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
-Based on the passage, what best explains the western blot results of type III secretion proteins in LuxS⁻ E. coli cells cultured in PCM?

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
-Based on the passage, what best explains the western blot results of type III secretion proteins in LuxS⁻ E. coli cells cultured in PCM?

Quizplus · Accepted Answer

11ecba2d_0f52_7788_a3bf_17f0c8edd7ed_MD0008_00 In quorum sensing, changes in gene expression are dependent on the cellular density of bacteria that produce and secrete the autoinducer, a signaling molecule that influences the activity of specific genes in nearby cells (AI-3, in this case). The amount of autoinducer in extracellular space correlates with bacterial population density. According to the passage, deficiency in the LuxS enzyme leads to decreased AI-3 expression. AI-3 generally acts on neighboring bacteria to induce expression of LEE genes. These genes encode the type III secretion proteins necessary to introduce EHEC-secreted toxins into the host cells to cause disease. Because LuxS⁻ mutants lack the LuxS enzyme and have insufficient AI-3 levels, these mutants lack type III secretion proteins, as shown in Figure 2. However, Figure 2 also shows that growing LuxS⁻ cells in PCM resulted in the expression of type III secretion proteins. Based on Figure 1, which shows that LEE genes can be expressed via a LuxS-independent method, the banding patterns in Figure 2 suggest that compounds (eg, epinephrine, norepinephrine) released by epithelial cells into the PCM activated the transcription of LEE genes despite the deficiency of AI-3. (Choice A) Figure 1 shows that secretin and gastrin affect the concentration of LEE mRNA at a level that is comparable to the control group, LuxS⁻ (ie, no significant change compared to the control). As a result, it would not be expected that these hormones rescue the production of type III secretion proteins in LuxS⁻ cells. (Choice B) The passage states that type III secretion proteins can be secreted only by bacteria, not by the intestinal epithelial cells. (Choice C) A reverse mutation for LuxS⁻ E. coli would result in the western blot results showing similar banding patterns for all three E. coli cultures. However, this is not the case as LuxS⁻ E. coli show a reduced banding pattern compared with the WT. Educational objective: Inducers are chemical substances (signals) that regulate gene expression. Specifically, secreted inducers can influence gene expression in other cells.

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