Group A Streptococcus Invasion Research Articles

The effects of air pollutants exposure on the transmission and severity of invasive infection caused by an opportunistic pathogen Streptococcus pyogenes

Currently, urbanization is associated with an increase in air pollutants that contribute to invasive pathogen infections by altering the host's innate immunity and antimicrobial resistance capability. Streptococcus pyogenes, also known as Group A Streptococcus (GAS), is a gram-positive opportunistic pathogen that causes a wide range of diseases, especially in children and immunosuppressed individuals. Diesel exhaust particle (DEP), a significant constituent of particulate matter (PM), are considered a prominent risk factor for respiratory illness and circulatory diseases worldwide. Several clinical and epidemiological studies have identified a close association between PM and the prevalence of viral and bacterial infections. This study investigated the role of DEP exposure in increasing pulmonary and blood bacterial counts and mortality during GAS M1 strain infection in mice. Thus, we characterized the upregulation of reactive oxygen species production and disruption of tight junctions in the A549 lung epithelial cell line due to DEP exposure, leading to the upregulation of GAS adhesion and invasion. Furthermore, DEP exposure altered the leukocyte components of infiltrated cells in bronchoalveolar lavage fluid, as determined by Diff-Quik staining. The results highlighted the DEP-related macrophage dysfunction, neutrophil impairment, and imbalance in pro-inflammatory cytokine production via the toll-like receptor 4/mitogen-activated protein kinase signaling axis. Notably, the tolerance of the GAS biofilms toward potent antibiotics and bacterial resistance against environmental stresses was also significantly enhanced by DEP. This study aimed to provide a better understanding of the physiological and molecular interactions between exposure to invasive air pollutants and susceptibility to invasive GAS infections.

FBXO2/SCF ubiquitin ligase complex directs xenophagy through recognizing bacterial surface glycan.

Xenophagy, also known as antibacterial selective autophagy, degrades invading bacterial pathogens such as group A Streptococcus (GAS) to defend cells. Although invading bacteria are known to be marked with ubiquitin and selectively targeted by xenophagy, how ubiquitin ligases recognize invading bacteria is poorly understood. Here, we show that FBXO2, a glycoprotein-specific receptor for substrate in the SKP1/CUL1/F-box protein (SCF) ubiquitin ligase complex, mediates recognition of GlcNAc side chains of the GAS surface carbohydrate structure and promotes ubiquitin-mediated xenophagy against GAS. FBXO2 targets cytosolic GAS through its sugar-binding motif and GlcNAc expression on the GAS surface. FBXO2 knockout resulted in decreased ubiquitin accumulation on intracellular GAS and xenophagic degradation of bacteria. Furthermore, SCF components such as SKP1, CUL1, and ROC1 are required for ubiquitin-mediated xenophagy against GAS. Thus, SCFFBXO2 recognizes GlcNAc residues of GAS surface carbohydrates and functions in ubiquitination during xenophagy.

Induction of cyclophilin A by influenza A virus infection facilitates group A Streptococcus coinfection.

During influenza A epidemics, bacterial coinfection is a major cause of increased morbidity and mortality. However, the roles of host factors in regulating influenza A virus (IAV)-triggered bacterial coinfection remain elusive. Cyclophilin A (CypA) is an important regulator of infection and immunity. Here, we show that IAV-induced CypA expression facilitates group A Streptococcus (GAS) coinfection both invitro and invivo. Upon IAV infection, CypA interacts with focal adhesion kinase (FAK) and inhibited E3 ligase cCbl-mediated, K48-linked ubiquitination of FAK, which positively regulates integrin α5 expression and actin rearrangement via the FAK/Akt signaling pathway to facilitate GAS colonization and invasion. Notably, CypA deficiency or inhibition by cyclosporine A significantly inhibits IAV-triggered GAS coinfection in mice. Collectively, thesefindings reveal that CypA is critical for GAS infection, and induction of CypA expression is another way for IAV to promote bacterial coinfection, suggesting that CypA is a promising therapeutic target for the secondary bacterial infection.

