Experimental prediction of the evolution of cefepime resistance from the CMY-2 AmpC beta-lactamase

Understanding of the evolutionary histories of many genes has not yet allowed us to predict the evolutionary potential of those genes. Intuition suggests that current biochemical activity of gene products should be a good predictor of the potential to evolve related activities; however, we have little evidence to support that intuition. Here we use our in vitro evolution method to evaluate biochemical activity as a predictor of future evolutionary potential. Neither the class C Citrobacter freundii CMY-2 AmpC beta-lactamase nor the class A TEM-1 beta-lactamase confer resistance to the beta-lactam antibiotic cefepime, nor do any of the naturally occurring alleles descended from them. However, the CMY-2 AmpC enzyme and some alleles descended from TEM-1 confer high-level resistance to the structurally similar ceftazidime. On the basis of the comparison of TEM-1 and CMY-2, we asked whether biochemical activity is a good predictor of the evolutionary potential of an enzyme. If it is, then CMY-2 should be more able than the TEMs to evolve the ability to confer higher levels of cefepime resistance. Although we generated CMY-2 evolvants that conferred increased cefepime resistance, we did not recover any CMY-2 evolvants that conferred resistance levels as high as the best cefepime-resistant TEM alleles.     

Neutral evolution of the sex-determining gene transformer in Drosophila

The amino acid sequence of the transformer (tra) gene exhibits an extremely rapid rate of evolution among Drosophila species, although the gene performs a critical step in sex determination. These changes in amino acid sequence are the result of either natural selection or neutral evolution. To differentiate between selective and neutral causes of this evolutionary change, analyses of both intraspecific and interspecific patterns of molecular evolution of tra gene sequences are presented. Sequences of 31 tra alleles were obtained from Drosophila americana. Many replacement and silent nucleotide variants are present among the alleles; however, the distribution of this sequence variation is consistent with neutral evolution. Sequence evolution was also examined among six species representative of the genus Drosophila. For most lineages and most regions of the gene, both silent and replacement substitutions have accumulated in a constant, clock-like manner. In exon 3 of D. virilis and D. americana we find evidence for an elevated rate of nonsynonymous substitution, but no statistical support for a greater rate of nonsynonymous relative to synonymous substitutions. Both levels of analysis of the tra sequence suggest that, although the gene is evolving at a rapid pace, these changes are neutral in function.     

Experimental prediction of the natural evolution of antibiotic resistance

The TEM family of beta-lactamases has evolved to confer resistance to most of the beta-lactam antibiotics, but not to cefepime. To determine whether the TEM beta-lactamases have the potential to evolve cefepime resistance, we evolved the ancestral TEM allele, TEM-1, in vitro and selected for cefepime resistance. After four rounds of mutagenesis and selection for increased cefepime resistance each of eight independent populations reached a level equivalent to clinical resistance. All eight evolved alleles increased the level of cefepime resistance by a factor of at least 32, and the best allele improved by a factor of 512. Sequencing showed that alleles contained from two to six amino acid substitutions, many of which were shared among alleles, and that the best allele contained only three substitutions.     

Gnarled-trunk evolutionary model of influenza A virus hemagglutinin

Human influenza A viruses undergo antigenic changes with gradual accumulation of amino acid substitutions on the hemagglutinin (HA) molecule. A strong antigenic mismatch between vaccine and epidemic strains often requires the replacement of influenza vaccines worldwide. To establish a practical model enabling us to predict the future direction of the influenza virus evolution, relative distances of amino acid sequences among past epidemic strains were analyzed by multidimensional scaling (MDS). We found that human influenza viruses have evolved along a gnarled evolutionary pathway with an approximately constant curvature in the MDS-constructed 3D space. The gnarled pathway indicated that evolution on the trunk favored multiple substitutions at the same amino acid positions on HA. The constant curvature was reasonably explained by assuming that the rate of amino acid substitutions varied from one position to another according to a gamma distribution. Furthermore, we utilized the estimated parameters of the gamma distribution to predict the amino acid substitutions on HA in subsequent years. Retrospective prediction tests for 12 years from 1997 to 2009 showed that 70% of actual amino acid substitutions were correctly predicted, and that 45% of predicted amino acid substitutions have been actually observed. Although it remains unsolved how to predict the exact timing of antigenic changes, the present results suggest that our model may have the potential to recognize emerging epidemic strains.     

