# Cholerae Autoinducer-1

Cholerae autoinducer-1 (CAI-1) has many important functions and applications within Vibrio cholerae. CAI-1 is a chemokine released by the bacterium V. cholerae and is a quorum sensing bacterium that is capable of producing a biofilm, and is fatal to humans upon contamination without immediate medical intervention; due to the cholera toxin with-which it produces. CAI-1 is known structurally as a (S)-3-hydroxytridecan and regulates expression of virulence factors. CAI-1 is a signaling molecule between individual V. cholerae microbes, that communicate to each other to react to the surrounding environment.

## Discovery

V. cholerae is the bacterium that houses the CAI-1 chemokine, and was incidentally discovered twice at different locations and by different people independent of the other. Once by Filippo Pacini in 1854 and again by Robert Koch in 1883. CAI-1 has no readily apparent recordable discovery date, but some of the earliest studies on autoinducers within and the quorum-sensing nature of V. cholerae was done by the Microbiology department of Harvard Medical school. Prior to the study of V. cholerae, researchers obtained data from a close relative, Vibrio harveyi. Interestingly, the accumulation of autoinducer in colonies of V. cholera decrease LuxO repression on hapR expression. Research seems indicative that hapR plays a role in protecting the cell from virulence factors, thereby helping the cell survive.

## Chemical Structure and Properties

CAI-1 is identified as (S)-3-hydroxytridecan. It is produced by multiple Vibrio species, functioning as an intra-genus signal. CAI-1 directly works with AI-2, which aids in interspecies communication. AI-2 is identified as (2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran borate. The two autoinducers are created by synthase CqsA and LuxS. CAI-1 has a molecular weight of 214.34 g/mol, with the formula C$_{13}$H$_{26}$O$_2$. It is synthesized by CqsA, a pyridoxal phosphate dependent amintotransferase enzyme, but its role is not fully known. CqsA can be identified by substrates (S)-2-aminobutyrate and deconyl coenzyme A.
Furthermore, an example of the effects of CAI-1 on it's capacity to grow and reproduce can be shown from the studies of the National Library of Medicine (NIH). NIH demonstrates that an abundance of autoinducer within the Vibrio cholerae genus can yield to the formation of a certain protease that upon activation leads to degradation of attachment sites, thereby allowing the bacterial strain to be free from restraints and be carried to another area for growth.

## Role in Biofilm Formation

CAI-1 plays a critical role in regulating biofilm formation in Vibrio cholerae by controlling the switch between biofilm production and dispersal through quorum sensing. At low cell densities and when CAI-1 levels are minimal, V. cholerae favors biofilm formation as a survival strategy in environmental reservoirs. Biofilms protect the bacteria from environmental stresses and allow them to persist in aquatic environments. As cell density increases and CAI-1 accumulates, it activates the quorum sensing pathways that inhibit further biofilm formation and promote dispersal, enabling the bacteria to spread and infect new hosts.
As part of the quorum sensing system, CAI-1 primarily functions to sense the presence of Vibrio species and integrates signals from multiple autoinducers. Research shows that while CAI-1 alone can initiate a response at low cell densities, both CAI-1 and the more abundantly produced autoinducer-2 (AI-2) must be present in sufficient quantities to fully repress biofilm formation. This dual autoinducer system allows V. cholerae to assess both its own population and the presence of other bacterial species in the environment, changing its biofilm behavior based on what’s around it.
These insights suggest that CAI-1 plays a key role in ensuring that V. cholerae only disperses from biofilms when sufficient bacterial density is achieved, and when the surrounding environment supports successful transmission and colonization. This makes CAI-1 a potential target for therapies aimed at disrupting biofilm formation to control cholera outbreaks.

## Role in Quorum Sensing

Quorum sensing is essentially the intercommunication between cells to interact and respond to it's environment.V. cholerae uses two autoinducers: cholera autoinducer-1 (CAI-1) and autoinducer-2 (AI-2). CAI-1 is the major quorum-sensing signal. Quorum sensing is the cell to cell communication that allows organisms to share information. This process can be used to control pathogenicity and biofilm formation, determine population size and modulate virulence mechanisms, and track changes in the cell or gene expression. Quorum sensing uses production, release, accumulation, and detection of autoinducers to do their work.
CAI-1 and AI-2 transduce information into a cell using a phosphorylation-dephosphorylation cascade affecting target gene expression. Specifically in V. cholerae, CqsS detects CAI-1 and LuxS detects AI-2. Information from these two autoinducers is transducer through LuxO to maintain levels of HapR transcription factor. HapR represses genes for biofilm formation and production of virulence factors. This is the goal of quorum sensing at high cell densities. At lower cell densities, without autoinducers, HapR is not produced.

## Signaling Pathway

In Vibrio cholerae, it is through quorum sensing that bacterial cells are able to behave and communicate based on the amount of bacterial cells present. When the concentration of CAI-1 reaches a certain limit, it binds to CqsS (a receptor for CAI-1). Transcription of virulence genes, including those responsible for the production of cholera toxin, is then activated because of the phosphorylation cascade caused by the binding of CqsS. Because of the ability for V. cholerae to adapt to its environment through CAI-1’s ability to facilitate communication, it increases the pathogenic abilities in the host.
The downstream signaling cascades that are triggered by CAI-1 binding to CqsS includes multiple regulatory pathways. CqsS undergoes autophosphorylation which involves the transfer of the phosphate group to LuxO, a response regulator. This activates LuxO, which inhibits the synthesis of HapR, a master regulator, through the expression of small regulatory RNAs (sRNAs). As the HapR levels decrease, there is upregulation of virulence factors and biofilm formation which enhances the ability for the bacteria to cause infection. The way that this system works allows for efficient adaptation to the host environment and for V. cholerae to infect more efficiently.

## Function in Vibrio cholerae Pathogenesis

CAI-1 is an important signaling molecule that has a role in regulating virulence factors, such as the cholera toxin, in Vibrio cholerae. This is done through a quorum sensing mechanism. As V. cholerae multiplies, genes of the virulence factors are expressed as amount of CAI-1 accumulates including the ones that encode for the cholera toxin. This toxin has the ability to disrupt electrolyte balance in intestinal epithelial cells which can lead to issues including severe diarrhea, which is known to be a common symptom of this toxin. In addition to the cholera toxin, there are other virulence factors such as surface adhesins, which are essential in helping the bacteria to adhere to the intestinal mucosa. This will consequently assist the bacteria in its pathogenic properties.
Because CAI-1 assists in facilitating biofilm formation, it plays a significant role in promoting colonization of the human gut. This provides a way for V. cholerae to avoid attacks from the host immune responses as well as any antibiotics. The signaling cascades initiated by CAI-1 enable V. cholerae to survive and infect in the harsh conditions of the gut. Observing the important role of CAI-1 in V. cholerae can highlight possible key targets for any future therapeutic intervention.