The symbiosis, or mutually beneficial relationship between organisms, between the Hawaiian bobtail squid and bioluminescent bacteria Vibrio fischeri gives researchers an opportunity to study and understand interactions between microorganisms and their hosts in a natural environment. V. fischeri colonizes a specialized organ in the bobtail squid meant for producing light (light organ). This begins a lifelong symbiosis in which each morning, the squid expels 95% of the bacteria and other waste, leaving 5% to keep growing in the organ. The researchers analyzed the proteins in the expelled bacteria and waste to understand how both the bacteria and the squid contribute to symbiosis. The researchers identified 1581 proteins, with 870 coming from the bacteria and 711 coming from the squid. Squid proteins showed the role of the immune system and reactive oxygen species (ROS), or chemically reactive compounds containing oxygen, in the symbiosis. Bacterial proteins gave the researchers a better understanding of how quorum sensing, bacterial movement, and detoxification of ROS in the light organ.
The researchers wanted to understand what kind of proteins were involved in the symbiotic relationship between the Hawaiian bobtail squid and Vibrio fischeri.
The symbiosis between the Hawaiian bobtail squid and bacteria Vibrio fischeri is a well-studied model for understanding interactions between microorganisms and their hosts. Soon after a bobtail squid is born, V. fischeri colonize a specialized light organ. In the light organ, the bacteria are connected to the external environment through pores and ducts, through which the squid expel bacteria daily. This expulsion is linked to the nocturnal hunting activity of the squid. At night, the bacteria are at their highest density and produce light which prevents the squid from casting a shadow, enabling them to be better predators and escape predators of their own. At dawn, the squid expel 95% of bacteria and the remaining bacteria grow again by the next night. This expulsion helps regulate the number and viability of bacteria in the light organ and increases the concentration of V. fischeri in the environment, which allow future squid to be colonized by the bacteria. The expulsion mechanism is pictured in Figure 1.
The substance expelled from the light organ is a thick, paste-like substance made up of bacteria and other waste from the host, such as shed hemocytes and epithelial cells. Previous studies have investigated the cellular and biochemical substances in this expulsion, as well as genetic expression involved in the expulsion process. Genetic analysis has shown there are a number of genes involved in the expulsion process, and that the expression of these genes was most active just before and after dawn when the expulsion occurs.
In this study, the researchers attempted to characterize the kinds of proteins (proteomics) involved in the expulsion process. Proteomic studies for the expulsion process are very few and limited, and have been focused on colonization of the light organ. Here, the researchers used multidimensional protein identification technology (MudPIT) in addition to 2D- and 3D-gel electrophoresis to describe squid and bacterial proteins involved in the expulsion process. Analyses showed immunity proteins and proteins involved in detoxification for the squid, and bacterial proteins involved in quorum sensing and movement. The analyses also showed that many proteins remain uncharacterized in the squid-bacteria relationship. Identification of squid and bacteria proteins are an important first step in understanding how these organisms maintain such a highly specific relationship.
Expulsion samples were analyzed using a number of proteomic techniques. 1D- and 2D gel electrophoresis revealed that the expulsion is made up of a complex mixture of numerous proteins from both the squid and bacteria. The researchers found a unique protein that was expressed by V. fischeri in the expulsion but not the culture, which was found to be a quorum sensing-regulated protein only expressed in the light organ. This analysis is shown in Figure 3.
The authors further identified proteins using MudPIT, and found 1581 unique proteins from both squid and bacteria. 870 of these proteins came from bacteria, and 676 came from squid. These numbers can be found in Table 2.
The researchers then organized all proteins to achieve a better understanding of what functions they have in maintaining the symbiotic relationship between squid and bacteria. They found that the 25 most common bacterial proteins were involved in stress responses, quorum sensing, movement, and signaling pathways, which have previously been associated with the squid/bacteria association (Table 4, Table S3). Common squid-associated proteins were involved in the innate immune system, oxidative stress, and signaling pathways (Table 5).
