Preferential Codon Research Articles

Synonymous codon usage influences the transmission of peste des petits ruminants (PPR) virus in camels.

Peste des petits ruminants virus (PPRV) is an infectious pathogen; causing highly contagious, acute febrile, and economically important disease of small ruminants. The virus is known to have intrinsic ability to adapt new hosts and to cross the species barrier. The incidence of PPR has already been reported in unusual host species such as camels, bovines, and wild animals from spill-over or natural infection. Still, there are elementary gaps in our knowledge of the extent of susceptibility of camel to PPRV and the adaptability of PPRV to camel. The present study delineates the potential role of preferential codon usage patterns responsible for adaptation, host immune evasion, and transmission of PPRV to unusual hosts like old world camel species namely, dromedary and bactrian camel. The results indicate codon usage of the PPRV genome is functioned by an interplay of mutational pressure and natural selection to exhort the adaptation and fitness of PPRV in probable hosts. The indices of natural selection like the relative codon deoptimization index (RCDI) and codon adaptation index (CAI) predict the ability of PPRV to adapt and evolve in camel species. The analysis also depicts the potential role of the CpG depletion mechanism employed by PPRV to evade host adaptive immune response. The report emphasizes the need for a comprehensive national PPR surveillance plan in unusual hosts like camels for the successful implementation of the PPR Global Eradication Programme (PPR- GEP).

Identification and Functional Implications of the E5 Oncogene Polymorphisms of Human Papillomavirus Type 16.

The persistence of the human papillomavirus type 16 (HPV16) infection on the cervical epithelium contributes to the progression of cervical cancer. Studies have demonstrated that HPV16 genetic variants may be associated with different risks of developing cervical cancer. However, the E5 oncoprotein of HPV16, which is related to several cellular mechanisms in the initial phases of the infection and thus contributes to carcinogenesis, is still little studied. Here we investigate the HPV16 E5 oncogene variants to assess the effects of different mutations on the biological function of the E5 protein. We detected and analyzed the HPV16 E5 oncogene polymorphisms and their phylogenetic relationships. After that, we proposed a tertiary structure analysis of the protein variants, preferential codon usage, and functional activity of the HPV16 E5 protein. Intra-type variants were grouped in the lineages A and D using in silico analysis. The mutations in E5 were located in the T-cell epitopes region. We therefore analyzed the interference of the HPV16 E5 protein in the NF-kB pathway. Our results showed that the variants HPV16E5_49PE and HPV16E5_85PE did not increase the potential of the pathway activation capacity. This study provides additional knowledge about the mechanisms of dispersion of the HPV16 E5 variants, providing evidence that these variants may be relevant to the modulation of the NF-κB signaling pathway.

Optimization of Pichia pastoris Expression System for High-Level Production of Margatoxin.

Margatoxin (MgTx) is a high-affinity blocker of voltage-gated potassium (Kv) channels. It inhibits Kv1.1–Kv1.3 ion channels in picomolar concentrations. This toxin is widely used to study physiological function of Kv ion channels in various cell types, including immune cells. Isolation of native MgTx in large quantities from scorpion venom is not affordable. Chemical synthesis and recombinant production in Escherichia coli need in vitro oxidative refolding for proper disulfide bond formation, resulting in a very low yield of peptide production. The Pichia pastoris expression system offers an economical approach to overcome all these limitations and gives a higher yield of correctly refolded recombinant peptides. In this study, improved heterologous expression of recombinant MgTx (rMgTx) in P. pastoris was obtained by using preferential codons, selecting the hyper-resistant clone against Zeocin, and optimizing the culturing conditions. About 36 ± 4 mg/L of >98% pure His-tagged rMgTx (TrMgTx) was produced, which is a threefold higher yield than has been previously reported. Proteolytic digestion of TrMgTx with factor Xa generated untagged rMgTx (UrMgTx). Both TrMgTx and UrMgTx blocked the Kv1.2 and Kv1.3 currents (patch-clamp) (K d for Kv1.2 were 64 and 14 pM, and for Kv1.3, 86 and 50 pM, respectively) with comparable potency to the native MgTx. The analysis of the binding kinetics showed that TrMgTx had a lower association rate than UrMgTx for both Kv1.2 and Kv1.3. The dissociation rate of both the analogues was the same for Kv1.3. However, in the case of Kv1.2, TrMgTx showed a much higher dissociation rate with full recovery of the block than UrMgTx. Moreover, in a biological functional assay, both peptides significantly downregulated the expression of early activation markers IL2R and CD40L in activated CD4+ TEM lymphocytes whose activation was Kv1.3 dependent. In conclusion, the authors report that the Pichia expression system is a powerful method to produce disulfide-rich peptides, the overexpression of which could be enhanced noticeably through optimization strategies, making it more cost-effective. Since the presence of the His-tag on rMgTx only mildly altered the block equilibrium and binding kinetics, recombinant toxins could be used in ion channel research without removing the tag and could thus reduce the cost and time demand for toxin production.