Multiple global suppressors of protein stability defects facilitate the evolution of extended-spectrum TEM β-lactamases

The introduction of extended-spectrum cephalosporins and β-lactamase inhibitors has driven the evolution of extended-spectrum β-lactamases (ESBLs) that possess the ability to hydrolyze these drugs. The evolved TEM ESBLs from clinical isolates of bacteria often contain substitutions that occur in the active site and alter the catalytic properties of the enzyme to provide an increased hydrolysis of extended-spectrum cephalosporins or an increased resistance to inhibitors. These active-site substitutions often result in a cost in the form of reduced enzyme stability. The evolution of TEM ESBLs is facilitated by mutations that act as global suppressors of protein stability defects in that they allow the enzyme to absorb multiple amino acid changes despite incremental losses in stability associated with the substitutions. The best-studied example is the M182T substitution, which corrects protein stability defects and is commonly found in TEM ESBLs or inhibitor-resistant variants from clinical isolates. In this study, a genetic selection for second-site mutations that could partially restore function to a severely destabilized primary mutant enabled the identification of A184V, T265M, R275Q, and N276D, which are known to occur in TEM ESBLs from clinical isolates, as suppressors of TEM-1 protein stability defects. Further characterization demonstrated that these substitutions increased the thermal stability of TEM-1 and were able to correct the stability defects of two different sets of destabilizing mutations. The acquisition of compensatory global suppressors of stability costs associated with active-site mutations may be a common mechanism for the evolution of novel protein function.     

Variable evolutionary rates in the molecular evolution of mammalian growth hormones

In mammals pituitary growth hormone (GH) shows a slow basal rate of evolution (0.22 ± 0.03 × 10^–9 substitutions/amino acid site/year) which appears to have increased by at least 25–50-fold on two occasions, during the evolution of primates (to at least 10.8 ± 1.3 X 10^–9 substitutions/amino acid site/year) and artiodactyl ruminants (to at least 5.6 ± 1.3 X 10^–9 substitutions/amino acid site/year). That these rate increases are real, and not due to inadvertent comparison of nonorthologous genes, was established by showing that features of the GH gene sequences that are not expressed as mature hormone do not show corresponding changes in evolutionary rate. Thus, analysis of nonsynonymous substitutions in the coding sequence for the mature protein confirmed the rate increases seen in the primate and ruminant GHs, but analysis of nonsynonymous substitutions in the signal peptide sequence, synonymous substitutions in the coding sequence for signal peptide or mature protein, and 5 and 3 untranslated sequences showed no statistically significant changes in evolutionary rate. Evidence that the increases in evolutionary rate are probably due to positive selection is provided by the observation that in the cases of both ruminant and primate GHs the periods of rapid evolution were followed by a return to a slow rate similar to the basal rate seen in other mammalian GHs. Differences between the biological properties of GHs have been identified which may relate to these periods of rapid adaptive molecular evolution. On the basis of sequence data currently available (but excluding rodent GHs which show an intermediate rate, the basis of which is not clear) for most (90%) of evolutionary time mammalian GHs have been in the slow phase of evolution, with possibly most of the few amino acid substitutions that have occurred being neutral in nature. But most (80%) of the amino acid substitutions that have been introduced into GH during the course of mammalian evolution have been accepted during the rapid phases and were adaptive in nature.     

Identification of amino acid substitutions that alter the substrate specificity of TEM-1 beta-lactamase.

