Detecting genetic gain and loss events in terms of protein domain: Method and implementation.

Continuous gain and loss of genes are the primary driving forces of bacterial evolution and environmental adaptation. Studying bacterial evolution in terms of protein domain, which is the fundamental function and evolutionary unit of proteins, can provide a more comprehensive understanding of bacterial differentiation and phenotypic adaptation processes. Therefore, we proposed a phylogenetic tree-based method for detecting genetic gain and loss events in terms of protein domains. Specifically, the method focuses on a single domain to trace its evolution process or on multiple domains to investigate their co-evolution principles. This novel method was validated using 122 Shigella isolates. We found that the loss of a significant number of domains was likely the main driving force behind the evolution of Shigella, which could reduce energy expenditure and preserve only the most essential functions. Additionally, we observed that simultaneously gained and lost domains were often functionally related, which can facilitate and accelerate phenotypic evolutionary adaptation to the environment. All results obtained using our method agree with those of previous studies, which validates our proposed method.

Spatio-temporal spread and evolution of Lassa virus in West Africa.

BACKGROUND: Lassa fever is a hemorrhagic disease caused by Lassa virus (LASV), which has been classified by the World Health Organization as one of the top infectious diseases requiring prioritized research. Previous studies have provided insights into the classification and geographic characteristics of LASV lineages. However, the factor of the distribution and evolution characteristics and phylodynamics of the virus was still limited. METHODS: To enhance comprehensive understanding of LASV, we employed phylogenetic analysis, reassortment and recombination detection, and variation evaluation utilizing publicly available viral genome sequences. RESULTS: The results showed the estimated the root of time of the most recent common ancestor (TMRCA) for large (L) segment was approximately 634 (95% HPD: [385879]), whereas the TMRCA for small (S) segment was around 1224 (95% HPD: [10301401]). LASV primarily spread from east to west in West Africa through two routes, and in route 2, the virus independently spread to surrounding countries through Liberia, resulting in a wider spread of LASV. From 1969 to 2018, the effective population size experienced two significant increased, indicating the enhanced genetic diversity of LASV. We also found the evolution rate of L segment was faster than S segment, further results showed zinc-binding protein had the fastest evolution rate. Reassortment events were detected in multiple lineages including sub-lineage IIg, while recombination events were observed within lineage V. Significant amino acid changes in the glycoprotein precursor of LASV were identified, demonstrating sequence diversity among lineages in LASV. CONCLUSION: This study comprehensively elucidated the transmission and evolution of LASV in West Africa, providing detailed insights into reassortment events, recombination events, and amino acid variations.

Enantioselective conjugate addition to nitroolefins catalysed by helical peptides with a single remote stereogenic centre.

Two short pentapeptides rich in α-aminoisobutyric acid (Aib) residues have been shown to act as enantioselective organocatalysts for the conjugate addition of nucleophiles to nitroolefins. An L-alanine terminated peptide, (Aib)4(L-Ala)NHtBu, which has neither functionalised sidechains nor a highly designed reactive site, used an exposed N-terminal primary amine and the amide bonds of the backbone to mediate catalysis. Folding of this peptide into a 310 helical structure was observed by crystallography. Folding into a helix relays the conformational preference of the chiral alanine residue at the C-terminus to the primary amine at the N-terminus, 0.9 nm distant. The chiral environment and defined shape produced by the 310 helix brings the amine site into proximity to two exposed amide NHs. Reaction scope studies implied that the amine acts as a Brønsted base and the solvent-exposed NH groups of the helix, shown to weakly bind ß-nitrostyrene, are needed to obtain an enantiomeric excess. Replacement of L-alanine with D-phenylalanine gave (Aib)4(D-Phe)NHtBu, a peptide that now catalysed the benchmark reaction with the opposite enantioselectivity. These studies show how achiral residues can play a key role in enantioselective catalysis by peptides through the promotion of folding.

Genetic characteristics of classical astroviruses in Shenzhen, China, 2016-2019.

