Person: Serohijos, Adrian
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Serohijos
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Adrian
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Serohijos, Adrian
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Publication Chromosomal Barcoding of E. Coli Populations Reveals Lineage Diversity Dynamics at High Resolution(Nature Publishing Group, 2020-03) Jasinska, Weronika; Manhart, Michael; Lerner, Jesse; Gauthier, Louis; Serohijos, Adrian; Bershtein, ShimonEvolutionary dynamics in large asexual populations is strongly influenced by multiple competing beneficial lineages, most of which segregate at very low frequencies. However, technical barriers to tracking a large number of these rare lineages in bacterial populations have so far prevented a detailed elucidation of evolutionary dynamics. Here, we overcome this hurdle by developing a chromosomal-barcoding technique that allows simultaneous tracking of ~450,000 distinct lineages in E. coli, which we use to test the effect of sub-inhibitory concentrations of common antibiotics on the evolutionary dynamics of low-frequency lineages. We find that populations lose lineage diversity at distinct rates corresponding to their antibiotic regimen. We also determine that some lineages have similar fates across independent experiments. By analyzing the trajectory dynamics, we attribute the reproducible fates of these lineages to the presence of pre-existing beneficial mutations, and we demonstrate how the relative contribution of pre-existing and de novo mutations varies across drug regimens. Finally, we reproduce the observed lineage dynamics by simulations. Altogether, our results provide both a valuable methodology for studying bacterial evolution as well as insights into evolution under sub-inhibitory antibiotic levels.Publication Protein Homeostasis Imposes a Barrier on Functional Integration of Horizontally Transferred Genes in Bacteria(Public Library of Science, 2015) Bershtein, Shimon; Serohijos, Adrian; Bhattacharyya, Sanchari; Manhart, Michael; Choi, Jeong-Mo; Mu, Wanmeng; Zhou, Jingwen; Shakhnovich, EugeneHorizontal gene transfer (HGT) plays a central role in bacterial evolution, yet the molecular and cellular constraints on functional integration of the foreign genes are poorly understood. Here we performed inter-species replacement of the chromosomal folA gene, encoding an essential metabolic enzyme dihydrofolate reductase (DHFR), with orthologs from 35 other mesophilic bacteria. The orthologous inter-species replacements caused a marked drop (in the range 10–90%) in bacterial growth rate despite the fact that most orthologous DHFRs are as stable as E.coli DHFR at 37°C and are more catalytically active than E. coli DHFR. Although phylogenetic distance between E. coli and orthologous DHFRs as well as their individual molecular properties correlate poorly with growth rates, the product of the intracellular DHFR abundance and catalytic activity (kcat/KM), correlates strongly with growth rates, indicating that the drop in DHFR abundance constitutes the major fitness barrier to HGT. Serial propagation of the orthologous strains for ~600 generations dramatically improved growth rates by largely alleviating the fitness barriers. Whole genome sequencing and global proteome quantification revealed that the evolved strains with the largest fitness improvements have accumulated mutations that inactivated the ATP-dependent Lon protease, causing an increase in the intracellular DHFR abundance. In one case DHFR abundance increased further due to mutations accumulated in folA promoter, but only after the lon inactivating mutations were fixed in the population. Thus, by apparently distinguishing between self and non-self proteins, protein homeostasis imposes an immediate and global barrier to the functional integration of foreign genes by decreasing the intracellular abundance of their products. Once this barrier is alleviated, more fine-tuned evolution occurs to adjust the function/expression of the transferred proteins to the constraints imposed by the intracellular environment of the host organism.Publication Stability of the Influenza Virus Hemagglutinin Protein Correlates with Evolutionary Dynamics(American Society for Microbiology, 2018) Klein, Eili Y.; Blumenkrantz, Deena; Serohijos, Adrian; Shakhnovich, Eugene; Choi, Jeong-Mo; Rodrigues, Joao; Smith, Brendan D.; Lane, Andrew P.; Feldman, Andrew; Pekosz, AndrewABSTRACT Protein thermodynamics are an integral determinant of viral fitness and one of the major drivers of protein evolution. Mutations in the influenza A virus (IAV) hemagglutinin (HA) protein can eliminate neutralizing antibody binding to mediate escape from preexisting antiviral immunity. Prior research on the IAV nucleoprotein