Aliivibrio Fischeri and the Role of Quorum Sensing

Introduction

By Elizabeth Matteri

Aliivibrio fischeri are gram-negative Proteobacteria that are capable of producing bioluminescence. Although previously termed Vibrio fischeri, researchers proposed a reclassification of the species and changed the name in 2007 (Urbanczyk et al. 2007). The bacteria, while found throughout the ocean, colonize the light organs of specific kinds of squid and fish species. They can also be found on decaying matter in the ocean (Schaefer et al. 1996, Boettcher et al. 1990).

Figure 1. The light organ of the Hawaiian Bobtail Squid (Euprymna scolopes) by Edward G. Ruby and Kyu-Ho Lee (1998). [7] http://www.ncbi.nlm.nih.gov/pmc/articles/PMC106330/

The most understood relationship that Aliivibrio fischeri forms is with the Hawaiian Bobtail squid (Euprymna scolopes). This squid is usually found near the shore, is nocturnal, and has two bilobed light organs on its ventral side (Wei et al. 1989). These light organs are made up of sacks, or crypts, that hold the Aliivibrio fischeri as well as two lenses and a reflector (Wei et al. 1989). This symbiotic relationship is a key predator avoidance strategy used by the squid, as the light that the bacteria emit is used to counterilluminate the squid against moonlight or starlight shining into the ocean, making them less visible to predators. In other words, the ventral side of the animal is brightened and appears more similar in color to the lighter water nearer to the surface, so a predator below the squid is less likely to notice its presence due to camouflage (Jones et al. 2004). Despite the symbiosis, it is important to note that both the bacteria and the Hawaiian Bobtail squid are capable of surviving independently of each other (Lupp et al. 2003).

Due to the many years of research that have focused on this symbiotic relationship, Aliivibrio fischeri and the Hawaiian Bobtail squid are model organisms for the ways in which bacteria can colonize a host and influence the development of a host (Visick et al. 2006). For many years, the way in which the bacteria would colonize the light organs of the squid was very poorly understood, but more recent studies are beginning to bring this process to light (Visick et al. 2006). Also, because of how the bacteria use quorum sensing as an integral part of their colonization of the host squid, Aliivibrio fischeri are a model organism used to better understand the role of quorum sensing in general, and they will hopefully lead to the development of quorum sensing inhibitor drugs in the future that can help prevent pathogenesis by other bacterial species (Visick et al. 2006). Not only this, but a quorum sensing inhibitory drug could provide an alternative form of treatment to antibiotics, as many human pathogens are becoming drug resistant—which would help save many lives (Suga et al. 2003).

Quorum Sensing and Bioluminescence

Quorum sensing is a type of signaling pathway that is used by gram-negative and certain gram-positive bacteria to regulate gene expression. In all forms of bacteria that use quorum sensing, the system is dependent on cell density (Schaefer et al. 1996). Gene regulation occurs as the bacteria release autoinducers, which are signaling molecules that increase in number with cell density. Quorum sensing can be used for a wide range of different processes including the formation of a symbiosis, virulence, the production of antibiotics, the formation of biofilms, motility, and sporulation, amongst others. Although many components of a quorum sensing pathway are unique, they all share the common aspect that they allow for communication that regulates gene expression, and further allows the behavior of all of the cells in a community to be impacted (Miller et al. 2001). Interestingly, the communication is not limited between bacterial cells, but can also impact the cells of a host organism (Visick et al. 2000).

The quorum sensing system in the relationship between Aliivibrio fischeri and the Hawaiian Bobtail squid is the best studied in the world, and is considered the model for understanding how a quorum sensing pathway works. The pathway regulates the genes that cause luminescence. In order for the systems to be successful the high density of bacterial cells in the light organs of the squid are essential (Schaefer et al. 1996). Overall, it is the lux operon that controls quorum sensing in these bacteria, which is made up of a number of different genes. Within this operon there are two units: luxCDABEG and luxR (Lupp et al. 2003). The two main genes involved in any quorum sensing pathway are the luxI and luxR genes. While the luxI gene is responsible for the synthesis of the autoinducer, called N-acyl homoserine lactone (HSL), the R gene is responsible for coding the transcription factor, which then responds to HSL. Aliivibrio fischeri have the genes termed luxI and luxR, which are responsible for synthesizing 3-oxohexanol HSL that then binds to the luxR product. The high cell density required for this pathway to work is necessary, because a critical concentration of the 3-ocohexanol HSL autoinducer needs to be built up for it to bind to the luxR product (Schaefer et al. 1996).