Human astrovirus (HAstV) is a single-stranded, positive-sense RNA virus and is the leading cause of viral gastroenteritis. However, despite its prevalence, astroviruses still remain one of the least studied enteroviruses. In this study, we sequenced 11 classical astrovirus strains from clinical samples collected in Shenzhen, China from 2016 to 2019, analyzed their genetic characteristics, and deposited them into GenBank. We conducted phylogenetic analysis using IQ-TREE software, with references to astrovirus sequences worldwide. The phylogeographic analysis was performed using the Bayesian Evolutionary Analysis Sampling Trees program, through Bayesian Markov Chain Monte Carlo sampling. We also conducted recombination analysis with the Recombination Detection Program. The newly sequenced strains were categorized as HAstV genotype 1, which is the predominant genotype in Shenzhen. Phylogeographic reconstruction indicated that HAstV-1 may have migrated from the United States to China, followed by frequent transmission between China and Japan. The recombination analysis revealed recombination events within and across genotypes, and identified a recombination-prone region that produced relatively uniform recombination breakpoints and fragment lengths. The genetic analysis of HAstV strains in Shenzhen addresses the current lack of astrovirus data in the region of Shenzhen and provides key insights to the evolution and transmission of astroviruses worldwide. These findings highlight the importance of improving surveillance of astroviruses.

Characterization and Diversity of Klebsiella pneumoniae Prophages.

Klebsiella pneumoniae is a common human commensal and opportunistic pathogen. In recent years, the clinical isolation and resistance rates of K. pneumoniae have shown a yearly increase, leading to a special interest in mobile genetic elements. Prophages are a representative class of mobile genetic elements that can carry host-friendly genes, transfer horizontally between strains, and coevolve with the host's genome. In this study, we identified 15,946 prophages from the genomes of 1437 fully assembled K. pneumoniae deposited in the NCBI database, with 9755 prophages on chromosomes and 6191 prophages on plasmids. We found prophages to be notably diverse and widely disseminated in the K. pneumoniae genomes. The K. pneumoniae prophages encoded multiple putative virulence factors and antibiotic resistance genes. The comparison of strain types with prophage types suggests that the two may be related. The differences in GC content between the same type of prophages and the genomic region in which they were located indicates the alien properties of the prophages. The overall distribution of GC content suggests that prophages integrated on chromosomes and plasmids may have different evolutionary characteristics. These results suggest a high prevalence of prophages in the K. pneumoniae genome and highlight the effect of prophages on strain characterization.

Evaluating the effect of SARS-CoV-2 spike mutations with a linear doubly robust learner.

Driven by various mutations on the viral Spike protein, diverse variants of SARS-CoV-2 have emerged and prevailed repeatedly, significantly prolonging the pandemic. This phenomenon necessitates the identification of key Spike mutations for fitness enhancement. To address the need, this manuscript formulates a well-defined framework of causal inference methods for evaluating and identifying key Spike mutations to the viral fitness of SARS-CoV-2. In the context of large-scale genomes of SARS-CoV-2, it estimates the statistical contribution of mutations to viral fitness across lineages and therefore identifies important mutations. Further, identified key mutations are validated by computational methods to possess functional effects, including Spike stability, receptor-binding affinity, and potential for immune escape. Based on the effect score of each mutation, individual key fitness-enhancing mutations such as D614G and T478K are identified and studied. From individual mutations to protein domains, this paper recognizes key protein regions on the Spike protein, including the receptor-binding domain and the N-terminal domain. This research even makes further efforts to investigate viral fitness via mutational effect scores, allowing us to compute the fitness score of different SARS-CoV-2 strains and predict their transmission capacity based solely on their viral sequence. This prediction of viral fitness has been validated using BA.2.12.1, which is not used for regression training but well fits the prediction. To the best of our knowledge, this is the first research to apply causal inference models to mutational analysis on large-scale genomes of SARS-CoV-2. Our findings produce innovative and systematic insights into SARS-CoV-2 and promotes functional studies of its key mutations, serving as reliable guidance about mutations of interest.

Comparative genomics reveals new insights into the evolution of the IncA and IncC family of plasmids.

