Vibrio cholerae VciB Promotes Iron Uptake via Ferrous Iron Transporters.
Mey et al. 2008. Journal of Bacteriology
The pathogenic gram-negative bacterium Vibrio cholerae infects 3-5 million people each year with the acute intestinal disease cholera. The ability of V. cholerae to survive in a mammalian host and its natural aquatic environment depends largely on its capability to acquire iron. Since nearly all organisms require iron for survival many have developed mechanisms to protect it from competitors or invaders like V. cholerae. In the human host lactoferrin, transferrin, heme, and other compounds bind iron with extremely high affinity in an attempt to control infection and protect iron supplies. To combat this V. cholerae has developed a number of iron transport mechanisms to steal iron away from iron-binding compounds within its host.
One V. cholerae iron transport system utilizes high-affinity iron chelators called siderophores. These molecules are synthesized by the cell and transported through the periplasm into the environment. They bind ferric iron with a greater affinity than the human iron-protecting compounds and are able to steal it from the host. Once bound to ferric iron, siderophores are then bound to outer membrane receptors where they are actively transported back into the periplasm by energy transduced from a variety of TonB dependent energy transduction systems.
Although siderophore facilitated iron transport is the primary mechanism of iron acquisition in iron replete environments there are many other iron-transport systems utilized by V. cholerae. Since siderophore use is limited to ferric iron (Fe3+) V. cholerae has employed techniques to capture ferrous iron (Fe2+). One such system is feoABC (Feo) which is comprised of three subunits. The primary subunit of feoABC is FeoB, which acts as a pore that allows ferrous iron to move across the inner membrane and into the cytoplasm of V. cholerae. The passage of iron into the cytoplasm must be closely regulated by V. cholerae due to the well documented toxicity of iron. The toxic effects of iron are caused by oxidative stress due its strong electron accepting capability. As a result a great deal of energy is spent controlling its concentration within the cell. Since small levels of iron are required for metabolism and DNA synthesis V. cholerae has developed a gene repressor known as Fur that is concentration controlled. As iron-levels increase in the cell Fur proteins are bound to iron which causes Fur to bind to promoter sequences upstream of iron-transport genes, effectively ceasing their activity.
Mey et al and many others hypothesize that many if not all major V. cholerae iron transport systems have been identified. Although this is the case many of these systems are not completely characterized and some other possible iron-transport systems may exist. The primary objective of Mey et al. in this research was to characterize a two-gene operon called vciAB, which had observable influence on iron acquisition systems in V. cholerae.
The inspiration to undergo this research was incited by mutational assays of V. cholerae
iron acquisition systems. As was previously mentioned, the primary iron acquisition system of V. cholerae under iron-limitation involves the use of siderophores. In order to identify and characterize other iron transport systems Mey et al utilized an E. coli mutant defective in siderophore transport. They then transformed a cosmid library from the classical strain of V. cholerae into this E. coli mutant and selected colonies that could survive in iron-replete media. Survival indicated the possession of at least one non-siderophore iron-transport system in the mutants. Dividing up the original cosmid showed the presence of an operon called vciAB that seemingly stimulated growth of the siderophore mutants in iron replete conditions. In order to initially characterize vciAB the researchers analyzed the sequence using a number of bioinformatic tools including BLAST searches against the National Center for Biotechnology (NCBI) and National Microbial Pathogen Data Resource (NMPDR) databases. By using the aforementioned tools they determined that vciA was similar to other TonB energy transduction dependent receptors in V. cholerae. These analyses signified that vciA was a potential iron-transport system with an unidentified iron-binding ligand. Further bioinformatic analysis indicated that vciA was most similar to a previously identified ferrichrome receptor FhuA. Ferrichrome is an iron sequestering compound secreted by a few genera of fungi. V. choleare has been shown to utilize this compound when it is in close proximity to the bacteria.
To test this hypothesized function mutational assays were performed on mutants lacking FhuA. When ferrichrome was supplied to these mutants no increase in growth was observed and it was concluded that vciA did not utilize ferrichrome as an iron source and further suggested that FhuA is the only ferrichrome receptor of V. cholerae. From everything I have read in the past this claim is very well supported by additional research. The second half of the vciAB operon, vciB, was also analyzed by bioinformatic tools. One striking result of this analysis was that the researchers found limited homologues to vciB in both the NCBI and NMPDR databases. This is very intriguing considering most other iron-acquisition systems in V. cholerae share similarity with gram-negative species of bacteria. For instance the siderophore secreted by V. cholerae, vibriobactin, is similar to that of the E. coli siderophore
enterobactin. Furthermore the ferrous iron transport system in V. cholerae is homologous to the main ferrous iron transport system in E. coli. Many other homologues to other iron transport systems exist in the V. cholerae genome so I find it unlikely that vciB is not a homologue to many other species as the authors claim. In their methods they do not include their parameters used for their bioinformatic tools, but one possibility is that the parameters for their search were too specific to identify potential homologues. Another possible explanation for this is that many
gram-negative bacteria genomes have not yet been sequenced. I would suggest revisiting this claim in a later paper if they have not done so already.