Figure 2. Aliivibrio fischeri showing bioluminescence. http://assets.inhabitat.com/wp-content/blogs.dir/1/files/2011/12/Vibrio-fischeri-Project-537x409.jpg

Once critical population density of the Aliivibrio fischeri population has been reached within the light organs of the squid, bioluminescence can occur. An enzyme called luciferase “uses molecular oxygen from the surrounding environment to oxidize both an aliphatic aldehyde and a reduced flavin mononucleotide” (Visick et al 2000, 4578). One of the products of this reaction is an intermediate that is very unstable, and this molecule is responsible for emitting a photon that is a blue-green color, thus producing bioluminescence (Visick et al. 2000). Although the squid do not have control over this reaction, they can control whether or not they show the light. This is done with a shutter that is located within the light organ (Wei et al. 1989).

Also, the squid can eject the bacteria from the light organ, which is actually done approximately every 24 hours. These expulsions can occur, because the light organ has pores that are open to the ocean water around them. Up to 90-95% of the Aliivibrio fischeri within the organ will be expelled by the host squid. The squid only do this expulsion at the onset of the daylight hours, when they return to hide in the sand after being in the open ocean water at night. Since the animals are hidden in the sand, bioluminescence is no longer necessary. After the squid has expelled the bacteria, it then needs to regrow them from the few remaining in the crypt, or from bacteria in the surrounding water in a 12 hour period, so that the light organ will be fully functional by the next night. Much about this process is not fully understood, since the metabolic costs of maintaining the bacteria in the crypts are not known. There is, however, the possibility that the squid expel the Aliivibrio fischeri in order to try and select a more competitive symbiont strain (there are many different strains of the bacteria) (Ruby et al. 1998).

Although the luxI gene is the main protein that synthesizes the autoinducer in the quorum sensing system of Aliivibrio fischeri, there is also a second, less understood gene termed AinS. In their study, Lupp et al. (2003) investigated some of the differences between both the luxI and AinS components of the quorum system. In order to do this, the researchers used both a strain with an AinS mutation, and a strain with an AinS-luxI mutation. The researchers found that while the luxI gene is what primarily drives the production of bioluminescence in the squid, the AinS protein was more important when the bacteria were being grown in culture. Lupp et al. (2005) discovered that the Ain gene allows the Aliivibiro fischeri bacteria to produce bioluminescence at lower densities. They found that mutant strains of Aliivibrio fischeri that contained the luxI gene, but not the AinS gene, were actually not able to colonize the host effectively (Lupp et al. 2003).

Quorum Sensing and Host Interactions

Figure 3. Image of the Hawaiian Bobtail Squid showing bioluminescence produced by Aliivibrio fischeri http://featuredcreature.com/wp-content/uploads/2012/10/51fb4cd584d7a2ce200809fee3d34d8b2.png

Not only are the luxI and luxR genes important for causing the production of bioluminescence, but they also play an important role in maintaining the symbiotic relationship between the Aliivibrio fischeri and the host squid. In fact, the ability for bioluminescence to occur in this symbiosis is dependent on cell-to-cell interactions between both the bacteria and the host, which thereby impacts host development (Visick et al. 2000). After hatching, the squid are born without any host bacteria, and must acquire them from the water around them. However, the exact mechanism by which the squid obtain Aliivibrio fischeri and not other bacteria is not yet fully understood (Wei et al. 1989).

