Ten years of pan-genome analyses Vernikos, George; Medini, Duccio; Riley, David R ...
Current opinion in microbiology,
02/2015, Letnik:
23
Journal Article
Recenzirano
Highlights • Next generation sequencing technologies have enabled large-scale genome analyses. • Pan-genome analyses provide a framework for predicting and modeling genomic diversity. • Analyses can ...be conducted at different scales, from single species to super kingdom level. • Datasets and technical implementations govern pan-genome analysis-derived conclusions.
From a common ancestor,
and
evolved in parallel into one of the most important pathogens and a mutualistic colonizer of humans, respectively. This evolutionary scenario provides a unique basis for ...studies of both infection-associated properties and properties important for harmonious coexistence with the host. We performed detailed comparisons of 60 genomes of
,
,
, the three
subspecies
,
, and
, and
Nonfunctional remnants of ancestral genes in both
and in
support the evolutionary model and the concept that evolutionary changes on both sides were required to reach their present relationship to the host. Confirmed by screening of >7,500 genomes, we identified 224 genes associated with virulence. The striking difference to commensal streptococci was the diversity of regulatory mechanisms, including regulation of capsule production, a significantly larger arsenal of enzymes involved in carbohydrate hydrolysis, and proteins known to interfere with innate immune factors. The exclusive presence of the virulence factors in
enhances their potential as vaccine components, as a direct impact on beneficial members of the commensal microbiota can be excluded. In addition to loss of these virulence-associated genes, adaptation of
to a mutualistic relationship with the host apparently required preservation or acquisition of 25 genes lost or absent from
Successful adaptation of
and other commensal streptococci to a harmonious relationship with the host relied on genetic stability and properties facilitating life in biofilms.
is one of the most important human pathogens but is closely related to
, with which humans live in harmony. The fact that the two species evolved from a common ancestor provides a unique basis for studies of both infection-associated properties and properties important for harmonious coexistence with the host. By detailed comparisons of genomes of the two species and other related streptococci, we identified 224 genes associated with virulence and 25 genes unique to the mutualistic species. The exclusive presence of the virulence factors in
enhances their potential as vaccine components, as a direct impact on beneficial members of the commensal microbiota can be excluded. Successful adaptation of
and other commensal streptococci to a harmonious relationship with the host relied on genetic stability and properties facilitating life in biofilms.
Bacterial genome sequencing has become so easy and accessible that the genomes of multiple strains of more and more individual species have been and will be generated. These data sets provide for in ...depth analysis of intra-species diversity from various aspects. The pan-genome analysis, whereby the size of the gene repertoire accessible to any given species is characterized together with an estimate of the number of whole genome sequences required for proper analysis, is being increasingly applied. Different models exist for the analysis and their accuracy and applicability depend on the case at hand. Here we discuss current models and suggest a new model of broad applicability, including examples of its implementation.
Acidithiobacillus ferrooxidans is a major participant in consortia of microorganisms used for the industrial recovery of copper (bioleaching or biomining). It is a chemolithoautrophic, ...gamma-proteobacterium using energy from the oxidation of iron- and sulfur-containing minerals for growth. It thrives at extremely low pH (pH 1-2) and fixes both carbon and nitrogen from the atmosphere. It solubilizes copper and other metals from rocks and plays an important role in nutrient and metal biogeochemical cycling in acid environments. The lack of a well-developed system for genetic manipulation has prevented thorough exploration of its physiology. Also, confusion has been caused by prior metabolic models constructed based upon the examination of multiple, and sometimes distantly related, strains of the microorganism.
The genome of the type strain A. ferrooxidans ATCC 23270 was sequenced and annotated to identify general features and provide a framework for in silico metabolic reconstruction. Earlier models of iron and sulfur oxidation, biofilm formation, quorum sensing, inorganic ion uptake, and amino acid metabolism are confirmed and extended. Initial models are presented for central carbon metabolism, anaerobic metabolism (including sulfur reduction, hydrogen metabolism and nitrogen fixation), stress responses, DNA repair, and metal and toxic compound fluxes.
