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.     

DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps

The innate immune response plays a crucial role in satisfactory host resolution of bacterial infection. In response to chemotactic signals, neutrophils are early responding cells that migrate in large numbers to sites of infection. The recent discovery of secreted neutrophil extracellular traps (NETs) composed of DNA and histones opened a novel dimension in our understanding of the microbial killing capacity of these specialized leukocytes. M1 serotype strains of the pathogen Group A Streptococcus (GAS) are associated with invasive infections including necrotizing fasciitis (NF) and express a potent DNase (Sda1). Here we apply a molecular genetic approach of allelic replacement mutagenesis, single gene complementation, and heterologous expression to demonstrate that DNase Sda1 is both necessary and sufficient to promote GAS neutrophil resistance and virulence in a murine model of NF. Live fluorescent microscopic cell imaging and histopathological analysis are used to establish for the first time a direct linkage between NET degradation and bacterial pathogenicity. Inhibition of GAS DNase activity with G-actin enhanced neutrophil clearance of the pathogen in vitro and reduced virulence in vivo. The results demonstrate a significant role for NETs in neutrophil-mediated innate immunity, and at the same time identify a novel therapeutic target against invasive GAS infection.     

A bacterial regulatory RNA attenuates virulence,spread and human host cell phagocytosis

Staphylococcus aureus pathogenesis is directed by regulatory proteins and RNAs. We report the case of an RNA attenuating virulence and host uptake, possibly to sustain commensalism. A S. aureus sRNA, SprC (srn_3610), reduced virulence and bacterial loads in a mouse infection model. S. aureus deleted for sprC became more virulent and increased bacterial dissemination in colonized animals. Conversely, inducing SprC expression lowered virulence and the bacterial load. Without sprC, S. aureus phagocytosis by monocytes and macrophages was higher, whereas bacteria were internalized at lower yields when SprC expression was stimulated. Without sprC, higher internalization led to a greater number of extracellular bacteria, facilitating colonization. SprC expression decreased after phagocytosis, concurring with the facilitated growth of bacteria lacking the sRNA in the presence of an oxidant. The major staphylococcal autolysin facilitates S. aureus uptake by human phagocytes. ATL proved to be negatively regulated by SprC. The SprC domains involved in pairing with atl mRNA were analyzed. The addition of ATL reduced phagocytosis of bacteria lacking sprC with no effects on wild-type bacterial uptake, implying that SprC influences phagocytosis, at least in part, by controlling ATL. Since the control of SprC on ATL was modest, other factors must contribute to atl regulation.     

Regulatory rewiring confers serotype‐specific hyper‐virulence in the human pathogen group A Streptococcus

Phenotypic heterogeneity is commonly observed between isolates of a given pathogen. Epidemiological analyses have identified that some serotypes of the group A Streptococcus (GAS) are non‐randomly associated with particular disease manifestations. Here, we present evidence that a contributing factor to the association of serotype M3 GAS isolates with severe invasive infections is the presence of a null mutant allele for the orphan kinase RocA. Through use of RNAseq analysis, we identified that the natural rocA mutation present within M3 isolates leads to the enhanced expression of more than a dozen immunomodulatory virulence factors, enhancing phenotypes such as hemolysis and NAD⁺ hydrolysis. Consequently, an M3 GAS isolate survived human phagocytic killing at a level 13‐fold higher than a rocA complemented derivative, and was significantly more virulent in a murine bacteremia model of infection. Finally, we identified that RocA functions through the CovR/S two‐component system as levels of phosphorylated CovR increase in the presence of functional RocA, and RocA has no regulatory activity following covR or covS mutation. Our data are consistent with RocA interfacing with the CovR/S two‐component system, and that the absence of this activity in M3 GAS potentiates the severity of invasive infections caused by isolates of this serotype.     

Insights into Streptococcus agalactiae PI-2b pilus biosynthesis and role in adherence to host cells

The core PI-2b pilus present in “hypervirulent” ST-17 Streptococcus agalactiae strains consists of three pilin subunits (Spb1, Ap1 and Ap2) assembled by sortase SrtC1 and cell-wall anchored by Srt2. Spb1 was shown to be the major pilin and Ap2 the anchor pilin. Ap1 is a putative adhesin. Two additional genes, orf and lep, are part of this operon. The contribution of Lep and Ap1 to the biogenesis of the PI-2b pilus was investigated. Concerning the role of PI-2b, we found that higher PI-2b expression resulted in higher adherence to human brain endothelial cells and higher phagocytosis by human THP1 macrophages.     

The expression of the let-7 small regulatory RNA is controlled by ecdysone during metamorphosis in Drosophila melanogaster

In Caenorhabditis elegans, the heterochronic pathway controls the timing of developmental events during the larval stages. A component of this pathway, the let-7 small regulatory RNA, is expressed at the late stages of development and promotes the transition from larval to adult (L/A) stages. The stage-specificity of let-7 expression, which is crucial for the proper timing of the worm L/A transition, is conserved in Drosophila melanogaster and other invertebrates. In Drosophila, pulses of the steroid hormone 20-hydroxyecdysone (ecdysone) control the timing of the transition from larval to pupal to adult stages. To test whether let-7 expression is regulated by ecdysone in Drosophila, we used Northern blot analysis to examine the effect of altered ecdysone levels on let-7 expression in mutant animals, organ cultures, and S2 cultured cells. Experiments were conducted to test the role of Broad-Complex (BR-C), an essential component in the ecdysone pathway, in let-7 expression. We show that ecdysone and BR-C are required for let-7 expression, indicating that the ecdysone pathway regulates the temporal expression of let-7 in Drosophila. These results demonstrate an interaction between steroid hormone signaling and the heterochronic pathway in insects.