In one particular study, aimed at trying to understand how the bacteria impact the host, Visick et al. (2000) introduced bacteria strains with mutations of either the luxA, the luxI, or the luxR gene. They found that even though colonization of the light organ was possible at first, each mutant strain produced less than 0.01% of the bioluminescence that the wild type parent strain normally produces. The researchers found that because these mutated strains could not properly maintain bioluminescence, the bacteria could not maintain a level of colonization that was normal. Lastly, Aliivibrio fischeri actually induce morphological changes to the squid host, and none of the mutant strains were capable of causing these changes. The wild type strain induces a swelling in epithelial cells around the light organ during colonization. In other words, the wild type Aliivibrio fischeri cause differentiation of host cells, and induce swelling by increasing the cytoplasm of the surrounding epithelial. This is beneficial for both the bacteria and the squid, because it causes the crypt of the light organ to shrink in size, making the bacteria pack closer together. As stated earlier, high population density in the organ is what allows for the bioluminescence to occur so brightly. Interestingly, the researchers found that the squid are extremely selective for Aliivirbrio fischeri out of all bacteria in the surrounding water, but they note that there are different strains that have either increased or decreased luminescence compared to others. They conclude that the different strains of bacteria compete with each other in order to colonize the light organ, and that those strains with greater luminescence will out compete others.

Similar to Visick et al.'s (2000) findings that luminescence is an important factor in the ability of Aliivibrio fischeri to successfully colonize the light organ of the host squid, Lupp et al. (2005) found that the quorum sensing pathway plays a crucial role in both early and late colonization. Lupp et al. (2005) were again interested in how both the lux and the ain quorum sensing pathways that Aliivibrio fischeri employ impact colonization. Interestingly, the researchers found that if they inactivated only the ain gene and not the lux gene, then the initiation of the symbiotic relationship between the bacteria and the host was delayed. Also, the lux system does not seem to be particularly important in the beginning stages of the symbiotic relationship, but plays a greater role in later colonization. This data reflects those that were collected by Visick et al. (2000). Both groups of researchers determined that colonization would occur normally if the lux pathway was removed, however, bioluminescence levels were reduced. According to Lupp et al. (2005) this gives strong evidence that the ain pathway is important for other reasons than bioluminescence. It is thought that the reason why the ain pathway is so crucial to the induction of colonization, because it codes for a number of genes that are involved in the motility of the Aliivibrio fischeri bacteria. Overall this study is a good indication that both quorum sensing pathways play an important role in maintaining a symbiotic relationship between the bacteria and the host squid.

Quorum Sensing and Pathogenesis in other Bacteria

Using Aliivibrio fischeri as a model organism for quorum sensing has had a far-reaching impact on the world of microbiology, because quorum sensing pathways are used by so many other species of bacteria. Interestingly, quorum sensing often plays a key role in pathogenesis, so gaining a better understanding of how the pathways work can lead to treatment possibilities in the future. Often quorum sensing is used by pathogens as the autoinducers can help to regulate virulence genes when the bacterial population has become large enough while in contact with a host (Visick et al. 2000, Suga et al. 2003).

Schaefer et al. (1996) conducted a study that could one day lead to quorum sensing inhibitory drugs that could help prevent disease. The researchers examined the ability of different luxR analogs to inhibit the proper binding of the autoinducer. For their experiment, they actually used E. coli as their study organism, and then determined if the Aliivibiro fischeri autoinducer could be used to turn on bioluminescence genes within the strain. The main purpose of this study was to learn more about the relationship between the autoinducer and the luxR gene, in order to see how an inhibitory drug might be used in the future. Schaefer et al. (1996) found that all of their analogs were able to bind with the luxR binding site, and one almost had the same affinity for the binding site as the real HSL autoinducer (however, not all analogs were nearly this successful). Many of the analogs were also able to bring about bioluminescence, although not as strongly as the original HSL. However, Schaefer et al. (1996) also found that five of the analogs that they used were able to inhibit bioluminescence by over 80% (Figure 4). In order to determine this, the they compared these five analogs E, J, L, M, Y to 20nM of the autoinducer with differing concentrations. First, they compared the analogs when no autoinducer was present, then in a 1:1 ration of analog to autoinducer, then a 5:1 ratio, and finally a 10:1 ratio. These analogs were chosen, since they were the ones that bound best with the luxR binding site. Overall, this study is particularly interesting, because it shows how the binding site of the luxR gene is a potential drug target.

