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Deciphering interplay between Salmonella invasion effectors.

Cain RJ, Hayward RD, Koronakis V - PLoS Pathog. (2008)

Bottom Line: Salmonellae deploy effectors that trigger localized actin reorganization to force their own entry into non-phagocytic host cells.Here we describe trans and cisbinary entry effector interplay (BENEFIT) screens that systematically examine functional associations between effectors following their delivery into the host cell.The results reveal extensive ordered synergistic and antagonistic relationships and their relative potency, and illuminate an unexpectedly sophisticated signaling network evolved through longstanding pathogen-host interaction.

View Article: PubMed Central - PubMed

Affiliation: University of Cambridge, Department of Pathology, Cambridge, United Kingdom.

ABSTRACT
Bacterial pathogens have evolved a specialized type III secretion system (T3SS) to translocate virulence effector proteins directly into eukaryotic target cells. Salmonellae deploy effectors that trigger localized actin reorganization to force their own entry into non-phagocytic host cells. Six effectors (SipC, SipA, SopE/2, SopB, SptP) can individually manipulate actin dynamics at the plasma membrane, which acts as a 'signaling hub' during Salmonella invasion. The extent of crosstalk between these spatially coincident effectors remains unknown. Here we describe trans and cisbinary entry effector interplay (BENEFIT) screens that systematically examine functional associations between effectors following their delivery into the host cell. The results reveal extensive ordered synergistic and antagonistic relationships and their relative potency, and illuminate an unexpectedly sophisticated signaling network evolved through longstanding pathogen-host interaction.

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Related in: MedlinePlus

Sip-Sop synergy revealed by cis BENEFIT screening.A. Schematic illustrating cis BENEFIT screening. Pair-wise combinations of effector-augmented (d-effector) S.typhimurium strains [enhanced effector shown in capitals, e.g. aceBp (dSopB) and aCebp (dSipC) produce increased levels of SopB and SipC, respectively] were mixed 50∶50 (overall MOI 50), where one strain additionally carries spectinomycin resistance (SpR). The mixed inoculum is used to infect cultured cells, and a reciprocal infection performed in parallel in which the opposing strain is spectinomycin resistant. After infection (60 min), extracellular bacteria are killed with gentamicin and infected cell lysate dilutions replica plated on LB agar (depicted white) and LB containing spectinomycin (grey). Overall invasion rate and invasion of the spectinomycin resistant strain are calculated by scoring colony-forming units. The invasion rate of the strain lacking the marker is calculated by subtracting invasion of the spectinomycin resistant strain from the overall value (X1, Y2). To correct for the mild influence of spectinomycin on invasion efficiency, invasion rates of individual strains are averaged with the parallel experiment i.e. [X1+X2Sp]÷2 = X and [Y1+Y2Sp]÷2 = Y. Each pair of infections were performed 4 times in triplicate. B. S.typhimurium SL1344 or effector-augmented (d-effector) strains were mixed pair wise (50∶50; MOI 50) for infection. Invasion of each strain was assessed using selectable markers after 60 min (Figure 4A). Results are the mean of four independent experiments each performed in triplicate Upper: Pie charts depict total invasion by each combination (size; combined %) and relative contribution of each strain (division; %). Lower: Tables show difference (%) in total invasion rate (left) and relative invasion rate of each strain (right) after correction. Shading denotes a significant increase (green) or no significant change (grey) in invasion rate (Mann Whitney U p<0.05).
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ppat-1000037-g004: Sip-Sop synergy revealed by cis BENEFIT screening.A. Schematic illustrating cis BENEFIT screening. Pair-wise combinations of effector-augmented (d-effector) S.typhimurium strains [enhanced effector shown in capitals, e.g. aceBp (dSopB) and aCebp (dSipC) produce increased levels of SopB and SipC, respectively] were mixed 50∶50 (overall MOI 50), where one strain additionally carries spectinomycin resistance (SpR). The mixed inoculum is used to infect cultured cells, and a reciprocal infection performed in parallel in which the opposing strain is spectinomycin resistant. After infection (60 min), extracellular bacteria are killed with gentamicin and infected cell lysate dilutions replica plated on LB agar (depicted white) and LB containing spectinomycin (grey). Overall invasion rate and invasion of the spectinomycin resistant strain are calculated by scoring colony-forming units. The invasion rate of the strain lacking the marker is calculated by subtracting invasion of the spectinomycin resistant strain from the overall value (X1, Y2). To correct for the mild influence of spectinomycin on invasion efficiency, invasion rates of individual strains are averaged with the parallel experiment i.e. [X1+X2Sp]÷2 = X and [Y1+Y2Sp]÷2 = Y. Each pair of infections were performed 4 times in triplicate. B. S.typhimurium SL1344 or effector-augmented (d-effector) strains were mixed pair wise (50∶50; MOI 50) for infection. Invasion of each strain was assessed using selectable markers after 60 min (Figure 4A). Results are the mean of four independent experiments each performed in triplicate Upper: Pie charts depict total invasion by each combination (size; combined %) and relative contribution of each strain (division; %). Lower: Tables show difference (%) in total invasion rate (left) and relative invasion rate of each strain (right) after correction. Shading denotes a significant increase (green) or no significant change (grey) in invasion rate (Mann Whitney U p<0.05).

Mentions: We next modified the BENEFIT screen to eliminate the imposed ordered effector activity and the concentration bias resulting from transfection. In this ‘cis’ screen, cultured cells were infected with pairwise mixtures of effector-augmented strains, in combination with each other or WT S.typhimurium (Figure 4A).


