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Pabio 552, Midterm Exam
Thursday May 4, 2006
Problem 1 (43 points total; Questions 1A - M). You have discovered a novel secretion system
used by a gram negative bacterial pathogen. The bacterium is endocytosed by host macrophages and
remains inside the endosome, where it secretes a bacterial protein toxin through a novel channel in
the endosomal membrane. To secrete this toxin, the bacterium first creates a channel that spans its
inner and outer membrane. This channel is capable of docking onto a channel in the endosomal
membrane. The bacterium then cotranslationally translocates the toxin through both channels
simultaneously and into the host cytosol. Thus, while the protein is being synthesized inside the
bacterial cytoplasm, it is also being translocated into the host cell cytoplasm. The protein toxin has
been identified and sequenced but it’s mechanism of action is not well understood.
Question 1A. A new graduate student has joined your lab and wants to know whether normal
uninfected macrophages ever use a system where channels in two different membranes align in
order to translocate a protein. What is your answer to that question? (one short paragraph; 3
points)
Yes. In higher eukaryotic cells, the best understood example of this occurs in mitochondria. As
mentioned in lecture and discussed in Alberts, p. 681, mitochondrial protein translocation goes
through the TOM and TIM channels at contact sites where these two channels are aligned.
Bacterial secretion systems won’t get credit, since the question asks about examples in normal
uninfected macrophages. Nuclear pore examples are okay but not quite as good as TOM/TIM.
In explaining your answer to the question in A, you realize you need to explain the topology of this
secretion mechanism.
Question 1B. Exactly how many structures (i.e. physical barriers) are crossed by this toxin
during secretion? Give ONE NUMBER (3 points).
I was also very liberal with this answer. If you showed all the relevant spaces in your diagram, I
gave you credit regardless of whether you counted the beginning and ending spaces.
Question 1C. How many topological spaces are crossed by this toxin? Give ONE NUMBER
(3 points)
Your diagram needed to show bacterium, bacterial plasma membrane, bacterial outer membrane,
endosomal membrane, cytosol, plasma membrane, and extracellular space.
Question 1D. Explain how you came up with those numbers by drawing a diagram. Label all
the structures and spaces, and indicate which spaces are the topological equivalent of
extracellular space and which are the topological equivalent of intracellular space (i.e. cytosol).
(one diagram, 3 points)
You are interested in the identity of the channel in the endosomal membrane. Others in the field had
hypothesized that a channel from the ER was enriched in the membranes of endosomes infected with
this bacterium and that translocation occurred through a known channel complex. An alternative
hypothesis is that the channel in the endosomal membrane is composed of a novel protein that had
not been recognized previously. Your postdoc discovers that it is very easy to purify these infected
endosomes by differential centrifugation. Furthermore, this bacterium infects macrophages at a very
high copy number, so that a large number of infected endosomes can be purified from cultured cells.
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Pabio 552, Midterm Exam
Thursday May 4, 2006
By performing biochemical comparisons of normal endosomes (from uninfected cells) versus
infected endosomes, your postdoc eventually identifies a protein present in the infected endosomes
and not in normal endosomes. This protein turns out to be a novel protein and is predicted to be a
multipass membrane protein, so the postdoc proposes that it could be the protein that forms the toxin
translocation channel in the host endosomal membrane.
The postdoc is leaving the lab and a new graduate student wants to take over the project. The
graduate student points out that in the absence of functional data, there is no evidence that the newly
isolated endosomal protein is actually the channel. The postdoc’s next experiment was to perform
immunofluorescence colocalization studies to show that the toxin colocalizes with the novel
endosomal protein.
Question 1E. Describe exactly how the immunofluorescence colocalization experiment should
be performed. Describe exactly what tools the postdoc would need to show colocalization of
the toxin with the novel endosomal membrane protein. (one paragraph, 2 points)
What I wanted here was 1) that you would either use antibodies directed against the toxin and the
channel, or make fluorescent protein fusion constructs. 2) If you opted for primary antibodies, I
wanted some details on secondary antibodies. 3) You also needed to propose to look at CELLS not
isolated organelles, since it is only in the context of the whole cell that you would be able to tell if
colocalization was in some compartments (i.e. endosome) and not others.