Innate immune system
Microbe-associated molecular patterns (MAMPs) and pattern recognition receptors (PRRs) are important components of the squid/bacteria association, and several were identified in the researchers’ analyses. MAMPs determine the specificity of the symbiotic relationship, ensuring that no other bacteria come in to the light organ and that V. fischeri doesn’t escape. Several proteins in both squid and bacteria were identified related to pattern recognition including EsPGRP2 and EsPGRP3.
Aside from MAMPs and PRRs, outer membrane proteins (OMPs), which are located on bacterial cell surfaces and mediate recognition between the two partners, were also identified. Understanding how OMPs differ in the light organ and the external environment may shed light on how the specificity of the squid/bacteria association is maintained.
In addition, proteins were also identified related to NFκB signaling, which plays an important role in mediating inflammation and opsonization.
Reactive oxygen and nitrogen stress response
The chemical environment inside the light organ may influence the maintenance of symbiosis and its specificity. Previous studies have shown that reactive oxygen species (ROS) are abundant in the light organ, which are thought to play a key role in the initiation and maintenance of the symbiotic relationship. Hypohalous acid, generated by an enzyme in the host, is thought to create an oxidative environment only V. fischeri can overcome to associate with the host, which V. fischeri may overcome using antioxidant proteins and enzymes.
Another role of ROS may be to maintain specificity of the symbiotic relationship by eliminating non-symbiotic bacteria and pathogens from colonizing the light organ. Proteins expressed by the bacteria show functions to protect themselves from ROS, including katA, AhpC, and thiol peroxidase, all which process ROS and turn them into non-toxic compounds.
On the other hand, reactive nitrogen species contribute to signaling and development of the symbiotic relationship. In young squid, the light organ contains high levels of nitric oxide (NO), which bacteria must overcome to colonize the light organ. Bacterial enzymes such as nitric oxide dioxygenase (Hmp) were detected in the researchers’ analyses, suggesting that V. fischeri can overcome nitrogen-related oxidative stress.
Finally, iron is also known to play an important role in the symbiotic relationship. The researchers identified several iron-binding proteins, suggesting a possible role for these proteins in regulating V. fischeri growth.
Quorum sensing regulates luminescence of V. fischeri, which helps the squid avoid predation in a mechanism called counterillumination. Lux proteins, which are involved in bioluminescence, were one of the most prevalent bacterial proteins.
Two-component signaling pathways are important mechanisms by which bacteria sense and interact with the external environment. The researchers found multiple proteins involved in these pathways, including GacA, ArcA, and CpxP.
Other Related Stresses
The researchers also found cold shock proteins, which often function in general stress responses and have yet to be described with respect to squid/bacteria association. They may play a role in maintaining high cell densities in the light organ and/or helping bacteria with the transition of moving from the light organ to the external environment.
When V. fischeri are expelled from the light organ, they fully regenerate their flagella, which help them move freely in the external environment. The researchers found several proteins related to flagellar structure and regulation, suggesting that the bacteria are generating flagella before being expelled from the light organ.
When in the light organ, the metabolism of V. fischeri fluctuate with the daily rhythm of the light organ. Previous studies have shown that the bacteria ferments chitin, a sugar, at night to obtain energy. The results of this study show an abundance of chitin-metabolizing proteins, supporting previous studies.
Squid were collected from shallow sand flats in Oahu, HI. They were kept in natural seawater in the laboratory, and squid were acclimated for at least 48 hours under laboratory conditions before samples were collected.
Exudate and central core collection
Expelled material was collected by dissecting the squid a few minutes before dawn, using artificial light to induce expulsion, and collecting the resulting expulsion.
Gel-based proteomic methods
The researchers used 1D- and 2D-polyacrylamide gel electrophoresis to analyze proteins from the expulsion.
Mass spectrometry proteomic methods
The researchers used multidimensional protein identification technology (MudPIT) and liquid chromatography tandem mass spectrometry to analyze proteins from the expulsion.
Characterizing proteins in the light organ allowed for the identification of a high number of squid and bacterial proteins, as well as furthered understanding of the symbiotic relationship between the Hawaiian bobtail squid and V. fischeri. The results of this study complement previous studies, but also identified some new proteins with unknown functions. Further research is necessary to fully understand the maintenance of symbiosis.