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A bacterial regulatory RNA attenuates virulence, spread and human host cell phagocytosis.

Le Pabic H, Germain-Amiot N, Bordeau V, Felden B - Nucleic Acids Res. (2015)

Bottom Line: A S. aureus sRNA, SprC (srn_3610), reduced virulence and bacterial loads in a mouse infection model.ATL proved to be negatively regulated by SprC.The SprC domains involved in pairing with atl mRNA were analyzed.

View Article: PubMed Central - PubMed

Affiliation: Inserm U835-Upres EA2311, Biochimie Pharmaceutique, Rennes University, 2 av. du prof. Léon Bernard, 35000 Rennes, France.

No MeSH data available.


Related in: MedlinePlus

SprC reduces S. aureus internalization by human macrophages and its absence augments phagocytosis and bacterial release from the host cells. (A) Number of S. aureus cells (CFU) after internalization. Newman, Newman-ΔsprC, Newman-ΔsprC pCN38 and Newman-ΔsprC pCN38ΩsprC cells were incubated with the THP1 macrophages (MOI 1:25) for 2 h. The non-phagocytosed bacteria were killed by culturing in medium containing 50 μg/ml gentamycin for 24 h. The medium was then replaced with fresh media for up to 4 days. (B) After 4 days, colony counts correspond to the bacteria that were released by the human macrophages. The data presented are the mean ± SD of three independent experiments realized in triplicates. The data are considered highly significant for P-values ≤0.01 (**). (C) Monitoring bacterial growth and SprC expression levels, by Northern blots (OD600nm = 2), in S. aureus strains Newman ΔsprC-pCN38 (pink) and Newman ΔsprC-pCN38ΩsprC (blue). The 5S rRNAs are the loading controls. Each blot presented is from identical experiments. (D) Inhibition of S. aureus strain Newman ΔsprC phagocytosis by the THP1 macrophages by adding increasing concentrations of purified ATL protein (right panel) whereas exogenous ATL has limited, if any, effects on S. aureus strain Newman internalization (left panel).
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Figure 5: SprC reduces S. aureus internalization by human macrophages and its absence augments phagocytosis and bacterial release from the host cells. (A) Number of S. aureus cells (CFU) after internalization. Newman, Newman-ΔsprC, Newman-ΔsprC pCN38 and Newman-ΔsprC pCN38ΩsprC cells were incubated with the THP1 macrophages (MOI 1:25) for 2 h. The non-phagocytosed bacteria were killed by culturing in medium containing 50 μg/ml gentamycin for 24 h. The medium was then replaced with fresh media for up to 4 days. (B) After 4 days, colony counts correspond to the bacteria that were released by the human macrophages. The data presented are the mean ± SD of three independent experiments realized in triplicates. The data are considered highly significant for P-values ≤0.01 (**). (C) Monitoring bacterial growth and SprC expression levels, by Northern blots (OD600nm = 2), in S. aureus strains Newman ΔsprC-pCN38 (pink) and Newman ΔsprC-pCN38ΩsprC (blue). The 5S rRNAs are the loading controls. Each blot presented is from identical experiments. (D) Inhibition of S. aureus strain Newman ΔsprC phagocytosis by the THP1 macrophages by adding increasing concentrations of purified ATL protein (right panel) whereas exogenous ATL has limited, if any, effects on S. aureus strain Newman internalization (left panel).

Mentions: The influence of SprC on S. aureus phagocytosis was also evaluated in human macrophages, whose primary roles are to ingest foreign particles and infectious microorganisms. Phagocytosis of S. aureus strain Newman-ΔsprC by PMA-induced THP1 macrophages was compared to an isogenic Newman strain. Host cell internalization of strain Newman-ΔsprC was significantly enhanced compared to strain Newman (Figure 5A). A trans-complemented Newman ΔsprC-pCN38ΩsprC strain was constructed. Northern blots confirmed the re-expression of SprC in strain Newman ΔsprC-pCN38ΩsprC, and its growth was identical to that of the strain Newman-ΔsprC-pCN38 (Figure 5C). Conversely, the phagocytosis of a trans-complemented strain (Newman ΔsprC-pCN38ΩsprC) was decreased compared to a Newman ΔsprC-pCN38 strain. Interestingly, bacterial release from the THP1 macrophages, after 4 days, was significantly increased in the absence of the sRNA, and reduced in the trans-complemented strains (Figure 5B). Therefore, these observations suggest that the bacteria lacking SprC could possess higher virulence by a higher uptake and release from the immune host cells.


