Today's journal club was a discussion of the paper
Self-Regulation of Candida albicans Population Size during GI Colonization." by White SJ, Rosenbach A, Lephart P, Nguyen D, Benjamin A, Tzipori S, Whiteway M, Mecsas J, and Kumamoto CA. in PLoS Pathogens 2007 Dec 7;3(12):e184.
For full disclosure I chose and presented this paper. I picked this paper for several reasons. First, I am a strong supporter of open access journals, such as PLoS pathogens so I wanted to advertise. Second, the area is of interest to me and hits on an important topic. Third, there are a couple of potential teaching points, one of which hits on a point we covered last week.
Candida albicans is a commensal (this effectively means the organism does not harm or benefit the host, although this is a point I'll try to touch on in another post) in essentially everyone.
C. albicans lives throughout your digestive system including your mouth, esophagus, and intestinal tract and in the vaginal tract.
C. albicans is generally well known as the causative agent of thrush (oral candidiasis) and vaginal yeast infections. It is also the primary cause of diaper rash (so antibiotic diapers generally are useless because they kill bacteria not fungi, but they market well to young mothers). However, unlike many organisms, you don't find
C. albicans in the environment, in other words,
C. albicans' natural niche is the human mucosa.
C. albicans does infect other mammals particular in a zoo environment, however it does not appear to naturally colonize these other mammals. While mucosal infections, like thrush and vaginitis, are problematic, they are generally not life threatening. However, if
C. albicans enters the bloodstream, it can disseminate to virtually every organ, including kidneys, liver, bones, heart, and brain, and kill you. This disseminated infection is called systemic candidiasis. Now these systemic infections require a host with an impaired immune system, including organ transplant patients and chemotherapy patients. Because
C. albicans can cause infections if conditions are favorable, it is considered an opportunistic pathogen. What's interesting, is that the
C. albicans that cause these systemic infections are the
C. albicans organisms that normally reside in you gut. It is the
C. albicans you are already carrying that causes these life-threatening infections. This is different from many other organisms, like
E. coli, where the flora within you is generally not disease causing but variants from the environment are. Ok, long introduction to get to the main problem being addressed in this paper. We know very little about how
C. albicans colonizes and grows as a commensal or how it escapes from the GI tract to the bloodstream as an opportunistic pathogen.
This paper uses a piglet model and mouse model to look at gene expression changes in
C. albicans when it is in the GI tract compared to a control laboratory environment. They identify several genes including
EFH1 which was expressed more in the GI tract, compared to the oral cavity or laboratory conditions. They then go on to study
EFH1 in a little more detail. However, I want to point out a couple of problems I had with the aspect of the paper. First, the number of samples used was very low and only from the pig model. In fact, they only used 2 oral samples and 1 intestinal sample! An n of 1 does not a strong position make. That's not to say the results are incorrect just tenuous at best. It would have made me happier if they had also looked in the mouse GI samples to corroborate the pig data. Second, they compare the
C. albicans from the pig model with laboratory grown cells. This is always going to present problems because the conditions are fundamentally different. Here, the onus is on the researchers to make things as close as possible, and I thought they fell short of the mark. For instance, the authors grew
C. albicans in the laboratory at 34°C whereas the body temperature of the pig is ~39°C and the mouse is ~37°C, so there is a 3-5 degree difference (yes, this can be significant). What bothers me here is that you can grow the cells in the laboratory at whatever temperature you want trivially. So Im left wondering, what the hell? Next, they grew the cells in he laboratory in a yeast extract, bacto-peptone, sucrose solution. Not what I expect the gut of a pig or mouse has. Some alternatives could be an extract from the grain/oats/or whatever the animals are fed or an infusion from an animal source, such as beef heart (commonly used) or even pig intestine! Finally, they use logarithmically grown cells in the lab, whereas the
C. albicans in the animals are almost certainly primarily in stationary phase. In fact, the authors go on to show that all but one of the genes they identified are expressed preferentially in stationary phase cells compared to logarithmically grown cells. In short, the part of the paper identified
C. albicans genes expressed in the animal because
C. albicans in the animal are not rapidly dividing.
The authors go on to characterize a gene called
EFH1, which encodes a transcription factor of unknown function. They take a genetic approach and delete the gene and then look to see the effect. Surprisingly, they find that the
efh1∆/∆ mutant colonizes the mouse intestinal tract better than the wild-type
EFH1/EFH1 strain. This is surprising because we generally think more is better. Thus, we expect that wild-type
C. albicans grows the best in the host and the only phenotype we would see is less growth in the host, not more. I mean how could a mutation make the cells grow better!?!?! There's lots of reasons, such as loss of this gene makes the cells grow better in this specific system (which is not relevant to the natural system), but in other important ways not addressed in this model the mutant is dead on arrival. So the positive selection observed in this model would be balanced by the strong negative selection under other conditions.
