Characterizing and deciphering the interaction of human platelets and platelet-derived proteins with Streptococcus pneumoniae and Staphylococcus aureus

Streptococcus pneumoniae (pneumococci) and Staphylococcus aureus (S. aureus) are human-specific commensals of the upper respiratory tract. Every individual is asymptomatically colonized with both bacteria at least once in their life-time. The opportunistic pathogens can affect further organs and invade into deeper tissue. The occupation of normally sterile niches of the human body with the bacteria can lead to local infections such as sinusitis, otitis media and abscesses, or to life-threatening diseases like pneumonia, meningitis or sepsis. A strong interaction between the bacterium and the respiratory epithelial cells is a prerequisite for a successful colonization. This interaction is ensured by bacterial surface proteins, so called adhesins. The binding of the adhesins to the epithelial lineage occurs predominantly indirectly via components of the extracellular matrix (ECM), but also directly to cellular receptors. Pneumococci and S. aureus bind to various ECM glycoproteins, amongst others: fibronectin, fibrinogen, vitronectin, and collagen. Also binding of both pathogens to human thrombospondin-1 has been described. Thrombospondin-1 is mainly stored in the α-granula of thrombocytes (platelets) and released into the circulation upon activation. However, thrombospondin-1 is also produced and secreted by other cell types like endothelial cells, macrophages, and fibroblasts, which gets subsequently incorporated as component into the ECM. So far, no thrombosponin-1-binding adhesins of pneumococci were identified. PspC, Hic, and PavB are important surface-localized virulence factors, which were shown to interact with human ECM and plasma proteins. PspC and Hic bind to vitronectin and factor H, which inhibits the complement cascade of the human immune system. PavB interacts with fibronectin and plasminogen, and a pavB-deficient mutant of S. pneumoniae showed diminished capacity in colonization in a mouse model. Among the surface proteins of S. aureus, only Eap was identified as thrombospondin-1-binding adhesin. Beyond colonization, pneumococci and S. aureus can enter the blood circulation, interact with platelets, and cause their activation. The aggregation of platelets, especially initiated by S. aureus, plays an important role in the clinic, because most of the septic patients develop thrombocytopenia. Surface localized factors of S. pneumoniae triggering platelet activation are unknown to date. In contrast, few proteins of S. aureus with potential to activate platelets, including Eap, were identified previously. This study identified the surface proteins PavB, PspC, and Hic of S. pneumoniae as specific ligands of the human thrombospondin-1. Flow cytometric, surface plasmon resonance spectroscopic and immunological analyses revealed interactions between the pneumococcal proteins and soluble as well as immobilized thrombospondin-1. The use of specific pneumococcal deletion mutants verified the importance of the three virulence factors as binding partners of soluble thrombospondin-1. The results suggest that pneumococci are capable of acquiring soluble thrombospondin-1 from blood as well as utilizing immobilized glycoprotein of the ECM as substrate for adhesion. Furthermore, the thrombospondin-1-binding domain within the pneumococcal proteins was analyzed by use of recombinant fragments of PavB, PspC, and Hic. The binding capacity of thrombospondin-1 increased proportionally with the amount of repetitive sequences in PavB and PspC, and the length of the α-helical region within the Hic molecule. The binding behavior of thrombospondin-1 towards PavB and PspC is comparable with that of the ECM proteins vitronectin and fibronectin, but is unique towards Hic. The localization of the binding domain of the adhesins within the thrompospondin-1 molecule occurred via use of glycosaminoglycans as competitive inhibitors for the interaction. The results suggest that the pneumococcal proteins Hic and PspC target the identical binding region within thrombospondin-1, which differs from the binding domain for PavB. However, all three virulence