Factors that affect Vibrio fisheri colonization to squid host

Symbiosis between animal tissue and bacterial cells plays an important regulating host’s tissue development, immune system development and nutrition. However the mechanics of selection of a particular bacterial species, out of the bacterial diversity in the environment, is not thoroughly understood. This study aims to understand the molecular determinants of microbiota selection by using Euprymna scolopes and Vibrio fischeri bacteria as a model.

E. scolopes inhabits seawater containing 10^6 bacteria per milliliter, with V. fischeri comprising at most 0.02% of the environmental population. E. scolopes hatch without symbionts, but then rapidly acquire environmental bacteria and proceed to select for V. fischeri in a “winnowing” process that ensures colonization by only the specific symbiont. The squid–Vibrio system thus presents an opportunity to investigate the processes that underlie acquisition of specific environmental microbes. This study applied a global forward genetic approach to identify animal colonization factors.

High-throughput insertion sequencing is used to identify the bacterial genes that are required during host colonization. They initially develop signature-tagged mutagenesis of V. fischeri strains to identify factors that are specifically defective for growth in an animal host. They then used insertion sequencing (INSeq) of mutagenize V. fischeri to E. scolopes squid isolate. In this experiment they identified essential bacterial genes in the V. fischeri that allows colonization of the squid host. They also showed a genome-wide identification of genes that are conditionally required for colonization of the squid host.

Development of mutant pool V. fischeri:

They developed a pool of mutant V. fischeri strains to identify colonization determinants. A library of over 41000 independent genomic sequences was used as the “input” library in this study. Using the INSeq high-throughput method, they have sequences and facilitated in identification of over 41000 independent genomic insertion sites.

With the mutagenesis approach, they have identified essential genes, for colonization of squid host, as those with reduced transposon insertion in the mutant population. They used E.coli as the model organism for which it best curated list of essential genes exists with V. fischeri. They mapped the V. fischeri genes that were orthologs of essential genes in E. coli, and found that there were no or relatively few transposon counts in V. fischeri. They thus classified these genes as “putative essential” in V. fischeri.

Identification of Squid colonization factors:

A concentration of 2 x 10⁵ cfu/mL of bacterial was used to study colonization factors of bacterial to squid. A defined 96 mutant library was allowed to colonize the squid and the individual animal outputs were sampled by INSeq. They observed that in most cases, colonization of mutants to squid host is proportional to the relative abundance of the input bacterial. They have observed over 75% of known mutant colonization and these genes were categorized by the COG (cluster of orthologous groups) classification. They concluded that the categories that included a large number of the colonization factors included those for signal transduction, motility, amino acid transport and metabolism and cell envelope integrity. The COG analysis highlighted broad-scale classes of genes that are required for squid colonization.

The experiment then followed with a competition assay of mutant strains and the wild-type strain in a 1:1 ratio. Nine strains have exhibited fitness deficits. These nine strains include mutants in genes encoding two copper efflux systems (CusC and CopA); two protein quality control factors (cytoplasmic chaperone DnaJ and periplasmic endoprotease DegS); an inner membrane protein predicted to assist in the secretion of auto transporters (TamB/YtfN); a predicted lysine 2,3-amino mutase (YjeK/EpmB); and three poorly characterized proteins (YdhC, YafD, and YhcB).

Influence of biofilm production in colonization:

V. fischeri must synthesize a symbiotic biofilm to passage through the squid ciliated epithelial field to the light organ pore. Biofilm assembly in vivo correlates strongly with the ability to form wrinkled colonies upon overexpression of the histidine kinase RscS. They have shown that colonization-defective mutants are unable to express wrinkled colony phenotype and hence exhibit defects in the biofilm assay. The degS and yhcB mutants exhibited modest defects in the wrinkled colony biofilm assay, and dnaJ, which encodes the cytoplasmic cochaperone for DnaK, exhibited no wrinkling in the assay.

DnaJ (Hsp40) is conserved from bacteria through metazoans as a cochaperone for the ATP-dependent DnaK (Hsp70) chaperone. These chaperones are required for heat tolerance. They predict that even in the absence of competition with wild-type strain, the dnaJ mutant would fail to colonize the light organ. DnaJ therefore influence biofilm development in the host mucociliary field during the initiation stage of squid colonization.

This study thus has shown novel genetic factors that are required for establishment of V. fischeri in its squid host. They have shown the influence of biofilm regulation, the prerequisites colonization factors and the effect of copper efflux system in the acquisition of beneficial microbes in the squid host.

John F. B. II, Mattias C. G., David C. C., Sarah J. Q., Celeste A. M., Randi F., Cheryl W., Andrew L. G., and Mark J. M. (2014) Global discovery of colonization determinants in the squid symbiont Vibrio fischeri. PNAS, 111, 48, 17284-17289.

http://www.pnas.org/content/111/48/17284.full