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

ABSTRACTTransformation of genetic material between bacteria was first observed in the 1920s usingStreptococcus pneumoniaeas a model organism. Since then, the mechanism of competence induction and transformation has been well characterized, mainly using planktonic bacteria or septic infection models. However, epidemiological evidence suggests that genetic exchange occurs primarily during pneumococcal nasopharyngeal carriage, which we have recently shown is associated with biofilm growth, and is associated with cocolonization with multiple strains. However, no studies to date have comprehensively investigated genetic exchange during cocolonizationin vitroandin vivoor the role of the nasopharyngeal environment in these processes. In this study, we show that genetic exchange during dual-strain carriagein vivois extremely efficient (10−2) and approximately 10,000,000-fold higher than that measured during septic infection (10−9). This high transformation efficiency was associated with environmental conditions exclusive to the nasopharynx, including the lower temperature of the nasopharynx (32 to 34°C), limited nutrient availability, and interactions with epithelial cells, which were modeled in a novel biofilm modelin vitrothat showed similarly high transformation efficiencies. The nasopharyngeal environmental factors, combined, were critical for biofilm formation and induced constitutive upregulation of competence genes and downregulation of capsule that promoted transformation. In addition, we show that dual-strain carriagein vivoand biofilms formedin vitrocan be transformed during colonization to increase their pneumococcal fitness and also, importantly, that bacteria with lower colonization ability can be protected by strains with higher colonization efficiency, a process unrelated to genetic exchange.IMPORTANCEAlthough genetic exchange between pneumococcal strains is known to occur primarily during colonization of the nasopharynx and colonization is associated with biofilm growth, this is the first study to comprehensively investigate transformation in this environment and to analyze the role of environmental and bacterial factors in this process. We show that transformation efficiency during cocolonization by multiple strains is very high (around 10−2). Furthermore, we provide novel evidence that specific aspects of the nasopharyngeal environment, including lower temperature, limited nutrient availability, and epithelial cell interaction, are critical for optimal biofilm formation and transformation efficiency and result in bacterial protein expression changes that promote transformation and fitness of colonization-deficient strains. The results suggest that cocolonization in biofilm communities may have important clinical consequences by facilitating the spread of antibiotic resistance and enabling serotype switching and vaccine escape as well as protecting and retaining poorly colonizing strains in the pneumococcal strain pool.

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The Transcriptional Regulator CpsY Is Important for Innate Immune Evasion in Streptococcus pyogenes

Infection and Immunity ◽

10.1128/iai.00925-16 ◽

2016 ◽

Vol 85 (3) ◽

Cited By ~ 2

Author(s):

Luis A. Vega ◽

Kayla M. Valdes ◽

Ganesh S. Sundar ◽

Ashton T. Belew ◽

Emrul Islam ◽

...

Keyword(s):

Streptococcus Pyogenes ◽

Immune Evasion ◽

Immune Cell ◽

Group A Streptococcus ◽

Transcriptional Regulator ◽

Innate Immune ◽

Content Type ◽

Human Host

ABSTRACTAs an exclusively human pathogen,Streptococcus pyogenes(the group A streptococcus [GAS]) has specifically adapted to evade host innate immunity and survive in multiple tissue niches, including blood. GAS can overcome the metabolic constraints of the blood environment and expresses various immunomodulatory factors necessary for survival and immune cell resistance. Here we present our investigation of one such factor, the predicted LysR family transcriptional regulator CpsY. The encoding gene,cpsY, was initially identified as being required for GAS survival in a transposon-site hybridization (TraSH) screen in whole human blood. CpsY is homologous with transcriptional regulators ofStreptococcus mutans(MetR),Streptococcus iniae(CpsY), andStreptococcus agalactiae(MtaR) that regulate methionine transport, amino acid metabolism, resistance to neutrophil-mediated killing, and survivalin vivo. Our investigation indicated that CpsY is involved in GAS resistance to innate immune cells of its human host. However, GAS CpsY does not manifest thein vitrophenotypes of its homologs in other streptococcal species. GAS CpsY appears to regulate a small set of genes that is markedly different from the regulons of its homologs. The differential expression of these genes depends on the growth medium, and CpsY modestly influences their expression. The GAS CpsY regulon includes known virulence factors (mntE,speB,spd,nga[spn],prtS[SpyCEP], andsse) and cell surface-associated factors of GAS (emm1,mur1.2,sibA[cdhA], andM5005_Spy0500). Intriguingly, the loss of CpsY in GAS does not result in virulence defects in murine models of infection, suggesting that CpsY function in immune evasion is specific to the human host.