factors seem to bind in the N-terminal part of thrombospondin-1. Two-dimensional gel electrophoresis, thrombospondin-1 overlay assay and subsequent mass spectrometric analysis identified AtlA of S. aureus as a surface localized interaction partner of human thrombospondin-1. Moreover, a vitronectin binding activity for AtlA was determined. Immunological and surface plasmon resonance binding studies with recombinant AtlA fragments revealed that interactions with both matrix proteins is mediated via the C-terminal located repeats R1R2 of the AtlA amidase domain. Binding of thrombospondin-1 and vitronectin occurred not simultaneously, due to a competitive inhibition. The second part of the study focused on the activation of human platelets by recombinant pneumococcal and staphylococcal proteins. In total, 28 proteins of S. pneumoniae and 52 proteins of S. aureus were incubated with human platelets. The activation of the cells was detected by flow cytometry using the activation markers P-selectin and the dimerization of the integrin αIIbβIII. The proteins CbpL, PsaA, PavA, and SP_0899 of S. pneumoniae induced platelet activation, however, the detailed mechanism has to be deciphered in further studies. Furthermore, the secreted proteins CHIPS, FLIPr, and AtlA of S. aureus were discovered as inductors for the activation of platelets. In addition, the domains of AtlA and Eap, crucial for platelet activation, were narrowed down. Interestingly, CHIPS, FLIPr, and Eap were described as inhibitors of neutrophil recruitment. Platelets are recently recognized as immune cells, due to the expression of immune receptors. The data obtained in this study highlight a comprehensive spectrum of effects of the S. aureus proteins towards different type of immune cells. Besides the activation of platelets in suspension buffer and plasma, the aggregation of platelets in whole blood was triggered by the proteins CHIPS, AtlA, and Eap. These results suggest a contribution of the proteins during the S. aureus-induced infectious endocarditis. Secretion of the platelet activating virulence factors, which were identified within this study, might represent a pathogenic strategy during S. aureus infection in which a direct contact between S. aureus and platelets is not required or even avoided. In conclusion, PavB, PspC, and Hic of S. pneumoniae and AtlA of S. aureus were identified as interaction partners of human thrombospondin-1. Furthermore, CHIPS, FLIPr, AtlA, and Eap were characterized as platelet activators. This study provides candidates for the development of protein-based vaccines, to prevent bacterial colonization and to neutralize secreted pathogenic factors.Streptococcus pneumoniae (Pneumokokken) und Staphylococcus aureus (S. aureus) sind Gram-positive humanspezifische Kommensalen des oberen Respirationstraktes. Mit sehr hoher Wahrscheinlichkeit ist jedes Individuum mindestens einmal im Leben asymptomatisch mit diesen Bakterien besiedelt. Die opportunistischen Pathogene können jedoch weitere Organe befallen und in tiefere Gewebsschichten vordringen. Die Besetzung von normalerweise sterilen Nischen des menschlichen Körpers mit beiden Erregern kann zu lokalen Entzündungen wie Sinusitis, Otitis Media und Abszessen oder zu lebensbedrohlichen Infektionen wie Pneumonie, Meningitis oder Sepsis führen. Eine erfolgreiche Kolonisierung setzt eine starke Interaktion zwischen dem Bakterium und den respiratorischen Epithelzellen voraus. Diese Wechselwirkung wird durch bakterielle Oberflächenproteine, so genannte Adhäsine, gewährleistet. Die Bindung der bakteriellen Adhäsine an Epithelzellen erfolgt überwiegend indirekt über Komponenten der extrazellulären Matrix (EZM) aber auch direkt an zelluläre Rezeptoren. Pneumokokken und S. aureus binden an zahlreiche EZM-Glykoproteine, darunter Fibronektin, Fibrinogen, Vitronektin und Kollagen. Es wurde jedoch auch eine Bindung beider Pathogene an humanes Thrombospondin-1 beschrieben. Thrombospondin-1 wird hauptsächlich in den α-Granula der Thrombozyten gespeichert und während einer Aktivierung in die Blutbahn sezerniert. Jedoch wird Thrombosponin-1 auch von weiteren Zellen wie Endothelzellen, Makrophagen und Fibroblasten produziert und sekretiert, welches daraufhin als Bestandteil