Question 1F. Given the grad student’s concern, what is the grad student’s most likely reaction
to immunofluorescence colocalization being the next experiment in the project? (a few
sentences, 3 points)
Immunofluorescence colocalization will simply show that the two proteins are in the same place at
the same time, but that does not establish that the endosomal protein is physically associated with
the channel or that it funcitons a channel. Since light microsocopy does not achieve high resolution,
you wouldn’t know if the endosomal protein was next to the channel protein or was in fact the
channel.
Question 1G. The astute grad student wants to perform three experiments to test the
hypothesis that the novel endosomal protein is critical for translocation of the bacterial toxin.
Describe what you think are the three most useful experiments for testing this hypothesis and
how each should be performed. Include critical controls and point out how each of your
experiments addresses the hypothesis, what important aspects of the hypothesis are not
addressed by each experiment, and whether you anticipate potential technical problems with
your proposed experiments. Note that it is very easy to make mutants in the genes encoded by this
bacterium, so assume you can engineer whatever bacterial mutations you want to make and you
don’t need to describe the methods for doing this (since this isn’t a genetics course). Also assume
availability of standard biochemical tools and reagents. (3 paragraphs, 6 points)
1. siRNA to knock down the novel channel protein.
This will not demonstrate that the toxin actually goes through the endosomal protein but that the
protein is required for translocation
2. coimmunoprecipitation of toxin and channel. This also does not demonstrate that the toxin goes
through the endosomal protein.
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Pabio 552, Midterm Exam
Thursday May 4, 2006
3. make a toxin mutant that does not fully translocate and gets stuck in the channel. Then crosslink
or immunoprecipitate toxin mutant with channel. You could also show that this toxin mutant
prevents translocation of the wild-type protein, assuming you can make a heterozygote with one
copy of wild-type and one copy of mutant protein.
I accepted a lot of other types of experiments. Come talk to me if you have questions on why you lost
points.
The graduate student actually comes up with a fairly novel approach to testing this hypothesis. First,
endosomes containing this channel are isolated from macrophages (as described above). The grad
student learned in a cell biology course that when membranes are sheared during isolation, they can
form both “outside out” and “inside out” vesicles. This is because when they are broken up during
homogenization they can reseal in either direction. Furthermore, the bacteria come out of these
endosomes during the homogenization process, so most resealed endosomal vesicles don’t contain
bacteria. Furthermore, a subsequent isolation step removes any endosomal vesicles that might
contain bacteria. The student realizes that vesicles with the reverse topology (the luminal side facing
out) could be useful for cell-free translocation experiments. (Note that you are not allowed to use
strategies involving these “inside out” vesicles in your answer to 1G above).
The student uses an antibody against a luminal epitope of the protein to purify these “inside out”
vesicles, and then sets up the following experiment. The student sets up a reticulocyte lysate
translation system that contains ribosomes, translation initiation and elongation factors, and
additional components required for translation (such as purified amino acids, 35S methionine, and
ATP/GTP). The student then transcribes a cDNA encoding the toxin protein into mRNA and
translates this in the reticulocyte lysate system in the presence and absence of the “inside out”
endosomal membranes, and analyzes the samples by SDS-PAGE.
The first time the student runs the samples, the 25 kD toxin protein appears as a smear of multiple
bands rather than a discrete band by SDS-PAGE.
Question 1 H: Describe three technical errors during sample preparation that could result in
failure to resolve a single discrete protein translation product by SDS-PAGE, assuming the
translation reaction was successful? (a few sentences, 2 points)
What I wanted was 1) no SDS in sample buffer or gel running buffer, leading to failure to completely
denature the protein; 2) no DTT in the sample buffer, leading to proteins not being fully reduced; 3)
proteins not boiled or heated so they weren’t fully denatured. I gave credit if students said that
proteases had acted on the proteins, but they also needed to have something about denaturation, but
they had to also talk about denaturation and reduction problems.
The second time, the student performs the experiment correctly and below is the autoradiogram of
the SDS-PAGE (Fig. 1). The toxin mRNA was translated for 2 hours, and the translation was
performed with or without endosomal membranes added at the start of translation (Mb: +/-); with or
without detergent added at the start of translation (Detergent: +/-); and with or without a protease
added at the end of the translation. Note that in lanes 8 – 10, the student translated a mutant of the
toxin that is missing N terminal hydrophobic amino acids. This mutant protein migrates at 23.5 kD.