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Relevance of Biofilm Models in Periodontal Research: From Static to Dynamic Systems

Microorganisms ◽

10.3390/microorganisms9020428 ◽

2021 ◽

Vol 9 (2) ◽

pp. 428

Author(s):

María Carmen Sánchez ◽

Andrea Alonso-Español ◽

Honorato Ribeiro-Vidal ◽

Bettina Alonso ◽

David Herrera ◽

...

Keyword(s):

Research Group ◽

Surface Composition ◽

Bacterial Colonization ◽

Implant Surface ◽

Biofilm Growth ◽

Biofilm Model ◽

Dental Implant Surface ◽

The Impact

Microbial biofilm modeling has improved in sophistication and scope, although only a limited number of standardized protocols are available. This review presents an example of a biofilm model, along with its evolution and application in studying periodontal and peri-implant diseases. In 2011, the ETEP (Etiology and Therapy of Periodontal and Peri-Implant Diseases) research group at the University Complutense of Madrid developed an in vitro biofilm static model using representative bacteria from the subgingival microbiota, demonstrating a pattern of bacterial colonization and maturation similar to in vivo subgingival biofilms. When the model and its methodology were standardized, the ETEP research group employed the validated in vitro biofilm model for testing in different applications. The evolution of this model is described in this manuscript, from the mere observation of biofilm growth and maturation on static models on hydroxyapatite or titanium discs, to the evaluation of the impact of dental implant surface composition and micro-structure using the dynamic biofilm model. This evolution was based on reproducing the ideal microenvironmental conditions for bacterial growth within a bioreactor and reaching the target surfaces using the fluid dynamics mimicking the salivary flow. The development of this relevant biofilm model has become a powerful tool to study the essential processes that regulate the formation and maturation of these important microbial communities, as well as their behavior when exposed to different antimicrobial compounds.

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Crucial Role of Nucleic Acid Sensing via Endosomal Toll-Like Receptors for the Defense of Streptococcus pyogenes in vitro and in vivo

Frontiers in Immunology ◽

10.3389/fimmu.2019.00198 ◽

2019 ◽

Vol 10 ◽

Cited By ~ 5

Author(s):

Anna Hafner ◽

Ulrike Kolbe ◽

Isabel Freund ◽

Virginia Castiglia ◽

Pavel Kovarik ◽

...

Keyword(s):

Nucleic Acid ◽

Streptococcus Pyogenes ◽

Crucial Role ◽

Toll Like Receptors

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Streptococcus pyogenes evades adaptive immunity through specific IgG glycan hydrolysis

Journal of Experimental Medicine ◽

10.1084/jem.20190293 ◽

2019 ◽

Vol 216 (7) ◽

pp. 1615-1629 ◽

Cited By ~ 2

Author(s):

Andreas Naegeli ◽

Eleni Bratanis ◽

Christofer Karlsson ◽

Oonagh Shannon ◽

Raja Kalluru ◽

...

