Natural selection and evolution of streptococcal virulence genes involved in tissue-specific adaptations

The molecular mechanisms underlying niche adaptation in bacteria are not fully understood. Primary infection by the pathogen group A streptococcus (GAS) takes place at either the throat or the skin of its human host, and GAS strains differ in tissue site preference. Many skin-tropic strains bind host plasminogen via the plasminogen-binding group A streptococcal M protein (PAM) present on the cell surface; inactivation of genes encoding either PAM or streptokinase (a plasminogen activator) leads to loss of virulence at the skin. Unlike PAM, which is present in only a subset of GAS strains, the gene encoding streptokinase (ska) is present in all GAS isolates. In this study, the evolution of the virulence genes known to be involved in skin infection was examined. Most genetic diversity within ska genes was localized to a region encoding the plasminogen-docking domain (beta-domain). The gene encoding PAM displayed strong linkage disequilibrium (P < 0.01) with a distinct phylogenetic cluster of the ska beta-domain-encoding region. Yet, ska alleles of distant taxa showed a history of intragenic recombination, and high intrinsic levels of recombination were found among GAS strains having different tissue tropisms. The data suggest that tissue-specific adaptations arise from epistatic coselection of bacterial virulence genes. Additional analysis of ska genes showed that approximately 4% of the codons underwent strong diversifying selection. Horizontal acquisition of one ska lineage from a commensal Streptococcus donor species was also evident. Together, the data suggest that new phenotypes can be acquired through interspecies recombination between orthologous genes, while constrained functions can be preserved; in this way, orthologous genes may provide a rich and ready source for new phenotypes and thereby play a facilitating role in the emergence of new niche adaptations in bacteria.     

The fibronectin-binding capacity and host cell adherence of Streptococcus pyogenes strains are discordant with each other

Surface exposed fibronectin-binding proteins (FBPs) play an important role in the adherence of Streptococcus pyogenes (group A streptococcus, GAS) to host cells. This pathogen expresses numerous FBPs, of which SfbI, SfbII and PrtF2 are major surface exposed FBPs. However, GAS strains differ in the genetic potential to express these proteins. To test whether this difference reflects in differences in fibronectin (Fn) binding, a set of circulating strains previously examined for adherence to host cells was used. The 68 distinct strains were isolated from throat, skin and blood. They were analyzed for (a) the presence of genes for SfbI, SfbII and PrtF2 and (b) the extent of Fn binding. The results suggest that strains possessing two or more of the genes for these FBPs bound Fn significantly more than strains possessing none or one of the genes. No correlation between the extent of Fn binding and the tissue site of isolation was found. Furthermore, together with our previous studies on adherence capacity of these GAS strains, we found no correlation between Fn binding ability and the avidity of the strains to adhere to epithelial cells. We suggest that while Fn binding is important for adhesion, for many GAS strains the extent of Fn binding is not the critical determinant of adherence.     

Genome sequence of a nephritogenic and highly transformable M49 strain of Streptococcus pyogenes

The 1,815,783-bp genome of a serotype M49 strain of Streptococcus pyogenes (group A streptococcus [GAS]), strain NZ131, has been determined. This GAS strain (FCT type 3; emm pattern E), originally isolated from a case of acute post-streptococcal glomerulonephritis, is unusually competent for electrotransformation and has been used extensively as a model organism for both basic genetic and pathogenesis investigations. As with the previously sequenced S. pyogenes genomes, three unique prophages are a major source of genetic diversity. Two clustered regularly interspaced short palindromic repeat (CRISPR) regions were present in the genome, providing genetic information on previous prophage encounters. A unique cluster of genes was found in the pathogenicity island-like emm region that included a novel Nudix hydrolase, and, further, this cluster appears to be specific for serotype M49 and M82 strains. Nudix hydrolases eliminate potentially hazardous materials or prevent the unbalanced accumulation of normal metabolites; in bacteria, these enzymes may play a role in host cell invasion. Since M49 S. pyogenes strains have been known to be associated with skin infections, the Nudix hydrolase and its associated genes may have a role in facilitating survival in an environment that is more variable and unpredictable than the uniform warmth and moisture of the throat. The genome of NZ131 continues to shed light upon the evolutionary history of this human pathogen. Apparent horizontal transfer of genetic material has led to the existence of highly variable virulence-associated regions that are marked by multiple rearrangements and genetic diversification while other regions, even those associated with virulence, vary little between genomes. The genome regions that encode surface gene products that will interact with host targets or aid in immune avoidance are the ones that display the most sequence diversity. Thus, while natural selection favors stability in much of the genome, it favors diversity in these regions.     