TEM-1 beta-lactamase is the most prevalent plasmid-mediated beta-lactamase in gram-negative bacteria. Recently, TEM beta-lactamase variants with amino acid substitutions in the active-site pocket of the enzyme have been identified in natural isolates with increased resistance to extended-spectrum cephalosporins. To identify other amino acid substitutions that alter the activity of TEM-1 towards extended-spectrum cephalosporins, we probed regions around the active-site pocket by random-replacement mutagenesis. This mutagenesis technique involves randomizing the DNA sequence of three to six codons in the blaTEM-1 gene to form a library containing all or nearly all of the possible substitutions for the region randomized. In total, 20 different residue positions that had been randomized were screened for amino acid substitutions that increased enzyme activity towards the extended-spectrum cephalosporin cefotaxime. Substitutions at positions 104, 168, and 238 in the TEM-1 beta-lactamase that resulted in increased enzyme activity towards extended-spectrum cephalosporins were found. In addition, small deletions in the loop containing residues 166 to 170 drastically altered the substrate specificity of the enzyme by increasing activity towards extended-spectrum cephalosporins while virtually eliminating activity towards ampicillin.     

Accelerated Evolution and Molecular Surface of Venom Phospholipase A2 Enzymes

Multiple phospholipase A₂ (PLA₂) isoenzymes found in a single snake venom induce a variety of pharmacological effects. These multiple forms are formed by gene duplication and accelerated evolution of exons. We examined the amino acid sequences of 127 snake venom PLA₂ enzymes and their homologues to study in which location most natural substitutions occur. Our data show that hot spots of amino acid substitutions in this group of proteins occur mostly on the surface. A logistic model correlating the substitution rates of each amino acid residue with their surface accessibility indicates that the probability of natural substitutions occurring in the fully exposed residue is 2.6–3.5 times greater than that of substitutions occurring in buried residues. These surface substitutions play a significant role in the evolution of new PLA₂ isoenzymes by altering the specificity of targeting to various tissues or cells, resulting in distinct pharmacological effects. Thus natural substitutions in PLA₂ enzymes, in contrast to popular belief, are not random substitutions but appear to be directed toward modifying the molecular surface. Received: 11 May 1998 / Accepted: 29 June 1998     

Interdependent MHC-DRB exon-plus-intron evolution in artiodactyls

Exon 2 sequences of an expressed MHC-DRB locus from sheep were examined for polymorphisms in both the antigen-binding regions and the adjacent intronic mixed simple tandem repeat. Twenty-one novel exon 2 Ovar-DRB alleles were identified. Short nucleotide motifs are extensively shared between certain exon 2 regions of Ovar-DRB alleles. The simple repeat variations, the number of different amino acids at usually polymorphic sites, and the number of silent substitutions were reduced in the intraspecies analyses of sheep DRB sequences, compared with those of cattle and goats. It was paradoxical that the abundance of different sheep alleles was similar to that of cattle and goats. This paradox may be explained by postulating a relatively small number of "ancient" alleles, with the present-day Ovar-DRB alleles being generated by reciprocal exchange of nucleotide motifs. At the antigen-binding sites, new combinations of amino acids were maintained in Ovar-DRB alleles by strong positive selection. In sheep--and less pronounced in goats and cattle--the DRB alleles can be divided into two groups. In one group, silent substitutions are increased when compared with the other. This suggests separate evolutionary pathways for certain groups of DRB alleles within a species. The simple repetitive sequences are also discussed with respect to the evolution of DRB alleles.      

On the nature of mutations in molecular evolution

In this work it is proposed that in evolution amino acid substitutions implying strong physico chemical and structural differences are more relevant and more frequent than substitutions between similar amino acids. This analysis is made over a group of protein families representing about 10 000 substitutions and as examples the evolutionary trees of fibrinopeptides A and calcitonins were constructed and compared.     

The combined effects of amino acid substitutions and indels on the evolution of structure within protein families

Background

In the process of protein evolution, sequence variations within protein families can cause changes in protein structures and functions. However, structures tend to be more conserved than sequences and functions. This leads to an intriguing question: what is the evolutionary mechanism by which sequence variations produce structural changes? To investigate this question, we focused on the most common types of sequence variations: amino acid substitutions and insertions/deletions (indels). Here their combined effects on protein structure evolution within protein families are studied.