Streptococcus pyogenes infects human endometrium by limiting the innate immune response.

Group A Streptococcus (GAS), a Gram-positive human-specific pathogen, yields 517,000 deaths annually worldwide, including 163,000 due to invasive infections and among them puerperal fever. Before efficient prophylactic measures were introduced, the mortality rate for mothers during childbirth was approximately 10%; puerperal fever still accounts for over 75,000 maternal deaths annually. Yet, little is known regarding the factors and mechanisms of GAS invasion and establishment in postpartum infection. We characterized the early steps of infection in an ex vivo infection model of the human decidua, the puerperal fever portal of entry. Coordinate analysis of GAS behavior and the immune response led us to demonstrate that (a) GAS growth was stimulated by tissue products; (b) GAS invaded tissue and killed approximately 50% of host cells within 2 hours, and these processes required SpeB protease and streptolysin O (SLO) activities, respectively; and (c) GAS impaired the tissue immune response. Immune impairment occurred both at the RNA level, with only partial induction of the innate immune response, and protein level, in an SLO- and SpeB-dependent manner. Our study indicates that efficient GAS invasion of the decidua and the restricted host immune response favored its propensity to develop rapid invasive infections in a gynecological-obstetrical context.

Cross-serotype protection against group A Streptococcal infections induced by immunization with SPy_2191

Group A Streptococcus (GAS) infection causes a range of diseases, but vaccine development is hampered by the high number of serotypes. Here, using reverse vaccinology the authors identify SPy_2191 as a cross-protective vaccine candidate. From 18 initially identified surface proteins, only SPy_2191 is conserved, surface-exposed and inhibits both GAS adhesion and invasion. SPy_2191 immunization in mice generates bactericidal antibodies resulting in opsonophagocytic killing of prevalent and invasive GAS serotypes of different geographical regions, including M1 and M49 (India), M3.1 (Israel), M1 (UK) and M1 (USA). Resident splenocytes show higher interferon-γ and tumor necrosis factor-α secretion upon antigen re-stimulation, suggesting activation of cell-mediated immunity. SPy_2191 immunization significantly reduces streptococcal load in the organs and confers ~76-92% protection upon challenge with invasive GAS serotypes. Further, it significantly suppresses GAS pharyngeal colonization in mice mucosal infection model. Our findings suggest that SPy_2191 can act as a universal vaccine candidate against GAS infections.

Group A Streptococcus NAD-Glycohydrolase Inhibits Caveolin 1-Mediated Internalization Into Human Epithelial Cells.

Group A Streptococcus (GAS) invades epithelial cells causing persistent infection. GAS has a variety of effector proteins that modulate host systems to affect their survival in host environments. The main effector proteins of GAS are NAD-glycohydrolase (Nga) and streptolysin O (SLO). Although Nga has NADase activity and shows SLO-dependent cytotoxicity, some clinical isolates harbor NADase-inactive subtypes of Nga, and the function of NADase-inactive Nga is still unclear. In this study, we found that deletion of nga enhanced the internalization of GAS into HeLa and Ca9-22 cells. Amino acid substitution of Nga R289K/G330D (NADase-inactive) does not enhance GAS invasion, suggesting that Nga may inhibit the internalization of GAS into host cells in an NADase-independent manner. Moreover, double deletion of slo and nga showed similar invasion percentages compared with wild-type GAS, indicating the important role of SLO in the inhibition of GAS invasion by Nga. Furthermore, enhanced internalization of the nga deletion mutant was not observed in Cav1-knockout HeLa cells. Altogether, these findings demonstrate an unrecognized NADase-independent function of Nga as a negative regulator of CAV1-mediated internalization into epithelial cells.

NLRX1 Negatively Regulates Group A Streptococcus Invasion and Autophagy Induction by Interacting With the Beclin 1-UVRAG Complex.

