Regulatory gene mutation: a driving force behind group a Streptococcus strain‐ and serotype‐specific variation

Data from multiple bacterial pathogens are consistent with regulator‐encoding genes having higher mutation frequencies than the genome average. Such mutations drive both strain‐ and type‐ (e.g., serotype, haplotype) specific phenotypic heterogeneity, and may challenge public health due to the potential of variants to circumvent established treatment and/or preventative regimes. Here, using the human bacterial pathogen the group A Streptococcus (GAS; S. pyogenes) as a model organism, we review the types and regulatory‐, phenotypic‐, and disease‐specific consequences of naturally occurring regulatory gene mutations. Strain‐specific regulator mutations that will be discussed include examples that transform isolates into hyper‐invasive forms by enhancing expression of immunomodulatory virulence factors, and examples that promote asymptomatic carriage of the organism. The discussion of serotype‐specific regulator mutations focuses on serotype M3 GAS isolates, and how the identified rewiring of regulatory networks in this serotype may be contributing to a decades old epidemiological association of M3 isolates with particularly severe invasive infections. We conclude that mutation plays an outsized role in GAS pathogenesis and has clinical relevance. Given the phenotypic variability associated with regulatory gene mutations, the rapid examination of these genes in infecting isolates may inform with respect to potential patient complications and treatment options.     

The Streptococcus pyogenes orphan protein tyrosine phosphatase,SP‐PTP,possesses dual specificity and essential virulence regulatory functions

Group A Streptococcus (GAS) is a human pathogen that causes high morbidity and mortality. GAS lacks a gene encoding tyrosine kinase but contains one encoding tyrosine phosphatase (SP‐PTP). Thus, GAS is thought to lack tyrosine phosphorylation, and the physiological significance of SP‐PTP is, therefore, questionable. Here, we demonstrate that SP‐PTP possesses dual phosphatase specificity for Tyr‐ and Ser/Thr‐phosphorylated GAS proteins, such as Ser/Thr kinase (SP‐STK) and the SP‐STK‐phosphorylated CovR and WalR proteins. Phenotypic analysis of GAS mutants lacking SP‐PTP revealed that the phosphatase activity per se positively regulates growth, cell division and the ability to adhere to and invade host cells. Furthermore, A549 human lung cells infected with GAS mutants lacking SP‐PTP displayed increased Ser‐/Thr‐/Tyr‐phosphorylation. SP‐PTP also differentially regulates the expression of ～50% of the total GAS genes, including several virulence genes potentially through the two‐component regulators, CovR, WalR and PTS/HPr regulation of Mga. Although these mutants exhibit attenuated virulence, a GAS mutant overexpressing SP‐PTP is hypervirulent. Our study provides the first definitive evidence for the presence and importance of Tyr‐phosphorylation in GAS and the relevance of SP‐PTP as an important therapeutic target.     

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.     

The small regulatory RNA FasX enhances group A Streptococcus virulence and inhibits pilus expression via serotype‐specific targets

Bacterial pathogens commonly show intra‐species variation in virulence factor expression and often this correlates with pathogenic potential. The group A Streptococcus (GAS) produces a small regulatory RNA (sRNA), FasX, which regulates the expression of pili and the thrombolytic agent streptokinase. As GAS serotypes are polymorphic regarding (a) FasX abundance, (b) the fibronectin, collagen, T‐antigen (FCT) region of the genome, which contains the pilus genes (nine different FCT‐types), and (c) the streptokinase‐encoding gene (ska) sequence (two different alleles), we sought to test whether FasX regulates pilus and streptokinase expression in a serotype‐specific manner. Parental, fasX mutant and complemented derivatives of serotype M1 (ska‐2, FCT‐2), M2 (ska‐1, FCT‐6), M6 (ska‐2, FCT‐1) and M28 (ska‐1, FCT‐4) isolates were compared. While FasX reduced pilus expression in each serotype, the molecular basis differed, as FasX bound, and inhibited the translation of, different FCT‐region mRNAs. FasX enhanced streptokinase expression in each serotype, although the degree of regulation varied. Finally, we established that the regulation afforded by FasX enhances GAS virulence, assessed by a model of bacteremia using human plasminogen‐expressing mice. Our data are the first to identify and characterize serotype‐specific regulation by an sRNA in GAS, and to show an sRNA directly contributes to GAS virulence.     

