Emanuel Hanski

Protein translocation into host epithelial cells by infecting enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) causes diarrhoea in young children. EPEC induces the formation of actin pedestal in infected epithelial cells. A type III protein secretion system and several proteins that are secreted by this system, including EspB, are involved in inducing the formation of the actin pedestals. We have demonstrated that contact of EPEC with HeLa cells is associated with the induction of production and secretion of EspB. Shortly after infection, EPEC initiates translocation of EspB, and EspB fused to the CyaA reporter protein (EspB–CyaA), into the host cell. The translocated EspB was distributed between the membrane and the cytoplasm of the host cell. Translocation was strongly promoted by attachment of EPEC to the host cell, and both attachment factors of EPEC, intimin and the bundle‐forming pili, were needed for full translocation efficiency. Translocation and secretion of EspB and EspB–CyaA were abolished in mutants deficient in components of the type III protein secretion system, including sepA and sepB mutants. EspB–CyaA was secreted but not translocated by an espB mutant. These results indicate that EspB is both translocated and required for protein translocation by EPEC.

Roles of integrins and fibronectin in the entry of Streptococcus pyogenes into cells via protein F1

Entry of group A streptococcus (GAS) into cells has been suggested as an important trait in GAS pathogenicity. Protein F1, a fibronectin (Fn) binding protein, mediates GAS adherence to cells and the extracellular matrix, and efficient cell internalization. We demonstrate that the cellular receptors responsible for protein F1‐mediated internalization of GAS are integrins capable of Fn binding. In HeLa cells, bacterial entry is blocked by anti‐β1 integrin monoclonal antibody. In the mouse cell line GD25, a β1 null mutant, the αvβ3 integrin promotes GAS entry. Internalization of these cells by GAS is blocked by a peptide that specifically binds to αvβ3 integrin. In both cell lines, entry of GAS requires the occupancy of protein F1 by Fn. Neither the 29 kDa nor the 70 kDa N‐terminal fragments or the 120 kDa cell‐binding fragment of Fn promote bacterial entry. Fn‐coated beads are taken up efficiently by HeLa cells. Both the entry of GAS via protein F1 and the uptake of Fn‐coated beads are blocked by anti‐β1 antibody but are unaffected by a large excess of soluble Fn. Internalization of HeLa cells by bacteria bearing increasing amounts of prebound Fn to protein F1 reveals a sigmoidal ultrasensitive curve. These suggest that the ability of particles to interact via Fn with multiple integrin sites plays a central role in their ability to enter cells.

EspH, a new cytoskeleton-modulating effector of enterohaemorrhagic and enteropathogenic Escherichia coli

Enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) are closely related pathogens. During infection, EPEC and EHEC use a type III secretion system (TTSS) to translocate effector proteins into the infected cells and thereby modify specific host functions. These include transient filopodium formation which is Cdc42‐dependent. Filopodia formation is followed by assembly of actin pedestals, the process enhanced by inhibition of Cdc42. We discovered that orf 18 of the enterocyte effacement locus encodes a new effector, which we termed EspH. We show that EspH is translocated efficiently into the infected cells by the TTSS and localizes beneath the EPEC microcolonies. Inactivation of espH resulted in enhanced formation of filopodia and attenuated the pedestals formation. Furthermore, overexpression of EspH resulted in strong repression of filopodium formation and heightened pedestal formation. We also demonstrate that overexpression of EspH by EHEC induces marked elongation of the typically flat pedestals. Similar pedestal elongation was seen upon infection of COS cells overexpressing EspH. EspH transiently expressed by the COS cells was localized to the membrane and disrupted the actin cytoskeletal structure. Our findings indicate that EspH is a modulator of the host actin cytoskeleton structure.

Protein F1 Is Required for Efficient Entry of Streptococcus pyogenes into Epithelial Cells

It was recently reported that strains of Streptococcus pyogenes are capable of inducing entry of the bacterium into epithelial cells; however, nothing is known regarding the gene(s) and the underlying mechanism(s) involved. Using isogenic mutants of S. pyogenes JRS4 strain that are defective in the expression of each of the surface proteins F1 and M6, it was demonstrated that both are required for efficient internalization. Expression of F1 on the surface of a poorly invading S. pyogenes strain significantly enhances its internalization efficiency. Protein F1-mediated internalization is inhibited by UR, the nonrepetitive fibronectin-binding domain of this protein, and to a lesser extent, by the repetitive fibronectin-binding domain, RD2. Polyclonal anti-human fibronectin antibodies completely abolish F1-mediated internalization; increasing fibronectin concentrations result in a significant enhancement of bacterial uptake. The findings shown here suggest that protein F1 mediates streptococcal internalization and that the M6 protein is required for more efficient entry of the bacterium.

