S. aureus can also be internalized by host cells and persist within these cells. In the present study, we comparatively analyzed survival and physiological adaptation of S. aureus HG001 after internalization by two human lung epithelial cell lines (S9 and A549), and human embryonic kidney cells (HEK 293). Combining enrichment of bacteria from host-pathogen assays by cell sorting and quantitation of the pathogen's proteome by mass spectrometry we characterized S. aureus adaptation during the initial phase between 2.5 h and 6.5 h post-infection. Starting with about 2 × 10(6) bacteria, roughly 1450 S. aureus proteins, including virulence factors and metabolic enzymes were identified by spectral comparison and classical database searches. Most of the bacterial adaptation reactions, such as decreased levels of ribosomal proteins and metabolic enzymes or increased amounts of proteins involved in arginine and lysine biosynthesis, enzymes coding for terminal oxidases and stress responsive proteins or activation of the sigma factor SigB were observed after internalization into any of the three cell lines studied. However, differences were noted in central carbon metabolism including regulation of fermentation and threonine degradation. Since these differences coincided with different intracellular growth behavior, complementary profiling of the metabolome of the different non-infected host cell types was performed. This revealed similar levels of intracellular glucose but host cell specific differences in the amounts of amino acids such as glycine, threonine or glutamate. With this comparative study we provide an impression of the common and specific features of the adaptation of S. aureus HG001 to specific host cell environments as a starting point for follow-up studies with different strain isolates and regulatory mutants.

No MeSH data available.


Related in: MedlinePlus