Keyword(s):

Streptococcus Pyogenes ◽

Vaccine Development ◽

Group A Streptococcus ◽

Invasive Infection ◽

Invasive Infections ◽

Life Threatening ◽

Group A ◽

Specific Igg

Streptococcus pyogenes (Group A streptococcus; GAS) is a human pathogen causing diseases from uncomplicated tonsillitis to life-threatening invasive infections. GAS secretes EndoS, an endoglycosidase that specifically cleaves the conserved N-glycan on IgG antibodies. In vitro, removal of this glycan impairs IgG effector functions, but its relevance to GAS infection in vivo is unclear. Using targeted mass spectrometry, we characterized the effects of EndoS on host IgG glycosylation during the course of infections in humans. Substantial IgG glycan hydrolysis occurred at the site of infection and systemically in the severe cases. We demonstrated decreased resistance to phagocytic killing of GAS lacking EndoS in vitro and decreased virulence in a mouse model of invasive infection. This is the first described example of specific bacterial IgG glycan hydrolysis during infection and thereby verifies the hypothesis that EndoS modifies antibodies in vivo. This mechanisms of immune evasion could have implications for treatment of severe GAS infections and for future efforts at vaccine development.

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Development and Characterization of an In Vivo Central Venous Catheter Candida albicans Biofilm Model

Infection and Immunity ◽

10.1128/iai.72.10.6023-6031.2004 ◽

2004 ◽

Vol 72 (10) ◽

pp. 6023-6031 ◽

Cited By ~ 244

Author(s):

D. Andes ◽

J. Nett ◽

P. Oschel ◽

R. Albrecht ◽

K. Marchillo ◽

...

Keyword(s):

Electron Microscopy ◽

Scanning Electron Microscopy ◽

Drug Resistance ◽

Venous Catheter ◽

In Vitro Models ◽

Central Venous ◽

Biofilm Growth ◽

Scanning Electron

ABSTRACT Biofilms represent a niche for microorganisms where they are protected from both the host immune system and antimicrobial therapies. Biofilm growth serves as an increasing source of clinical infections. Candida infections are difficult to manage due to their persistent nature and associated drug resistance. Observations made in biofilm research have generally been limited to in vitro models. Using a rat central venous catheter model, we characterized in vivo Candida albicans biofilm development. Time-course quantitative culture demonstrated a progressive increase in the burden of viable cells for the first 24 h of development. Fluorescence and scanning electron microscopy revealed a bilayered architecture. Adjacent to the catheter surface, yeast cells were densely embedded in an extracellular matrix. The layer adjacent to the catheter lumen was less dense. The outermost surface of the biofilm contained both yeast and hyphal forms, and the extracellular material in which they were embedded appeared fibrous. These architectural features were similar in many respects to those described for in vitro models. However, scanning electron microscopy also revealed host cells embedded within the biofilm matrix. Drug susceptibility was determined by using two assays and demonstrated a biofilm-associated drug resistance phenotype. The first assay demonstrated continued growth of cells in the presence of supra-MIC antifungal drug concentrations. The second assay demonstrated reduced susceptibility of biofilm-grown cells following removal from the biofilm structure. Lastly, the model provided sufficient nucleic material for study of differential gene expression associated with in vivo biofilm growth. Two fluconazole efflux pumps, CDR1 and CDR2, were upregulated in the in vivo biofilm-associated cells. Most importantly, the studies described provide a model for further investigation into the molecular mechanisms of C. albicans biofilm biology and drug resistance. In addition, the model provides a means to study novel drug therapies and device technologies targeted to the control of biofilm-associated infections.

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Studies of Pseudomonas aeruginosa Mutants Indicate Pyoverdine as the Central Factor in Inhibition of Aspergillus fumigatus Biofilm

Journal of Bacteriology ◽

10.1128/jb.00345-17 ◽

2017 ◽

Vol 200 (1) ◽

Cited By ~ 33

Author(s):

Gabriele Sass ◽

Hasan Nazik ◽

John Penner ◽

Hemi Shah ◽

Shajia Rahman Ansari ◽

...