Group A Streptococcus (GAS) can invade epithelial cells; however, these bacteria are targeted and eventually destroyed by autophagy. Members of the Nod-like receptor (NLR) family are thought to be critical for the autophagic response to invasive bacteria. However, the intracellular sensors within host cells that are responsible for bacterial invasion and the induction of autophagy are largely unknown. Thus, our aim was to examine the role of one such NLR, namely NLRX1, in invasion and autophagy during GAS infection. We found that GAS invasion was markedly increased in NLRX1 knockout cells. This led to the potentiation of autophagic processes such as autophagosome and autolysosome formation. NLRX1 was found to interact with Beclin 1 and UVRAG, members of Beclin1 complex, and knockout of these proteins inhibited invasion and autophagy upon GAS infection. Especially, NLRX1 interacted with Beclin 1 via its NACHT domain and this interaction was responsible for the NLRX1-mediated inhibition of invasion and autophagic processes including autophagosome and autolysosome formation during GAS infection. These findings demonstrate that NLRX1 functions as a negative regulator to inactivate the Beclin 1–UVRAG complex, which regulates invasion and autophagy during GAS infection. Thus, our study expands our knowledge of the role of NLRX1 during bacterial invasion and autophagy and could lead to further investigations to understand pathogen–host cell interactions, facilitating novel targeted therapeutics.

Hypervirulent Group A Streptococcus of Genotype emm3 Invades the Vascular System in Pulmonary Infection of Mice.

Natural mutations of the two-component regulatory system CovRS are frequently associated with invasive group A Streptococcus (GAS) isolates and lead to the enhancement of virulence gene expression, innate immune evasion, systemic dissemination, and virulence. How CovRS mutations enhance systemic dissemination is not well understood. A hypervirulent GAS isolate of the emm3 genotype, MGAS315, was characterized using a mouse model of pulmonary infection to understand systemic dissemination. This strain has a G1370T mutation in the sensor kinase covS gene of CovRS. Intratracheal inoculation of MGAS315 led to the lung infection that displayed extensive Gram staining at the alveolar ducts, alveoli, and peribronchovascular and perivascular interstitium. The correction of the covS mutation did not alter the infection at the alveolar ducts and alveoli but prevented GAS invasion of the peribronchovascular and perivascular interstitium. Furthermore, the covS mutation allowed MGAS315 to disrupt and degrade the smooth muscle and endothelial layers of the blood vessels, directly contributing to systemic dissemination. It is concluded that hypervirulent emm3 GAS covS mutants can invade the perivascular interstitium and directly attack the vascular system for systemic dissemination.

Caveolin 1 restricts Group A Streptococcus invasion of nonphagocytic host cells.

Caveolae are composed of 2 major proteins, caveolin 1 (CAV1) and cavin 1 or polymerase transcript release factor I (CAVIN1). Here, we demonstrate that CAV1 levels modulate invasion of Group A Streptococcus (GAS) into nonphagocytic mammalian cells. GAS showed enhanced internalisation into CAV1-knockout mouse embryonic fibroblasts and CAV1 knockdown human epithelial HEp-2 cells, whereas overexpression of CAV1 in HEp-2 cells reduced GAS invasion. This effect was not dependent on the expression of the GAS fibronectin binding protein SfbI, which had previously been implicated in caveolae-mediated uptake. Nor was this effect dependent on CAVIN1, as knockout of CAVIN1 in mouse embryonic fibroblasts resulted in reduced GAS internalisation. Although CAV1 restricted GAS invasion into host cells, we observed only minimal association of invading GAS (strain M1T15448 ) with CAV1 by immunofluorescence and very low association of invading M1T15448 with caveolae by transmission electron microscopy. These observations suggest that physical interaction with caveolae is not needed for CAV1 restriction of invading GAS. An indirect mechanism of action is also consistent with the finding that changing membrane fluidity reverses the increased invasion observed in CAV1-null cells. Together, these results suggest that CAV1 protects host cells against GAS invasion by a caveola-independent mechanism.

