Group A Streptococcus Biofilm 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.

An optimised GAS-pharyngeal cell biofilm model

Group A Streptococcus (GAS) causes 700 million infections and accounts for half a million deaths per year. Biofilm formation has been implicated in both pharyngeal and dermal GAS infections. In vitro, plate-based assays have shown that several GAS M-types form biofilms, and multiple GAS virulence factors have been linked to biofilm formation. Although the contributions of these plate-based studies have been valuable, most have failed to mimic the host environment, with many studies utilising abiotic surfaces. GAS is a human specific pathogen, and colonisation and subsequent biofilm formation is likely facilitated by distinct interactions with host tissue surfaces. As such, a host cell-GAS model has been optimised to support and grow GAS biofilms of a variety of GAS M-types. Improvements and adjustments to the crystal violet biofilm biomass assay have also been tailored to reproducibly detect delicate GAS biofilms. We propose 72 h as an optimal growth period for yielding detectable biofilm biomass. GAS biofilms formed are robust and durable, and can be reproducibly assessed via staining/washing intensive assays such as crystal violet with the aid of methanol fixation prior to staining. Lastly, SEM imaging of GAS biofilms formed by this model revealed GAS cocci chains arranged into three-dimensional aggregated structures with EPS matrix material. Taken together, we outline an efficacious GAS biofilm pharyngeal cell model that can support long-term GAS biofilm formation, with biofilms formed closely resembling those seen in vivo.

Assessing the Role of Pharyngeal Cell Surface Glycans in Group A Streptococcus Biofilm Formation.

Group A Streptococcus (GAS) causes 700 million infections and accounts for half a million deaths per year. Antibiotic treatment failure rates of 20–40% have been observed. The role host cell glycans play in GAS biofilm formation in the context of GAS pharyngitis and subsequent antibiotic treatment failure has not been previously investigated. GAS serotype M12 GAS biofilms were assessed for biofilm formation on Detroit 562 pharyngeal cell monolayers following enzymatic removal of all N-linked glycans from pharyngeal cells with PNGase F. Removal of N-linked glycans resulted in an increase in biofilm biomass compared to untreated controls. Further investigation into the removal of terminal mannose and sialic acid residues with α1-6 mannosidase and the broad specificity sialidase (Sialidase A) also found that biofilm biomass increased significantly when compared to untreated controls. Increases in biofilm biomass were associated with increased production of extracellular polymeric substances (EPS). Furthermore, it was found that M12 GAS biofilms grown on untreated pharyngeal monolayers exhibited a 2500-fold increase in penicillin tolerance compared to planktonic GAS. Pre-treatment of monolayers with exoglycosidases resulted in a further doubling of penicillin tolerance in resultant biofilms. Lastly, an additional eight GAS emm-types were assessed for biofilm formation in response to terminal mannose and sialic acid residue removal. As seen for M12, biofilm biomass on monolayers increased following removal of terminal mannose and sialic acid residues. Collectively, these data demonstrate that pharyngeal cell surface glycan structures directly impact GAS biofilm formation in a strain and glycan specific fashion.

The Arginine Deiminase Pathway Impacts Antibiotic Tolerance during Biofilm-Mediated Streptococcus pyogenes Infections.

Bacterial biofilms are responsible for a variety of serious human infections and are notoriously difficult to treat due to their recalcitrance to antibiotics. Further work is necessary to elicit a full understanding of the mechanism of this antibiotic tolerance. The arginine deiminase (ADI) pathway is responsible for bacterial pH maintenance and is highly expressed during biofilm growth in multiple bacterial species. Using the group A Streptococcus (GAS) as a model human pathogen, the ADI pathway was demonstrated to contribute to biofilm growth. The inability of antibiotics to reduce GAS populations when in a biofilm was demonstrated by in vitro studies and a novel animal model of nasopharyngeal infection. However, disruption of the ADI pathway returned GAS biofilms to planktonic levels of antibiotic sensitivity, suggesting the ADI pathway is influential in biofilm-related antibiotic treatment failure and provides a new strategic target for the treatment of biofilm infections in GAS and potentially numerous other bacterial species.IMPORTANCE Biofilm-mediated bacterial infections are a major threat to human health because of their recalcitrance to antibiotic treatment. Through the study of Streptococcus pyogenes, a significant human pathogen that is known to form antibiotic-tolerant biofilms, we demonstrated the role that a bacterial pathway known for responding to acid stress plays in biofilm growth and antibiotic tolerance. This not only provides some insight into antibiotic treatment failure in S. pyogenes infections but also, given the widespread nature of this pathway, provides a potentially broad target for antibiofilm therapies. This discovery has the potential to impact the treatment of many different types of recalcitrant biofilm infections.

Adaptation of the group A Streptococcus adhesin Scl1 to bind fibronectin type III repeats within wound-associated extracellular matrix: implications for cancer therapy.