Keyword(s):

Cystic Fibrosis ◽

Pseudomonas Aeruginosa ◽

Aspergillus Fumigatus ◽

Fungal Pathogens ◽

Human Pathogens ◽

Biofilm Growth ◽

Content Type ◽

Iron Deprivation

ABSTRACT Pseudomonas aeruginosa and Aspergillus fumigatus are common opportunistic bacterial and fungal pathogens, respectively. They often coexist in airways of immunocompromised patients and individuals with cystic fibrosis, where they form biofilms and cause acute and chronic illnesses. Hence, the interactions between them have long been of interest and it is known that P. aeruginosa can inhibit A. fumigatus in vitro. We have approached the definition of the inhibitory P. aeruginosa molecules by studying 24 P. aeruginosa mutants with various virulence genes deleted for the ability to inhibit A. fumigatus biofilms. The ability of P. aeruginosa cells or their extracellular products produced during planktonic or biofilm growth to affect A. fumigatus biofilm metabolism or planktonic A. fumigatus growth was studied in agar and liquid assays using conidia or hyphae. Four mutants, the pvdD pchE, pvdD, lasR rhlR, and lasR mutants, were shown to be defective in various assays. This suggested the P. aeruginosa siderophore pyoverdine as the key inhibitory molecule, although additional quorum sensing-regulated factors likely contribute to the deficiency of the latter two mutants. Studies of pure pyoverdine substantiated these conclusions and included the restoration of inhibition by the pyoverdine deletion mutants. A correlation between the concentration of pyoverdine produced and antifungal activity was also observed in clinical P. aeruginosa isolates derived from lungs of cystic fibrosis patients. The key inhibitory mechanism of pyoverdine was chelation of iron and denial of iron to A. fumigatus. IMPORTANCE Interactions between human pathogens found in the same body locale are of vast interest. These interactions could result in exacerbation or amelioration of diseases. The bacterium Pseudomonas aeruginosa affects the growth of the fungus Aspergillus fumigatus. Both pathogens form biofilms that are resistant to therapeutic drugs and host immunity. P. aeruginosa and A. fumigatus biofilms are found in vivo, e.g., in the lungs of cystic fibrosis patients. Studying 24 P. aeruginosa mutants, we identified pyoverdine as the major anti-A. fumigatus compound produced by P. aeruginosa. Pyoverdine captures iron from the environment, thus depriving A. fumigatus of a nutrient essential for its growth and metabolism. We show how microbes of different kingdoms compete for essential resources. Iron deprivation could be a therapeutic approach to the control of pathogen growth.

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Streptococcus pyogenes Arginine and Citrulline Catabolism Promotes Infection and Modulates Innate Immunity

Infection and Immunity ◽

10.1128/iai.00916-13 ◽

2013 ◽

Vol 82 (1) ◽

pp. 233-242 ◽

Cited By ~ 24

Author(s):

Zachary T. Cusumano ◽

Michael E. Watson ◽

Michael G. Caparon

Keyword(s):

Nitric Oxide ◽

Innate Immunity ◽

Streptococcus Pyogenes ◽

Murine Model ◽

Acid Stress ◽

Arginine Deiminase ◽

Content Type ◽

Cutaneous Tissue

ABSTRACTA bacterium's ability to acquire nutrients from its host during infection is an essential component of pathogenesis. For the Gram-positive pathogenStreptococcus pyogenes, catabolism of the amino acid arginine via the arginine deiminase (ADI) pathway supplements energy production and provides protection against acid stressin vitro. Its expression is enhanced in murine models of infection, suggesting an important rolein vivo. To gain insight into the function of the ADI pathway in pathogenesis, the virulence of mutants defective in each of its enzymes was examined. Mutants unable to use arginine (ΔArcA) or citrulline (ΔArcB) were attenuated for carriage in a murine model of asymptomatic mucosal colonization. However, in a murine model of inflammatory infection of cutaneous tissue, the ΔArcA mutant was attenuated but the ΔArcB mutant was hyperattenuated, revealing an unexpected tissue-specific role for citrulline metabolism in pathogenesis. When mice defective for the arginine-dependent production of nitric oxide (iNOS−/−) were infected with the ΔArcA mutant, cutaneous virulence was rescued, demonstrating that the ability ofS. pyogenesto utilize arginine was dispensable in the absence of nitric oxide-mediated innate immunity. This work demonstrates the importance of arginine and citrulline catabolism and suggests a novel mechanism of virulence by whichS. pyogenesuses its metabolism to modulate innate immunity through depletion of an essential host nutrient.