After performing bioinformatic tests on the vciAB operon the researchers tested the
growth of mutants lacking either vciA or vciB. Through growth assays (Figure 1) they found that vciA was relatively unimportant for growth in an E. coli enterobactin defective (ARM110) mutant carrying V. cholerae iron transport genes under iron-replete conditions. On the other hand vciB alone stimulated growth of ARM110 suggesting that vciB was important for growth, but vciA was largely unimportant for iron-acquisition by this operon alone. This was indeed interesting considering vciA was similar to a TonB dependent receptor, but it is likely that vciA is interacting with iron-acquisition pathways due to the presence of a Fur box just upstream of its coding sequence (Figure 1). This is something the authors should have expounded upon in their study, but did not do so. Since VciA failed to perform as a dedicated TonB receptor for VciB, but was similar in sequence to other TonB receptors, the authors thought that perhaps another TonB receptor present in E. coli was able to compensate for the loss of VciA. To test this hypothesis the authors utilized an E. coli mutant defective in TonB synthesis. What they found was that even when E. coli was defective in the TonB energy transduction pathway the presence of VciB allowed E. coli to survive in an iron-limiting environment (Figure 2A). Here they used an iron chelator called EDDA to create iron-limiting conditions. They understandably concluded that these results showed that VciB did not require a TonB dependent receptor to transport iron across the outer membrane. Furthermore, this assay showed that when iron was not limited in the environment (0 μg/ml EDDA) there was no significant influence on growth by the presence of VciB suggesting that it is only important in iron-limiting environments. This is indeed important
information considering that in the natural aquatic environment of V. cholerae iron exists primarily in an insoluble ferric form which significantly limits its availability. Furthermore, in the animal intestine iron is protected by a plethora or compounds which severely limit the amount of free iron that V. cholerae can acquire.
Since VciB did not follow many of the originally proposed models, the authors attempted to categorize its function as a complete stand-alone iron transport system. To do this they transformed a plasmid containing vciB into a mutant strain of Shigella flexneri (SM193w) that lacks iron transport systems necessary for growth in LB-media. The utilization of such a strain may seem a bit unconventional, but the utilization of this strain allows the research team to easily eliminate the presence of a number of natural iron-transporting systems in V. cholerae and E. coli. They found that the presence of vciB did not stimulate growth of SM193w which showed that VciB could not act as an iron-transporting mechanism by itself. To further confirm these results VciB was also tested for its ability to stimulate growth of an E. coli mutant which lacked
most iron transport mechanisms. The results of growth assays were similar for the E. coli mutant, confirming the inability of VciB to act as a stand-alone iron transport system. These assays do strongly suggest that it can not function by itself, but further analysis using V. cholerae should be performed when more complete information exists regarding the full extent of iron-transport systems utilized by V. cholerae.
To this point Mey et al. were largely unsuccessful in elucidating the function of VciB.
They hypothesized that VciB must be acting on another iron-transport system by up-regulating its function. They focused their attention on the ferrous iron transport system Feo. A mutation was introduced into ARM110 that disrupted the pore forming subunit of Feo, feoB. They found that in iron limiting conditions the presence of VciB was unable to stimulate growth of the feoB subunit (Figure 2B). Since it was unable to stimulate growth of feoB mutants it is most likely that VciB affects the function of ferrous iron transporters in some way. To further test these results the researchers analyzed the ability of VciB to stimulate a variety of both ferrous and ferric iron
transporters. They found that it stimulated not only Feo, but also a ferrous iron transporter in S. flexneri called Sit. VciB was not able to stimulate the activity of ferric iron transporters in V. cholerae or H. influenzae suggesting that it was acting solely on ferrous iron transport systems (Figure 3). Further supporting this claim the researchers found that the accumulation of radio- labeled iron was nearly threefold higher in S. flexneri containing Feo and VciB. However when feoB was removed from S. flexneri the accumulation of radio-labeled iron was not influenced by the presence of VciB (Figure 4).
In order to better understand the in vivo functionality of VciB Mey et al. inoculated
infant mice with a strain of V. cholerae containing VciB and a strain without VciB. They
hypothesized that VciB may play a role in virulence because ferrous iron is generally the most prevalent form of iron in the intestine. However, they found no significant influence on the virulence of V. cholerae in this model. It seems that the presence of other iron acquisition systems in V. cholerae influence were masking the effect of VciB in vivo. In the future mutations to the primary iron-acquisition systems should be created, and VciB should be re-tested for its functionality. I am relatively surprised the authors did not perform these assays.
This research provides some interesting insight on iron transport in V. cholerae, but
seems to be lacking some general relevance. Firstly, as they mentioned towards the beginning of the paper few homologues exist to the vciAB operon. In order to strengthen their study they should have eased their parameters with regards to gap penalties and looked for more possible homologues. Overall I found their methods sound, but there were areas they could have expanded their study. For instance, their iron limiting conditions were 5 μg/ml and 50 μg/ml. A few more concentrations between 5 and 50 would have been useful to determine the amount of resistance to iron-replete conditions VciB provides V. cholerae with. Another limiting factor of
this research was the inability to determine any in vivo role of VciB. Understandably this was only a first attempt, but the use of mutants lacking other systems would have supported this research more completely.
Insofar as their discussion is concerned I agree with most of their interpretations of the
data presented here. They present three hypotheses regarding the function of VciB. Out of these three hypotheses I agree that the most likely hypothesis is that VciB acts as a ferric iron reductase in the periplasm. Since it likely contains a periplasmic loop it is likely that this loop comes into contact with ferric iron and reduces it to ferrous iron. This would then allow the Feo system to uptake ferrous iron in greater amounts. They also try to explain VciA to little avail. It is interesting that VciA had no influence on the acquisition of iron, but since it is similar to other TonB dependent ligand receptors it is likely that VciA is a candidate for future studies.
In conclusion this research is interesting, but without more detail it seems very limited in scope. While it may be the first such report of a protein that directly interacts with ferrous iron transport systems, its insight may be limited to a small subset of bacteria. Without more data and more extensive studies I feel that the research is somewhat incomplete, but does not lack promise. More extensive mutational assays may shed light on the function of vciAB in vivo and provide more relevance to this study.