Modification of Transfer RNA Levels Affects Cyclin Aggregation and the Correct Duplication of Yeast Cells.

Codon usage bias (the preferential use of certain synonymous codons (optimal) over others is found at the organism level (intergenomic) within specific genomes (intragenomic) and even in certain genes. Whether it is the result of genetic drift due to GC/AT content and/or natural selection is a topic of intense debate. Preferential codons are mostly found in genes encoding highly-expressed proteins, while lowly-expressed proteins usually contain a high proportion of rare (lowly-represented) codons. While optimal codons are decoded by highly expressed tRNAs, rare codons are usually decoded by lowly-represented tRNAs. Whether rare codons play a role in controlling the expression of lowly- or temporarily-expressed proteins is an open question. In this work we approached this question using two strategies, either by replacing rare glycine codons with optimal counterparts in the gene that encodes the cell cycle protein Cdc13, or by overexpression the tRNAGly that decodes rare codons from the fission yeast, Schizosaccharomyces pombe. While the replacement of synonymous codons severely affected cell growth, increasing tRNA levels affected the aggregation status of Cdc13 and cell division. These lead us to think that rare codons in lowly-expressed cyclin proteins are crucial for cell division, and that the overexpression of tRNA that decodes rare codons affects the expression of proteins containing these rare codons. These codons may be the result of the natural selection of codons in genes that encode lowly-expressed proteins.

Transit From Autotrophism to Heterotrophism: Sequence Variation and Evolution of Chloroplast Genomes in Orobanchaceae Species.

The family Orobanchaceae including autotrophic, hemiparasitic, and holoparasitic species, is becoming a key taxa to study the evolution of chloroplast genomes in different lifestyles. But the early evolutionary trajectory in the transit from autotrophism to hemiparasitism still maintains unclear for the inadequate sampling. In this study, we compared 50 complete chloroplast genomes in Orobanchaceae, containing four newly sequenced plastomes from hemiparasitic Pedicularis, to elucidate the sequence variation patterns in the evolution of plastomes. Contrasted to the sequence and structural hypervariabilities in holoparasites, hemiparasitic plastomes exhibited high similarity to those of autotrophs in gene and GC contents. They are generally characterized with functional or physical loss of ndh/tRNA genes and the inverted small-single-copy region. Gene losses in Orobanchaceae were lineage-specific and convergent, possibly related to structural reconfiguration and expansion/contraction of the inverted region. Pseudogenization of ndh genes was unique in hemiparasites. At least in Pedicularis, the ndhF gene might be most sensitive to the environmental factors and easily pseudogenized when autotrophs transit to hemiparasites. And the changes in gene contents and structural variation potentially deeply rely on the feeding type. Selective pressure, together with mutational bias, was the dominant factor of shaping the codon usage patterns. The relaxed selective constraint, potentially with genome-based GC conversion (gBGC) and preferential codon usage, drive the fluctuation of GC contents among taxa with different lifestyles. Phylogenetic analysis in Orobanchaceae supported that parasitic species were single-originated while holoparasites were multiple-originated. Overall, the comparison of plastomes provided a good opportunity to understand the evolution process in Orobanchaceae with different lifestyles.

Evolution of Codon Usage Bias in Diatoms.