Incompatibility groups IncA and IncC plasmids are of great concern due to their ability to disseminate antibiotic resistance in bacteria via conjugative transfer. A deep understanding of their genomic structures and evolutionary characteristics is of great significance for improving our knowledge about its multidrug-resistance evolution and dissemination. However, current knowledge of their backbone structure, features of core functional modules and the characteristics of variable regions is based on a few plasmids, which highlights the need for a comprehensive systematic study. The present study thoroughly compared and analysed 678 IncA and IncC plasmid genomes. We found that their core functional genes were occasionally deficient and sometimes existed as multiple functional copies/multiple families, which resulted in much diversity. The phylogeny of 13 core functional genes corresponded well to the plasmid subtypes. The conjugative transfer system gained diverse complexity and exhibited many previously unnoticed types with multiple combinations. The insertion of mobile genetic elements (MGEs) in plasmids varied between types and was present in 4 insertion spots in different types of plasmids with certain types of transposons, integrons and insertion sequences. The impact of gene duplication, deletion, the insertion of MGEs, genome rearrangement and recombination resulted in the complex dynamic variable backbone of IncA and IncC plasmids. And IncA and IncC plasmids were more complex than their closest relative SXT/R391 integrative conjugative elements (ICEs), which included nearly all of the diversity of SXT/R391 in key systems. Our work demonstrated a global and systematic view of the IncA and IncC plasmids and provides many new insights into their genome evolution.

A Standardized Framework for Better Understanding of Phenotypic Differences within Bacterial Phyla Based on Protein Domain.

We propose a standardized framework to classify target species based on their protein domains, which can be utilized in different contexts, like eukaryotes and prokaryotes. In this study, by applying the framework to the bacterial kingdom as an implementation example and comparing the results with the current taxonomy standards at the phylum level, we came to the conclusion that the sequence of domains rather than the content of domains in a protein and the presence of one domain rather than the number of occurrences of one domain play more important roles in deciding bacterial phenotypes as well as matching the current taxonomy. In addition, the comparison also helps us to better focus on the species that conflict with the current phylum category, as well as to further investigate their phenotypic or genotypic differences. IMPORTANCE A 3-step framework was designed which can be applied to clustering species based on their protein domains, and different candidate models are proposed in each step for better adaptation of various scenarios. We show its implementation for the bacterial kingdom as an example, which helps us to find the most appropriate model combination that will best reflect the relationship between domains and phenotypes in this context. In addition, identifying species that are distant in the results but should be closely related phylogenetically can help us to focus on the mismatch for better understanding of their key phenotypic or genotypic differences.

Inactivated Rabies Virus Vectored MERS-Coronavirus Vaccine Induces Protective Immunity in Mice, Camels, and Alpacas.

Middle East respiratory syndrome coronavirus (MERS-CoV) is an emergent coronavirus that has caused frequent zoonotic events through camel-to-human spillover. An effective camelid vaccination strategy is probably the best way to reduce human exposure risk. Here, we constructed and evaluated an inactivated rabies virus-vectored MERS-CoV vaccine in mice, camels, and alpacas. Potent antigen-specific antibody and CD8+ T-cell responses were generated in mice; moreover, the vaccination reduced viral replication and accelerated virus clearance in MERS-CoV-infected mice. Besides, protective antibody responses against both MERS-CoV and rabies virus were induced in camels and alpacas. Satisfyingly, the immune sera showed broad cross-neutralizing activity against the three main MERS-CoV clades. For further characterization of the antibody response induced in camelids, MERS-CoV-specific variable domains of heavy-chain-only antibody (VHHs) were isolated from immunized alpacas and showed potent prophylactic and therapeutic efficacies in the Ad5-hDPP4-transduced mouse model. These results highlight the inactivated rabies virus-vectored MERS-CoV vaccine as a promising camelid candidate vaccine.

Characterizing the Core Internal Gene Pool of H9N2 Responsible for Continuous Reassortment With Other Influenza A Viruses.