Results

Sequence-structure correlation analysis on 75 homologous structure families (from SCOP) that contain 20 or more non-redundant structures shows that in most of these families there is, statistically, a bilinear correlation between the amount of substitutions and indels versus the degree of structure variations. Bilinear regression of percent sequence non-identity (PNI) and standardized number of gaps (SNG) versus RMSD was performed. The coefficients from the regression analysis could be used to estimate the structure changes caused by each unit of substitution (structural substitution sensitivity, SSS) and by each unit of indel (structural indel sensitivity, SIDS). An analysis on 52 families with high bilinear fitting multiple correlation coefficients and statistically significant regression coefficients showed that SSS is mainly constrained by disulfide bonds, which almost have no effects on SIDS.

Conclusions

Structural changes in homologous protein families could be rationally explained by a bilinear model combining amino acid substitutions and indels. These results may further improve our understanding of the evolutionary mechanisms of protein structures.     

Frequent and widespread parallel evolution of protein sequences

Understanding the patterns and causes of protein sequence evolution is a major challenge in evolutionary biology. One of the critical unresolved issues is the relative contribution of selection and genetic drift to the fixation of amino acid sequence differences between species. Molecular homoplasy, the independent evolution of the same amino acids at orthologous sites in different taxa, is one potential signature of selection; however, relatively little is known about its prevalence in eukaryotic proteomes. To quantify the extent and type of homoplasy among evolving proteins, we used phylogenetic methodology to analyze 8 genome-scale data matrices from clades of different evolutionary depths that span the eukaryotic tree of life. We found that the frequency of homoplastic amino acid substitutions in eukaryotic proteins was more than 2-fold higher than expected under neutral models of protein evolution. The overwhelming majority of homoplastic substitutions were parallelisms that involved the most frequently exchanged amino acids with similar physicochemical properties and that could be reached by a single-mutational step. We conclude that the role of homoplasy in shaping the protein record is much larger than generally assumed, and we suggest that its high frequency can be explained by both weak positive selection for certain substitutions and purifying selection that constrains substitutions to a small number of functionally equivalent amino acids.     

Broadening of neutralization activity to directly block a dominant antibody-driven SARS-coronavirus evolution pathway

Phylogenetic analyses have provided strong evidence that amino acid changes in spike (S) protein of animal and human SARS coronaviruses (SARS-CoVs) during and between two zoonotic transfers (2002/03 and 2003/04) are the result of positive selection. While several studies support that some amino acid changes between animal and human viruses are the result of inter-species adaptation, the role of neutralizing antibodies (nAbs) in driving SARS-CoV evolution, particularly during intra-species transmission, is unknown. A detailed examination of SARS-CoV infected animal and human convalescent sera could provide evidence of nAb pressure which, if found, may lead to strategies to effectively block virus evolution pathways by broadening the activity of nAbs. Here we show, by focusing on a dominant neutralization epitope, that contemporaneous- and cross-strain nAb responses against SARS-CoV spike protein exist during natural infection. In vitro immune pressure on this epitope using 2002/03 strain-specific nAb 80R recapitulated a dominant escape mutation that was present in all 2003/04 animal and human viruses. Strategies to block this nAb escape/naturally occurring evolution pathway by generating broad nAbs (BnAbs) with activity against 80R escape mutants and both 2002/03 and 2003/04 strains were explored. Structure-based amino acid changes in an activation-induced cytidine deaminase (AID) "hot spot" in a light chain CDR (complementarity determining region) alone, introduced through shuffling of naturally occurring non-immune human VL chain repertoire or by targeted mutagenesis, were successful in generating these BnAbs. These results demonstrate that nAb-mediated immune pressure is likely a driving force for positive selection during intra-species transmission of SARS-CoV. Somatic hypermutation (SHM) of a single VL CDR can markedly broaden the activity of a strain-specific nAb. The strategies investigated in this study, in particular the use of structural information in combination of chain-shuffling as well as hot-spot CDR mutagenesis, can be exploited to broaden neutralization activity, to improve anti-viral nAb therapies, and directly manipulate virus evolution.     