Codon usage bias (CUB)—preferential use of one of the synonymous codons, has been described in a wide range of organisms from bacteria to mammals, but it has not yet been studied in marine phytoplankton. CUB is thought to be caused by weak selection for translational accuracy and efficiency. Weak selection can overpower genetic drift only in species with large effective population sizes, such as Drosophila that has relatively strong CUB, while organisms with smaller population sizes (e.g., mammals) have weak CUB. Marine plankton species tend to have extremely large populations, suggesting that CUB should be very strong. Here we test this prediction and describe the patterns of codon usage in a wide range of diatom species belonging to 35 genera from 4 classes. We report that most of the diatom species studied have surprisingly modest CUB (mean Effective Number of Codons, ENC = 56), with some exceptions showing stronger codon bias (ENC = 44). Modest codon bias in most studied diatom species may reflect extreme disparity between astronomically large census and modest effective population size (Ne), with fluctuations in population size and linked selection limiting long-term Ne and rendering selection for optimal codons less efficient. For example, genetic diversity (pi ~0.02 at silent sites) in Skeletonema marinoi corresponds to Ne of about 10 million individuals, which is likely many orders of magnitude lower than its census size. Still, Ne ~107 should be large enough to make selection for optimal codons efficient. Thus, we propose that an alternative process—frequent changes of preferred codons, may be a more plausible reason for low CUB despite highly efficient selection for preferred codons in diatom populations. The shifts in the set of optimal codons should result in the changes of the direction of selection for codon usage, so the actual codon usage never catches up with the moving target of the optimal set of codons and the species never develop strong CUB. Indeed, we detected strong shifts in preferential codon usage within some diatom genera, with switches between preferentially GC-rich and AT-rich 3rd codon positions (GC3). For example, GC3 ranges from 0.6 to 1 in most Chaetoceros species, while for Chaetoceros dichaeta GC3 = 0.1. Both variation in selection intensity and mutation spectrum may drive such shifts in codon usage and limit the observed CUB. Our study represents the first genome-wide analysis of CUB in diatoms and the first such analysis for a major phytoplankton group.

Global regulator BldA regulates morphological differentiation and lincomycin production in Streptomyces lincolnensis.

Global regulator BldA, the only tRNA for a rare leucine codon UUA, is best known for its ability to affect morphological differentiation and secondary metabolism in the genus Streptomyces. In this study, we confirmed the regulatory function of the bldA gene (Genbank accession no. EU124663.1) in Streptomyces lincolnensis. Disruption of bldA hinders the sporulation and lincomycin production, that can recur when complemented with a functional bldA gene. Western blotting assays demonstrate that translation of the lmbB2 gene which encodes a L-tyrosine hydroxylase is absolutely dependent on BldA; however, mistranslation of the lmbU gene which encodes a cluster-situated regulator (CSR) is observed in a bldA mutant. Intriguingly, when the preferential cognate codon CTG was used, the expression level of LmbU was not the highest compared to the usage of rare codon TTA or CTA, indicating the rare codon in this position is significant for the regulation of lmbU expression. Moreover, replacement of TTA codons in both genes with another leucin codon in the bldA mutant did not restore lincomycin production. Thus, we believe that the bldA gene regulates lincomycin production via controlling the translation of not only lmbB2 and lmbU, but also the other TTA-containing genes. In conclusion, the present study demonstrated the importance of the bldA gene in morphological differentiation and lincomycin production in S. lincolnensis.

Constitutive expression of the active fragment of human vasostatin Vs30 in Pichia pastoris SMD1168H

Vasostatin 30 (Vs30) is an active fragment derived from the N-terminal region (135–164 aa) of human calreticulin and has the ability to inhibit angiogenesis. In this work, the expression of Vs30 was performed using a protease-deficient strain of the methylotrophic yeast Pichia pastoris. The vs30 gene was optimized for P. pastoris preferential codon usage and inserted into constitutive expression vector pGAPZαA. In addition, a plasmid with four copies of the expression cassette was obtained and transformed into P. pastoris. The flask fermentation conditions were: culture volume of 25 mL in 250 mL baffled flasks at 28 °C, pH 6 and harvest time of 48 h. Up to 21.07 mg/L Vs30 were attained and purified by ultrafiltration with a 30-kDa cut-off membrane and the recovery was 49.7%. Bioactivity of Vs30 was confirmed by the inhibition of cell proliferation, as well as the inhibition of the capillary-like structures formation of EA.hy926 cells in vitro. This work constitutes the first report on the expression of Vs30 in Pichia pastoris using a constitutive promoter and multi-copy approach such as strategies to improve the recombinant Vs30 expression.