Disorazoles Block Group A Streptococcal Invasion into Epithelial Cells Via Interference with the Host Factor Ezrin

Bacterial pathogens use invasion into human cells asa strategy to escape not only the host's immune response, but also anti-bacterial treatment. This often leads to persistence and enables reinitiation of the infection process at a later time point. Here, we show that a family of myxobacterial metabolites,disorazoles, block invasion of group A Streptococcus (GAS) into human epithelial cells. Mechanistically, disorazoles target ezrin, a host protein involved in linking microfilaments to the membrane, and affect invasion most likely by interfering with dynamic phosphorylation of ezrin. Overall, our study suggests ezrin as a new factor in two different GAS invasion pathways, independent of the already known CD44 pathway, and that disorazoles are promising "pathoblocker" compounds aimed at this additional invasion mechanism.

The IL-8 protease SpyCEP is detrimental for Group A Streptococcus host-cells interaction and biofilm formation.

SpyCEP-mediated chemokine degradation translates into more efficient spreading and increased severity of invasive Group A Streptococcus (GAS) infections, due to impaired neutrophil recruitment to the site of infection. SpyCEP is markedly up-regulated in invasive as compared to colonizing GAS isolates raising the question whether SpyCEP expression hinders bacterial attachment and thus colonization of the host. To address this question we used a molecular approach involving the use of homologous GAS strains either expressing or not SpyCEP or expressing an enzymatically inactive variant of SpyCEP. We found that expression of enzymatically functional SpyCEP lowered GAS adherence and invasion potential toward various epithelial and endothelial cells. SpyCEP also blunted biofilm formation capacity. Our data indicate that expression of SpyCEP decreases colonization and thus might be detrimental for the spreading of GAS.

The innate immune response elicited by Group A Streptococcus is highly variable among clinical isolates and correlates with the emm type.

Group A Streptococcus (GAS) infections remain a significant health care problem due to high morbidity and mortality associated with GAS diseases, along with their increasing worldwide prevalence. Macrophages play a key role in the control and clearance of GAS infections. Moreover, pro-inflammatory cytokines production and GAS persistence and invasion are related. In this study we investigated the correlation between the GAS clinical isolates genotypes, their known clinical history, and their ability to modulate innate immune response. We constituted a collection of 40 independent GAS isolates representative of the emm types currently prevalent in France and responsible for invasive (57.5%) and non-invasive (42.5%) clinical manifestations. We tested phagocytosis and survival in mouse bone marrow-derived macrophages and quantified the pro-inflammatory mediators (IL-6, TNF-α) and type I interferon (INF-β) production. Invasive emm89 isolates were more phagocytosed than their non-invasive counterparts, and emm89 isolates more than the other isolates. Regarding the survival, differences were observed depending on the isolate emm type, but not between invasive and non-invasive isolates within the same emm type. The level of inflammatory mediators produced was also emm type-dependent and mostly invasiveness status independent. Isolates of the emm1 type were able to induce the highest levels of both pro-inflammatory cytokines, whereas emm89 isolates induced the earliest production of IFN-β. Finally, even within emm types, there was a variability of the innate immune responses induced, but survival and inflammatory mediator production were not linked.

Active and Passive Immunizations with the Streptococcal Esterase Sse Protect Mice against Subcutaneous Infection with Group A Streptococci

The human pathogen group A Streptococcus (GAS) produces many secreted proteins that play important roles in GAS pathogenesis, including hydrolases that degrade proteins and nucleic acids. This study targets another kind of hydrolase, carboxylic esterase, with the objectives of identifying GAS esterase and determining whether it is a protective antigen. The putative esterase gene SPy1718 was cloned, and the recombinant protein (Sse) was prepared. Sse was detected in GAS culture supernatant, and patients with streptococcal pharyngitis seroconverted to Sse, indicating that Sse was produced in vivo and in vitro. Sse hydrolyzes p-nitrophenyl butyrate, and the residue (178)Ser is critical for this esterase activity. There are two Sse variant complexes according to the available genome databases, consistent with the previous finding of two antigenic Sse variants. Complex I includes serotypes M1, M2, M3, M5, M6, M12, and M18, whereas M4, M28, and M49 belong to complex II. Sse variants share >98% identity in amino acid sequence within each complex but have about 37% variation between the two groups. Active immunization with M1 Sse significantly protects mice against lethal subcutaneous infection with virulent M1 and M3 strains and inhibits GAS invasion of mouse skin tissue. Passive immunization with anti-Sse antiserum also significantly protects mice against subcutaneous GAS infection, indicating that the protection is mediated by Sse-specific antibodies. The results suggest that Sse plays an important role in tissue invasion and is an antigen protective in subcutaneous infection against GAS strains of more than one serotype.