SummaryThe human‐adapted pathogen group A Streptococcus (GAS) utilizes wounds as portals of entry into host tissue, wherein surface adhesins interact with the extracellular matrix, enabling bacterial colonization. The streptococcal collagen‐like protein 1 (Scl1) is a major adhesin of GAS that selectively binds to two fibronectin type III (FnIII) repeats within cellular fibronectin, specifically the alternatively spliced extra domains A and B, and the FnIII repeats within tenascin‐C. Binding to FnIII repeats was mediated through conserved structural determinants present within the Scl1 globular domain and facilitated GAS adherence and biofilm formation. Isoforms of cellular fibronectin that contain extra domains A and B, as well as tenascin‐C, are present for several days in the wound extracellular matrix. Scl1‐FnIII binding is therefore an example of GAS adaptation to the host's wound environment. Similarly, cellular fibronectin isoforms and tenascin‐C are present in the tumor microenvironment. Consistent with this, FnIII repeats mediate GAS attachment to and enhancement of biofilm formation on matrices deposited by cancer‐associated fibroblasts and osteosarcoma cells. These data collectively support the premise for utilization of the Scl1‐FnIII interaction as a novel method of anti‐neoplastic targeting in the tumor microenvironment.

Streptolysin-induced endoplasmic reticulum stress promotes group A Streptococcal host-associated biofilm formation and necrotising fasciitis.

Group A Streptococcus (GAS) is a human pathogen that causes infections ranging from mild to fulminant and life-threatening. Biofilms have been implicated in acute GAS soft-tissue infections such as necrotising fasciitis (NF). However, most in vitro models used to study GAS biofilms have been designed to mimic chronic infections and insufficiently recapitulate in vivo conditions along with the host-pathogen interactions that might influence biofilm formation. Here, we establish and characterise an in vitro model of GAS biofilm development on mammalian cells that simulates microcolony formation observed in a mouse model of human NF. We show that on mammalian cells, GAS forms dense aggregates that display hallmark biofilm characteristics including a 3D architecture and enhanced tolerance to antibiotics. In contrast to abiotic-grown biofilms, host-associated biofilms require the expression of secreted GAS streptolysins O and S (SLO, SLS) that induce endoplasmic reticulum (ER) stress in the host. In an in vivo mouse model, the streptolysin null mutant is attenuated in both microcolony formation and bacterial spread, but pretreatment of soft-tissue with an ER stressor restores the ability of the mutant to form wild-type-like microcolonies that disseminate throughout the soft tissue. Taken together, we have identified a new role of streptolysin-driven ER stress in GAS biofilm formation and NF disease progression.

In vivo expression of Streptococcus pyogenes immunogenic proteins during tibial foreign body infection.

Group A streptococcus (GAS) is an important human pathogen that causes a number of diseases with a wide range of severities. While all known strains of GAS are still sensitive to penicillin, there have been reports of antibiotic treatment failure in as many as 20% to 40% of cases. Biofilm formation has been implicated as a possible cause for these failures. A biofilm is a microbially derived, sessile community where cells grow attached to a surface or as a bacterial conglomerate and surrounded by a complex extracellular matrix. While the ability of group A streptococcus to form biofilms in the laboratory has been shown, there is a lack of understanding of the role of GAS biofilms during an infection. We hypothesized that during infections, GAS exhibits a biofilm phenotype, complete with unique protein expression. To test this hypothesis, a rabbit model of GAS osteomyelitis was developed. A rabbit was inoculated with GAS using an infected indwelling device. Following the infection, blood and tissue samples were collected. Histological samples of the infected tibia were prepared, and the formation of a biofilm in vivo was visualized using peptide nucleic acid fluorescent in situ hybridization (PNA-FISH) and confocal microscopy. In addition, Western blotting with convalescent rabbit serum detected cell wall proteins expressed in vitro under biofilm and planktonic growth conditions. Immunogenic proteins were then identified using matrix-assisted laser desorption ionization-time of flight tandem mass spectrometry (MALDI-TOF/TOF MS). These identities, along with the in vivo results, support the hypothesis that GAS forms biofilms during an infection. This unique phenotype should be taken into consideration when designing a vaccine or any other treatment for group A streptococcus infections.

Streptococcus pyogenes Biofilm Growth In Vitro and In Vivo and Its Role in Colonization, Virulence, and Genetic Exchange

Group A streptococcus (GAS) commonly colonizes the oropharynx and nonintact skin. However, colonization has been little studied and the role of biofilm formation is unclear, as biofilm experiments to date have not been conducted under conditions that mimic the host environment. In this study we grew GAS biofilms on human keratinocytes under various environmental conditions and used this model to evaluate colonization, invasive disease and natural transformation. GAS grown on epithelial cells, but not biofilms grown on abiotic surfaces, produced biofilms with characteristics similar to in vivo colonization. These biofilm bacteria showed a 100-fold higher bacterial burden of nasal-associated lymphoid tissue in mice than broth-grown bacteria, and were not virulent during septic infection, which was attributed in part to down-regulation of genes typically involved in localized and invasive disease. We also showed for the first time that GAS were naturally transformable when grown in biofilms and during colonization of NALT in vivo. These findings provide novel model systems to study biofilm formation of GAS in vitro and in vivo, suggest an important role for biofilm formation during GAS colonization, and provide an explanation for the known genome diversity within the GAS population.