Synonymous codon changes at the 5′-end of the gene strongly impact the heterologous protein expression in Escherichia coli

This study describes the impact of 5′-end codon modulation on the expression of a heterologous gene, human granulocyte colony stimulating factor (GCSF), in Escherichia coli. Fourteen different constructs (pGCSF-01 to pGCSF-14) carrying single or multiple synonymous substitutions at +2, +3 and further down from +4 to +7 codons, were prepared and their expression was monitored in E. coli BL21 Codon-Plus (DE3) RIPL using a strong T7 lac-promoter based expression system. A single nucleotide change at +2 Thr codon (ACC→ACA) either alone or in combination with +3 Pro codon (CCC/CCT/CCA) resulted in the expression enhancement of an otherwise poorly expressed native-GCSF, to a level that corresponded to 45–50% of the total E. coli BL21 CodonPlus (DE3) RIPL cellular proteins. The differences in GCSF expression amongst different constructs could be attributed to the preferential or non-preferential codon usage, reduced number of G/C nucleotides and the stability of mRNA secondary structure formed near the 5′-end coding region. The expression of GCSF achieved was in the form of biologically inactive inclusion bodies that were solubilized using mild concentration of a non-ionic surfactant and refolded by a simplified, step-dialysis approach. Biological activity of the purified GCSF, assessed in induced neutropenic mice, was similar to the commercially available preparation of the GCSF analog (filgrastim).

Expression of a codon-optimized β-glucosidase from Cellulomonas flavigena PR-22 in Saccharomyces cerevisiae for bioethanol production from cellobiose.

Bioethanol is one of the main biofuels produced from the fermentation of saccharified agricultural waste; however, this technology needs to be optimized for profitability. Because the commonly used ethanologenic yeast strains are unable to assimilate cellobiose, several efforts have been made to express cellulose hydrolytic enzymes in these yeasts to produce ethanol from lignocellulose. The C. flavigenabglA gene encoding β-glucosidase catalytic subunit was optimized for preferential codon usage in S. cerevisiae. The optimized gene, cloned into the episomal vector pRGP-1, was expressed, which led to the secretion of an active β-glucosidase in transformants of the S. cerevisiae diploid strain 2-24D. The volumetric and specific extracellular enzymatic activities using pNPG as substrate were 155 IU L-1 and 222IU g-1, respectively, as detected in the supernatant of the cultures of the S. cerevisiae RP2-BGL transformant strain growing in cellobiose (20g L-1) as the sole carbon source for 48h. Ethanol production was 5g L-1 after 96h of culture, which represented a yield of 0.41g g-1 of substrate consumed (12g L-1), equivalent to 76% of the theoretical yield. The S. cerevisiae RP2-BGL strain expressed the β-glucosidase extracellularly and produced ethanol from cellobiose, which makes this microorganism suitable for application in ethanol production processes with saccharified lignocellulose.

Lectin I from Bauhinia variegata (BVL-I) expressed by Pichia pastoris inhibits initial adhesion of oral bacteria in vitro

