Antagonistic evolution of an antibiotic and its molecular chaperone: how to maintain a vital ectosymbiosis in a highly fluctuating habitat

Abstract

Evolution of antimicrobial peptides (AMPs) has been shown to be driven by recurrent duplications and balancing/positive selection in response to new or altered bacterial pathogens. We use Alvinella pompejana, the most eurythermal animal known on Earth, to decipher the selection patterns acting on AMP in an ecological rather than controlled infection approach. The preproalvinellacin multigenic family presents the uniqueness to encode a molecular chaperone (BRICHOS) together with an AMP (alvinellacin) that controls the vital ectosymbiosis of Alvinella. In stark contrast to what is observed in the context of the Red queen paradigm, we demonstrate that exhibiting a vital and highly conserved ecto-symbiosis in the face of thermal fluctuations has led to a peculiar selective trend promoting the adaptive diversification of the molecular chaperone of the AMP, but not of the AMP itself. Because BRICHOS stabilizes beta-stranded peptides, this polymorphism likely represents an eurythermal adaptation to stabilize the structure of alvinellacin, thus hinting at its efficiency to select and control the epibiosis across the range of temperatures experienced by the worm; Our results fill some knowledge gaps concerning the function of BRICHOS in invertebrates and offer perspectives for studying immune genes in an evolutionary ecological framework.

Figure 1

Gene diversification of preproalvinellacin. (A) Coalescence tree of alleles found in two well-recaptured individuals of Alvinella pompejana (white) and its sister species Alvinella caudata (black), and (B) molecular evidence that it comes from tandemly repeated gene duplications in A. caudata. (A) Reconstruction of lineages without recombination was performed on the 3′ region on all nucleotide sites by Maximum Likelihood using the K2P model in MEGA 5.0. Allele coverage: A. caudata: two individuals with 15 clone recaptures each, A. pompejana: two individuals with 50 clone recaptures each. (B) Agarose gel electrophoresis with a 1 kb DNA ladder showing the amplification of the 1300 bp 5′ region of the A. caudata preproalvinellacin (complete gene: 2000 bp) using the 5′ primers (arrows). Longer extra bands indicate the co-amplification of two and three linked genes as summarized by the boxes representing the tandemly-duplicated gene and the position of the PCR products.

Figure 2

Evolution of genetic diversity and K_a/K_s along the preproalvinellacin gene and its corresponding coding sequence: (A) active alvinellacin is cleaved from a larger proteic precursor (i.e. preproalvinellacin). In contrast to all described AMPs, the preproalvinellacin family harbors the pattern of a BRICHOS containing protein: a hydrophobic domain (the signal peptide), a propiece with a linker and a BRICHOS domain and a C-terminal region with β-sheet propensities (alvinellacin). (B) Sliding window of the overall nucleotidic diversity (θπ) along the intronic and exonic regions of the gene in A. pompejana (black) and A. caudata (grey), (C) between-paralog K_a/K_s mean (dashed line/left) and the average within-paralog π_a/π_s (solid line/right) along the coding sequence of the gene. Sliding window length = 50 bp, step size = 10 bp. Introns are colored in grey (5 introns); exons are colored as follows: orange: BRICHOS domain, green: AMP domain.

Figure 3

Splits Tree reticulated network of all alleles encoded by the preproalvinellacin multigenic family in A. pompejana: (A) the NeigborNet network together with (B) the schematic reconstruction of natural intergenic recombinants using the NJ tree topology obtained with the MEGA 5 software and the HKY model of substitutions. Sequences from the 5′ region were used in the two reconstruction methods and the six paralogs (Parx) as well as their intergenic recombinants (Rx) are represented by boxes in which the red, dark green, light green, blue, yellow and purple colors represent the genetic paralogous background and their proportions in recombinants, darker colors, the portions of the recombining alleles which accumulated their own set of mutations, and bars correspond to indel polymorphisms (including alleles with the 34 codons deletion in Par5).

Figure 4

Ancestral reconstruction of polymorphic amino-acid replacements in the BRICHOS domain and the mature AMP in Alvinella pompejana (Ap) using the aaML package of PaML4.0 and Alvinella caudata (Ac) as an outgroup to orientate mutations. Positions of amino-acid replacements are labeled according to the start (methionine) codon. The Bayesian probability of occurrence of an amino-acid in the ancestral sequence is given in brackets. The tree topology and associated bootstrap values (obtained from 1000 replicates) was obtained by the ML method in Mega 5.0 using the full sequences of the 3′ region of the gene. Sequence labels represent the individual number and the clone number and are representative of the 6 paralogous clades (excluding natural recombinants) and subsequently used in the mapping of the BRICHOS mutations (see BRICHOS amino-acid alignment in Fig. S1).

Figure 5

Upregulation of expression of the preproalvinellacin gene and the molecular chaperone hsp 70 in Alvinella pompejana submitted to thermal or pressure stress. (A) The level of transcription is significantly higher for both genes in “not re-pressurized animals” compared to those re-pressurized immediately after raising from −2500 m at the in situ pressure of 250 bars in the DESEARES vessel. (B) In animals kept under in situ pressure in the BALIST device, thermal stress led to an up-regulation of the two genes. P-values from Student’s tests were calculated versus the control treatment (normalized to 1), based on the experimental measures performed in triplicates (**p < 0.01, *p < 0.05).