in die EZM eingebaut wird. Für Pneumokokken wurde bisher kein Thrombospondin-1-bindendes Adhäsin identifiziert. PspC, Hic und PavB sind wichtige oberflächenlokalisierte Virulenzfaktoren, für die bereits Interaktionen mit anderen humanen EZM- und Plasmaproteinen beschrieben wurde. PspC sowie Hic binden unter anderem Vitronektin und Faktor H, wodurch die Komplementkaskade des angeborenen humanen Immunsystems inhibiert wird. PavB interagiert mit Fibronektin und Plasminogen und eine PavB-defiziente Mutante von S. pneumoniae zeigte eine reduzierte Kolonisierungsfähigkeit im Mausmodell. Unter den Oberflächenproteinen von S. aureus wurde bisher nur Eap als Thrombospondin-1-bindendes Adhäsin identifiziert. Über die Kolonisierung hinaus können Pneumokokken und S. aureus in die Blutzirkulation gelangen, mit Thrombozyten interagieren und eine Aktivierung dieser veranlassen. Die Thrombozytenaggregation, insbesondere ausgelöst durch S. aureus, spielt in der Klinik eine bedeutende Rolle, da der überwiegende Anteil der septischen Patienten eine Thrombozytopenie entwickelt. Bisher sind noch keine oberflächenlokalisierte Faktoren von S. pneumoniae bekannt, welche eine Thrombozytenaktivierung auslösen können. Dahingegen wurden für S. aureus bereits einige Proteine, unter anderem Eap, mit Thrombozyten-aktivierungspotential identifiziert. In der vorliegenden Arbeit konnte gezeigt werden, dass die Oberflächenproteine PavB, PspC und Hic von S. pneumoniae spezifische Liganden des humanen Thrombospondin-1 sind. In durchflusszytometrischen, oberflächenresonanzspektroskopischen sowie immunologischen Analysen wurde eine Interaktion mit löslichem sowie immobilisiertem Thrombospondin-1 nachgewiesen. Die Verwendung von spezifischen Pneumokokken-Deletionsmutanten zeigte, dass diese drei Virulenzfaktoren bedeutende Bindungspartner für lösliches Thrombospondin-1 sind. Die Ergebnisse deuten daraufhin, dass Pneumokokken in der Lage sind, in der Blutbahn befindliches lösliches Thrombospondin-1 zu akquirieren und immobilisiertes Glykoprotein der EZM als Substrat für die Adhäsion zu verwenden. Des Weiteren wurde die Bindungsdomäne von Thrombospondin-1 innerhalb der Pneumokokken-Proteine unter Verwendung von rekombinanten Teilfragmenten von PavB, PspC und Hic analysiert. Die Bindungskapazität von Thrombospondin-1 nahm hierbei proportional zur Anzahl der repetitiven Domänen in PavB und PspC und der Länge der α-helikalen Region im Hic Molekül zu. Das Bindungsverhalten von Thrombospondin-1 gegenüber PavB und PspC ist somit vergleichbar mit den EZM-Proteinen Vitronektin und Fibronektin, ist jedoch gegenüber Hic individuell. Die Eingrenzung der Bindungsdomäne der Adhäsine im Thrombospondin-1-Molekül erfolgte unter Verwendung von Glykosaminoglykanen als kompetitive Inhibitoren für die Interaktion. Die Ergebnisse legen nahe, dass die Pneumokokken-Proteine Hic und PspC die gleiche Bindungsstelle im Thrombospondin-1 besetzen, welche sich jedoch von der Bindungsregion von PavB unterscheidet. Jedoch scheinen alle drei Virulenzfaktoren im N-terminalen Bereich des Thrombospondin-1 zu binden. Mittels zweidimensionaler Gelelektrophorese, Thrombospondin-1-Overlay Assay und anschließender massenspektometrischer Analyse konnte AtlA von S. aureus als oberflächen-lokalisierter Interaktionspartner des humanen Thrombospondin-1 identifiziert werden. Darüber hinaus wurde eine Bindungsaktivität für Vitronektin im AtlA ermittelt. Immunologische und oberflächenresonanzspektroskopischen Bindungsstudien mit rekombinanten AtlA Teilfragmenten zeigten, dass die Interaktion mit beiden Matrixproteinen über die C-terminal gelegenen Repeats R1R2 der AtlA Amidaseregion vermittelt wird. Die Bindung von Thrombospondin-1 und Vitronektin erfolgte des Weiteren nicht simultan, da die Interaktion jeweils kompetitiv inhibiert werden konnte. Im zweiten Teil der Arbeit wurde die Aktivierung von humanen Thrombozyten mit rekombinanten Pneumokokken- und S. aureus-Proteinen ermittelt. Insgesamt wurden 28 Proteine von S. pneumoniae und 52 Proteine von S. aureus mit humanen Thrombozyten inkubiert. Die Aktivierung der Zellen wurde anhand der Aktivierungsmarker P-Selektin und der Dimerisierung des Integrins αIIbβIII mittels Durchflusszytometrie analysiert. Hierbei