Interspecies Communication among Commensal and Pathogenic Streptococci

ABSTRACTQuorum sensing (QS) regulates diverse and coordinated behaviors in bacteria, including the production of virulence factors, biofilm formation, sporulation, and competence development. It is now established that some streptococci utilize Rgg-type proteins in concert with short hydrophobic peptides (SHPs) to mediate QS, and sequence analysis reveals that several streptococcal species contain highly homologous Rgg/SHP pairs. In group A streptococcus (GAS), two SHPs (SHP2 and SHP3 [SHP2/3]) were previously identified to be important in GAS biofilm formation. SHP2/3 are detected by two antagonistic regulators, Rgg2 and Rgg3, which control expression of the shp genes. In group B streptococcus (GBS), RovS is a known virulence gene regulator and ortholog of Rgg2, whereas no apparent Rgg3 homolog exists. Adjacent to rovS is a gene (shp1520) encoding a peptide nearly identical to SHP2. Using isogenic mutant strains and transcriptional reporters, we confirmed that RovS/SHP1520 comprise a QS circuit in GBS. More important, we performed experiments demonstrating that production and secretion of SHP1520 by GBS can modulate Rgg2/3-regulated gene expression in GAS in trans; likewise, SHP2/3 production by GAS can stimulate RovS-mediated gene regulation in GBS. An isolate of Streptococcus dysgalactiae subsp. equisimilis also produced a secreted factor capable of simulating the QS circuits of both GAS and GBS, and sequencing confirms the presence of an orthologous Rgg2/SHP2 pair in this species as well. To our knowledge, this is the first documented case of bidirectional signaling between streptococcal species in coculture and suggests a role for orthologous Rgg/SHP systems in interspecies communication between important human pathogens.

The group A streptococcal collagen‐like protein‐1, Scl1, mediates biofilm formation by targeting the extra domain A‐containing variant of cellular fibronectin expressed in wounded tissue

Wounds are known to serve as portals of entry for group A Streptococcus (GAS). Subsequent tissue colonization is mediated by interactions between GAS surface proteins and host extracellular matrix components. We recently reported that the streptococcal collagen-like protein-1, Scl1, selectively binds the cellular form of fibronectin (cFn) and also contributes to GAS biofilm formation on abiotic surfaces. One structural feature of cFn, which is predominantly expressed in response to tissue injury, is the presence of a spliced variant containing extra domain A (EDA/EIIIA). We now report that GAS biofilm formation is mediated by the Scl1 interaction with EDA-containing cFn. Recombinant Scl1 proteins that bound cFn also bound recombinant EDA within the C-C' loop region recognized by the α(9)β(1) integrin. The extracellular 2-D matrix derived from human dermal fibroblasts supports GAS adherence and biofilm formation. Altogether, this work identifies and characterizes a novel molecular mechanism by which GAS utilizes Scl1 to specifically target an extracellular matrix component that is predominantly expressed at the site of injury in order to secure host tissue colonization.

Dispersal of Group A Streptococcal Biofilms by the Cysteine Protease SpeB Leads to Increased Disease Severity in a Murine Model

Group A Streptococcus (GAS) is a Gram-positive human pathogen best known for causing pharyngeal and mild skin infections. However, in the 1980's there was an increase in severe GAS infections including cellulitis and deeper tissue infections like necrotizing fasciitis. Particularly striking about this elevation in the incidence of severe disease was that those most often affected were previously healthy individuals. Several groups have shown that changes in gene content or regulation, as with proteases, may contribute to severe disease; yet strains harboring these proteases continue to cause mild disease as well. We and others have shown that group A streptococci (MGAS5005) reside within biofilms both in vitro and in vivo. That is to say that the organism colonizes a host surface and forms a 3-dimensional community encased in a protective matrix of extracellular protein, DNA and polysaccharide(s). However, the mechanism of assembly or dispersal of these structures is unclear, as is the relationship of these structures to disease outcome. Recently we reported that allelic replacement of the streptococcal regulator srv resulted in constitutive production of the streptococcal cysteine protease SpeB. We further showed that the constitutive production of SpeB significantly decreased MGAS5005Δsrv biofilm formation in vitro. Here we show that mice infected with MGAS5005Δsrv had significantly larger lesion development than wild-type infected animals. Histopathology, Gram-staining and immunofluorescence link the increased lesion development with lack of disease containment, lack of biofilm formation, and readily detectable levels of SpeB in the tissue. Treatment of MGAS5005Δsrv infected lesions with a chemical inhibitor of SpeB significantly reduced lesion formation and disease spread to wild-type levels. Furthermore, inactivation of speB in the MGAS5005Δsrv background reduced lesion formation to wild-type levels. Taken together, these data suggest a mechanism by which GAS disease may transition from mild to severe through the Srv mediated dispersal of GAS biofilms.