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Mapping of the interactions between partition proteins Delta and Omega of plasmid pSM19035 from Streptococcus pyogenes

Microbiology ◽

10.1099/mic.0.045369-0 ◽

2011 ◽

Vol 157 (4) ◽

pp. 1009-1020 ◽

Cited By ~ 5

Author(s):

Michal Dmowski ◽

Grazyna Jagura-Burdzy

Keyword(s):

Streptococcus Pyogenes ◽

Dna Sequences ◽

Copy Number ◽

Terminal Part ◽

Partition System ◽

Plasmid Partition ◽

Nucleoprotein Complex ◽

Interaction Domains

Formation of the segrosome, a nucleoprotein complex crucial for proper functioning of plasmid partition systems, involves interactions between specific partition proteins (ParA-like and ParB-like), ATP and specific DNA sequences (the centromeric sites). Although partition systems have been studied for many years, details of the segrosome formation are not yet clear. Organization of the pSM19035-encoded partition system is unique; in contrast with other known par systems, here, the δ and ω genes do not constitute an operon. Moreover, Omega [a ParB-like protein which has a Ribbon-Helix-Helix (RHH) structure] recognizes multiple centromeric sequences located in the promoters of δ, ω and copS (copy-number control gene). The ParA-like protein Delta is a Walker-type ATPase. In this work, we identify the interaction domains and requirements for dimerization and hetero-interactions of the Delta and Omega proteins of pSM19035 plasmid. The RHH structures are involved in Omega dimerization in vivo and its N-terminal unstructured part is indispensable for association with Delta, both in vivo and in vitro. Omega does not need to form dimers to interact with Delta. ATP binding is not required for Delta dimerization but is important for interaction with Omega in vivo. The in vitro interaction between Delta and Omega depends on ATP but does not require the presence of specific DNA segments (the centromere) recognized by Omega. The C-terminal part of the Delta protein (aa 198–284) is indispensable for interaction with Omega. Delta most probably interacts with Omega as a dimer since two amino acid substitutions in a conserved region between the A′ and B motifs abolish both the dimerization of Delta and its interaction with Omega.

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Effect of Simulated Gastrointestinal Conditions on Biofilm Formation bySalmonella1,4,[5],12:i:-

The Scientific World JOURNAL ◽

10.1155/2014/153956 ◽

2014 ◽

Vol 2014 ◽

pp. 1-5 ◽

Cited By ~ 6

Author(s):

R. Seixas ◽

M. Gabriel ◽

J. Machado ◽

L. Tavares ◽

F. Bernardo ◽

...

Keyword(s):

Gastrointestinal Tract ◽

Biofilm Formation ◽

Contributing Factors ◽

Biofilm Growth ◽

Biofilm Production ◽

Gastrointestinal Conditions ◽

Mueller Hinton ◽

Mueller Hinton Broth

SalmonellaTyphimurium 1,4,[5],12:i:- is a major serovar responsible for human salmonellosis whose biofilm-forming ability, influenced by environmental conditions like those found in the gastrointestinal tract, is one of the main contributing factors to its ability to persist in the host and thus one of the main causes of chronic relapsing infections. Most studies to evaluate biofilm formation are performed in microtiter assays using standard media. However, no reports are available on the ability of this serovar to produce biofilm underin vitrosimulated gastrointestinal conditions which better correlate with the environment found in the gastrointestinal tract. To address this, a modified biofilm assay simulating intestinal fluid was conceived to assess the biofilm formation of 133SalmonellaTyphimurium 1,4,[5],12:i:- isolates with and without agitation and at three different time points (24 h, 48 h, and 72 h). The results were then compared to the existing microtiter method using conventional biofilm growth medium (Mueller Hinton Broth). Statistical analysis revealed significant differences in the results obtained between the three protocols used. The simulated human intestinal environment impaired biofilm production demonstrating that conditions like pH, agitation or the presence of enzymes can influence biofilm production. Therefore, results fromin vitrosimulation ofin vivoconditions may contribute to unravelling factors relating to biofilm formation and persistence in the context of the human host.

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