Virulence factors in Streptococcus pyogenes

Während meiner Dissertation lag mein Interesse in der Erweiterung des Verständnisses der Pathogenität des bekannten humanen Pathogens Streptococcus pyogenes, auch bekannt als Gruppe A Streptococcus (GAS). Dieses Manuskript ist in vier Teile unterteilt: 1) die Rolle von Superantigen-codierenden lysogenen Bakteriophagen in Erwerb und Verbreitung von Virulenzgenen in Streptococci, 2) der Identifikation von small RNAs mit potentiellen regulatorischen Funktionen in GAS, 3) der Rolle des interspezifischen Kommunikationssystems luxS/AI-2 in GAS Metabolismus und Überleben unter Stresskonditionen und 4) der Rolle von Streptolysinen in der angeborenen Immunantwort von Makrophagen auf GAS Infektion. 1) Seit den späten 1980er Jahren, war ein signifikanter Anstieg von schweren Formen durch GAS verursachter Krankheiten im Menschen zu beobachten. Eine Hypothese um dieses Phänomen zu erklären, beruht auf einer gesteigerten Virulenz des Pathogens selbst. In GAS codieren Prophagen für einen oder mehr putative oder bekannte Virulenzfaktoren, einschließlich Superantigenen. Im ersten Teil meiner Dissertation wird die Identifikation des induzierbaren Prophagen F149, welcher für das Superantigen SSA in einem klinischen Isolat von GAS des Serotyps M12 codiert, beschrieben. Durch lysogene Konversion war der Phage zwischen klinischen Isolaten von verschiedenen GAS M-Serotypen (M1, M3, M5, M12, M19, M28 und M94), als auch in Gruppe C Streptococcus equisimilis (GCSE), ex-vivo transferierbar. Unsere Ergebnisse zeigen, dass horizontaler Transfer von lysogenen Phagen zwischen GAS durch die M-Typ-Barriere möglich ist und unterstützen das Konzept der interspezifischen lysogenen Toxin-Konversion. Somit würde lysogene Konversion es GAS ermöglichen, sich effizienter an die wechselnden Bedingungen im Wirt anzupassen, was wiederum zu potentiell konkurrenzfähigeren, virulenteren Klonen führen würde. 2) Kürzlich erschienene Studien zeigen, dass small RNAs (sRNAs) in Bakterien eine signifikante Rolle in der Regulation der Genexpression spielen. Jedoch sind Informationen über die Rolle von sRNAs in S. pyogenes relativ begrenzt. Aus diesem Grund war unser Ziel im zweiten Abschnitt meiner Arbeit die Identifikation neuer sRNAs in S. pyogenes. Zu diesem Zweck wurde ein in silico Verfahren angewandt, fokussierend auf die intergenen Regionen des Genoms von M1-Serotyp GAS. Von 178 identifizierten putativen Loci wurden 90 zur weiteren Analyse ausgewählt, wobei 29 (32%) dieser sRNAs unter normalen Wachstumsbedingungen exprimiert werden. Die Expression der Kandidaten wurde verifiziert in den Serotypen M1, M3, M5 und M49, ihre 5’ und 3’ Enden wurden determiniert, ihre Stabilität bestimmt und ihre in silico Sekundärstruktur wurde vorhergesagt. Gefunden wurden 5 Riboswitches, 6 leader elements, 4 funktionelle RNAs, 8 T-Boxen und 5 neue sRNAs. Zwei dieser sRNA Kandidaten befinden sich in der 5’ UTR von Riboflavin- und Tryptophan- Transportergenen und scheinen diese Gene durch trankriptionelle oder translationelle Attenuierungsmechansimen zu kontrollieren. Das zeigt die Wichtigkeit von sRNAs in der Kontrolle der Aufnahme essentieller Metaboliten in Bakterien durch regulatorische Genexpression. Zusätzlich wurde eine in der intergenen Region identifizierte sRNA SpyRNA049 auf ihre potentielle Rolle in GAS-Pathogenität weiter analysiert. 3) Frühere Studien zeigten, dass das interspezifische Zell-Zell-Kommunikationssystem luxS/AI-2 eine Rolle in Metabolismus und Pathogenität spielt. Im dritten Abschnitt meiner Dissertation war das Ziel, die Rolle des luxS/AI-2 Systems in den S. pyogenes Serotypen M1 und M19 zu verstehen. Unsere Ergebnisse lassen auf eine limitierte Rolle von luxS/AI-2 im S-Adenosylmethioninzyklus schließen. Überlebensassays mit luxS-Mutanten sowie luxS-Expression und AI-2-ähnliche Aktivitätsassays im leicht sauren Milieu demonstrierten die Beteiligung des luxS/AI-2 Systems in der Stressadaptation von GAS. Interaktionsanalyse mit Wirtszellen zeigte, dass die luxS-Mutanten im Vergleich zum Wildtyp in höherem Ausmaß in Epithelzellen und Makrophagen überlebten. Alles in allem zeigen unsere Daten, dass das luxS/AI-2 System nicht nur in die Regulation der Virulenzfaktorexpression involviert ist, sondern auch für Bakterien einen Vorteil darstellt, in herausfordernden Wirtsumgebungen zu überleben. 