Contribution of exogenous genetic elements to the group A Streptococcus metagenome

Variation in gene content among strains of a bacterial species contributes to biomedically relevant differences in phenotypes such as virulence and antimicrobial resistance. Group A Streptococcus (GAS) causes a diverse array of human infections and sequelae, and exhibits a complex pathogenic behavior. To enhance our understanding of genotype-phenotype relationships in this important pathogen, we determined the complete genome sequences of four GAS strains expressing M protein serotypes (M2, M4, and 2 M12) that commonly cause noninvasive and invasive infections. These sequences were compared with eight previously determined GAS genomes and regions of variably present gene content were assessed. Consistent with the previously determined genomes, each of the new genomes is approximately 1.9 Mb in size, with approximately 10% of the gene content of each encoded on variably present exogenous genetic elements. Like the other GAS genomes, these four genomes are polylysogenic and prophage encode the majority of the variably present gene content of each. In contrast to most of the previously determined genomes, multiple exogenous integrated conjugative elements (ICEs) with characteristics of conjugative transposons and plasmids are present in these new genomes. Cumulatively, 242 new GAS metagenome genes were identified that were not present in the previously sequenced genomes. Importantly, ICEs accounted for 41% of the new GAS metagenome gene content identified in these four genomes. Two large ICEs, designated 2096-RD.2 (63 kb) and 10750-RD.2 (49 kb), have multiple genes encoding resistance to antimicrobial agents, including tetracycline and erythromycin, respectively. Also resident on these ICEs are three genes encoding inferred extracellular proteins of unknown function, including a predicted cell surface protein that is only present in the genome of the serotype M12 strain cultured from a patient with acute poststreptococcal glomerulonephritis. The data provide new information about the GAS metagenome and will assist studies of pathogenesis, antimicrobial resistance, and population genomics.     

Variations in the distribution of genes encoding virulence and extracellular proteins in group A streptococcus are largely restricted to 11 genomic loci

Group A streptococcus (GAS) is a human pathogen associated with a wide range of human diseases that vary in symptoms and clinical severity. In this report we describe the use of a targeted low density array representing genes encoding classical virulence factors, purported virulence factors and other extracellular proteins to examine differences in the genetic profiles of 68 clinical GAS isolates. Of the 226 genes on the array (encoding 217 virulence factors or putative extracellular proteins and nine positive control house-keeping proteins) 62 had distributions that were statistically associated with specific GAS M-types. While 32 of these genes were bacteriophage related, the remaining 30 have not previously been described as bacteriophage associated. We show that these 'non-bacteriophage related' genes are found in 11 loci located in five greater chromosomal regions, often near classical GAS virulence factors, and often accompanied by genes associated with mobile genetic elements (MGEs). Many of these loci also demonstrated genetic variation within strains of the same M-type, suggesting these regions to be recombinatorial and mutational hotspots. Evidence for acquisition of genes from other species is also apparent in these loci. Our data suggests that imprecise recombination events involving MGEs not only result in acquisition of new genes, but can also result in deletion of flanking chromosomal genes. Thus MGE related events would appear to be the major contributor to variation of discrete virulence loci, which could account for the disease causing propensity of individual strains. We believe that profiling of the 11 loci could be a meaningful tool in epidemiological GAS typing studies.     

