Elucidating the role of innate immunity in E. scolopes/V. fisheri symbiosis using high throughput molecular techniques

The Euprymna scolopes/Vibrio fisheri symbiosis is a well-studied model system and much effort has gone into elucidating the ‘dialog’ between microbe and host that initiates and maintains the exclusivity of the relationship. In a world where everything is everywhere, E. scolopes actively selects and promotes growth of V. fisheri against a background concentration of 10⁶ microbes per ml of seawater!

Collins et al. used high throughput liquid chromatography and tandem mass spectrometry (LC-MS/MS) to characterise the transcriptome and proteome of circulating haemocytes in adult E. scolopes. Invertebrates lack an adaptive immune system and haemocytes function much as phagocytes by engulfing microorganisms. Real-time PCR (qRT-PCR) was used to compare expression of several innate immunity genes between symbiotic and cured (V. fisheri free) hosts to understand molecular mechanisms by which the haemocyte response may change due to colonisation.

‘Cured’ haemocytes were produced by injecting E.scolopes with antiobiotics to remove V. fisheri. However, the indiscriminate effects of antibiotic treatment on the normal biota of E. scolopes was not considered. Antibiotic treatment is likely to compromise the normal function of the immune system and would likely affect immune gene expression, at the very least making E.scolopes more prone to infection. Perhaps an alternate method of producing ‘cured’ haemocytes should be found.

As one would expect the transcriptome and proteome results correlated well (but not exactly). 279.9 Mb reads were identified in the transcriptome whilst 702 unique proteins were also identified. Much of the transcriptome and peptides identified were predicted to possess a cytoskeletal function. This relates well with the function of haemocytes; when activated they migrate through tissues and quickly change shape in order to ‘phagocytose’ particles.

Recognition of microbe associated molecular patterns (MAMPs- expressed for example on the lipopolysaccharide membrane of gram-negative bacteria) by host pattern recognition receptors (PRRs) is fundamental in allowing the host to differentiate between beneficial and harmful microbes. A novel PRR was identified (EsPGRP5) and was one of the most abundant transcripts in the haemocytes. Five EsPGRPs have been identified, and are important during the onset of symbiosis. EsPGRP5 was down-regulated 4.7 fold in naïve haemocytes from cured hosts demonstrating that V. fisheri regulates gene expression in E. scolopes. The presence of evolutionarily conserved MAMPs expressed by Vibrio fisheri  (e.g. tracheal cytotoxin) has been shown to be important in the early development of the light organ.

Interestingly a protein similar to cephalotoxin was identified in both the proteome and transcriptome. This is the first report of cephalotoxin not being used for prey capture in cephalopods although its function in haemocytes remains unknown. Nitric oxide synthase (NOS), important in creating antimicrobial reactive nitrogen species experienced a 16-fold increase in naïve haemocytes suggesting a feedback mechanism for the reduction in NOS for colonised hosts.

We know that E. scolopes expels its symbionts at dawn and you would expect this breakdown in symbiosis to be exhibited though differences in expression in the transcriptome and proteome of the haemocytes. This study used haemocytes harvested from a ‘variety’ of time points to generate an overview of the symbiosis. It would be informative to look at different time points to understand the internal dialog between host and symbiont during expulsion and re-colonisation to elucidate the factors that mediate this process.

Overall I thought this study was a fascinating first foray into the applications of transcriptomic and proteomic analysis when looking at symbioses. The sheer amount of data produced by this method is daunting and knowing which data are robust and useful may be difficult to identify. The discrepancy between the resolved proteome and transcriptome is perhaps an indicator that the techniques used lack accuracy.

Primary Reference: 
Collins, A. J., Schleicher, T. R., Rader, B.A., and Nyholm, S. V. (2012). Understanding the role of host hemocytes in a squid/vibrio symbiosis using transcriptomics and proteomics. Frontiers in immunology, 3.