Lectins are non-immune proteins that reversibly bind to carbohydrates in a specific manner. Bauhinia variegata lectin I (BVL-I) is a Gal/GalNAc-specific, single-chain lectin isolated from Bauhinia variegata seeds that has been implicated in the inhibition of bacterial adhesion and the healing of damaged skin. Since the source of the native protein (nBVL) is limited, this study aimed to produce recombinant BVL-I in Pichia pastoris (rBVL-Ip). The coding sequence for BVL-I containing preferential codons for P. pastoris was cloned into the pPICZαB plasmid. A single expressing clone was selected and fermented, resulting in the secretion and glycosylation of the protein. Fed-batch fermentation in 7L-scale was performed, and the recombinant lectin was purified from culture supernatant, resulting in a yield of 1.5mg/L culture. Further, rBVL-Ip was compared to nBVL and its recombinant version expressed in Escherichia coli BL21 (DE3) (rBVL-Ie). Although it was expressed as a monomer, rBVL-Ip retained its biological activity since it was able to impair the initial adhesion of Streptococcus mutans and S. sanguinis in an in vitro model of biofilm formation and bacterial adhesion. In summary, rBVL-Ip produced in Pichia pastoris represents a viable alternative to large-scale production, encouraging further biological application studies with this lectin.

Web application for automatic prediction of gene translation elongation efficiency.

Expression efficiency is one of the major characteristics describing genes in various modern investigations. Expression efficiency of genes is regulated at various stages: transcription, translation, posttranslational protein modification and others. In this study, a special EloE (Elongation Efficiency) web application is described. The EloE sorts the organism's genes in a descend order on their theoretical rate of the elongation stage of translation based on the analysis of their nucleotide sequences. Obtained theoretical data have a significant correlation with available experimental data of gene expression in various organisms. In addition, the program identifies preferential codons in organism's genes and defines distribution of potential secondary structures energy in 5´ and 3´ regions of mRNA. The EloE can be useful in preliminary estimation of translation elongation efficiency for genes for which experimental data are not available yet. Some results can be used, for instance, in other programs modeling artificial genetic structures in genetically engineered experiments.

EloE: Web application for estimation of gene translation elongation efficiency

Many modern investigations study important gene characteristic such as the efficiency of its expression. As is known, it is determined at the levels of transcription, translation, posttranslational modification, etc. The EloE (Elongation Efficiency) program, which sorts the organism genes in order of decreasing their supposed translation elongation rate based on the analysis of their nucleotide sequences, is presented in the work. The obtained theoretical data are significantly correlated to the available experimental data on gene expression in different organisms (for example, S. cerevisiae and H. pylori). The program also detects preferential codons in the organism genome and constructs the distribution of the stability of potential secondary structures near the mRNA 5′- and 3′-ends. The program can be used for preliminarily estimating the level of gene expression in the studied organism (for which the experimental data are not available yet). The results of EloE can be transferred in third-party program instruments that model artificial genetic constructions for genetically engineered experiments.

A web application for automatic prediction of gene translation elongation efficiency

Summary Expression efficiency is one of the major characteristics describing genes in various modern investigations. Expression efficiency of genes is regulated at various stages: transcription, translation, posttranslational protein modification and others. In this study, a special EloE (Elongation Efficiency) web application is described. The EloE sorts the organism’s genes in a descend order on their theoretical rate of the elongation stage of translation based on the analysis of their nucleotide sequences. Obtained theoretical data have a significant correlation with available experimental data of gene expression in various organisms. In addition, the program identifies preferential codons in organism’s genes and defines distribution of potential secondary structures energy in 5´ and 3´ regions of mRNA. The EloE can be useful in preliminary estimation of translation elongation efficiency for genes for which experimental data are not available yet. Some results can be used, for instance, in other programs modeling artificial genetic structures in genetically engineered experiments. The EloE web application is available at http://www-bionet.sscc.ru:7780/EloE.

Gene expression level: is it an important factor in codon optimization for overexpression of recombinant proteins?

Background Microalgae are becoming a viable model for the expression of recombinant proteins. However, suitable levels of expression for commercial production have not yet been obtained. One way to increase the heterologous protein production is the codon optimization of the target gene so that it presents the preferential codons used by the host organism according to its own pool of transfer RNA (tRNA). For codon optimization the Codon Usage Database (CUD; http://www.kazusa.or.jp/codon) has been used. The database provides codon frequency tables based on gene transcripts, regardless of their level of expression. Therefore, the aim of this study was to determine if there are changes in the frequency of codons used when taking into account the level of expression of genes, considering the 50 most expressed genes and 50 least expressed genes from the microalgae Chlamydomonas reinhardtii.