Genetics of dispersal

Dispersal is a process of central importance for the ecological and evolutionary dynamics of populations and communities, because of its diverse consequences for gene flow and demography. It is subject to evolutionary change, which begs the question, what is the genetic basis of this potentially complex trait? To address this question, we (i) review the empirical literature on the genetic basis of dispersal, (ii) explore how theoretical investigations of the evolution of dispersal have represented the genetics of dispersal, and (iii) discuss how the genetic basis of dispersal influences theoretical predictions of the evolution of dispersal and potential consequences. Dispersal has a detectable genetic basis in many organisms, from bacteria to plants and animals. Generally, there is evidence for significant genetic variation for dispersal or dispersal‐related phenotypes or evidence for the micro‐evolution of dispersal in natural populations. Dispersal is typically the outcome of several interacting traits, and this complexity is reflected in its genetic architecture: while some genes of moderate to large effect can influence certain aspects of dispersal, dispersal traits are typically polygenic. Correlations among dispersal traits as well as between dispersal traits and other traits under selection are common, and the genetic basis of dispersal can be highly environment‐dependent. By contrast, models have historically considered a highly simplified genetic architecture of dispersal. It is only recently that models have started to consider multiple loci influencing dispersal, as well as non‐additive effects such as dominance and epistasis, showing that the genetic basis of dispersal can influence evolutionary rates and outcomes, especially under non‐equilibrium conditions. For example, the number of loci controlling dispersal can influence projected rates of dispersal evolution during range shifts and corresponding demographic impacts. Incorporating more realism in the genetic architecture of dispersal is thus necessary to enable models to move beyond the purely theoretical towards making more useful predictions of evolutionary and ecological dynamics under current and future environmental conditions. To inform these advances, empirical studies need to answer outstanding questions concerning whether specific genes underlie dispersal variation, the genetic architecture of context‐dependent dispersal phenotypes and behaviours, and correlations among dispersal and other traits.     

Comparison of the evolutionary dynamics of symbiotic and housekeeping loci: a case for the genetic coherence of rhizobial lineages

In prokaryotes, lateral gene transfer across chromosomal lineages may be mediated by plasmids, phages, transposable elements, and other accessory DNA elements. However, the importance of such transfer and the evolutionary forces that may restrict gene exchange remain largely unexplored in native settings. In this study, tests of phylogenetic congruence are employed to explore the range of horizontal transfer of symbiotic (sym) loci among distinct chromosomal lineages of native rhizobia, the nitrogen-fixing symbiont of legumes. Rhizobial strains isolated from nodules of several host plant genera were sequenced at three loci: symbiotic nodulation genes (nodB and nodC), the chromosomal housekeeping locus glutamine synthetase II (GSII), and a portion of the 16S rRNA gene. Molecular phylogenetic analysis shows that each locus generally subdivides strains into the same major groups, which correspond to the genera Rhizobium, Sinorhizobium, and Mesorhizobium. This broad phylogenetic congruence indicates a lack of lateral transfer across major chromosomal subdivisions, and it contrasts with previous studies of agricultural populations showing broad transfer of sym loci across divergent chromosomal lineages. A general correspondence of the three rhizobial genera with major legume groups suggests that host plant associations may be important in the differentiation of rhizobial nod and chromosomal loci and may restrict lateral transfer among strains. The second major result is a significant incongruence of nod and GSII phylogenies within rhizobial subdivisions, which strongly suggests horizontal transfer of nod genes among congenerics. This combined evidence for lateral gene transfer within, but not between, genetic subdivisions supports the view that rhizobial genera are "reproductively isolated" and diverge independently. Differences across rhizobial genera in the specificity of host associations imply that the evolutionary dynamics of the symbiosis vary considerably across lineages in native settings.      

Spatial and ecological population genetic structures within two island‐endemic Aeonium species of different niche width

