Although most community-acquired methicillinresistant S. aureus (CA-MRSA) bacter
ID: 78396 • Letter: A
Question
Although most community-acquired methicillinresistant S. aureus (CA-MRSA) bacteria are not as resistant to antibiotics as the hospital-acquired MRSA strains, the recently identified strains that we hear about in the news appear to be more virulent. A group of researchers has recently discovered that bacterial cultures of a particularly virulent strain of CA-MRSA (called USA300) cause apoptosis in leukocytes and neutrophils, as evidenced by release of cytochrome c from mitochondria, followed by cytokine release and cell death. Antibodies generated against bacterial culture medium from the USA300 strain identified two proteins of 33 kDa and 44 kDa on Western blots.
To determine the localization of these two proteins, researchers performed sodium dodecyl sulfatepolyacrylamide gel electrophoresis and Western blot analysis of lysates from the bacterial cell pellets and the bacterial culture medium, as well as lysates of human neutrophils treated with the USA300 strain. The results from Western blot analysis are shown in the left blot in Figure 1. The researchers then applied the cell-free bacterial culture medium to the neutrophils and performed subcellular fractionation of the treated neutrophils. The results fromWestern blot analysis are shown in the right blot in Figure 1.
A. Provide an interpretation (with rationale) of the results shown in Figure 1.
B. Predict (with rationale) possible functions for the two proteins, which have been named LukS and LukF (LukS is the 33-kDa protein, and LukF is the 44-kDa protein). Provide at least one additional (different) experiment that could be performed to confirm your prediction.
C. The researchers speculate that the two proteins, LukS and LukF, are two subunits of an A-B-type toxin. Based on the results shown in Figure 1, do you agree with this interpretation? Provide your rationale. Provide an experiment that would allow you to verify your answer.
44 kDa 33 kDa Neutrophil subcellular fraction Figure 1 Western blots using Bacterial Culture Neutrophil against USA300- antibodies cells medium lysates Cytosol Membrane Mitochondria secreted proteins (continued)Explanation / Answer
A) In the lane for the bacterial cells both antibodies of 44 kDA and 33 kDa are absent .
And both of these proteins are present in the lanes corresponding to culture medium and neutrophil lysates.
In the western blot analysis using the cell free bacterial culture medium to the neutrophils and for which sub- cellular fractionation of the treated neutrophils was done, it was observed that ,
a) 44 KDa protien was found to be in the membrane part as well as the mitochondria.
b) 33 KDa of the protein molecule was found to be in the cytosol as well as the mitochondria. . Mitochondria is an organelle which is double bound membrane . The 33 kDA molecule is highly localised in the membrane part of the mitochondria part of the cell. , A smaller amount of 33 kDA protein is found to be in the cytosol part of the cell.
2) Prediction of the functions:
By the observations, it could be predicted that these proteins function as pore forming proteins in the membrane which lead to release of contents of the organelles ( mainly the mitochondria ). These act together as as subunits assembling in the host defence cells.
3) The two proteins, LukS and LukF seem to be like two subunits of an A-B-type toxin. Even though both of these act as together , there is difference in the concentration of these proteins , observed in the membrane as well as the cytoplasm. Therfore it could be predicted that ,one of the protein component "A" could be the "active" portion, and the "B" component is usually the "binding" portion. The active A component could be mostly present in the cytoplasmic region , where as the binding B component could be largely present in the membrane region.
An experiment , in which fluorescent tagged antibodies generated against Luk S and LukF components parts of the proteins could be generated . After allowing sufficient time for the binding process, these could be detected by immunofluorescence method.
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