Bioinformatics analysis provides a valuable platform for gene discovery and functional prediction that helps explain the activity of A. ferrooxidans in industrial bioleaching and its role as a primary producer in acidic environments. An analysis of the genome of the type strain provides a coherent view of its gene content and metabolic potential.
Capsular polysaccharide (CPS), which surrounds the bacteria, is one of the most significant and multifaceted contributors to
virulence. Capsule prevents entrapment in mucus during colonization, traps ...water to protect against desiccation, can serve as an energy reserve, and protects the bacterium against complement-mediated opsonization and immune cell phagocytosis. To date, 100 biochemically and serologically distinct capsule types have been identified for
; 20 to 30 of which have well-defined propensity to cause opportunistic human infection. Among these, serotype 3 is perhaps the most problematic as serotype 3 infections are characterized as having severe clinical manifestations including empyema, bacteremia, cardiotoxicity, and meningitis; consequently, with a fatality rate of 30%-47%. Moreover, serotype 3 resists antibody-mediated clearance despite its inclusion in the current 13-valent conjugate vaccine formulation. This review covers the role of capsule in pneumococcal pathogenesis and the importance of serotype 3 on human disease. We discuss how serotype 3 capsule synthesis and presentation on the bacterial surface is distinct from other serotypes, the biochemical and physiological properties of this capsule type that facilitate its ability to cause disease, and why existing vaccines are unable to confer protection. We conclude with discussion of the clonal properties of serotype 3 and how these have changed since introduction of the 13-valent vaccine in 2000.
Reverse vaccinology accelerates the discovery of potential vaccine candidates (PVCs) prior to experimental validation. Current programs typically use one bacterial proteome to identify PVCs through a ...filtering architecture using feature prediction programs or a machine learning approach. Filtering approaches may eliminate potential antigens based on limitations in the accuracy of prediction tools used. Machine learning approaches are heavily dependent on the selection of training datasets with experimentally validated antigens (positive control) and non-protective-antigens (negative control). The use of one or few bacterial proteomes does not assess PVC conservation among strains, an important feature of vaccine antigens.
We present ReVac, which implements both a panoply of feature prediction programs without filtering out proteins, and scoring of candidates based on predictions made on curated positive and negative control PVCs datasets. ReVac surveys several genomes assessing protein conservation, as well as DNA and protein repeats, which may result in variable expression of PVCs. ReVac's orthologous clustering of conserved genes, identifies core and dispensable genome components. This is useful for determining the degree of conservation of PVCs among the population of isolates for a given pathogen. Potential vaccine candidates are then prioritized based on conservation and overall feature-based scoring. We present the application of ReVac, applied to 69 Moraxella catarrhalis and 270 non-typeable Haemophilus influenzae genomes, prioritizing 64 and 29 proteins as PVCs, respectively.
ReVac's use of a scoring scheme ranks PVCs for subsequent experimental testing. It employs a redundancy-based approach in its predictions of features using several prediction tools. The protein's features are collated, and each protein is ranked based on the scoring scheme. Multi-genome analyses performed in ReVac allow for a comprehensive overview of PVCs from a pan-genome perspective, as an essential pre-requisite for any bacterial subunit vaccine design. ReVac prioritized PVCs of two human respiratory pathogens, identifying both novel and previously validated PVCs.
With the exponential increase in the number of bacterial taxa with genome sequence data, a new standardized method to assign species designations is needed that is consistent with classically ...obtained taxonomic analyses. This is particularly acute for unculturable, obligate intracellular bacteria with which classically defined methods, like DNA-DNA hybridization, cannot be used, such as those in the
. In this study, we generated nucleotide-based core genome alignments for a wide range of genera with classically defined species, as well as those within the
. We created a workflow that uses the length, sequence identity, and phylogenetic relationships inferred from core genome alignments to assign genus and species designations that recapitulate classically obtained results. Using this method, most classically defined bacterial genera have a core genome alignment that is ≥10% of the average input genome length. Both
and
fail to meet this criterion, indicating that the taxonomy of these genera should be reexamined. Consistently, genomes from organisms with the same species epithet have ≥96.8% identity of their core genome alignments. Additionally, these core genome alignments can be used to generate phylogenomic trees to identify monophyletic clades that define species and neighbor-network trees to assess recombination across different taxa. By these criteria,
organisms are delineated into species different from the currently used supergroup designations, while
organisms are delineated into 9 distinct species, compared to the current 27 species. By using core genome alignments to assign taxonomic designations, we aim to provide a high-resolution, robust method to guide bacterial nomenclature that is aligned with classically obtained results.