suggests that protein stability may constrain seasonal IAV evolution; however, the role of stability in shaping the evolutionary dynamics of the HA protein has not been explored. We used the full coding sequence of 9,797 H1N1pdm09 HA sequences and 16,716 human seasonal H3N2 HA sequences to computationally estimate relative changes in the thermal stability of the HA protein between 2009 and 2016. Phylogenetic methods were used to characterize how stability differences impacted the evolutionary dynamics of the virus. We found that pandemic H1N1 IAV strains split into two lineages that had different relative HA protein stabilities and that later variants were descended from the higher-stability lineage. Analysis of the mutations associated with the selective sweep of the higher-stability lineage found that they were characterized by the early appearance of highly stabilizing mutations, the earliest of which was not located in a known antigenic site. Experimental evidence further suggested that H1N1 HA stability may be correlated with in vitro virus production and infection. A similar analysis of H3N2 strains found that surviving lineages were also largely descended from viruses predicted to encode more-stable HA proteins. Our results suggest that HA protein stability likely plays a significant role in the persistence of different IAV lineages. IMPORTANCE: One of the constraints on fast-evolving viruses, such as influenza virus, is protein stability, or how strongly the folded protein holds together. Despite the importance of this protein property, there has been limited investigation of the impact of the stability of the influenza virus hemagglutinin protein—the primary antibody target of the immune system—on its evolution. Using a combination of computational estimates of stability and experiments, our analysis found that viruses with more-stable hemagglutinin proteins were associated with long-term persistence in the population. There are two potential reasons for the observed persistence. One is that more-stable proteins tolerate destabilizing mutations that less-stable proteins could not, thus increasing opportunities for immune escape. The second is that greater stability increases the fitness of the virus through increased production of infectious particles. Further research on the relative importance of these mechanisms could help inform the annual influenza vaccine composition decision process.Publication Contribution of Selection for Protein Folding Stability in Shaping the Patterns of Polymorphisms in Coding Regions(Oxford University Press, 2013) Serohijos, Adrian; Shakhnovich, EugeneThe patterns of polymorphisms in genomes are imprints of the evolutionary forces at play in nature. In particular, polymorphisms have been extensively used to infer the fitness effects of mutations and their dynamics of fixation. However, the role and contribution of molecular biophysics to these observations remain unclear. Here, we couple robust findings from protein biophysics, enzymatic flux theory, the selection against the cytotoxic effects of protein misfolding, and explicit population dynamics simulations in the polyclonal regime. First, we recapitulate results on the dynamics of clonal interference and on the shape of the DFE, thus providing them with a molecular and mechanistic foundation. Second, we predict that if evolution is indeed under the dynamic equilibrium of mutation–selection balance, the fraction of stabilizing and destabilizing mutations is almost equal among single-nucleotide polymorphisms segregating at high allele frequencies. This prediction is proven true for polymorphisms in the human coding region. Overall, our results show how selection for protein folding stability predominantly shapes the patterns of polymorphisms in coding regions.Publication Influenza A H1N1 Pandemic Strain Evolution – Divergence and the Potential for Antigenic Drift Variants(Public Library of Science, 2014) Klein, Eili Y.; Serohijos, Adrian; Choi, Jeong-Mo; Shakhnovich, Eugene; Pekosz, AndrewThe emergence of a novel A(H1N1) strain in 2009 was the first influenza pandemic of the genomic age, and unprecedented surveillance of the virus provides the opportunity to better understand the evolution of influenza. We examined changes in the nucleotide coding regions and the amino acid sequences of the hemagglutinin (HA), neuraminidase (NA), and nucleoprotein (NP) segments of the A(H1N1)pdm09 strain using publicly available data. We calculated the nucleotide and amino acid hamming distance from the vaccine strain A/California/07/2009 for each sequence. We also estimated Pepitope–a measure of antigenic diversity based on changes in the epitope regions–for each isolate. Finally, we compared our results to A(H3N2) strains collected over the same period. Our analysis found