Ok, we have an interesting unexpected phenotype, what to do now? From my last
journal club , you probably know the answer is a complementation test. Yes, put a wild-type copy of the
EFH1 gene into the
efh1∆/∆ mutant and look to see if the phenotype is restored. Well the authors did this and found that the
efh1∆/∆ +EFH1 strain actuall grew worse than the wild-type strain. In other words, based on growth in the intestine of a mouse
efh1∆/∆ >
EFH1/EFH1 >
efh1∆/∆ +EFH1. In the perfect world, you would expect
efh1∆/∆ >
EFH1/EFH1 =
efh1∆/∆ +EFH1. So why the discrepancy? Well for one the researchers used a strong promoter to express the complementing
EFH1. So the amount of
EFH1 mRNA, and thus Efh1 protein, will be different. This could be the issue and is the one favored by the authors. It provides evidence that suggests the amount of Efh1 protein controls how much
C. albicans growth occurs in the intestine. This is really cool!
With a really cool result comes a higher bar than would be seen with an expected result. So do the authors reach a higher bar? In my opinion no. For starters they never showed that the amount of Efh1 protein (or mRNA for that matter) is actually any different under these conditions. This seems like a fairly obvious requirement. Also, the genetics were not as rigorous as I would expect. First, they introduced their complementation construct into the endogenous locus and it didn't work. They note that others have seen this problem, which means its ok. Nice sloppy reasoning there, "others couldnt figure it out, so why should we." They also use express
EFH1 from the promoter of another gene. This is often done, but its usually for a scientific reason. The authors provide no justification for this, which is odd. Finally, the strains they compare are not appropriate.
Their mutant strain has the following genotype:
arg4∆/∆ his1∆/∆ ura3∆/∆ efh1::ARG4/efh1::HIS1 leu2::URA3/LEU2. This strain is deleted for
ARG4,
HIS1, and
URA3, two markers
ARG4 and
HIS1 are used to disrupt
EFH1 and
URA3 is introduced into the
LEU2 locus to make the strain prototrophic again. Since
C. albicans is diploid this strain has 1 copy of
ARG4,
HIS,
URA3, and
LEU2; 0 copies of
EFH1.
Their overexpression strain has the following genotype:
arg4∆/∆ his1∆/∆ ura3∆/∆ efh1::ARG4/efh1::HIS1 adh1::P-EFH1::URA3/ADH1.
This strain is deleted for
ARG4,
HIS1, and
URA3, two markers
ARG4 and
HIS1 are used to disrupt
EFH1 and
URA3 is introduced into the
ADH1 locus along with the complementing
EFH1 allele. Since
C. albicans is diploid this strain has 1 copy of
ARG4,
HIS,
URA3,
ADH1, and
EFH1; 2 copies of
LEU2. Also, note that
URA3 is expressed from different sites in the genome.
So the mutant and complemented strain they are comparing differ in
EFH1 (important, this is what you are testing);
LEU2 and
ADH1 copy numbers and site of
URA3 integration (inportant, these can confound your results since they are genetic differences). So we have the authors hypothesis Efh1 protein levels control
C. albicans growth levels in the intestine. But we can consider other hypotheses, how about loss of one copy of
ADH1 reduces
C. albicans growth in the intestine and there is no complementation whatsoever. If we inhibit cell wall synthesis in the
efh1∆/∆ mutant, growth will be reduced but that doesn't mean Efh1 protein promotes cell wall synthesis. Based on the way the experiment was done, I do not find a strong evidence that the authors actually did get any complementation. This gets back to an earlier premise I made in the first journal club, when you get unexpected or novel results, the bar is higher to ensure you are likely correct. This paper establishes some new approaches to study
C. abicans intestinal colonization, but fails to reach the level of rigor to establish the surprising result that Efh1 protein controls the levels of growth of
C. albicans in the intestine.
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