Paxillin phosphorylation: bifurcation point downstream of integrin-linked kinase (ILK) in streptococcal invasion

Efficient group A streptococcus (GAS) invasion of mammalian cells requires fibronectin (Fn) binding proteins, such as M1 and PrtF1/SfbI, that bridge bacteria to integrins and activate cellular signalling for ingestion. Previous studies of GAS invasion, mediated by both proteins, suggest a common signalling pathway. However, distinct cellular morphological changes at the port of bacterial entry suggest that different signals are also induced. Here we report that paxillin is phosphorylated in response to Fn-bound GAS that express either M1 or PrtF1/SfbI protein, but is not phosphorylated in response to a mutant deficient in both proteins. Inhibition of paxillin phosphorylation by a tyrosine kinase inhibitor, PP2, or by expression of a dominant negative form of paxillin significantly reduced invasion by M1+ but did not affect ingestion of PrtF1/SfbI+ strains. In contrast, another tyrosine inhibitor, genistein, did not significantly prevent paxillin phosphorylation and had no effect on ingestion of the M1+ strain, but reduced PrtF1/SfbI-mediated entry. This suggests that paxillin phosphorylation is induced by both proteins but only required for M1-mediated invasion. A bifurcation point, downstream of integrin-linked kinase (ILK) and phosphoinositide 3-kinase, likely accounts for the distinct morphological changes. Furthermore, ILK activity is indispensable for M1-induced paxillin recruitment and phosphorylation.

Streptococcal modulation of cellular invasion via TGF-β1 signaling

Group A Streptococcus (GAS) and other bacterial pathogens are known to interact with integrins as an initial step in a complex pathway of bacterial ingestion by host cells. Efficient GAS invasion depends on the interaction of bound fibronectin (Fn) with integrins and activation of integrin signaling. TGF-beta1 regulates expression of integrins, Fn, and other extracellular matrix proteins, and positively controls the integrin signaling pathway. Therefore, we postulated that TGF-beta1 levels could influence streptococcal invasion of mammalian cells. Pretreatment of HEp-2 cells with TGF-beta1 increased their capacity to ingest GAS when the bacteria expressed fibronectin-binding proteins (M1 or PrtF1). Western blots revealed significant induction of alpha5 integrin and Fn expression by HEp-2 cells in response to TGF-beta1. Increased ingestion of streptococci by these cells was blocked by a specific inhibitor of the TGF-beta1 receptor I and antibodies directed against alpha5 integrin and Fn, indicating that increased invasion depends on TGF-beta1 up-regulation of both the alpha5 integrin and Fn. The capacity of TGF-beta1 to up-regulate integrin expression and intracellular invasion by GAS was reproduced in primary human tonsil fibroblasts, which could be a source of TGF-beta1 in chronically infected tonsils. The relationship between TGF-beta1 and GAS invasion was strengthened by the observation that TGF-beta1 production was stimulated in GAS-infected primary human tonsil fibroblasts. These findings suggest a mechanism by which GAS induce a cascade of changes in mammalian tissue leading to elevated expression of the alpha5beta1 receptor, enhanced invasion, and increased opportunity for survival and persistence in their human host.