The Crassulacean genus Aeonium is a well‐known example for plant species radiation on oceanic archipelagos. However, while allopatric speciation among islands is documented for this genus, the role of intra‐island speciation due to population divergence by topographical isolation or ecological heterogeneity has not yet been addressed. The aim of this study was to investigate intraspecific genetic structures and to identify spatial and ecological drivers of genetic population differentiation on the island scale. We analyzed inter simple sequence repeat variation within two island‐endemic Aeonium species of La Palma: one widespread generalist that covers a large variety of different habitat types (Ae. davidbramwellii) and one narrow ecological specialist (Ae. nobile), in order to assess evolutionary potentials on this island. Gene pool differentiation and genetic diversity patterns were associated with major landscape structures in both species, with phylogeographic implications. However, overall levels of genetic differentiation were low. For the generalist species, outlier loci detection and loci–environment correlation approaches indicated moderate signatures of divergent selection pressures linked to temperature and precipitation variables, while the specialist species missed such patterns. Our data point to incipient differentiation among populations, emphasizing that ecological heterogeneity and topographical structuring within the small scales of an island can foster evolutionary processes. Very likely, such processes have contributed to the radiation of Aeonium on the Canary Islands. There is also support for different evolutionary mechanisms between generalist and specialist species.     

MALDI-TOF mass spectrometry as a tool for differentiation of invasive and noninvasive Streptococcus pyogenes isolates

A novel mass spectral fingerprinting and proteomics approach using MALDI-TOF MS was applied to detect and identify protein biomarkers of group A Streptococcus (GAS) strains. Streptococcus pyogenes ATCC 700294 genome strain was compared with eight GAS clinical isolates to explore the ability of MALDI-TOF MS to differentiate isolates. Reference strains of other bacterial species were also analyzed and compared with the GAS isolates. MALDI preparations were optimized by varying solvents, matrices, plating techniques, and mass ranges for S. pyogenes ATCC 700294. Spectral variability was tested. A subset of common, characteristic, and reproducible biomarkers in the range of 2000-14 000 Da were detected, and they appeared to be independent of the culture media. Statistical analysis confirmed method reproducibility. Random Forest analysis of all selected GAS isolates revealed differences among most of them, and summed spectra were used for hierarchical cluster analysis. Specific biomarkers were found for each strain, and invasive GAS isolates could be differentiated. GAS isolates from cases of necrotizing fasciitis were clustered together and were distinct from isolates associated with noninvasive infections, despite their sharing the same emm type. Almost 30% of the biomarkers detected were tentatively identified as ribosomal proteins.     

Convergence and divergence in lizard colour polymorphisms

Colour polymorphic species are model systems for examining the evolutionary processes that generate and maintain discrete phenotypic variation in natural populations. Lizards have repeatedly evolved strikingly similar polymorphic sexual signals in distantly related lineages, providing an opportunity to examine convergence and divergence in colour polymorphism, correlated traits and associated evolutionary processes. Herein, we synthesise the extensive literature on lizard colour polymorphisms in both sexes, including recent advances in understanding of the underlying biochemical, cellular and genetic mechanisms, and correlated behavioural, physiological and life-history traits. Male throat, head or ventral colour morphs generally consist of red/orange, yellow and white/blue morphs, and sometimes mixed morphs with combinations of two colours. Despite these convergent phenotypes, there is marked divergence in correlated behavioural, physiological and life-history traits. We discuss the need for coherence in morph classification, particularly in relation to ‘mixed’ morphs. We highlight future research directions such as the genetic basis of convergent phenotypes and the role of environmental variation in the maintenance of polymorphism. Research in this very active field promises to continue to provide novel insights with broad significance to evolutionary biologists.     

Emerging model systems in eco-evo-devo: the environmentally responsive spadefoot toad

Spadefoot toads have emerged as a model system for addressing fundamental questions in ecological and evolutionary developmental biology (eco-evo-devo). Their tadpoles produce a wide range of adaptive phenotypes in direct response to diverse environmental stimuli. Such phenotypic plasticity offers an excellent opportunity to examine how an organism's ecology affects its development as well as how an organism's development influences its ecology and evolution. By characterizing and understanding the interconnectedness between an organism's environment, its development responses, and its ecological interactions in natural populations, such research promises to clarify further the role of the environment in not only selecting among diverse phenotypes, but also creating such phenotypes in the first place.     