Temperature adaptation of lactate dehydrogenase. Structural, functional and genetic aspects

Comparison of the primary structures of thermophilic, mesophilic and psychrophilic lactate dehydrogenase (LDH) reveals a multitude of temperature-related amino acid substitutions. In the substitutions amino acid residues occurring preferentially in thermophilic, mesophilic (psychrophilic) LDH were found. On this basis, amino acid residues could be classified in an order from typical thermophilic (thermostabilizing) to typical mesophilic (thermolabilizing, increasing dynamics of the enzyme molecule) residues. The temperature-dependent ratio between thermostabilizing and thermolabilizing amino acid residues forms the basis for the specific structural and functional properties of thermophilic or mesophilic LDH. It is interesting that there appears to be a relationship between this order from thermophilic to mesophilic amino acid residues and the type of bases coding for these individual residues in the translation step of protein biosynthesis. Temperature-related amino acid substitutions are based on temperature-related base substitutions. A possible mechanism of temperature adaptation of LDH through alternative selection of thermophilic and mesophilic amino acid residues at the level of tRNA (anticodon)-mRNA (codon) interactions is discussed. These temperature-adaptation processes are evolutionary events in which the evolution and structure of the genetic code are involved.     

The function of the Saccharomyces cerevisiae iso-1-cytochrome c gene is independent of the codon at invariant residue Phe82 when the gene is present on a low-copy-number vector.

Phe82 is the most studied invariant residue of cytochrome c. However, the physiological relevance of amino acid substitutions at this position is unclear because previous studies were either performed in vitro (i.e. using purified protein) or in yeast where the gene for the protein is present on a multi-copy vector. Multi-copy vectors yield a level of cytochrome c in yeast that is greater than the wild-type level. Oligodeoxyribonucleotide-directed mutagenesis was used to change the codon for Phe82 to that of the other 19 naturally occurring amino acids as well as the amber stop codon. The alleles are present on a yeast shuttle phagemid containing the CEN6 gene which ensures a vector copy number of one to two in yeast. All the missense alleles support growth under conditions requiring a functional iso-1-cytochrome c. However the F82C, F82P, and F82R variants grow at a significantly lower rate. After selection for function, phagemids were rescued from the transformants and the identity of the mutation verified. It is concluded that all 20 amino acids are capable of supporting function. Reasons for the evolutionary invariance of Phe82 are discussed.     

Heterotachy and functional shift in protein evolution

Study of structure/function relationships constitutes an important field of research, especially for modification of protein function and drug design. However, the fact that rational design (i.e. the modification of amino acid sequences by means of directed mutagenesis, based on knowledge of the three-dimensional structure) appears to be much less efficient than irrational design (i.e. random mutagenesis followed by in vitro selection) clearly indicates that we understand little about the relationships between primary sequence, three-dimensional structure and function. The use of evolutionary approaches and concepts will bring insights to this difficult question. The increasing availability of multigene family sequences that has resulted from genome projects has inspired the creation of novel in silico evolutionary methods to predict details of protein function in duplicated (paralogous) proteins. The underlying principle of all such approaches is to compare the evolutionary properties of homologous sequence positions in paralogs. It has been proposed that the positions that show switches in substitution rate over time--i.e., 'heterotachous sites'--are good indicators of functional divergence. However, it appears that heterotachy is a much more general process, since most variable sites of homologous proteins with no evidence of functional shift are heterotachous. Similarly, it appears that switches in substitution rate are as frequent when paralogous sequences are compared as when orthologous sequences are compared. Heterotachy, instead of being indicative of functional shift, may more generally reflect a less specific process related to the many intra- and inter-molecular interactions compatible with a range of more or less equally viable protein conformations. These interactions will lead to different constraints on the nature of the primary sequences, consistently with theories suggesting the non-independence of substitutions in proteins. However, a specific type of amino acid variation might constitute a good indicator of functional divergence: substitutions occurring at positions that are generally slowly evolving. Such substitutions at constrained sites are indeed much more frequent soon after gene duplication. The identification and analysis of these sites by complementing structural information with evolutionary data may represent a promising direction to future studies dealing with the functional characterization of an ever increasing number of multi-gene families identified by complete genome analysis.     