Although Aliivibrio fischeri is not harmful to humans, we now know of a number of different pathogens that employ quorum sensing that are harmful. These include, but are not limited to Pseudomonas aeruginosa, Burkholderia cepacia, Salmonella typhimurium, and Yersinia enterocolitica. The quorum sensing system of Pseudomonas aeruginosa is one of the best understood quorum sensing systems employed by bacteria that harm humans (Suga et al. 2003).

In their review, Suga et al. (2003) explore the quorum sensing pathway of P. aeruginosa bacteria and potential molecular drug targets that could be developed to help prevent pathogenesis. Similar to all other quorum sensing pathways, both a protein that can synthesize an N-acyl-homoserine lactone (asyl-HSL) is necessary as well as a protein that the produced autoinducer can bind to (called simply the R protein, in this case). Unlike the quorum sensing pathway of Aliivibrio fischeri, the R proteins of P. aeruginosa cause genes responsible for coding virulence factors to be induced. Another key difference between the two quorum sensing systems, is that P. aeruginosa has two autoinducers instead of just the 3-oxohexanoyl HSL. These are N-93-oxo-dodecanoyl)-HSL and N-butanoyl HSL. According to the researchers, approximately 600 genes of P. aeruginosa are controlled by the quorum sensing pathway, including the virulence genes, and other genes that allow the bacteria to survive within the host.

Suga et al. (2003) focused their review on both agonisst and antagonists of the autoinducers in an attempt to better understand how quorum sensing inhibitory drugs could be synthesized. They conclude, based on a number of different studies, that a number of different agonists that are effective autoinducer analogs have been developed. Although these are helpful in terms of learning more about the interactions between the autoinducers and the luxR protein, a better inhibitory drug might be made using autoinducer analog antagonists (although they are currently posing more of a challenge to synthesize). Although it did not function in wild type P. aeruginosa, an antagonist was able to disrupt the quorum sensing system and thereby also the architecture of the biofilm made by the bacteria in a strain that had exogenous autodinducers. In conclusion, the authors indicate, as did Schaefer et al. (1996) in their experiment, that the best target of an inhibitory drug would be creating an analog autoinducers that disrupts binding to the R protein. Although some compounds like the antagonist that disrupted the architecture of the biofilm that P. aeruginosa produce, a drug that could entirely prevent the biofilm would be more promising. Also, it would be essential that the synthesized drug could disrupt the quorum sensing pathway in a wild type strain.

Suga et al. (2003) suggest that because the quorum sensing of P. aeruginosa is used to create a biofilm, which then makes antibiotics less effective, inhibiting the production of a biofilm by inhibiting the quorum sensing pathway could lead to a new an more effective drug. The development of this kind of drug is particularly important, because more strains of P. aeruginosa are evolving antibiotics resistance. According to the Centers for Disease Control and Prevention, multidrug resistant strains of P. aeruginosa are responsible for up to 400 deaths in the United States currently, and this number is expected to increase in the future unless a new treatment can be designed. A quorum sensing inhibiting drug has the potential to truly alter how not only P. aeruginosa bacterial infections are treated, but could also lead to an overall shift in how any bacteria that employs quorum sensing to code its virulence genes is treated.

Conclusion

The relationship between Aliivibrio fischeri and the Hawaiian Bobtail squid has opened doors for scientists who wish not only to understand how quorum sensing can impact the relationship between bacteria and a host, but also for the study of medicine and the possibility for inhibition of pathogenesis. By continuing to study this symbiosis, which seems to act as an infection in some ways, but does not actually harm the host, new insights can be gained about how the communication between different cells impacts a host. Although much is already known about this system, there are still many only partially answered questions that must be addressed in the future, including how the squid are able to be so selective for just Aliivibiro fischeri strains, and what exactly allows the bacteria to colonize the light organs effectively. Most importantly, the development of quorum sensing inhibitory drugs could bring about dramatic changes in the way that some human affecting pathogens are treated, as more strains of harmful bacteria are evolving to be antibiotic resistant (CDC, 2014).

References

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Edited by student of Joan Slonczewski for BIOL 238 Microbiology, 2009, Kenyon College.