Vibrio cholerae is an infamous bacteria, first identified in 1854, though the effects of the microbe had been observed for decades and three cholera pandemics before the causative agent of the plague was observed. For thirty more years, this microbe would not be linked to the disease it caused in humans until 1884, when Robert Koch insisted the comma-shaped bacteria were linked to the disease that had ravaged communities around the world. This discovery is intimately tied to the history of epidemiology as the idea that the disease could be contracted from contaminated water led to some of the earliest environmental tracking related to disease. You can learn more about the discovery of V. cholerae as a pathogen in this PBS broadcast.
An artist’s rendition of V. cholerae, with the flagellum (tail) and pili (hairlike filaments) visible on the cell. [source]
Cholera is an infection caused by a toxin from the Vibrio cholerae bacteria when it colonizes a person’s small intestine, leading to severe gastrointestinal symptoms, most commonly diarrhea which can result in deadly dehydration. The disease affects millions of people worldwide and seven cholera pandemics, including an ongoing one across Central and Eastern Africa, have occurred. Transmission of the bacteria from the environment to humans is often through contaminated food or water, since V. cholerae thrives in freshwater often along with populations of plankton or aquatic copepods.
We often think of bacteria as inert, passive denizens of the environment around us, existing on all surfaces but at the mercy of the larger macroscopic world. However, the reality is quite the opposite, because in order to survive, bacteria have to acutely sense changes in the environment around them and act accordingly. Bacteria accomplish this sensing through the use of signal transduction pathways. Environmental change is sensed via a chemical interaction with a receptor protein on the surface of the bacteria, which eventually triggers changes in gene expression, causing the whole organism to react.
Vibrio cholerae is a master of the aforementioned environmental sensing, which allows it to survive in vastly different contexts, from water surfaces to the intensely acidic environment of a person’s stomach. Vibrio is also proficient at horizontal gene transfer, a method of exchanging DNA between individual bacteria instead of between parent and offspring. Genes that create more virulent strains of Vibrio can be acquired through the environment, and the bacteria have a specific mechanism that gives them a higher chance of acquiring beneficial DNA from their environment, using their top-of-the-line sensing abilities. You can watch a video to learn more about the different types of horizontal gene transfer here.
Because the bacteria proliferates in aquatic environments rich with other organisms such as plankton and crustaceans, the presence of chitin in the water is a telltale sign that other beneficial and genetically-unique strains of cholera may be present. Chitin is a material commonly found in the protective shells or skeletons of water-dwelling invertebrates. Different strains of Vibrio can also form biofilms, large 3-dimensional communities of interlocked bacteria, on surfaces rich in chitin. Thus, Vibrio has devised a way to sense the presence of chitin and uses it to change its entire gene expression profile to make the bacteria capable of, or competent for natural transformation. Transformation is the uptake of free DNA from the environment into the bacteria’s own genome. This is a method of horizontal gene transfer, where beneficial genes from one individual can be added into the genome of another individual cell without the process of cloning through cellular division.
The bacteria accomplishes this impressive task with two transmembrane proteins called ChiS and TfoS. Transmembrane proteins are positioned within the cell membrane, and are able to access and sense both the internal and external environment of the cell.
ChiS activates Vibrio’s “chitin utilization program,” which allows the bacteria to take in chitin from the environment and use it in catabolism to gain energy products from the molecules. However, it was also shown to work with TfoS to activate the genes for transformation, and researchers at Indiana University set out to discover exactly how this two-protein gene regulation system works.
The researchers engineered bacterial strains that were lacking either of these proteins and compared them to a wild-type strain with both, and they also fluorescently tagged the promoter region for the creation of a regulatory RNA (PtfoR) that activates genes for natural transformation. They then exposed the bacteria to artificially high chitin levels, conditions that would hypothetically induce competency in the cells. They used fluorescence microscopy and imaging to quantify the expression of PtfoR which would tell them whether the downstream genes for natural transformation were going to be expressed.
They first examined whether ChiS was involved in PtfoR activation or just chitin catabolism in Vibrio cholerae, and the researchers found that in a strain lacking the ChiS protein, no PftoR fluorescence was seen, signifying the absence of PtfoR. They also found that transformation would not occur in strains lacking ChiS, demonstrating that the protein is necessary to make the bacteria competent for DNA uptake. Next, they wanted to figure out whether ChiS and TfoS worked together directly or had an indirect influence on one another.
This figure is intimidating, but let’s break it down step by step. The two proteins were tagged with two different fluorescent markers, and one of them is fused to a protein from Caulobacter called PopZ, which localizes to the pole of the cell, taking the fused protein with it. If the two Vibrio proteins bind each other directly, then the unfused protein will also relocate to the pole when PopZ is added. The system is analogous to a fishing hook (PopZ) with bait (ChiS/TfoS) and a fish biting onto the hook (TfoS/ChiS and localization of the proteins).
TfoS was tagged with red Cherry fluorescence and ChiS was fused with the PopZ protein. In the middle column, localization to the poles can be seen after PopZ introduction in both the fluorescence intensity quantification (bottom) and in the red fluorescence shining brighter in the poles in the 2nd row. These results allowed the researchers to conclude that ChiS and TfoS localized together, meaning they directly bind with each other as part of their mechanism of action in the bacteria.
The POLAR assay the researchers performed is a little bit like molecular fishing.
The researchers performed many more experiments to uncover the specifics of how exactly these two proteins regulate PtfoR expression in the presence of chitin. They discovered that the two proteins are completely inactive without environmental chitin binding, supporting that the proteins are sensors in environmental response to chitin by the bacteria. They also managed to uncover where in the cell the DNA promoter binds to ChiS (at the bacterial membrane) and then found out that TfoS subsequently binds to ChiS, localizing all 3 of these factors at the membrane. TfoS then also binds to PtfoR and induces transcription of the small RNA that allows for gene expression changes that make the bacteria competent for natural transformation.
The value of uncovering mechanisms behind horizontal gene transfer such as the transmembrane transcriptional regulators examined in this study is vast. Understanding how bacteria are able to respond to their environment and how they incorporate other cells’ DNA into their own genome is a key target for drug development against bacterial infections. It also allows scientists to better understand the environments that are conducive for gene uptake, which can lead to antibiotic resistance, an ongoing global health crisis. The researchers also named studies of other biomolecule interactions (ie. DNA) at the membrane as a future direction for understanding environmental sensing.
Vibrio cholerae is a successful bacteria and pathogen precisely because it is so well attuned to changes in the environment, and this pathway of interacting proteins is just one of countless examples why the bacteria continues to thrive in waterways and cause disease throughout the world. It takes advantage of the presence of other Vibrio bacteria, taking up free DNA from the environment, which can be beneficial for survival and even cause new pathogenic strains to be created. Even the smallest forms of life on Earth must sense the changes occurring around them and improvise, adapt, and overcome to ensure their survival in the microbial world.
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