Integrin-linked kinase is an essential link between integrins and uptake of bacterial pathogens by epithelial cells

Entry of Streptococcus pyogenes or group A streptococcus (GAS) into host cells is mediated by fibronectin bound to surface proteins, M1 or PrtF1, forming a bridge to alpha5beta1 integrins. This interaction leads to cytoskeletal rearrangement and uptake of streptococci. We postulated that integrin-linked kinase (ILK), which directly associates with integrins, is the universal link between integrins and several bacterial pathogens. We showed that inhibition of ILK expression by siRNA silencing, or ILK kinase activity by chemical inhibitors or expression of a dominant negative form of ILK reduced M1-mediated invasion of epithelial cells up to 80%. To evaluate the ILK requirement for PrtF1-mediated GAS invasion, a M1-PrtF1+ recombinant strain within the M1 background was constructed. Inhibition of ILK kinase activity also significantly reduced invasion of epithelial cells by this recombinant and wild-type strain JRS4 that expresses PrtF1. In addition, impaired ILK kinase activity results in significant reduction of integrin-dependent invasion mediated by invasins of two other important pathogens, Staphylococcus aureus and Yersinia spp. This study suggests that bacterial pathogens evolved different molecules and strategies to exploit the host integrin signalling pathway for their survival.

Extracellular group A Streptococcus induces keratinocyte apoptosis by dysregulating calcium signalling

Group A Streptococcus (GAS) colonizes the oropharynx and damaged skin. To cause local infection or severe invasive syndromes the bacteria must gain access into deeper tissues. Host cell death may facilitate this process. GAS internalization has been identified to induce apoptosis. We now report an alternate mechanism of GAS-mediated apoptosis of primary human keratinocytes, initiated by extracellular GAS and involving dysregulation of intracellular calcium to produce endoplasmic reticulum stress. Two bacterial virulence factors are required for effective induction of apoptosis by extracellular GAS: (i) hyaluronic acid capsule that inhibits bacterial internalization and (ii) secreted cytolysin, streptolysin O (SLO), that forms transmembrane pores that permit extracellular calcium influx into the cytosol. Induction of keratinocyte apoptosis by wild-type GAS was accompanied by cell detachment and loss of epithelial integrity, a phenomenon not observed with GAS deficient in capsule or SLO. We propose that cell signalling initiated by extracellular GAS compromises the epithelial barrier by inducing premature keratinocyte differentiation and apoptosis, thereby facilitating GAS invasion of deeper tissues.

Two distinct genotypes of prtF2, encoding a fibronectin binding protein, and evolution of the gene family in Streptococcus pyogenes.

The group A Streptococcus (GAS) is an important pathogen that is responsible for a wide range of human diseases. Fibronectin binding proteins (FBPs) play an important role in promoting GAS adherence and invasion of host cells. The prtF2 gene encodes an FBP and is present in approximately 60% of GAS strains. In the present study we examined 51 prtF2-positive GAS strains isolated from the Northern Territory of Australia, and here we describe two genotypes of prtF2 which are mutually exclusive. The two genotypes have been identified previously as pfbp and fbaB. We show that these genotypes map to the same chromosomal location within the highly recombinatorial fibronectin-collagen-T antigen (FCT) locus, indicating that they arose from a common ancestor, and in this study these genotypes were designated the pfbp type and the fbaB type. Phylogenetic analysis of seven pfbp types, 14 fbaB types, and 11 prtF2-negative GAS strains by pulsed-field gel electrophoresis (PFGE) produced 32 distinct PFGE patterns. Interpretation of evolution based on the PFGE dendrogram by parsimony suggested that the pfbp type had a recent origin compared to the fbaB type. A comparison of multiple DNA sequences of the pfbp and fbaB types revealed a mosaic pattern for the amino-terminal region of the pfbp types. The fbaB type is generally conserved at the amino terminus but varies in the number of fibronectin binding repeats in the carboxy terminus. Our data also suggest that there is a possible association of the pfbp genotype with sof (84.2%), while the fbaB genotype was found in a majority of the GAS strains negative for sof (90.6%), indicating that these two prtF2 subtypes may be under different selective pressures.