Reassortment among avian influenza viruses is the main source of novel avian influenza virus subtypes. Studies have shown that the H9N2 virus often donates internal segments to generate novel reassortant avian influenza viruses, acting as a reassortment template. However, the characteristics of the internal pattern of reassortment remain unclear. In this article, we first defined the core gene pool of the internal segments of the H9N2 virus that provide templates for reassortment. We used genetic distance and sequence similarity to define typical clusters in the core gene pool. Then, we analyzed the phylogenetic relationships, feature vector distances, geographic distributions and mutation sites of strains related to the core gene pool. Strains in the same typical clusters have close phylogenetic relationships and feature vector distances. We also found that these typical clusters can be divided into three categories according to their main geographic distribution area. Furthermore, typical clusters in the same geographic area contain some common mutation patterns. Our results suggest that typical clusters in the core gene pool affect the reassortment events of the H9N2 virus in many respects, such as geographic distribution and amino acid mutation sites.

Reassortment Network of Influenza A Virus.

Influenza A virus (IAV) genomes are composed of eight single-stranded RNA segments. Genetic exchange through reassortment of the segmented genomes often endows IAVs with new genetic characteristics, which may affect transmissibility and pathogenicity of the viruses. However, a comprehensive understanding of the reassortment history of IAVs remains lacking. To this end, we assembled 40,296 whole-genome sequences of IAVs for analysis. Using a new clustering method based on Mean Pairwise Distances in the phylogenetic trees, we classified each segment of IAVs into clades. Correspondingly, reassortment events among IAVs were detected by checking the segment clade compositions of related genomes under specific environment factors and time period. We systematically identified 1,927 possible reassortment events of IAVs and constructed their reassortment network. Interestingly, minimum spanning tree of the reassortment network reproved that swine act as an intermediate host in the reassortment history of IAVs between avian species and humans. Moreover, reassortment patterns among related subtypes constructed in this study are consistent with previous studies. Taken together, our genome-wide reassortment analysis of all the IAVs offers an overview of the leaping evolution of the virus and a comprehensive network representing the relationships of IAVs.

Characterization of Burkholderia cepacia Complex Core Genome and the Underlying Recombination and Positive Selection.

Recombination and positive selection are two key factors that play a vital role in pathogenic microorganisms' population adaptation and diversification. The Burkholderia cepacia complex (Bcc) represents bacterial species with high similarity, which can cause severe infections among cases suffering from the chronic granulomatous disorder and cystic fibrosis (CF). At present, no genome-wide study has been carried out focusing on investigating the core genome of Bcc associated with the two evolutionary forces. The general characteristics of the core genome of Bcc species remain scarce as well. In this study, we explored the core orthologous genes of 116 Bcc strains using comparative genomic analysis and studied the two adaptive evolutionary forces: recombination and positive selection. We estimated 1005 orthogroups consisting entirely of single copy genes. These single copy orthologous genes in some Cluster of Orthologous Groups (COG) categories showed significant differences in the comparison of several evolutionary properties, and the encoding proteins were relatively simple and compact. Our findings showed that 5.8% of the core orthologous genes strongly supported recombination; in the meantime, 1.1% supported positive selection. We found that genes involved in protein synthesis as well as material transport and metabolism are favored by selection pressure. More importantly, homologous recombination contributed more genetic variation to a large number of genes and largely maintained the genetic cohesion in Bcc. This high level of recombination between Bcc species blurs their taxonomic boundaries, which leads Bcc species to be difficult or impossible to distinguish phenotypically and genotypically.

Genome-based classification of Burkholderia cepacia complex provides new insight into its taxonomic status.

BACKGROUND: Accurate classification of different Burkholderia cepacia complex (BCC) species is essential for therapy, prognosis assessment and research. The taxonomic status of BCC remains problematic and an improved knowledge about the classification of BCC is in particular needed. METHODS: We compared phylogenetic trees of BCC based on 16S rRNA, recA, hisA and MLSA (multilocus sequence analysis). Using the available whole genome sequences of BCC, we inferred a species tree based on estimated single-copy orthologous genes and demarcated species of BCC using dDDH/ANI clustering. RESULTS: We showed that 16S rRNA, recA, hisA and MLSA have limited resolutions in the taxonomic study of closely related bacteria such as BCC. Our estimated species tree and dDDH/ANI clustering clearly separated 116 BCC strains into 36 clusters. With the appropriate reclassification of misidentified strains, these clusters corresponded to 22 known species as well as 14 putative novel species. CONCLUSIONS: This is the first large-scale and systematic study of the taxonomic status of the BCC and could contribute to further insights into BCC taxonomy. Our study suggested that conjunctive use of core phylogeny based on single-copy orthologous genes, as well as pangenome-based dDDH/ANI clustering would provide a preferable framework for demarcating closely related species. REVIEWER: This article was reviewed by Dr. Xianwen Ren.

