Although most community-acquired methicillinresistant S. aureus (CA-MRSA) bacter
ID: 78021 • 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. LukF is the 44-kDa protein is a membrane associated protein
LukS is the 33-kDa protein is a mitocondria localized protein.
May be this protein are Exo-toxins in nature so they are absent in bacterial cell fractionation.
B. LukF probably make cell membrane more permeable.
LukS probably helps in release of cytocrome from mitochondria
Sequencing the both protein might help. If it is getting localized to mitochondria it will have a mitochondria localization signal sequence.
For membrane bound protein it shoud have a hydrophobic patch to get inserted in the membrane.
C. As their cellular localization is different as seen from western blot result it is dificult to draw that conclusion. If they are sub-unit they would have higer degree of affinity to each other. So immoilizing one unit in a surface using another one (lebeled in some kind of flurophore to easy to measure) as its ligand we can measure their binding affinity to each-other.
Related Questions
drjack9650@gmail.com
Navigate
Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.