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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

Protein groups displaying increased levels after internalization by all three cell lines. 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. 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,B) Proteins involved in synthesis of arginine and lysine, (C) terminal oxidases, (D) flavohaemoglobins, and (E) methionine sulfoxide reductase, (F) bacitracin stress response, (G) colicin V and bacteriocin production, (H) choline and betaine uptake, and (I) the ESAT-6 secretion system.
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Figure 6: Protein groups displaying increased levels after internalization by all three cell lines. 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. 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,B) Proteins involved in synthesis of arginine and lysine, (C) terminal oxidases, (D) flavohaemoglobins, and (E) methionine sulfoxide reductase, (F) bacitracin stress response, (G) colicin V and bacteriocin production, (H) choline and betaine uptake, and (I) the ESAT-6 secretion system.

Mentions: Because S. aureus also needs to adapt its protein inventory to the special conditions of the intracellular environment, we looked for pathways commonly displaying increased protein levels after entering the different cell lines, compared to non-adherent control bacteria exposed to the same medium and thus nutrient supply conditions. Some amino acid biosynthesis pathways, such as arginine (5 of 14, Figure 6A) and lysine (9 of 11, Figure 6B) biosynthesis, displayed increased protein levels for all cell lines which might be an adaptation to lower amino acids levels inside host cells vs. the cell culture medium. Having the metabolome data at hand, we wanted to make an effort to validate this hypothesis. For A549 cells we could directly compare the intracellular and extracellular concentrations because the cell volume of these cells has previously been reported (Jiang et al., 2010). However, intracellular lysine levels of uninfected host cells were similar to those in the supernatant, and arginine levels could not be measured because of technical reasons. Metabolite levels might differ in infected host cells, but since only a small proportion of host cells indeed carried S. aureus we could not assess metabolite levels in this sub-group specifically. Striking differences between the extracellular and host cell concentrations were observed for glycine (intracellular 2.3 mmol/L, extracellular 0.1 mmol/L), threonine (intracellular 4.7 mmol/L, extracellular 0.9 mmol/L), and glutamate (intracellular 16.0 mmol/L, extracellular 0.5 mmol/L) (see Supplementary Material Figure 1).


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)

Protein groups displaying increased levels after internalization by all three cell lines. 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. 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,B) Proteins involved in synthesis of arginine and lysine, (C) terminal oxidases, (D) flavohaemoglobins, and (E) methionine sulfoxide reductase, (F) bacitracin stress response, (G) colicin V and bacteriocin production, (H) choline and betaine uptake, and (I) the ESAT-6 secretion system.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Protein groups displaying increased levels after internalization by all three cell lines. 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. 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,B) Proteins involved in synthesis of arginine and lysine, (C) terminal oxidases, (D) flavohaemoglobins, and (E) methionine sulfoxide reductase, (F) bacitracin stress response, (G) colicin V and bacteriocin production, (H) choline and betaine uptake, and (I) the ESAT-6 secretion system.
Mentions: Because S. aureus also needs to adapt its protein inventory to the special conditions of the intracellular environment, we looked for pathways commonly displaying increased protein levels after entering the different cell lines, compared to non-adherent control bacteria exposed to the same medium and thus nutrient supply conditions. Some amino acid biosynthesis pathways, such as arginine (5 of 14, Figure 6A) and lysine (9 of 11, Figure 6B) biosynthesis, displayed increased protein levels for all cell lines which might be an adaptation to lower amino acids levels inside host cells vs. the cell culture medium. Having the metabolome data at hand, we wanted to make an effort to validate this hypothesis. For A549 cells we could directly compare the intracellular and extracellular concentrations because the cell volume of these cells has previously been reported (Jiang et al., 2010). However, intracellular lysine levels of uninfected host cells were similar to those in the supernatant, and arginine levels could not be measured because of technical reasons. Metabolite levels might differ in infected host cells, but since only a small proportion of host cells indeed carried S. aureus we could not assess metabolite levels in this sub-group specifically. Striking differences between the extracellular and host cell concentrations were observed for glycine (intracellular 2.3 mmol/L, extracellular 0.1 mmol/L), threonine (intracellular 4.7 mmol/L, extracellular 0.9 mmol/L), and glutamate (intracellular 16.0 mmol/L, extracellular 0.5 mmol/L) (see Supplementary Material Figure 1).

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