Benfang Lei's research on heme acquisition in Gram-positive pathogens and bacterial pathogenesis

Benfang Lei's laboratory conducts research on pathogenesis of human pathogen Group A Streptococcus (GAS) and horse pathogen Streptococcus equi (S. equi). His current research focuses on heme acquisition in Gram-positive pathogens and molecular mechanism of GAS and S. equi pathogenesis. Heme is an important source of essential iron for bacterial pathogens. Benfang Lei and colleagues identified the first cell surface heme-binding protein in Gram-positive pathogens and the heme acquisition system in GAS, demonstrated direct heme transfer from one protein to another, demonstrated an experimental pathway of heme acquisition by the Staphylococcus aureus Isd system, elucidated the activated heme transfer mechanism, and obtained evidence for a chemical mechanism of direct axial ligand displacement during the Shp-to-HtsA heme transfer reaction. These findings have considerably contributed to the progress that has been made over recent years in understanding the heme acquisition process in Gram-positive pathogens. Pathogenesis of GAS is mediated by an abundance of extracellular proteins, and pathogenic role and functional mechanism are not known for many of these virulence factors. Lei laboratory identified a secreted protein of GAS as a CovRS-regulated virulence factor that is a protective antigen and is critical for GAS spreading in the skin and systemic dissemination. These studies may lead to development of novel strategies to prevent and treat GAS infections.     

Mutual Exclusivity of Hyaluronan and Hyaluronidase in Invasive Group A Streptococcus

A recent analysis of group A Streptococcus (GAS) invasive infections in Australia has shown a predominance of M4 GAS, a serotype recently reported to lack the antiphagocytic hyaluronic acid (HA) capsule. Here, we use molecular genetics and bioinformatics techniques to characterize 17 clinical M4 isolates associated with invasive disease in children during this recent epidemiology. All M4 isolates lacked HA capsule, and whole genome sequence analysis of two isolates revealed the complete absence of the hasABC capsule biosynthesis operon. Conversely, M4 isolates possess a functional HA-degrading hyaluronate lyase (HylA) enzyme that is rendered nonfunctional in other GAS through a point mutation. Transformation with a plasmid expressing hasABC restored partial encapsulation in wild-type (WT) M4 GAS, and full encapsulation in an isogenic M4 mutant lacking HylA. However, partial encapsulation reduced binding to human complement regulatory protein C4BP, did not enhance survival in whole human blood, and did not increase virulence of WT M4 GAS in a mouse model of systemic infection. Bioinformatics analysis found no hasABC homologs in closely related species, suggesting that this operon was a recent acquisition. These data showcase a mutually exclusive interaction of HA capsule and active HylA among strains of this leading human pathogen.     

Phenotypic variation and genomic variation in insect virulence traits reveal patterns of intraspecific diversity in a locust-specific fungal pathogen

Intraspecific pathogen diversity is crucial for understanding the evolution and maintenance of adaptation in host–pathogen interactions. Traits associated with virulence are often a significant source of variation directly impacted by local selection pressures. The specialist fungal entomopathogen, Metarhizium acridum, has been widely implemented as a biological control agent of locust pests in tropical regions of the world. However, few studies have accounted for natural intraspecific phenotypic and genetic variation. Here, we examine the diversity of nine isolates of M. acridum spanning the known geographic distribution, in terms of (1) virulence towards two locust species, (2) growth rates on three diverse nutrient sources, and (3) comparative genomics to uncover genomic variability. Significant variability in patterns of virulence and growth was shown among the isolates, suggesting intraspecific ecological specialization. Different patterns of virulence were shown between the two locust species, indicative of potential host preference. Additionally, a high level of diversity among M. acridum isolates was observed, revealing increased variation in subtilisin-like proteases from the Pr1 family. These results culminate in the first in-depth analysis regarding multiple facets of natural variation in M. acridum, offering opportunities to understand critical evolutionary drivers of intraspecific diversity in pathogens.     

Strain variation in an emerging iridovirus of warm-water fishes

Although iridoviruses vary widely within and among genera with respect to their host range and virulence, variation within iridovirus species has been less extensively characterized. This study explores the nature and extent of intraspecific variation within an emerging iridovirus of North American warm-water fishes, largemouth bass virus (LMBV). Three LMBV isolates recovered from three distinct sources differed genetically and phenotypically. Genetically, the isolates differed in the banding patterns generated from amplified fragment length polymorphism analysis but not in their DNA sequences at two loci of different degrees of evolutionary stability. In vitro, the isolates replicated at identical rates in cell culture, as determined by real-time quantitative PCR of viral particles released into suspension. In vivo, the isolates varied over fivefold in virulence, as measured by the rate at which they induced mortality in juvenile largemouth bass. This variation was reflected in the viral loads of exposed fish, measured using real-time quantitative PCR; the most virulent viral strain also replicated to the highest level in fish. Together, these results justify the designation of these isolates as different strains of LMBV. Strain variation in iridoviruses could help explain why animal populations naturally infected with iridovirus pathogens vary so extensively in their clinical responses to infection. The results of this study are especially relevant to emerging iridoviruses of aquaculture systems and wildlife.