Passage
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a bacterium responsible for causing foodborne illnesses. EHEC virulence, or the ability to cause disease, is acquired through quorum sensing, a form of bacterial cell-cell communication dependent on population density. EHEC O157:H7 bacteria monitor population density by detecting changes in the concentration of autoinducer-3 (AI-3) , an extracellular signaling molecule secreted by many bacterial species. AI-3 generally acts on neighboring bacteria to activate the genes on the enterocyte effacement (LEE) locus. These genes code for type III secretion proteins (EspA, EspB, EspD, Tir) , which promote entry of Shiga-like toxins secreted by EHEC into host intestinal epithelial cells. This ultimately leads to cell death, lesions, and symptoms such as bloody diarrhea and abdominal cramps.It has been reported that a mutation in S-ribosylhomocysteine lyase (LuxS) , an enzyme involved in quorum sensing, may result in diminished production of AI-3. However, researchers predict that certain peptide hormones can activate LEE gene transcription in the LuxS. To test this hypothesis, mutant EHEC bacteria deficient in LuxS (LuxS⁻) were cultured and separately exposed to the hormones epinephrine (E) , norepinephrine (NE) , gastrin (G) , and secretin (S) at 37°C. Total RNA was extracted from the treated bacterial cultures, and LEE mRNA was quantified (Figure 1) .
Figure 1 Concentration of LEE mRNA in mutant LuxS⁻ bacteria supplemented with purified peptide hormones (Note: C = control.) Western blot was performed to compare the presence and concentration of type III secretion proteins from wild-type (WT) , LuxS⁻, and LuxS⁻ cultured in preconditioned media (PCM) . PCM was generated by culturing uninfected intestinal epithelial cells in growth media for 24 hours. After 24 hours, the cells were removed and the PCM was used to supplement the growth of LuxS⁻ bacteria.
Figure 2 Western blot results of type III secretion proteins isolated from WT, mutant LuxS⁻, and LuxS⁻ in PCM
-Which proteins identified in Figure 2 have the shortest amino acid sequence and the greatest concentration in WT cells, respectively?

Question

Passage
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a bacterium responsible for causing foodborne illnesses. EHEC virulence, or the ability to cause disease, is acquired through quorum sensing, a form of bacterial cell-cell communication dependent on population density. EHEC O157:H7 bacteria monitor population density by detecting changes in the concentration of autoinducer-3 (AI-3) , an extracellular signaling molecule secreted by many bacterial species. AI-3 generally acts on neighboring bacteria to activate the genes on the enterocyte effacement (LEE) locus. These genes code for type III secretion proteins (EspA, EspB, EspD, Tir) , which promote entry of Shiga-like toxins secreted by EHEC into host intestinal epithelial cells. This ultimately leads to cell death, lesions, and symptoms such as bloody diarrhea and abdominal cramps.It has been reported that a mutation in S-ribosylhomocysteine lyase (LuxS) , an enzyme involved in quorum sensing, may result in diminished production of AI-3. However, researchers predict that certain peptide hormones can activate LEE gene transcription in the LuxS. To test this hypothesis, mutant EHEC bacteria deficient in LuxS (LuxS⁻) were cultured and separately exposed to the hormones epinephrine (E) , norepinephrine (NE) , gastrin (G) , and secretin (S) at 37°C. Total RNA was extracted from the treated bacterial cultures, and LEE mRNA was quantified (Figure 1) .

Figure 1 Concentration of LEE mRNA in mutant LuxS⁻ bacteria supplemented with purified peptide hormones (Note: C = control.) Western blot was performed to compare the presence and concentration of type III secretion proteins from wild-type (WT) , LuxS⁻, and LuxS⁻ cultured in preconditioned media (PCM) . PCM was generated by culturing uninfected intestinal epithelial cells in growth media for 24 hours. After 24 hours, the cells were removed and the PCM was used to supplement the growth of LuxS⁻ bacteria.

Figure 2 Western blot results of type III secretion proteins isolated from WT, mutant LuxS⁻, and LuxS⁻ in PCM
-Which proteins identified in Figure 2 have the shortest amino acid sequence and the greatest concentration in WT cells, respectively?

Quizplus · Accepted Answer

11ecba2d_0f51_6617_a3bf_b5e1ed2f5b61_MD0008_00 Western blot is a laboratory technique that first separates proteins by size (gel electrophoresis) and then transfers them to a blotting membrane. A primary antibody specific for the protein of interest is used as a probe. The membrane is washed to clear unbound antibodies and then treated with a secondary antibody that specifically binds to the primary antibody. Depending on the type of secondary antibody used, detection of the proteins of interest is achieved either by fluorescence or by a reporter enzyme. In general, proteins with the shortest amino acid sequences have lower molecular weights and migrate quickly through the gel. Band intensity (thickness) demonstrates the relative levels of expression of the target proteins. In this experiment, EspA, EspB, EspD, and Tir were separated by gel electrophoresis according to their molecular weights. Figure 2 shows the relative size and expression levels of the type III secretion proteins isolated from WT, mutant LuxS⁻, and LuxS⁻ in PCM. The results demonstrate that Tir traveled farthest, indicating it had the lowest molecular weight and therefore the shortest amino acid sequence. The EspB protein had the greatest concentration in WT cells, as indicated by the increased intensity of its bands in Figure 2. (Choice A) EspA has the second-lowest molecular weight, as it migrated less than Tir. Tir demonstrated less signal intensity than EspB. (Choices C and D) EspB has the greatest molecular weight as it travelled the shortest distance compared with the other proteins. The EspD and EspA bands demonstrated less signal intensity compared with EspB bands. Educational objective: Western blot involves protein separation by gel electrophoresis, transfer onto a blotting membrane, and detection with protein-specific antibodies. Proteins with low molecular weight migrate farthest, and protein concentration is generally proportional to band intensity.

Passage Enterohemorrhagic Escherichia Coli (EHEC) O157:H7 Is a Bacterium Responsible for for Causing