that the mean hamming distance for the HA protein of the A(H1N1)pdm09 strain increased from 3.6 (standard deviation [SD]: 1.3) in 2009 to 11.7 (SD: 1.0) in 2013, while the mean hamming distance in the coding region increased from 7.4 (SD: 2.2) in 2009 to 28.3 (SD: 2.1) in 2013. These trends are broadly similar to the rate of mutation in H3N2 over the same time period. However, in contrast to H3N2 strains, the rate of mutation accumulation has slowed in recent years. Our results are notable because, over the course of the study, mutation rates in H3N2 similar to that seen with A(H1N1)pdm09 led to the emergence of two antigenic drift variants. However, while there has been an H1N1 epidemic in North America this season, evidence to date indicates the vaccine is still effective, suggesting the epidemic is not due to the emergence of an antigenic drift variant. Our results suggest that more research is needed to understand how viral mutations are related to vaccine effectiveness so that future vaccine choices and development can be more predictive.Publication The Influence of Selection for Protein Stability on dN/dS Estimations(Oxford University Press, 2014) Dasmeh, Pouria; Serohijos, Adrian; Kepp, Kasper P.; Shakhnovich, EugeneUnderstanding the relative contributions of various evolutionary processes—purifying selection, neutral drift, and adaptation—is fundamental to evolutionary biology. A common metric to distinguish these processes is the ratio of nonsynonymous to synonymous substitutions (i.e., dN/dS) interpreted from the neutral theory as a null model. However, from biophysical considerations, mutations have non-negligible effects on the biophysical properties of proteins such as folding stability. In this work, we investigated how stability affects the rate of protein evolution in phylogenetic trees by using simulations that combine explicit protein sequences with associated stability changes. We first simulated myoglobin evolution in phylogenetic trees with a biophysically realistic approach that accounts for 3D structural information and estimates of changes in stability upon mutation. We then compared evolutionary rates inferred directly from simulation to those estimated using maximum-likelihood (ML) methods. We found that the dN/dS estimated by ML methods (ωML) is highly predictive of the per gene dN/dS inferred from the simulated phylogenetic trees. This agreement is strong in the regime of high stability where protein evolution is neutral. At low folding stabilities and under mutation-selection balance, we observe deviations from neutrality (per gene dN/dS > 1 and dN/dS < 1). We showed that although per gene dN/dS is robust to these deviations, ML tests for positive selection detect statistically significant per site dN/dS > 1. Altogether, we show how protein biophysics affects the dN/dS estimations and its subsequent interpretation. These results are important for improving the current approaches for detecting positive selection.Publication Protein Quality Control Acts on Folding Intermediates to Shape the Effects of Mutations on Organismal Fitness(Elsevier BV, 2012) Bershtein, Shimon; Mu, Wanmeng; Serohijos, Adrian; Zhou, Jingwen; Shakhnovich, EugeneWhat are the molecular properties of proteins that fall on the radar of protein quality control (PQC)? Here we mutate the E. coli’s gene encoding dihydrofolate reductase (DHFR) and replace it with bacterial orthologous genes to determine how components of PQC modulate fitness effects of these genetic changes. We find that chaperonins GroEL/ES and protease Lon compete for binding to molten globule intermediate of DHFR, resulting in a peculiar symmetry in their action: overexpression of GroEL/ES and deletion of Lon both restore growth of deleterious DHFR mutants and most of the slow-growing orthologous DHFR strains. Kinetic steady-state modeling predicts and experimentation verifies that mutations affect fitness by shifting the flux balance in cellular milieu between protein production, folding, and degradation orchestrated by PQC through the interaction with folding intermediates.Publication Protein Homeostasis Imposes a Barrier on Functional Integration of Horizontally Transferred Genes in Bacteria(Public Library of Science, 2015) Bershtein, Shimon; Serohijos, Adrian; Bhattacharyya, Sanchari; Manhart, Michael; Choi, Jeong-Mo; Mu, Wanmeng; Zhou, Jingwen; Shakhnovich, EugeneHorizontal gene transfer (HGT) plays a central role in bacterial evolution, yet the molecular and cellular constraints on functional integration of the foreign genes are poorly understood. Here we performed inter-species replacement of the chromosomal folA gene, encoding an essential metabolic enzyme dihydrofolate reductase (DHFR), with orthologs from 35 other mesophilic bacteria. The orthologous inter-species replacements