Deciphering interplay between Salmonella invasion effectors.

Cain RJ, Hayward RD, Koronakis V - PLoS Pathog. (2008)

Sip-Sop synergy revealed by cis BENEFIT screening.A. Schematic illustrating cis BENEFIT screening. Pair-wise combinations of effector-augmented (d-effector) S.typhimurium strains [enhanced effector shown in capitals, e.g. aceBp (dSopB) and aCebp (dSipC) produce increased levels of SopB and SipC, respectively] were mixed 50∶50 (overall MOI 50), where one strain additionally carries spectinomycin resistance (SpR). The mixed inoculum is used to infect cultured cells, and a reciprocal infection performed in parallel in which the opposing strain is spectinomycin resistant. After infection (60 min), extracellular bacteria are killed with gentamicin and infected cell lysate dilutions replica plated on LB agar (depicted white) and LB containing spectinomycin (grey). Overall invasion rate and invasion of the spectinomycin resistant strain are calculated by scoring colony-forming units. The invasion rate of the strain lacking the marker is calculated by subtracting invasion of the spectinomycin resistant strain from the overall value (X1, Y2). To correct for the mild influence of spectinomycin on invasion efficiency, invasion rates of individual strains are averaged with the parallel experiment i.e. [X1+X2Sp]÷2 = X and [Y1+Y2Sp]÷2 = Y. Each pair of infections were performed 4 times in triplicate. B. S.typhimurium SL1344 or effector-augmented (d-effector) strains were mixed pair wise (50∶50; MOI 50) for infection. Invasion of each strain was assessed using selectable markers after 60 min (Figure 4A). Results are the mean of four independent experiments each performed in triplicate Upper: Pie charts depict total invasion by each combination (size; combined %) and relative contribution of each strain (division; %). Lower: Tables show difference (%) in total invasion rate (left) and relative invasion rate of each strain (right) after correction. Shading denotes a significant increase (green) or no significant change (grey) in invasion rate (Mann Whitney U p<0.05).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2268969&req=5

ppat-1000037-g004: Sip-Sop synergy revealed by cis BENEFIT screening.A. Schematic illustrating cis BENEFIT screening. Pair-wise combinations of effector-augmented (d-effector) S.typhimurium strains [enhanced effector shown in capitals, e.g. aceBp (dSopB) and aCebp (dSipC) produce increased levels of SopB and SipC, respectively] were mixed 50∶50 (overall MOI 50), where one strain additionally carries spectinomycin resistance (SpR). The mixed inoculum is used to infect cultured cells, and a reciprocal infection performed in parallel in which the opposing strain is spectinomycin resistant. After infection (60 min), extracellular bacteria are killed with gentamicin and infected cell lysate dilutions replica plated on LB agar (depicted white) and LB containing spectinomycin (grey). Overall invasion rate and invasion of the spectinomycin resistant strain are calculated by scoring colony-forming units. The invasion rate of the strain lacking the marker is calculated by subtracting invasion of the spectinomycin resistant strain from the overall value (X1, Y2). To correct for the mild influence of spectinomycin on invasion efficiency, invasion rates of individual strains are averaged with the parallel experiment i.e. [X1+X2Sp]÷2 = X and [Y1+Y2Sp]÷2 = Y. Each pair of infections were performed 4 times in triplicate. B. S.typhimurium SL1344 or effector-augmented (d-effector) strains were mixed pair wise (50∶50; MOI 50) for infection. Invasion of each strain was assessed using selectable markers after 60 min (Figure 4A). Results are the mean of four independent experiments each performed in triplicate Upper: Pie charts depict total invasion by each combination (size; combined %) and relative contribution of each strain (division; %). Lower: Tables show difference (%) in total invasion rate (left) and relative invasion rate of each strain (right) after correction. Shading denotes a significant increase (green) or no significant change (grey) in invasion rate (Mann Whitney U p<0.05).
Mentions: We next modified the BENEFIT screen to eliminate the imposed ordered effector activity and the concentration bias resulting from transfection. In this ‘cis’ screen, cultured cells were infected with pairwise mixtures of effector-augmented strains, in combination with each other or WT S.typhimurium (Figure 4A).

Bottom Line: Salmonellae deploy effectors that trigger localized actin reorganization to force their own entry into non-phagocytic host cells.Here we describe trans and cisbinary entry effector interplay (BENEFIT) screens that systematically examine functional associations between effectors following their delivery into the host cell.The results reveal extensive ordered synergistic and antagonistic relationships and their relative potency, and illuminate an unexpectedly sophisticated signaling network evolved through longstanding pathogen-host interaction.

View Article: PubMed Central - PubMed

Affiliation: University of Cambridge, Department of Pathology, Cambridge, United Kingdom.

ABSTRACT
Bacterial pathogens have evolved a specialized type III secretion system (T3SS) to translocate virulence effector proteins directly into eukaryotic target cells. Salmonellae deploy effectors that trigger localized actin reorganization to force their own entry into non-phagocytic host cells. Six effectors (SipC, SipA, SopE/2, SopB, SptP) can individually manipulate actin dynamics at the plasma membrane, which acts as a 'signaling hub' during Salmonella invasion. The extent of crosstalk between these spatially coincident effectors remains unknown. Here we describe trans and cisbinary entry effector interplay (BENEFIT) screens that systematically examine functional associations between effectors following their delivery into the host cell. The results reveal extensive ordered synergistic and antagonistic relationships and their relative potency, and illuminate an unexpectedly sophisticated signaling network evolved through longstanding pathogen-host interaction.

Show MeSH
Related in: MedlinePlus