A bacterial regulatory RNA attenuates virulence, spread and human host cell phagocytosis.

Le Pabic H, Germain-Amiot N, Bordeau V, Felden B - Nucleic Acids Res. (2015)

SprC reduces S. aureus internalization by human macrophages and its absence augments phagocytosis and bacterial release from the host cells. (A) Number of S. aureus cells (CFU) after internalization. Newman, Newman-ΔsprC, Newman-ΔsprC pCN38 and Newman-ΔsprC pCN38ΩsprC cells were incubated with the THP1 macrophages (MOI 1:25) for 2 h. The non-phagocytosed bacteria were killed by culturing in medium containing 50 μg/ml gentamycin for 24 h. The medium was then replaced with fresh media for up to 4 days. (B) After 4 days, colony counts correspond to the bacteria that were released by the human macrophages. The data presented are the mean ± SD of three independent experiments realized in triplicates. The data are considered highly significant for P-values ≤0.01 (**). (C) Monitoring bacterial growth and SprC expression levels, by Northern blots (OD600nm = 2), in S. aureus strains Newman ΔsprC-pCN38 (pink) and Newman ΔsprC-pCN38ΩsprC (blue). The 5S rRNAs are the loading controls. Each blot presented is from identical experiments. (D) Inhibition of S. aureus strain Newman ΔsprC phagocytosis by the THP1 macrophages by adding increasing concentrations of purified ATL protein (right panel) whereas exogenous ATL has limited, if any, effects on S. aureus strain Newman internalization (left panel).
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Figure 5: SprC reduces S. aureus internalization by human macrophages and its absence augments phagocytosis and bacterial release from the host cells. (A) Number of S. aureus cells (CFU) after internalization. Newman, Newman-ΔsprC, Newman-ΔsprC pCN38 and Newman-ΔsprC pCN38ΩsprC cells were incubated with the THP1 macrophages (MOI 1:25) for 2 h. The non-phagocytosed bacteria were killed by culturing in medium containing 50 μg/ml gentamycin for 24 h. The medium was then replaced with fresh media for up to 4 days. (B) After 4 days, colony counts correspond to the bacteria that were released by the human macrophages. The data presented are the mean ± SD of three independent experiments realized in triplicates. The data are considered highly significant for P-values ≤0.01 (**). (C) Monitoring bacterial growth and SprC expression levels, by Northern blots (OD600nm = 2), in S. aureus strains Newman ΔsprC-pCN38 (pink) and Newman ΔsprC-pCN38ΩsprC (blue). The 5S rRNAs are the loading controls. Each blot presented is from identical experiments. (D) Inhibition of S. aureus strain Newman ΔsprC phagocytosis by the THP1 macrophages by adding increasing concentrations of purified ATL protein (right panel) whereas exogenous ATL has limited, if any, effects on S. aureus strain Newman internalization (left panel).
Mentions: The influence of SprC on S. aureus phagocytosis was also evaluated in human macrophages, whose primary roles are to ingest foreign particles and infectious microorganisms. Phagocytosis of S. aureus strain Newman-ΔsprC by PMA-induced THP1 macrophages was compared to an isogenic Newman strain. Host cell internalization of strain Newman-ΔsprC was significantly enhanced compared to strain Newman (Figure 5A). A trans-complemented Newman ΔsprC-pCN38ΩsprC strain was constructed. Northern blots confirmed the re-expression of SprC in strain Newman ΔsprC-pCN38ΩsprC, and its growth was identical to that of the strain Newman-ΔsprC-pCN38 (Figure 5C). Conversely, the phagocytosis of a trans-complemented strain (Newman ΔsprC-pCN38ΩsprC) was decreased compared to a Newman ΔsprC-pCN38 strain. Interestingly, bacterial release from the THP1 macrophages, after 4 days, was significantly increased in the absence of the sRNA, and reduced in the trans-complemented strains (Figure 5B). Therefore, these observations suggest that the bacteria lacking SprC could possess higher virulence by a higher uptake and release from the immune host cells.

Bottom Line: A S. aureus sRNA, SprC (srn_3610), reduced virulence and bacterial loads in a mouse infection model.ATL proved to be negatively regulated by SprC.The SprC domains involved in pairing with atl mRNA were analyzed.

View Article: PubMed Central - PubMed

Affiliation: Inserm U835-Upres EA2311, Biochimie Pharmaceutique, Rennes University, 2 av. du prof. Léon Bernard, 35000 Rennes, France.

No MeSH data available.


Related in: MedlinePlus