4) Im Allgemeinen werden Bakterien von den Immunzellen des Wirts durch pattern recognition receptors, wie Toll-like receptors (TLRs), erkannt. Über die Erkennung von GAS durch Makrophagen ist jedoch wenig bekannt. Die Ergebnisse, die im vierten Teil meiner Dissertation beschrieben werden, lassen darauf schließen, dass Makrophagen S. pyogenes unabhängig von den TLRs die typisch für gram-positive Pathogene sind (TLR2, TLR4 und TLR9 sowie ihre Kombinationen), erkennen. Jedoch waren die in Makrophagen beobachteten Signalereignisse auf GAS-Infektion immer noch abhängig vom TLR-Adaptor MyD88. Überraschenderweise war die IFN-Produktion unabhängig von den GAS-Cytolysinen SLS und SLO, die oft von anderen gram-positiven Bakterien benötigt werden. Alles in allem lassen unsere Ergebnisse darauf schließen, dass GAS durch einen MyD88-abhängigen Rezeptor, anders als die üblicherweise verwendeten, erkannt wird und lassen einen neuen Typ der bakteriellen Erkennung für die Produktion von TypI IFN erkennen.During my thesis work, I have been interested in extending our understanding of the pathogenicity of the notorious human pathogen Streptococcus pyogenes, also called group A streptococcus (GAS). This thesis manuscript is divided into four parts: 1) role of superantigen-encoding lysogenic bacteriophages in the acquisition and spread of virulence genes among streptococci, 2) the identification of small RNAs with potential regulatory functions in GAS, 3) the role of the interspecies communication system luxS/AI-2 in GAS metabolism and survival under stress conditions and 4) the role of streptolysins in the innate immune responses of macrophages upon GAS infection. 1) Since the late 1980s, there has been a significant reemergence of severe forms of human diseases caused by GAS. One hypothesis to explain this phenomenon has focused on an enhanced virulence of the pathogen itself. In GAS, prophages encode one or more putative or established virulence factors including superantigens. The first part of my thesis describes the identification of an inducible prophage F149 encoding superantigen SSA in an M12 GAS clinical isolate. By lysogenic conversion the phage was transferable ex-vivo among GAS clinical isolates of various M serotypes (M1, M3, M5, M12, M19, M28 and M94) as well as of Group C Streptococcus equisimilis (GCSE). Our data indicate that horizontal transfer of lysogenic phage among GAS can occur across the M-type barrier and provide further support for the concept of interspecies lysogenic toxin-conversion. Therefore, lysogenic conversion would allow GAS to more efficiently adapt to the changing host conditions, thus potentially leading to fitter and more virulent clones. 2) Recent studies show that in bacteria small RNAs (sRNAs) play a significant role in the regulation of gene expression. However, information regarding sRNAs in S. pyogenes is rather limited. Thus, in the second part of my thesis, our aim was to identify novel sRNAs in S. pyogenes. For this purpose, we used an in silico approach focusing on the intergenic regions of the GAS M1 serotype genome. From 178 identified putative loci, 90 were further selected for analysis and 29 (32%) of these sRNAs showed expression under normal growth conditions. Expression of the candidates was verified in M1, M3, M5 and M49 serotypes, their 5’ and 3’ ends were mapped, their stability was determined and their in silico secondary structures were predicted. We found 5 riboswitches, 6 leader elements, 4 functional RNAs, 8 T-boxes and 5 novel sRNAs. Two of the sRNA candidates residing in the 5’ UTR of the riboflavin and tryptophan transporter genes seemed to control the transporter genes via transcriptional or translational attenuation mechanisms. This shows the importance of sRNAs in the control of the uptake of essential metabolites by regulating gene expression in bacteria. In addition, one sRNA SpyRNA049 identified in the intergenic region, was further analyzed for its potential role in GAS pathogenicity. 3) Previous studies showed that the interspecies luxS/AI-2 cell-cell communication system plays a role in metabolism and pathogenicity. In the third part of my thesis we aimed at understanding the role of the luxS/AI-2 system in S. pyogenes serotypes M1 and M19. Our results indicated a limited role of luxS/AI-2 in the activated methyl cycle metabolism. Survival assays with luxS mutants as well as luxS expression and AI-2 like activity assays under low acidic conditions demonstrated the involvement of luxS/AI-2 system in the adaptation of GAS to stress. Interaction analysis with host cells showed that the luxS mutants survived in epithelial cells and macrophages to a higher degree compared to the wild-type. Overall, our data show that the luxS/AI-2 system is not only involved in the regulation of virulence factor expression but also provides bacteria an advantage to survive in challenging host environments. 4) Bacteria are generally recognized by host immune cells by the pattern recognition receptors like toll-like receptors (TLRs). However, little is known about the recognition of GAS by macrophages. Data described in the fourth part of my thesis suggest that S. pyogenes is recognized by macrophages independently of the most commonly TLRs used to recognize gram-positive pathogens, TLR2, TLR4 and TLR9 and their combination. However, the signaling events observed in macrophages upon GAS infection were still dependent on the TLR-adaptor MyD88. Surprisingly, IFN production was independent of the GAS cytolysins, SLS and SLO, often required in other gram-positive bacteria. Overall our data indicate that GAS is recognized by a MyD88-dependent receptor other than those commonly used to detect bacteria and reveal a new type of bacterial recognition for the production of type I IFN