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Pabio 552, Midterm Exam
Thursday May 4, 2006
Question 1I: Approximately how many amino acids are present in the mutant versus the wildtype protein? (one number, 2 points)
227 amino acids in the wild-type; 213 in the mutant – about 14 removed. I wanted you to use
a number for average amino acid molecular weight that was somewhere between 110 and 135
daltons. So you should have come up with 11 – 14 amino acids. Anything out of that range needed
to have a pretty good explanation to not lose some points.
Question 1J: Draw out a simple cartoon that illustrates what is happening in each lane of the
gel in Fig. 1. State the four most important facts you know about this protein because of the
results of the experiment in Fig. 1. (11 simple cartoons, four sentences, 7 points)
The protein translocates through the endosomal vesicle and becomes protected from
protease.
The protein has an N terminal signal that is required for translocation.
The N terminal signal does not appear to be cleaved during translocation.
The protein will translocate through the endosomal channel even in the absence of the
bacterial membrane channel.
Most students had correct answers for the first two points (although some might have lost credit for
not making it clear that complete protection occurs at least for some toxin proteins). Many people
missed the last two points and lost credit as a result.
Question 1K: What simple variation of this experiment (i.e. using no additional reagents
besides those used in Fig. 1) will give you important additional data? (one paragraph, 4 points)
What I wanted was something we had gone over very carefully in class: in order to
demonstrate that a protein translocates only CO-TRANSLATIONALLY, you simply add the
membranes at the end of translation (i.e. at 2 hours rather than at the start of translation). If
translocation with protease protection occurs with membranes added at 2 hours, then translocation
can occur POST-TRANSLATIONALLY. If it fails to occur, then translocation only occurs cotranslationally. This experiment requires no additional reagents.
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Pabio 552, Midterm Exam
Thursday May 4, 2006
If you gave me an experiment that required additional reagents (i.e. purified channel
proteins), you lost points.
If you tried to use addition of protease at different times to determine if the process was co or
post-translational, you also lost points. Proteases added during translation will cleave the newlysynthesized protein as it emerges from the ribosome and will therefore completely prevent
translation and translocation.
Question 1L: What more complicated variation of this approach (i.e. using additional
technique and reagents) could answer in a direct way whether the protein is necessary and
sufficient for translocation of the bacterial toxin. (one paragraph, 5 points)
It is not known how the toxin acts. When the toxin acts, cells undergo morphologic changes
suggestive of cytoskeletal rearrangements. Purified toxin is inactive when added to cells but
transfected toxin cDNA is toxic to cells. In a bioinformatics screen, you find that the toxin has two
SH2 domains. A colleague finds that Gleevec, an Abl-kinase inhibitor, blocks the action of the
transfected toxin. Another colleague finds that a point mutation of a tyrosine within the toxin
(tyrosine mutated to leucine) results in a toxin that is completely inactive upon transfection. This
leads you to the hypothesis that the toxin binds via SH2 domains to a cellular kinase that in turn
activates the toxin by phosphorylating the toxin at this tyrosine residue. You are interested in testing
your hypothesis by examining whether the toxin becomes phosphorylated when expressed in cells.
A colleague tells you not to bother because when she incubated infected cells with dATP [32P] for
6 hours, and then immunoprecipitated the wild-type toxin, she found that the toxin was not
phosphorylated. You are not deterred, and ask a new student in your lab to try the experiment with
cellular extracts. The student adds dATP [32P] to extracts of infected cells and still finds no
phosphorylation of the wild-type toxin.
Question 1M: What experiment would you do next to test the hypothesis that the toxin is
phosphorylated in cells? (a few sentences, 3 points)
Your colleague should have used 32P inorganic. Your student should have used dATP [ 32P].
What you should do is repeat the student’s experiment with dATP [ 32P].
You had to be paying close attention in Jonathan Cooper’s paper discussion to get this right.
Someone in class asked why 32-P ATP was used in one experiment and 32-P inorganic in another.
He explained that ATP is not taken up by cells. So you can only use that to label extracts. To label
cells you must use 32 P labeled inorganic phosphate (which cells will take up). In a different class,
we touched on the issue of which phosphate needs to be labeled to transfer a labeled phosphate to a
protein in a phosphorylation reaction. It is the GAMMA phosphate (the one on the end) that is
transferred (and therefore should be labeled), not the ALPHA phosphate. So ideally people should
have explained both of these issues. If you explained one well, you probably got most credit or full
credit, depending on what you proposed to do next.