A streptococcal protease that degrades CXC chemokines and impairs bacterial clearance from infected tissues

Group A Streptococcus (GAS) causes the life‐threatening infection in humans known as necrotizing fasciitis (NF). Infected subcutaneous tissues from an NF patient and mice challenged with the same GAS strain possessed high bacterial loads but a striking paucity of infiltrating polymorphonuclear leukocytes (PMNs). Impaired PMN recruitment was attributed to degradation of the chemokine IL‐8 by a GAS serine peptidase. Here, we use bioinformatics approach coupled with target mutagenesis to identify this peptidase as ScpC. We show that SilCR pheromone downregulates scpC transcription via the two‐component system—SilA/B. In addition, we demonstrate that in vitro, ScpC degrades the CXC chemokines: IL‐8 (human), KC, and MIP‐2 (both murine). Furthermore, using a murine model of human NF, we demonstrate that ScpC, but not the C5a peptidase ScpA, is an essential virulence factor. An ScpC‐deficient mutant is innocuous for untreated mice but lethal for PMN‐depleted mice. ScpC degrades KC and MIP‐2 locally in the infected skin tissues, inhibiting PMN recruitment. In conclusion, ScpC represents a novel GAS virulence factor functioning to directly inactivate a key element of the host innate immune response.

Protein F2, a novel fibronectin‐binding protein from Streptococcus pyogenes, possesses two binding domains

Binding of the group A streptococcus (GAS) to respiratory epithelium is mediated by the fibronectin (Fn)‐binding adhesin, protein F1. Previous studies have suggested that certain GAS strains express Fn‐binding proteins that are different from protein F1. In this study, we have cloned, sequenced, and characterized a gene (prtF2) from GAS strain 100076 encoding a novel Fn‐binding protein, termed protein F2. Insertional inactivation of prtF2 in strain 100076 abolishes its high‐affinity Fn binding. prtF2‐related genes exist in most GAS strains that lack prtF1 (encoding protein F1) but bind Fn with high affinity. These observations suggest that protein F2 is a major Fn‐binding protein in GAS. Protein F2 is highly homologous to Fn‐binding proteins from Streptococcus dysgalactiae and Strep‐tococcus equisimilis, particularly in its carboxy‐terminal portion. Two domains are responsible for Fn binding by protein F2. One domain (FBRD) consists of three consecutive repeats, whereas the other domain (UFBD) resides on a non‐repeated stretch of approximately 100 amino acids and is located 100 amino acids amino‐terminal of FBRD. Each of these domains is capable of binding Fn when expressed as a separate protein. In strain 100076, protein F2 activity is regulated in response to alterations in the concentration of atmospheric oxygen.

Effect of a bacterial pheromone peptide on host chemokine degradation in group A streptococcal necrotising soft-tissue infections

BACKGROUND Necrotising soft-tissue infections due to group A streptococcus (GAS) are rare (about 0.2 cases per 100000 people). The disease progresses rapidly, causing severe necrosis and hydrolysis of soft tissues. Histopathological analysis of necrotic tissue debrided from two patients (one with necrotising fasciitis and one with myonecrosis) showed large quantities of bacteria but no infiltrating neutrophils. We aimed to investigate whether the poor neutrophil chemotaxis was linked with the ability of group A streptococcus (GAS) to degrade host chemokines. METHODS We did RT-PCR, ELISA, and dot-blot assays to establish whether GAS induces synthesis of interleukin 8 mRNA, but subsequently degrades the released chemokine protein. Class-specific protease inhibitors were used to characterise the protease that degraded the chemokine. We used a mouse model of human soft-tissue infections to investigate the pathogenic relevance of GAS chemokine degradation, and to test the therapeutic effect of a GAS pheromone peptide (SilCR) that downregulates activity of chemokine protease. FINDINGS The only isolates from the necrotic tissue were two beta-haemolytic GAS strains of an M14 serotype. A trypsin-like protease released by these strains degraded human interleukin 8 and its mouse homologue MIP2. When innoculated subcutaneously in mice, these strains produced a fatal necrotic soft-tissue infection that had reduced neutrophil recruitment to the site of injection. The M14 GAS strains have a missense mutation in the start codon of silCR, which encodes a predicted 17 aminoacid pheromone peptide, SilCR. Growth of the M14 strain in the presence of SilCR abrogated chemokine proteolysis. When SilCR was injected together with the bacteria, abundant neutrophils were recruited to the site of infection, bacteria were cleared without systemic spread, and the mice survived. The therapeutic effect of SilCR was also obtained in mice challenged with M1 and M3 GAS strains, a leading cause of invasive infections. INTERPRETATION The unusual reduction in neutrophils in necrotic tissue of people with GAS soft-tissue infections is partly caused by a GAS protease that degrades interleukin 8. In mice, degradation can be controlled by administration of SilCR, which downregulates GAS chemokine protease activity. This downregulation increases neutrophil migration to the site of infection, preventing bacterial spread and development of a fulminant lethal systemic infection.