Biotechnical paving of recombinant enterocin A as the candidate of anti-Listeria agent.

BackgroundEnterocin A is a classic IIa bacteriocin isolated firstly from Enterococcus faecium CTC492 with selective antimicrobial activity against Listeria strains. However, the application of enterocin A as an anti-Listeria agent has been limited due to its very low native yield. The present work describes high production of enterocin A through codon optimization strategy and its character study.ResultsThe gene sequence of enterocin A was optimized based on preferential codon usage in Pichia pastoris to increase its expression efficiency. The highest anti-Listeria activity reached 51,200 AU/ml from 180 mg/l of total protein after 24 h of induction in a 5-L fermenter. Recombinant enterocin A (rEntA), purified by gel filtration chromatography, showed very strong activity against Listeria ivanovii ATCC 19119 with a low MIC of 20 ng/ml. In addition, the rEntA killed over 99% of tested L. ivanovii ATCC19119 within 4 h when exposed to 4 × MIC (80 ng/ml). Moreover, it showed high stability under a wide pH range (2–10) and maintained full activity after 1 h of treatment at 80°C within a pH range of 2–8. Its antimicrobial activity was enhanced at 25 and 50 mM NaCl, while 100–400 mM NaCl had little effect on the bactericidal ability of rEntA.ConclusionThe EntA was successfully expressed in P. pastoris, and this feasible system could pave the pre-industrial technological path of rEntA as a competent candidate as an anti-Listeria agent. Furthermore, it showed high stability under wide ranges of conditions, which could be potential as the new candidate of anti-Listeria agent.

Natural Selection Determines Synonymous Codon Usage Patterns of Neuraminidase (NA) Gene of the Different Subtypes of Influenza A Virus in Canada

Synonymous codon usage patterns of neuraminidase (NA) gene of 64 subtypes (one is a mixed subtype) of influenza A virus found in Canada were analyzed. In total, 1422 NA sequences were analyzed. Among the subtypes, H1N1 is the prevailing one with 516 NCBI accession records, followed by H3N2, H3N8, and H4N6. The year of 2009 has the highest report records for the NA sequences in Canada, corresponding to the 2009 pandemic event. Correspondence analysis on the RSCU values of the four major subtypes showed that they had distinct clustering patterns in the two-dimensional scatter plot, indicating that different subtypes of IAV utilized different preferential codons. This subtype clustering pattern implied the important influence of natural selection, which could be further evidenced by an extremely flattened regression line in the neutrality plot (GC12 versus G3s plot) and a significant phylogenetic signal on the distribution of different subtypes in the clades of the phylogenetic tree (λ statistic). In conclusion, different subtypes of IAV showed an evolutionary differentiation on choosing different optimal codons. Natural selection played a deterministic role to structure IAV codon usage patterns in Canada.

Aptamers as a Sensitive Tool to Detect Subtle Modifications in Therapeutic Proteins

Therapeutic proteins are derived from complex expression/production systems, which can result in minor conformational changes due to preferential codon usage in different organisms, post-translational modifications, etc. Subtle conformational differences are often undetectable by bioanalytical methods but can sometimes profoundly impact the safety, efficacy and stability of products. Numerous bioanalytical methods exist to characterize the primary structure of proteins, post translational modifications; protein-substrate/protein/protein interactions and functional bioassays are available for most proteins that are developed as products. There are however few analytical techniques to detect changes in the tertiary structure of proteins suitable for use during drug development and quality control. For example, x-ray crystallography and NMR are impractical for routine use and do not capture the heterogeneity of the product. Conformation-sensitive antibodies can be used to map proteins. However the development of antibodies to represent sufficient epitopes can be challenging. Other limitations of antibodies include limited supply, high costs, heterogeneity and batch to batch variations in titer. Here we provide proof-of-principle that DNA aptamers to thrombin can be used as surrogate antibodies to characterize conformational changes. We show that aptamers can be used in assays using either an ELISA or a label-free platform to characterize different thrombin products. In addition we replicated a heat-treatment procedure that has previously been shown to not affect protein activity but can result in conformational changes that have serious adverse consequences. We demonstrate that a panel of aptamers (but not an antibody) can detect changes in the proteins even when specific activity is unaffected. Our results indicate a novel approach to monitor even small changes in the conformation of proteins which can be used in a routine drug-development and quality control setting. The technique can provide an early warning of structural changes during the manufacturing process that could have consequential outcomes downstream.