caused a marked drop (in the range 10–90%) in bacterial growth rate despite the fact that most orthologous DHFRs are as stable as E.coli DHFR at 37°C and are more catalytically active than E. coli DHFR. Although phylogenetic distance between E. coli and orthologous DHFRs as well as their individual molecular properties correlate poorly with growth rates, the product of the intracellular DHFR abundance and catalytic activity (kcat/KM), correlates strongly with growth rates, indicating that the drop in DHFR abundance constitutes the major fitness barrier to HGT. Serial propagation of the orthologous strains for ~600 generations dramatically improved growth rates by largely alleviating the fitness barriers. Whole genome sequencing and global proteome quantification revealed that the evolved strains with the largest fitness improvements have accumulated mutations that inactivated the ATP-dependent Lon protease, causing an increase in the intracellular DHFR abundance. In one case DHFR abundance increased further due to mutations accumulated in folA promoter, but only after the lon inactivating mutations were fixed in the population. Thus, by apparently distinguishing between self and non-self proteins, protein homeostasis imposes an immediate and global barrier to the functional integration of foreign genes by decreasing the intracellular abundance of their products. Once this barrier is alleviated, more fine-tuned evolution occurs to adjust the function/expression of the transferred proteins to the constraints imposed by the intracellular environment of the host organism.Publication Isolation and Analysis of Rare Norovirus Recombinants from Coinfected Mice Using Drop-Based Microfluidics(American Society for Microbiology, 2015) Zhang, Huidan; Cockrell, Shelley K.; Kolawole, Abimbola O.; Rotem, Assaf; Serohijos, Adrian; Chang, Connie B.; Tao, Ye; Mehoke, Thomas S.; Han, Yulong; Lin, Jeffrey S.; Giacobbi, Nicholas S.; Feldman, Andrew B.; Shakhnovich, Eugene; Weitz, David; Wobus, Christiane E.; Pipas, James M.Human noroviruses (HuNoVs) are positive-sense RNA viruses that can cause severe, highly infectious gastroenteritis. HuNoV outbreaks are frequently associated with recombination between circulating strains. Strain genotyping and phylogenetic analyses show that noroviruses often recombine in a highly conserved region near the junction of the viral polyprotein (open reading frame 1 [ORF1]) and capsid (ORF2) genes and occasionally within the RNA-dependent RNA polymerase (RdRP) gene. Although genotyping methods are useful for tracking changes in circulating viral populations, they report only the dominant recombinant strains and do not elucidate the frequency or range of recombination events. Furthermore, the relatively low frequency of recombination in RNA viruses has limited studies to cell culture or in vitro systems, which do not reflect the complexities and selective pressures present in an infected organism. Using two murine norovirus (MNV) strains to model coinfection, we developed a microfluidic platform to amplify, detect, and recover individual recombinants following in vitro and in vivo coinfection. One-step reverse transcriptase PCR (RT-PCR) was performed in picoliter drops with primers that identified the wild-type and recombinant progenies and scanned for recombination breakpoints at ∼1-kb intervals. We detected recombination between MNV strains at multiple loci spanning the viral protease, RdRP, and capsid ORFs and isolated individual recombinant RNA genomes that were present at a frequency of 1/300,000 or higher. This study is the first to examine norovirus recombination following coinfection of an animal and suggests that the exchange of RNA among viral genomes in an infected host occurs in multiple locations and is an important driver of genetic diversity.Publication Merging molecular mechanism and evolution: theory and computation at the interface of biophysics and evolutionary population genetics(Elsevier BV, 2014) Serohijos, Adrian; Shakhnovich, EugeneThe variation among sequences and structures in nature is both determined by physical laws and by evolutionary history. However, these two factors are traditionally investigated by disciplines with different emphasis and philosophy—molecular biophysics on one hand and evolutionary population genetics in another. Here, we review recent theoretical and computational approaches that address the critical need to integrate these two disciplines. We first articulate the elements of these integrated approaches. Then, we survey their contribution to our mechanistic understanding of molecular evolution, the polymorphisms in coding region, the distribution of fitness effects (DFE) of mutations, the observed folding stability of proteins in nature, and the distribution of protein folds in genomes.