Limits...
Two TIR-like domain containing proteins in a newly emerging zoonotic Staphylococcus aureus strain sequence type 398 are potential virulence factors by impacting on the host innate immune response.

Patterson NJ, Günther J, Gibson AJ, Offord V, Coffey TJ, Splitter G, Monk I, Seyfert HM, Werling D - Front Microbiol (2014)

Bottom Line: This does not, however, explain the apparent ease with which it crosses species-barriers.To assess the role of both proteins as potential virulence factors knock-out mutants were created.Our data suggest that both proteins may act as factors contributing to the enhanced ability of ST398 to cross species-barriers.

View Article: PubMed Central - PubMed

Affiliation: Molecular Immunology Group, Department of Pathology and Pathogen Biology, Royal Veterinary College Hatfield, UK.

ABSTRACT
Staphylococcus aureus, sequence type (ST) 398, is an emerging pathogen and the leading cause of livestock-associated methicillin-resistant S. aureus infections in Europe and North America. This strain is characterized by high promiscuity in terms of host-species and also lacks several traditional S. aureus virulence factors. This does not, however, explain the apparent ease with which it crosses species-barriers. Recently, TIR-domain containing proteins (Tcps) which inhibit the innate immune response were identified in some Gram-negative bacteria. Here we report the presence of two proteins, S. aureus TIR-like Protein 1 (SaTlp1) and S. aureus TIR-like Protein 2 (SaTlp2), expressed by ST398 which contain domain of unknown function 1863 (DUF1863), similar to the Toll/IL-1 receptor (TIR) domain. In contrast to the Tcps in Gram-negative bacteria, our data suggest that SaTlp1 and SaTlp2 increase activation of the transcription factor NF-κB as well as downstream pro-inflammatory cytokines and immune effectors. To assess the role of both proteins as potential virulence factors knock-out mutants were created. These showed a slightly enhanced survival rate in a murine infectious model compared to the wild-type strain at one dose. Our data suggest that both proteins may act as factors contributing to the enhanced ability of ST398 to cross species-barriers.

No MeSH data available.


Related in: MedlinePlus

Impact of SaTlp1 and SaTlp2 on NF-κB activation in two different cell types. Cells were transfected as described, and relative luciferase units were analyzed. Graphs showing the RLU per μg protein in lysates from (A) HEK293-boTLR2 cells stimulated with 100 ng ml-1 FSL-1 and (B) primary bovine mammary epithelial cells (pbMECs) stimulated with 30 μg ml-1 heat-killed E. coli. Cells were transfected with plasmids indicated. Error bars represent the standard error of the mean for each condition (±SEM) of three independent repeats performed in triplicates. Control: medium alone; NFkB alone: cells transfected with NFkB coding plasmid only; invTcpB: cells transfected with a plasmid in which the TcpB sequence was inserted in reverse direct; SaTlp1: cels transfected with plasmid coding for SaTlp1; SaTlp2: cells transfected with plasmid coding for SaTlp2; SaTlp1+2: cells transfected with both plasmids. Significant fold changes are denoted by asterisks in the figure (*p < 0.05; ***p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4260764&req=5

Figure 3: Impact of SaTlp1 and SaTlp2 on NF-κB activation in two different cell types. Cells were transfected as described, and relative luciferase units were analyzed. Graphs showing the RLU per μg protein in lysates from (A) HEK293-boTLR2 cells stimulated with 100 ng ml-1 FSL-1 and (B) primary bovine mammary epithelial cells (pbMECs) stimulated with 30 μg ml-1 heat-killed E. coli. Cells were transfected with plasmids indicated. Error bars represent the standard error of the mean for each condition (±SEM) of three independent repeats performed in triplicates. Control: medium alone; NFkB alone: cells transfected with NFkB coding plasmid only; invTcpB: cells transfected with a plasmid in which the TcpB sequence was inserted in reverse direct; SaTlp1: cels transfected with plasmid coding for SaTlp1; SaTlp2: cells transfected with plasmid coding for SaTlp2; SaTlp1+2: cells transfected with both plasmids. Significant fold changes are denoted by asterisks in the figure (*p < 0.05; ***p < 0.001).

Mentions: To assess whether the presence of the Tcp-like proteins in this S. aureus strain interferes with the activation of the innate immune response, we tested whether the identified proteins would exhibit similar effects as described for Tcps of other bacteria which seem to inhibit TLR signaling. We analyzed whether transfection of HEK cells expressing boTLR2 with SaTlp1 and SaTlp2 affected the downstream activity of the transcription factor NF-κB after stimulation with the TLR2 ligand FSL-1 (Figure 3A). The levels of NF-κB-induced luciferase were significantly up-regulated following transfection with SaTlp1 and SaTlp2 plasmids (both p < 0.001) when compared to the negative control. Simultaneous transfection of both plasmids also up-regulated NF-κB activation (p < 0.001). In the HEK-boTLR2 system these plasmids up-regulated NF-κB-dependent luciferase between 4.1 (SaTlp1) and 6.1 (SaTlp1/2) fold following stimulation compared to the negative control. In contrast, transfection of HEK-boTLR2 cells with TcpB which had been shown before to block TLR-dependent NF-κB activation (Radhakrishnan et al., 2009) did not induce significant NF-κB stimulation, confirming these data. FSL-1 failed to activate NF-κB in HEK293 not expressing TLR2 (Data not shown).