The Sulfate-Rich and Extreme Saline Sediment of the Ephemeral Tirez Lagoon: A Biotope for Acetoclastic Sulfate-Reducing Bacteria and Hydrogenotrophic Methanogenic Archaea

Our goal was to examine the composition of methanogenic archaea (MA) and sulfate-reducing (SRP) and sulfur-oxidizing (SOP) prokaryotes in the extreme athalassohaline and particularly sulfate-rich sediment of Tirez Lagoon (Spain). Thus, adenosine-5′-phosphosulfate (APS) reductase α (aprA) and methyl coenzyme M reductase α (mcrA) gene markers were amplified given that both enzymes are specific for SRP, SOP, and MA, respectively. Anaerobic populations sampled at different depths in flooded and dry seasons from the anoxic sediment were compared qualitatively via denaturing gradient gel electrophoresis (DGGE) fingerprint analysis. Phylogenetic analyses allowed the detection of SRP belonging to Desulfobacteraceae, Desulfohalobiaceae, and Peptococcaceae in ∂-proteobacteria and Firmicutes and SOP belonging to Chromatiales/Thiotrichales clade and Ectothiorhodospiraceae in γ-proteobacteria as well as MA belonging to methylotrophic species in Methanosarcinaceae and one hydrogenotrophic species in Methanomicrobiaceae. We also estimated amino acid composition, GC content, and preferential codon usage for the AprA and McrA sequences from halophiles, nonhalophiles, and Tirez phylotypes. Even though our results cannot be currently conclusive regarding the halotolerant strategies carried out by Tirez phylotypes, we discuss the possibility of a plausible “salt-in” signal in SRP and SOP as well as of a speculative complementary haloadaptation between salt-in and salt-out strategies in MA.

Adaptive Evolution of the Chlamydia trachomatis Dominant Antigen Reveals Distinct Evolutionary Scenarios for B- and T-cell Epitopes: Worldwide Survey

Background Chlamydia trachomatis is one of the most disseminated human pathogens, for which no vaccine is available yet. Understanding the impact of the host pressure on pathogen antigens is crucial, but so far it was only assessed for highly-restricted geographic areas. We aimed to evaluate the evolutionary picture of the chlamydial key antigen (MOMP), which is one of the leading multi-subunit vaccine candidates, in a worldwide basis.Methodology/Principal FindingsUsing genetics, molecular evolution methods and mathematical modelling, we analyzed all MOMP sequences reported worldwide, composed by 5026 strains from 33 geographic regions of five continents. Overall, 35.9% of variants were detected. The evolutionary pattern of MOMP amino acid gains/losses was found to differ from the remaining chromosome, reflecting the demanding constraints of this porin, adhesin and dominant antigen. Amino acid changes were 4.3-fold more frequent in host-interacting domains (P<10−12), specifically within B-cell epitopes (P<10−5), where 25% of them are at fixation (P<10−5). According to the typical pathogen-host arms race, this rampant B-cell antigenic variation likely represents neutralization escape mutants, as some mutations were previously shown to abrogate neutralization of chlamydial infectivity in vitro. In contrast, T-cell clusters of diverse HLA specificities are under purifying selection, suggesting a strategy that may lead to immune subversion. Moreover, several silent mutations are at fixation, generating preferential codons that may influence expression, and may also reflect recombination-derived ‘hitchhiking-effect’ from favourable nonsilent changes. Interestingly, the most prevalent C. trachomatis genotypes, E and F, showed a mutation rate 22.3-fold lower than that of the remainder (P<10−20), suggesting more fitted antigenic profiles.Conclusions/SignificanceGlobally, the adaptive evolution of the C. trachomatis dominant antigen is likely driven by its complex pathogenesis-related function and reflects distinct evolutionary antigenic scenarios that may benefit the pathogen, and thus should be taking into account in the development of a MOMP-based vaccine.