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Pabio 552, Midterm Exam
Thursday May 4, 2006
Problem 2 (37 points total; Questions 2A - I): You discover a novel innate immunity pathway
unique to rodents in which the presence of double stranded RNA in the cytosol leads to activation of
a novel transcription factor called XTI. XTI leads to expression of a newly identified cellular
RNAse that cleaves double stranded RNA in the cytoplasm. You test the effectiveness of this
pathway against the murine virus that you study and are disappointed to find that the novel RNase is
not expressed when cells are infected with the murine virus. Thus, it appears that the murine virus
is able to inhibit this innate immune defense.
Your first hypothesis for how the virus blocks this pathway is that the virus modifies XTI so that it is
not active as a transcription factor and therefore fails to activate expression of the RNase. Since noone has studied XTI localization in cells, you ask a student in the lab to use a newly available
antibody against XTI to examine XTI labeling in uninfected and infected cells, at day 1 and day 2
post infection (Fig. 2).
Question 2A: How do you interpret the results of Fig. 2? Hint: Your student says that a
careful bioinformatics analysis of XTI revealed an explanation for the result obtained. (a few
sentences, 3 points).
XTI has an NES and an NLS, i.e. it is a shuttling protein. Apparently, the NES has a stronger effect
than the NLS. So in fact a very small amount does get into the nucleus in some situations, but it is
too small to detect given the strong NES. However, in the case of transcription factors, even a small
amount of nuclear localization can have powerful effects. In addition, as many of you pointed out,
the best way to see the nuclear import of XTI would be to induce expression of double stranded DNA
in the cytoplasm.
Question 2B: A few weeks later your student repeats the experiment with an important
change, and has a much more interesting result that suggests that indeed there is a defect in
XTI import in infected cells but not in normal cells. What did the student do to obtain this
result? (a few sentences, 3 points)
The student used bioinformatics to find the putative NES, and then mutated that in order to
“unmask” the NLS. This would allow you to see instances of nuclear import very clearly. Only one
student suggested this – so that person received a generous dose of extra credit. Many of you came
up with the suggestion of expressing double stranded RNA to cause XTI to translocate in larger
quantities to the nucleus. If you explained that reasonably well, you received full credit even though
it wasn’t quite what I had in mind. We didn’t do a paper that covered NES and NLS mutations, so
to be fair, you didn’t have as much exposure to this concept as previous years’ students have had.
Hence I didn’t take off points if your logic was reasonable.
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Pabio 552, Midterm Exam
Thursday May 4, 2006
The student wants to confirm the data using a heterokaryon assay for nuclear export/import. You
have GFP tagged XTI as well as untagged XTI constructs in your lab. You also have cells with large
nuclei that are conducive to heterokaryon experiments and can be infected. The student tries the
heterokaryon assay and eventually does it correctly with the proper controls, and finds excellent
confirmation of the previous result, i.e. that infected cells have a defect in nuclear import of XTI.
Question 2C: Describe exactly how the successful heterokaryon experiment was performed,
and draw out the results obtained with uninfected vs. infected cells. (one paragraph and some
simple diagrams, 6 points).
This is also something we covered only briefly in class, but I was hoping you all would read up on
this assay and figure it out. What I looked for was 1) the nucleus of one cell gets INJECTED with a
fluorescent fusion XTI protein (i.e. GFP-XTI); 2) this nucleus also gets injected with a marker
protein (i.e. RFP-marker) that has NO NES and therefore stays in the injected nucleus to mark what
was injected. You then fuse this to another cell using PEG. If GFP-XTI has an export signal, it will
end up in the cytoplasm; if it has an import signal it was also end up in the NON-INJECTED
nucleus. The later should not happen in WT virus infected cells, but will happen with mutant virus.
If you didn’t point out that ONLY one nucleus gets injected with the tagged XTI, you lost a point. If
you didn’t point out that you need to mark the injected nucleus with a marker protein, you also lost a
point. Not that this experiment does NOT involve transfection.
Next you examine whether Importin- is involved in nuclear import of XTI in normal cells, and if so
whether that interaction is inhibited in infected cells. You perform an immunoprecipitation with
antibodies to Importin- under native conditions, and then immunoblot with antibodies to XTI.
Below is the immunoblot you obtained when this experiment was performed on normal cells
(uninfected), cells infected with wild-type virus (WT virus), and cells infected with a mutant virus
(mutant virus) that is not pathogenic (Fig. 3). Next to immunoprecipitation (IP) lanes are lanes
showing an equivalent aliquot of the total cell lysate that was programmed into the IP.