Two TIR-like domain containing proteins in a newly emerging zoonotic Staphylococcus aureus strain sequence type 398 are potential virulence factors by impacting on the host innate immune response.

Patterson NJ, Günther J, Gibson AJ, Offord V, Coffey TJ, Splitter G, Monk I, Seyfert HM, Werling D - Front Microbiol (2014)

Impact of SaTlp1 and SaTlp2 on NF-κB activation in two different cell types. Cells were transfected as described, and relative luciferase units were analyzed. Graphs showing the RLU per μg protein in lysates from (A) HEK293-boTLR2 cells stimulated with 100 ng ml-1 FSL-1 and (B) primary bovine mammary epithelial cells (pbMECs) stimulated with 30 μg ml-1 heat-killed E. coli. Cells were transfected with plasmids indicated. Error bars represent the standard error of the mean for each condition (±SEM) of three independent repeats performed in triplicates. Control: medium alone; NFkB alone: cells transfected with NFkB coding plasmid only; invTcpB: cells transfected with a plasmid in which the TcpB sequence was inserted in reverse direct; SaTlp1: cels transfected with plasmid coding for SaTlp1; SaTlp2: cells transfected with plasmid coding for SaTlp2; SaTlp1+2: cells transfected with both plasmids. Significant fold changes are denoted by asterisks in the figure (*p < 0.05; ***p < 0.001).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4260764&req=5

Figure 3: Impact of SaTlp1 and SaTlp2 on NF-κB activation in two different cell types. Cells were transfected as described, and relative luciferase units were analyzed. Graphs showing the RLU per μg protein in lysates from (A) HEK293-boTLR2 cells stimulated with 100 ng ml-1 FSL-1 and (B) primary bovine mammary epithelial cells (pbMECs) stimulated with 30 μg ml-1 heat-killed E. coli. Cells were transfected with plasmids indicated. Error bars represent the standard error of the mean for each condition (±SEM) of three independent repeats performed in triplicates. Control: medium alone; NFkB alone: cells transfected with NFkB coding plasmid only; invTcpB: cells transfected with a plasmid in which the TcpB sequence was inserted in reverse direct; SaTlp1: cels transfected with plasmid coding for SaTlp1; SaTlp2: cells transfected with plasmid coding for SaTlp2; SaTlp1+2: cells transfected with both plasmids. Significant fold changes are denoted by asterisks in the figure (*p < 0.05; ***p < 0.001).
Mentions: To assess whether the presence of the Tcp-like proteins in this S. aureus strain interferes with the activation of the innate immune response, we tested whether the identified proteins would exhibit similar effects as described for Tcps of other bacteria which seem to inhibit TLR signaling. We analyzed whether transfection of HEK cells expressing boTLR2 with SaTlp1 and SaTlp2 affected the downstream activity of the transcription factor NF-κB after stimulation with the TLR2 ligand FSL-1 (Figure 3A). The levels of NF-κB-induced luciferase were significantly up-regulated following transfection with SaTlp1 and SaTlp2 plasmids (both p < 0.001) when compared to the negative control. Simultaneous transfection of both plasmids also up-regulated NF-κB activation (p < 0.001). In the HEK-boTLR2 system these plasmids up-regulated NF-κB-dependent luciferase between 4.1 (SaTlp1) and 6.1 (SaTlp1/2) fold following stimulation compared to the negative control. In contrast, transfection of HEK-boTLR2 cells with TcpB which had been shown before to block TLR-dependent NF-κB activation (Radhakrishnan et al., 2009) did not induce significant NF-κB stimulation, confirming these data. FSL-1 failed to activate NF-κB in HEK293 not expressing TLR2 (Data not shown).

Bottom Line: This does not, however, explain the apparent ease with which it crosses species-barriers.To assess the role of both proteins as potential virulence factors knock-out mutants were created.Our data suggest that both proteins may act as factors contributing to the enhanced ability of ST398 to cross species-barriers.

View Article: PubMed Central - PubMed

Affiliation: Molecular Immunology Group, Department of Pathology and Pathogen Biology, Royal Veterinary College Hatfield, UK.

ABSTRACT
Staphylococcus aureus, sequence type (ST) 398, is an emerging pathogen and the leading cause of livestock-associated methicillin-resistant S. aureus infections in Europe and North America. This strain is characterized by high promiscuity in terms of host-species and also lacks several traditional S. aureus virulence factors. This does not, however, explain the apparent ease with which it crosses species-barriers. Recently, TIR-domain containing proteins (Tcps) which inhibit the innate immune response were identified in some Gram-negative bacteria. Here we report the presence of two proteins, S. aureus TIR-like Protein 1 (SaTlp1) and S. aureus TIR-like Protein 2 (SaTlp2), expressed by ST398 which contain domain of unknown function 1863 (DUF1863), similar to the Toll/IL-1 receptor (TIR) domain. In contrast to the Tcps in Gram-negative bacteria, our data suggest that SaTlp1 and SaTlp2 increase activation of the transcription factor NF-κB as well as downstream pro-inflammatory cytokines and immune effectors. To assess the role of both proteins as potential virulence factors knock-out mutants were created. These showed a slightly enhanced survival rate in a murine infectious model compared to the wild-type strain at one dose. Our data suggest that both proteins may act as factors contributing to the enhanced ability of ST398 to cross species-barriers.

No MeSH data available.


Related in: MedlinePlus