Properties of artificial evolution of proteins and peptides]

In vitro evolution is used to study protein sequences, structures, and interactions and to obtain proteins with new properties. To analyze the specific features of this process in experiments with phage display, we studied the amino acid composition of selected sequences, constructed a matrix of amino acid substitutions, and identified pairs of coadaptive substitutions. Amino acid frequency proved to be tightly associated with the number of corresponding codons; numerous correlated substitutions were found.     

Highly pathogenic avian influenza virus subtype H5N1 escaping neutralization: more than HA variation

Influenza A viruses are one of the major threats in modern health care. Novel viruses arise due to antigenic drift and antigenic shift, leading to escape from the immune system and resulting in a serious problem for disease control. In order to investigate the escape process and to enable predictions of escape, we serially passaged influenza A H5N1 virus in vitro 100 times under immune pressure. The generated escape viruses were characterized phenotypically and in detail by full-genome deep sequencing. Mutations already found in natural isolates were detected, evidencing the in vivo relevance of the in vitro-induced amino acid substitutions. Additionally, several novel alterations were triggered. Altogether, the results imply that our in vitro system is suitable to study influenza A virus evolution and that it might even be possible to predict antigenic changes of influenza A viruses circulating in vaccinated populations.     

Adaptive evolution after duplication of penaeidin antimicrobial peptides

Penaeidin antimicrobial peptides in penaeid shrimps are an important component of their innate immune system that provides immunity against infection caused by several gram-positive bacteria and filamentous fungal species. Despite the knowledge on the identification and characterization of these peptides in penaeid shrimps, little is known about the evolutionary pattern of these peptides and the underlying genetic mechanisms that maintain high sequence diversities in the penaeidin gene family. Based on the phylogenetic analyses and maximum likelihood-based codon substitution analyses, here we present the convincing evidence that multiple copies of penaeidins have evolved by gene duplication, and positive Darwinian selection (adaptive evolution) is the likely cause of accelerated rate of amino acid substitutions among these duplicated genes. While the average ratio of non-synonymous to synonymous substitutions (omega) for the entire coding region of both active domains is 0.9805, few codon sites showed significantly higher omega (3.73). The likelihood ratio tests that compare models incorporating positive selection (omega>1) at certain codon sites with models not incorporating positive selection (omega<1), failed to reject (p=0) the evidence of positive Darwinian selection. The rapid adaptive evolution of this gene family might be directed by the pathogens and the faster rate of amino acid substitutions in the N-terminal proline-rich and C-terminal cysteine-rich domains could be due to their direct involvement in the protection against pathogens. When the host expose to different habitats/environment an accelerated rate of amino acid substitutions in both the active domains may also be expected.     

Adaptive evolution in ecological communities

Understanding how natural selection drives evolution is a key challenge in evolutionary biology. Most studies of adaptation focus on how a single environmental factor, such as increased temperature, affects evolution within a single species. The biological relevance of these experiments is limited because nature is infinitely more complex. Most species are embedded within communities containing many species that interact with one another and the physical environment. To understand the evolutionary significance of such ecological complexity, experiments must test the evolutionary impact of interactions among multiple species during adaptation. Here we highlight an experiment that manipulates species composition and tracks evolutionary responses within each species, while testing for the mechanisms by which species interact and adapt to their environment. We also discuss limitations of previous studies of adaptive evolution and emphasize how an experimental evolution approach can circumvent such shortcomings. Understanding how community composition acts as a selective force will improve our ability to predict how species adapt to natural and human-induced environmental change.