Discrete colour polymorphism in the tawny dragon lizard (Ctenophorus decresii) and differences in signal conspicuousness among morphs

Intraspecific colour variation is common in nature and can vary from the coexistence of discrete colour variants in polymorphic species to continuous variation. Whether coloration is continuous or discrete is often ambiguous and many species exhibit a combination of the two. The nature of the variation (discrete or continuous) has implications for both the genetic basis of the colour variation and the evolutionary processes generating and maintaining it. Consequently, it is important to qualify the existence of discrete morphs, particularly in relation to the animal's visual system. In this study, we quantified male throat colour variation in Ctenophorus decresii tawny dragon lizard and tested for morphological and ecological correlates of the colour variants. We confirmed that discrete throat colour morphs can be defined based on colour and pattern analyses independent of the human visual system. We also found that the colour variants differed in their conspicuousness from the background, to the lizard's visual system, which has implications for signalling. However, the morphs did not differ in morphology or microhabitat use, which suggests that these characteristics are not involved in the evolutionary maintenance of the polymorphism.     

Bacteriophage content of M49 strains of Streptococcus pyogenes

Bacteriophages are common autonomous migrating mobile genetic elements in group A Streptococcus (GAS) and are often associated with the carriage of various virulence genes, including toxins, mitogens and enzymes. Two collections of GAS type M49 strains isolated from invasive (22 strains) and noninvasive (16 strains) clinical cases have been studied for the presence of phage and phage-associated virulence genes. All the GAS strains carried from at least two to six phage genomes as determined by the number of known phage integrase genes found. A sampling of the invasive M49 strains showed that they belonged to the same multilocus sequence typing type, carried two specific integrase genes ( int 5 and int 7), and contained the toxin genes spe A, spe H and spe I. Other invasive strains lacking this gene profile carried the prophage integrating in mutL–mutS region and inducing the 'mutator' phenotype. We suggest that this specific phage-related virulence gene constellation might be an important factor increasing M49 GAS pathogenicity.     

A new rapid and cost-effective method for detection of phages, ICEs and virulence factors encoded by Streptococcus pyogenes

Streptococcus pyogenes (group A Streptococcus, GAS) is a human pathogen that causes diseases of various intensity, from mild strep throat to life threatening invasive infections and postinfectional sequelae. S. pyogenes encodes multiple, often phage encoded, virulence factors and their presence is related to severity of the disease. Acquisition of mobile genetic elements, carrying virulence factors, as phages or ICEs (integrative and cojugative elements) has been shown previously to promote selection of virulent clones. We designed the system of eight low volume multi- and one singleplex PCR reactions to detect genes encoding twenty virulence factors (spd3, sdc, sdaB, sdaD, speB, spyCEP, scpA, mac, sic, speL, K, M, C, I, A, H, G, J, smeZ and ssa) and twenty one phage and ICE integration sites described so far for S. pyogenes. Classification of strains based on the phage and virulence factors absence or presence, correlates with PFGE MLST and emm typing results. We developed a novel, fast and cost effective system that can be used to detect GAS virulence factors. Moreover, this system may become an alternative and effective system to differentiate between GAS strains.     

Molecular evolution of a multigene family in group A streptococci

The emm genes are members of a gene family in group A streptococci (GAS) that encode for antiphagocytic cell-surface proteins and/or immunoglobulin-binding proteins. Previously sequenced genes in this family have been named "emm," "fcrA," "enn," "arp," "protH," and "mrp"; herein they will be referred to as the "emm gene family." The genes in the emm family are located in a cluster occupying 3-6 kb between the genes mry and scpA on the chromosome of Streptococcus pyogenes. Most GAS strains contain one to three tandemly arranged copies of emm-family genes in the cluster, but the alleles within the cluster vary among different strains. Phylogenetic analysis of the conserved sequences at the 3' end of these genes differentiates all known members of this family into four evolutionarily distinct emm subfamilies. As a starting point to analyze how the different subfamilies are related evolutionarily, the structure of the emm chromosomal region was mapped in a number of diverse GAS strains by using subfamily-specific primers in the polymerase chain reaction. Nine distinct chromosomal patterns of the genes in the emm gene cluster were found. These nine chromosomal patterns support a model for the evolution of the emm gene family in which gene duplication followed by sequence divergence resulted in the generation of four major-gene subfamilies in this locus.