Question 2D: What are the two most important things that you can conclude from the
experiment in Fig. 3? (2 sentences, 4 points)
Importin does not associate with XTI in cells infected with WT virus (but not mutant virus).
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Pabio 552, Midterm Exam
Thursday May 4, 2006
XTI appears to be post-translationally modified in cells infected with WT virus (but not mutant
virus).
If you talked about splicing, you lost some points (but not a lot). While possible, that is much less
likely than post-translational modifications (which have also been a major topic in this course).
If you thought that the other bands represented completely different proteins, you lost a lot of points.
Because they are recognized by antibody to XTI, they must be variants of XTI. The most likely
scenario is post-translational modification.
Question 2E: Present a hypothetical and plausible model for how you think the virus blocks
nuclear import of XTI. (1 paragraph, diagrams are acceptable but make sure to explain in
words as well, 4 points).
The virus ubiquitinates or sumoylates XTI, preventing it from being recognized by importin.
Ubiquitin and sumo are about 8 kD, so that fits with the size of the modification that is shown. This
is straight out of a paper we did for discussion, so you should have all recognized this. If you talked
about phosphorylation, you lost some points, since an 8 kD shift would be highly unlikely with
phosphorylation (as you saw in discussion papers, those shifts were very small). If you
hypothesized glycosylation, you lost a lot of points, since large sugars are ONLY put on in the ER (a
point that was heavily emphasized in class).
Question 2F: Describe the next two experiments you would perform to better understand the
result obtained in Fig. 3. Include controls and variations. Describe exactly what hypothesis
each experiment tests, what results are possible, and how you will interpret your results. (Less
than 1 page, diagrams are encouraged, 8 points)
A variety of different experiments were acceptable; come talk to me if you don’t understand why you
lost points.
Viruses often use multiple different mechanisms to ensure one important downstream effect. A
colleague of yours discovers that nuclear traffic of at least three other cellular proteins that use
Importin- is reduced during infection with this virus.
Question 2G: Does your model in Question 2E provide a simple and direct explanation for how
import of other cellular proteins is inhibited, or do you need to look for another mechanism of
viral action? Explain your answer. (a few sentences, 2 points)
Either answer sufficed here; it was your logic I assessed.
Your colleague performs immunoprecipitations for Ran-GAP and found that it is present at
markedly lower levels in virus infected cells, while Ran-GEF levels were unaffected.
Question 2H: Present the most likely (i.e. the simplest) hypothesis for how reduced Ran-GAP
levels could reduce nuclear import of multiple cellular proteins. (1 paragraph, diagrams are
acceptable but make sure to explain in words as well, 3 points).
You needed to explain that RanGAP promotes hydrolysis of GTP in RanGTP, thereby releasing the
exported cargo and allowing Ran to be loaded with GDP and used for import from the cytosol to the
nucleus.
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Pabio 552, Midterm Exam
Thursday May 4, 2006
Question 2I: Describe one experiment you would perform to test the hypothesis you described
in 2H. Describe the possible results, and how you will interpret your results. (~1/2 page,
diagrams are encouraged, 4 points)
A variety of different experiments were acceptable; come talk to me if you don’t understand why you
lost points.
Problem 3 (20 points total; Questions 3A-G): Listeria monocytogenes and Shigella flexneri are
environmental pathogens.
Question 3A: Compare and contrast the mechanisms by which these two species invade a host
cell. (~1/2 page, 3 points)
Shigella – “trigger” mechanism; direct contact with host cell not necessary; secretes plasmid encoded
invasions or modulators via TTSS which are translocated into host cell; leads to induction of lamellipodia
(ruffles) of host cell membrane and subsequent surround of cell and engulfment into endosome
Listeria – “zipper” mechanism; direct contact with host cell is required; bacterial adhesions bind receptors on
host cell membrane leading to formation of host cell envelope around bacterial cell with subsequent
engulment into endosome
Both mechanisms involve endosome formation via actin polymerization
Answers that included the above were generally given full credit.
The invasion strategies of these organisms are coupled with their ability to activate host cell actin
polymerization for their own motility within the host cell.
Question 3B. Compare and contrast the mechanisms by which each species does this. (~1/2 page, 3
points)
Shigella IcsA recruits and binds N-WASP; IcsA activates N-WASP which then binds and activates Arp2/3,
stimulating nucleation of actin.