On the Free Bridge Across the Digital Divide: Assessing the Quality of Facebook’s Free Basics Service

Free Basics is an initiative backed by Facebook to provide users in developing countries free mobile Internet access to selected services. Despite its wide-spread deployment and its potential impact on bridging the digital divide, to date, few studies have rigorously measured the quality of the free Internet service offered by Free Basics. In this short paper, we characterize the quality of the Free Basics service offered in Pakistan and South Africa along three dimensions: (i) the selection of accessible Web services, (ii) the functionality of those services, and (iii) the network performance for those services. While preliminary, our findings show that data-driven studies are essential for having more informed public debates on the pros and cons of the current design of the Free Basics service

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

Screening and identification of aquatic bacteriocinogenic Bacillus strains inhibiting clinical MRSA and VRE from Pakistan

Objective: The purpose of current study is to screen and identify novel bacteriocinogenic strains from marine, fresh and estuarine water bodies which can serve as alternatives to the antibiotics, as the antibiotic resistance is on the rise.Methodology: Altogether seven fresh, brackish and marine water samples from various regions of Sindh were collected All the aquatic bacterial strains were isolated by standard plate count and overlay methods. The bacteriocinogenic strains were screened by spot agar, stab and overlay, cross streak and agar well-diffusion techniques.Results: Out of the total 146 isolates, 41 (28%) were bacteriocinogenic, among which 28 (68%) were isolated from freshwater, 12 (29%) from marine and 1 (3%) from brackish water. The most significant bacteriocinogenic strains amongst the isolates were identified as B coagulans, B simplex, B macerans and B circulans. When tested against 17 significant pathogens, these strains could inhibit various clinical drug resistant Gram positive pathogens like Staphylococcus aureus and Methicillin resistant Staphylococcus aureus (MRSA), Streptococcus pyogenes, S agalactiae and Vancomycin resistant Enterococcus (VRE) and C diptheriae. Furthermore, Escherichia coli and Klebsiella spp. were observed to be sensitive against two bacteriocinogenic strains of B coagulans. Furthermore, 59% of the aquatic isolates were sensitive against one of the  bacteriocinogenic strain indicating competitive antagonism phenomenon.Conclusion: This research study reveals the bacteriocinogenic potential of indigenous aquatic isolates against drug resistant clinical pathogens as well as against fish pathogens

Phylogenetic group B2 expressed significant biofilm formation among drug-resistant uropathogenic Escherichia coli