A Type III Effector NleF from EHEC Inhibits Epithelial Inflammatory Cell Death by Targeting Caspase-4.

Enterohemorrhagic E. coli (EHEC) is a highly pathogenic bacterial strain capable of inducing severe gastrointestinal disease. Here, we show that EHEC uses the T3SS effector NleF to counteract the host inflammatory response by dampening caspase-4-mediated inflammatory epithelial cell death and by preventing the production of IL-1ß. The other two inflammatory caspases, caspase-1 and caspase-5, are not involved in EHEC ΔnleF-induced inflammatory cell death. We found that NleF not only interrupted the heterodimerization of caspase-4-p19 and caspase-4-p10, but also inhibited the interaction of caspase-1 and caspase-4. The last four amino acids of the NleF carboxy terminus are essential in inhibiting caspase-4-dependent inflammatory cell death.

Genetic Characteristic and Global Transmission of Influenza A H9N2 Virus.

The H9N2 virus has been demonstrated to donate its genes to other subtypes of influenza A virus, forming new reassortant virus which may infect human beings. Understanding the genetic characteristic and the global transmission patterns of the virus would guide the prevention and control of potentially emerging avian influenza A virus. In this paper, we hierarchically classified the evolution of the H9N2 virus into three main lineages based on the phylogenetic characteristics of the virus. Due to the distribution of sampling locations, we named the three lineages as Worldwide lineage, Asia-Africa lineage, and China lineage. Codon usage analysis and selective positive site analysis of the lineages further showed the lineage-specific evolution of the virus. We reconstructed the transmission routes of the virus in the three lineages through phylogeography analysis, by which several epicenters for migration of the virus were identified. The hierarchical classification of the lineages implied a possible original seeding process of the virus, starting from the Worldwide lineages to the Asian-Africa lineages and to the China lineages. In the process of H9N2 virus global transmission, the United States was the origin of the virus. China Mainland, Hong Kong SAR, Japan, and Korea were important transfer centers. Based on both the transmission route and the distribution of the hosts in each lineage, we concluded that the wild birds' migration has contributed much to the long-distance global spread of the virus, while poultry trade and people's lifestyle may have contributed to the relatively short-distance transmission in some areas of the Asia and Africa.

Evolution of bopA Gene in Burkholderia: A Case of Convergent Evolution as a Mechanism for Bacterial Autophagy Evasion.

Autophagy is an important defense mechanism targeting intracellular bacteria to restrict their survival and growth. On the other hand, several intracellular pathogens have developed an antiautophagy mechanism to facilitate their own replication or intracellular survival. Up to now, no information about the origin or evolution of the antiautophagic genes in bacteria is available. BopA is an effector protein secreted by Burkholderia pseudomallei via the type three secretion system, and it has been shown to play a pivotal role in their escape from autophagy. The evolutionary origin of bopA was examined in this work. Sequence similarity searches for BopA showed that no homolog of BopA was detected in eukaryotes. However, eukaryotic linear motifs were detected in BopA. The phylogenetic tree of the BopA proteins in our analysis is congruent with the species phylogeny derived from housekeeping genes. Moreover, there was no obvious difference in GC content values of bopA gene and their respective genomes. Integrated information on the taxonomic distribution, phylogenetic relationships, and GC content of the bopA gene of Burkholderia revealed that this gene was acquired via convergent evolution, not from eukaryotic host through horizontal gene transfer (HGT) event. This work has, for the first time, characterized the evolutionary mechanism of bacterial evasion of autophagy. The results of this study clearly demonstrated the role of convergent evolution in the evolution of how bacteria evade autophagy.