Listeria ActA mimics host N-WASP to activate Arp2/3, stimulating nucleation of actin filaments.
Nancy Freitag has received an unusual cell line (DH cells) from another investigator. She infects
these cells with wildtype Listeria and notices, to her surprise, that although the Listeria cells can
penetrate and proliferate within this strange cell line, they are unable to spread from cell to cell. The Listeria
cell line is a wildtype, but it seems to be displaying a phenotype similar to an ActA mutant. As a rotating
student in Nancy’s lab, she challenges you to figure out what is going on. The first thing you do is stain
infected and uninfected DH cells with fluorescently labeled phalloidin, but you get no signal in DH cells,
although a normal phalloidin signal is seen in parallel control cells. However, an actin immunoblot reveals the
same amount of actin in control cells, and infected as well as uninfected DH cells.
Question 3C: Give two hypotheses for what is happening. (1 paragraph, 3 points)
Nancy has challenged you to figure out why the Listeria will not spread from cell to cell. The phalloidin stain
you do should tell you that there is no F actin which is a hint that the G actin is not forming F actin – i.e. that
no actin polymerization is taking place. The immunoblot told you that total actin is the same in all cell types –
another hint that something is wrong with actin polymerization. Hypotheses that were based on something
being wrong with the DH cell line’s ability to polymerize actin were given full credit. Ideally, things like an
increase or imbalance in thyomsin beta 4 or profilin (proteins involved in G actin regulation) etc. were
considered good hypotheses.
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Pabio 552, Midterm Exam
Thursday May 4, 2006
Question 3D: Design at least two experiments to test one of your hypotheses. Include expected
results and how you would interpret them. (~1/2page, 3 points)
Dependent on answer to 3C.
Ampicomplexan gliding motility and invasion into host cells is a tightly coupled active process
involving parasite cell actin polymerization. The complex nature of this process involves several
organelles unique to ampicomplexans, including micronemes and rhoptries.
Question 3E: Describe the role of micronemes in Toxoplasma gondii motiliy and host cell
invasion. (~1/2 page, 2 points)
T. gondii micronemes secrete adhesive proteins from the apical end of the parasite cell which are then “rolled
over” / translocated to the posterior end allowing the parasite to move along its substrate; upon contact with a
host cell, secretion of microneme proteins is upregulated, particularly that of MIC2, a transmembrane protein
that forms a connection between the host cell receptor and the cytoskeleton of the parasite in order to
facilitate motility and invasion. The C terminal domain of MIC2 is bound to a complex in the parasite
cytoplasm that contains aldolase, an enzyme involved in the cross-linking of actin filaments. The actin
interacts with MyoA, a single headed myosin molecule within the parasite that is linked to the inner membrane
complex and microtubules of the parasite. As MyoA changes orientation, motility and invasion result.
Question 3F: Of the three invasive forms of Plasmodium falciparum, one of them appears to lack
rhoptries. Which form is this and why does it tolerate the lack of rhoptries? (~1/2 page, 3 points)
Rhoptries secrete proteins called ROPs as the parasite enters the host cell. These are involved in the
formation of the parasitophorous vacuole, a protective niche for the parasite as it develops. It is necessary to
avoid lysosomal degradation within the host. P. falciparum ookinetes do not have rhoptries. They pass
through cells but do not set up the parasitophorous vacuole because they do not remain intracellular. Since
they don’t need the vacuole, they don’t need the rhoptries.
Question 3G. Describe two ways Neisseria gonorrhoeae use IgA proteases. (~1/2 page, 3 points)
The predominant antibody on human mucosal surfaces is human IgA 1, which can bind and agglutinate
potential pathogens. IgA 1 proteases secreted by N. gonorrhoeae are capable of cleaving the hinge region of
human IgA1, separating the antigen-binding fragment (Fab) and constant regions (Fc) and impeding
clearance of the bacteria/helping promote its colonization.
IgA 1 protease also cleaves LAMP1, a membrane spanning glycoprotein of lysosomes. Neisseria pilus and
porins complexes are able to alter host cell Ca++ levels (porins “poke” through cell membrane and cause
influx of Ca++ from extracellular environment), causing exocytosis of both lysosomes and endosomes,
resulting in the redistribution of LAMP 1 to the cell surface where it is cleaved by the bacteria. This leads to
lysosomal modification and promotion of intracellular survival of bacteria.
Other answers were acceptable, these were just a couple of the possibilities.
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