With the increasing availability of genome sequences, we sought to develop and apply a robust, portable, and high-resolution method for the assignment of genera and species designations that can recapitulate classically defined taxonomic designations. Using cutoffs derived from the lengths and sequence identities of core genome alignments along with phylogenetic analyses, we sought to evaluate or reevaluate genus- and species-level designations for diverse taxa, with an emphasis on the order
, where species designations have been applied inconsistently. Our results indicate that the
genus has an overabundance of species designations, that the current
and
genus designations are both too broad and need to be divided, and that there are clear demarcations of
species that do not align precisely with the existing supergroup designations.
The bacterium Streptococcus pneumoniae is one of the leading causes of fatal infections affecting humans. Intriguingly, phylogenetic analysis shows that the species constitutes one evolutionary ...lineage in a cluster of the otherwise commensal Streptococcus mitis strains, with which humans live in harmony. In a comparative analysis of 35 genomes, including phylogenetic analyses of all predicted genes, we have shown that the pathogenic pneumococcus has evolved into a master of genomic flexibility while lineages that evolved into the nonpathogenic S. mitis secured harmonious coexistence with their host by stabilizing an approximately 15%-reduced genome devoid of many virulence genes. Our data further provide evidence that interspecies gene transfer between S. pneumoniae and S. mitis occurs in a unidirectional manner, i.e., from S. mitis to S. pneumoniae. Import of genes from S. mitis and other mitis, anginosus, and salivarius group streptococci ensured allelic replacements and antigenic diversification and has been driving the evolution of the remarkable structural diversity of capsular polysaccharides of S. pneumoniae. Our study explains how the unique structural diversity of the pneumococcal capsule emerged and conceivably will continue to increase and reveals a striking example of the fragile border between the commensal and pathogenic lifestyles. While genomic plasticity enabling quick adaptation to environmental stress is a necessity for the pathogenic streptococci, the commensal lifestyle benefits from stability. Importance: One of the leading causes of fatal infections affecting humans, Streptococcus pneumoniae, and the commensal Streptococcus mitis are closely related obligate symbionts associated with hominids. Faced with a shortage of accessible hosts, the two opposing lifestyles evolved in parallel. We have shown that the nonpathogenic S. mitis secured harmonious coexistence with its host by stabilizing a reduced genome devoid of many virulence genes. Meanwhile, the pathogenic pneumococcus evolved into a master of genomic flexibility and imports genes from S. mitis and other related streptococci. This process ensured antigenic diversification and has been driving the evolution of the remarkable structural diversity of capsular polysaccharides of S. pneumoniae, which conceivably will continue to increase and present a challenge to disease prevention.
Streptococcus pyogenes (Group A Streptococcus, GAS) remains a major public health burden worldwide, infecting over 750 million people leading to over 500,000 deaths annually. GAS pathogenesis is ...complex, involving genetically distinct GAS strains and multiple infection sites. To overcome fastidious genetic manipulations and accelerate pathogenesis investigations in GAS, we developed a mariner-based system (Krmit) for en masse monitoring of complex mutant pools by transposon sequencing (Tn-seq). Highly saturated transposant libraries (Krmit insertions in ca. every 25 nucleotides) were generated in two distinct GAS clinical isolates, a serotype M1T1 invasive strain 5448 and a nephritogenic serotype M49 strain NZ131, and analyzed using a Bayesian statistical model to predict GAS essential genes, identifying sets of 227 and 241 of those genes in 5448 and NZ131, respectively. A large proportion of GAS essential genes corresponded to key cellular processes and metabolic pathways, and 177 were found conserved within the GAS core genome established from 20 available GAS genomes. Selected essential genes were validated using conditional-expression mutants. Finally, comparison to previous essentiality analyses in S. sanguinis and S. pneumoniae revealed significant overlaps, providing valuable insights for the development of new antimicrobials to treat infections by GAS and other pathogenic streptococci.
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