The IL-8 Protease SpyCEP/ScpC of Group A Streptococcus Promotes Resistance to Neutrophil Killing

Interleukin-8 (IL-8) promotes neutrophil-mediated host defense through its chemoattractant and immunostimulatory activities. The Group A Streptococcus (GAS) protease SpyCEP (also called ScpC) cleaves IL-8, and SpyCEP expression is strongly upregulated in vivo in the M1T1 GAS strains associated with life-threatening systemic disease including necrotizing fasciitis. Coupling allelic replacement with heterologous gene expression, we show that SpyCEP is necessary and sufficient for IL-8 degradation. SpyCEP decreased IL-8-dependent neutrophil endothelial transmigration and bacterial killing, the latter by reducing neutrophil extracellular trap formation. The knockout mutant lacking SpyCEP was attenuated for virulence in murine infection models, and SpyCEP expression conferred protection to coinfecting bacteria. We also show that the zoonotic pathogen Streptococcus iniae possesses a functional homolog of SpyCEP (CepI) that cleaves IL-8, promotes neutrophil resistance, and contributes to virulence. By inactivating the multifunctional host defense peptide IL-8, the SpyCEP protease impairs neutrophil clearance mechanisms, contributing to the pathogenesis of invasive streptococcal infection.

Protein F: an adhesin of Streptococcus pyogenes binds fibronectin via two distinct domains

The binding of Streptococcus pyogenes to fibronectin (FN) enables the adherence of this pathogen to target epithelial cells, which is the first necessary step for initiation of infection. Binding is mediated by a bacterial surface protein termed protein F. Here we provide the complete structure of protein F and identify two domains responsible for binding to fibronectin. The first domain is located towards the C‐terminal end of the molecule and is composed of five repeats of 37 amino acids that are completely repeated four times and a fifth time partially. The second domain is adjacent to the first domain and is located on the /V‐terminal side of it. It is composed of a single stretch of 43 amino acids. Protein F expressed in Escherichia coli completely blocked the binding of fibronectin to S. pyogenes. However, mutant proteins that contained only one or the other of the two domains were only capable of partial blockage of binding. Complete blockage of binding of fibronectin could be achieved when a protein extract containing the N‐terminal domain was mixed in a binding reaction with a protein extract containing the C‐terminal domain. Similarly, a purified recombinant protein containing the two domains only, blocked the binding completely. In contrast, a purified recombinant protein containing just the C‐terminal domain, blocked the binding partially. A clone exclusively expressing the C‐terminal domain, completely blocked the binding of the 30 kDa N‐terminal fragment of fibronectin to S. pyogenes, whereas a clone expressing the N‐terminal domain failed to block the binding of this FN fragment. Thus, the two FN‐binding domains of protein F are necessary for maximal bacterial binding and act in concert to enhance the binding to fibronectin. The possibility that the two domains bind to two different regions on the fibronectin molecule is discussed.

Characterization of biofilm formation by clinically relevant serotypes of group A streptococci.

ABSTRACT Streptococcus pyogenes (group A streptococcus [GAS]) is a frequent cause of purulent infections in humans. As potentially important aspects of its pathogenicity, GAS was recently shown to aggregate, form intratissue microcolonies, and potentially participate in multispecies biofilms. In this study, we show that GAS in fact forms monospecies biofilms in vitro, and we analyze the basic parameters of S. pyogenes in vitro biofilm formation, using Streptococcus epidermidis as a biofilm-positive control. Of nine clinically important serotype strains, M2, M6, M14, and M18 were found to significantly adhere to coated and uncoated polystyrene surfaces. Fibronectin and collagen types I and IV best supported primary adherence of serotype M2 and M18 strains, respectively, whereas serotype M6 and M14 strains strongly bound to uncoated polystyrene surfaces. Absorption measurements of safranin staining, as well as electron scanning and confocal laser scanning microscopy, documented that primary adherence led to subsequent formation of three-dimensional biofilm structures consisting of up to 46 bacterial layers. Of note, GAS isolates belonging to the same serotype were found to be very heterogeneous in their biofilm-forming behavior. Biofilm formation was equally efficient under static and continuous flow conditions and consisted of the classical three steps, including partial disintegration after long-term incubation. Activity of the SilC signaling peptide as a component of a putative quorum-sensing system was found to influence the biofilm structure and density of serotype M14 and M18 strains. Based on the presented methods and results, standardized analyses of GAS biofilms and their impact on GAS pathogenicity are now feasible.