SB-BB

Passage 5

Some gram-negative bacteria use a type III secretion system (T3SS) to inject host cells with cytosolic effector proteins, which facilitate entry into host cells. Vibrio parahaemolyticus (VP), a motile bacterium with a single flagellum, uses a T3SS that injects the effector VopC. VopC contains a catalytic domain that irreversibly activates host cell GTPase Rac through the deamidation of the side chain of the residue at position 61. Activated Rac alters the actin cytoskeleton.

Experiment 1

Host cells were incubated in cell growth media containing VP for two hours. The cells were rinsed and incubated in new media containing an antibiotic effective against a wide range of bacteria, including VP. At hourly intervals, samples of host cells were collected and rinsed, and intracellular bacterial levels were measured (Figure 1).

Figure 1 Levels of intracellular bacteria at each time point

Experiment 2

To confirm that VopC activates Rac, a mutant strain of VP lacking the VopC gene was created (VPΔvopC). Plasmids encoding wild-type VopC or catalytically inactive VopC (VopCX) were transfected into VPΔvopC to create strains VPΔvopC + VopC and VPΔvopC + VopCX, respectively. Host cells were exposed to each strain, and GTP-bound Rac was measured by Western blot analysis (Figure 2).

Figure 2 Rac activation by VopC (Note: U indicates uninfected; aldolase is a constantly expressed protein in the host cell.)

Adapted from L. Zhang et al., “Type III Effector VopC Mediates Invasion for VibrioSpecies.” Cell Reports. VibrioSpecies.” Cell Reports.

Questions

34. In Figure 1, which statement best explains the data trend of an increase followed by a decrease in bacterial cell count?

  1. The number of host cellsin the sample increased until carrying capacity was reached.
  2. Infection with VP is maximal at 5 hours of exposure.
  3. VP replicates inside the host cell before causing host cell lysis.
  4. Rate of VP entry into host cells increases then decreases over time.

35. The amino acid residue at position 61 in Rac is most likely:

  1. glutamine.
  2. leucine.
  3. arginine.
  4. tyrosine.

36. What is the independent variable in Experiment 1?

  1. Length of time host cells were exposed to the bacteria
  2. Amount of cells collected in each sample
  3. Time at which the cell samples were collected
  4. Amount of intracellular bacteria measured at each time point

37. A Rac variant, in which the residue at position 61 was replaced with an alanine (Rac61A), was synthesized. Wild-type Rac and Rac61A were incubated separately with VopC. To obtain data to support that VopC modifies Rac at residue 61, the samples should be analyzed for the presence of which compound?

  1. CO2
  2. NH3
  3. O2
  4. H2S

38. Why was the total amount of Rac measured in Experiment 2?

  1. To confirm that the antibody used to detect GTP–Rac was working in each sample
  2. To determine if the same amount of each sample was loaded on the gel
  3. To act as a control for comparing rates of Rac activation by VopC
  4. To determine if VopC affects Rac expression levels

39. Under normal conditions in the absence of VP infection, what is the structure of the molecule that binds and activates Rac?

Answers

Question 34 Solution: The correct answer is C. This is a Biology question that falls under the content category “The structure, growth, physiology, and genetics of prokaryotes and viruses.“ The answer to this question is C because in Experiment 1, the host cells are exposed to VP for only two hours, then washed and incubated in VP-free media containing an antibiotic. Thus, the levels of intracellular bacteria (VP that has entered the host cell) measured at each time point represent the amount of VP in the host cells at that time point. The increasing levels at hours 3 and 4 suggest that VP is replicating inside the host cell. The sudden drop in intracellular bacterial levels is consistent with the hypothesis that the host cells are beginning to lyse, as the bacteria that had previously been inside the cell are rinsed away or killed by the antibiotic. This prevents any further infection or reinfection by VP or other extracellular bacteria, eliminating options B and D. Option A is incorrect because the amount of intracellular bacteria is being measured, not the number of host cells. It is a Data-based and Statistical Reasoning question because you are asked to interpret patterns in data to draw a conclusion.
Question 35 Solution: The correct answer is A. This is a Biochemistry question that falls under the content category “Structure and function of proteins and their constituent amino acids.“ The answer to this question is A because according to the passage, VopC removes an amide from the side chain of the amino acid residue at position 61 in Rac via a deamidation reaction. Of the options, only glutamine contains a side chain with an amide group. It is a Knowledge of Scientific Concepts and Principles question because you are asked to recall structural characteristics of amino acids.
Question 36 Solution: The correct answer is C. This is a Biology question that falls under the content category “The structure, growth, physiology, and genetics of prokaryotes and viruses.” The answer to this question is C because the independent variable in an experiment is that which is varied by the researcher in order to determine the effect (the dependent variable). In Experiment 1, the time at which cell samples are collected is the independent variable. It is a Reasoning about the Design and Execution of Research question because you are asked to communicate an understanding of experimental design to identify the independent variable in an experiment.
Question 37 Solution: The correct answer is B. This is a Biochemistry question that falls under the content category “Structure and function of proteins and their constituent amino acids.” The answer to this question is B because a deamidation reaction releases NH3, thus, if NH3 is detected in the sample containing wild-type Rac but not Rac61A, this will provide data to support that VopC deamidates Rac at residue 61. It is a Reasoning about the Design and Execution of Research question because you are asked to demonstrate an understanding of how to design an experiment that will provide data relevant to a specific investigation.
Question 38 Solution: The correct answer is D. This is a Biology question that falls under the content category “Transmission of genetic information from the gene to the protein.” The answer to this question is D because in order to determine how the presence or absence of VopC affects Rac activation, it is important to know if the total amount of Rac is affected by the strain being used. If the Rac expression is upregulated in one strain and not another, this will affect interpretation of the gel results. An antibody specific to GTP–Rac will not detect total Rac (A), aldolase is the loading control (B), and no information about the rate of activation is provided by the data (C). It is a Reasoning about the Design and Execution of Research question because you are asked to reason through the design of a given experiment to determine why a specific variable was measured.
Question 39 Solution: The correct answer is A. This is a Biochemistry question that falls under the content category “Transmission of genetic information from the gene to the protein.” The answer to this question is A because this is the structure of GTP. GTPases, like Rac, are activated by GTP. It is a Knowledge of Scientific Concepts and Principles question because you are asked to recall a characteristic of a common enzyme and the structure of the nucleotide that activates it.