Biofilm is an important virulent marker attributed to the development of urinary tract infections (UTIs) by uropathogenic E. coli (UPEC). Drug-resistant and biofilm-producing UPEC are highly problematic causing catheter-associated or recurrent UTIs with significant morbidity and mortality. The aim of the current study was to investigate the prevalence of biofilm formation and phylogenetic groups in drug-resistant UPEC to predict their ability to cause disease. This prospective study was conducted at the Department of Microbiology, University of Karachi from January to June 2019. A total of 50 highly drug-resistant UPEC were selected for this study. UPEC isolates were screened to form biofilm by Congo-red agar (CRA) and microtiter plate (MTP) technique. The representative biofilm-producing isolates were analysed by scanning electron microscopy (SEM) monitoring. Phylogenetic analysis was done by PCR method based on two preserved genes; chuA, yjaA and TspE4-C2 DNA fragment. On CRA 34 (68%) UPEC were slime producers, while on MTP 20 (40%) were strong biofilm producers, 19 (38%) moderate and 11 (22%) were low to negligible biofilm producers. Molecular typing confirmed that phylogenetic group B2 was prevalent in drug resistant UPEC strains. Pathogenic strains belonged to phylogenetic group B2 and D were found to have greater biofilm forming ability as compare to non-pathogenic commensal strains that belonged to phylogenetic group A. Our results indicate that biofilm formation vary in drug resistant UPEC belonged to different phylogenetic groups. This study indicates possible link between in vitro biofilm formation and phylogenetic groups of UPEC, therefore this knowledge might be helpful to predict the pathogenic potential of UPEC and help design strategies for controlling UTIs

Lysogenic Transfer of Group A Streptococcus Superantigen Gene among Streptococci

A group A Streptococcus (GAS) isolate, serotype M12, recovered from a patient with streptococcal toxic shock syndrome was analyzed for superantigen-carrying prophages, revealing phi149, which encodes superantigen SSA. Sequence analysis of the att-L proximal region of phi149 showed that the phage had a mosaic nature. Remarkably, we successfully obtained lysogenic conversion of GAS clinical isolates of various M serotypes (M1, M3, M5, M12, M19, M28, and M94), as well as of group C Streptococcus equisimilis (GCSE) clinical isolates, via transfer of a recombinant phage phi149::Km(r). Phage phi149::Km(r) from selected lysogenized GAS and GCSE strains could be transferred back to M12 GAS strains. Our data indicate that horizontal transfer of lysogenic phages among GAS can occur across the M-type barrier; these data also provide further support for the hypothesis that toxigenic conversion can occur via lysogeny between species. Streptococci might employ this mechanism specifically to allow more efficient adaptation to changing host challenges, potentially leading to fitter and more virulent clones

Functional analysis of the group A streptococcal luxS/AI-2 system in metabolism, adaptation to stress and interaction with host cells.

BACKGROUND: The luxS/AI-2 signaling pathway has been reported to interfere with important physiological and pathogenic functions in a variety of bacteria. In the present study, we investigated the functional role of the streptococcal luxS/AI-2 system in metabolism and diverse aspects of pathogenicity including the adaptation of the organism to stress conditions using two serotypes of Streptococcus pyogenes, M1 and M19. RESULTS: Exposing wild-type and isogenic luxS-deficient strains to sulfur-limited media suggested a limited role for luxS in streptococcal activated methyl cycle metabolism. Interestingly, loss of luxS led to an increased acid tolerance in both serotypes. Accordingly, luxS expression and AI-2 production were reduced at lower pH, thus linking the luxS/AI-2 system to stress adaptation in S. pyogenes. luxS expression and AI-2 production also decreased when cells were grown in RPMI medium supplemented with 10% serum, considered to be a host environment-mimicking medium. Furthermore, interaction analysis with epithelial cells and macrophages showed a clear advantage of the luxS-deficient mutants to be internalized and survive intracellularly in the host cells compared to the wild-type parents. In addition, our data revealed that luxS influences the expression of two virulence-associated factors, the fasX regulatory RNA and the virulence gene sibA (psp). CONCLUSION: Here, we suggest that the group A streptococcal luxS/AI-2 system is not only involved in the regulation of virulence factor expression but in addition low level of luxS expression seems to provide an advantage for bacterial survival in conditions that can be encountered during infections