konnte gezeigt werden, dass die Proteine CbpL, PsaA, PavA und SP_0899 von S. pneumoniae eine Aktivierung der Thrombozyten induzieren, welche jedoch noch im Detail in zukünftigen Studien weiter untersucht werden muss. Des Weiteren wurden die sekretierten Proteine CHIPS, FLIPr und AtlA von S. aureus als neue Induktoren der Thrombozytenaktivierung identifiziert. Zusätzlich wurde die Domäne in AtlA und Eap, welche die Aktivierung der Thrombozyten veranlasst, lokalisiert. Interessanterweise wurden CHIPS, FLIPr und Eap bereits als Inhibitoren der Rekrutierung von neutrophilen Granulozyten beschrieben. Thrombozyten werden aufgrund der Expression von Immunrezeptoren seit kurzem ebenfalls als Immunzellen anerkannt. Mit den generierten Daten konnte ein umfassendes Wirkungsspektrum der S. aureus-Proteine auf diverse Immunzellen hervorgehoben werden. Neben der Aktivierung der Thrombozyten in Suspensionspuffer und Plasma konnte auch eine Aggregation der Thrombozyten in Vollblut durch die Proteine hervorgerufen werden. Diese Ergebnisse deuten des Weiteren auf einen Beitrag der Proteine während der S. aureus-induzierten infektiösen Endokarditis hin. Die Sekretion der Virulenz-faktoren von S. aureus könnte eine neue Pathogenitätsstrategie darstellen, in der ein Kontakt zwischen Thrombozyt und S. aureus nicht zwingend notwendig ist oder sogar vermieden wird. Zusammenfassend konnten PavB, PspC und Hic von S. pneumoniae und AtlA von S. aureus als Interaktionspartner des humanen Thrombospondin-1 identifiziert werden. Des Weiteren wurden CHIPS, FLIPr, AtlA sowie Eap als Thrombozyten-Aktivatoren charakterisiert. Die vorliegende Arbeit liefert Kandidaten zur Protein-basierten Impfstoffentwicklung, wodurch die bakterielle Kolonisierung verhindert oder sekretierte Pathogenitätsfaktoren neutralisiert werden könnten

Contribution of Human Thrombospondin-1 to the Pathogenesis of Gram-Positive Bacteria

A successful colonization of different compartments of the human host requires multifactorial contacts between bacterial surface proteins and host factors. Extracellular matrix proteins and matricellular proteins such as thrombospondin-1 play a pivotal role as adhesive substrates to ensure a strong interaction with pathobionts like the Gram-positive Streptococcus pneumoniae and Staphylococcus aureus. The human glycoprotein thrombospondin-1 is a component of the extracellular matrix and is highly abundant in the bloodstream during bacteremia. Human platelets secrete thrombospondin-1, which is then acquired by invading pathogens to facilitate colonization and immune evasion. Gram-positive bacteria express a broad spectrum of surface-exposed proteins, some of which also recognize thrombospondin-1. This review highlights the importance of thrombospondin-1 as an adhesion substrate to facilitate colonization, and we summarize the variety of thrombospondin-1-binding proteins of S. pneumoniae and S. aureus

Genomic Evidence of mcr-1.26 IncX4 Plasmid Transmission between Poultry and Humans

ABSTRACT Colistin is still commonly used and misused in animal husbandry driving the evolution and dissemination of transmissible plasmid-mediated colistin resistance (mcr). mcr-1.26 is a rare variant and, so far, has only been detected in Escherichia coli obtained from a hospitalized patient in Germany in 2018. Recently, it was also notified in fecal samples from a pigeon in Lebanon. We report on the presence of 16 colistin-resistant, mcr-1.26-carrying extended-spectrum beta-lactamase (ESBL)-producing and commensal E. coli isolated from poultry samples in Germany, of which retail meat was the most common source. Short- and long-read genome sequencing and bioinformatic analyses revealed the location of mcr-1.26 exclusively on IncX4 plasmids. mcr-1.26 was identified on two different IncX4 plasmid types of 33 and 38 kb and was associated with an IS6-like element. Based on the genetic diversity of E. coli isolates, transmission of the mcr-1.26 resistance determinant is mediated by horizontal transfer of IncX4 plasmids, as confirmed by conjugation experiments. Notably, the 33-kb plasmid is highly similar to the plasmid reported for the human sample. Furthermore, we identified the acquisition of an additional beta-lactam resistance linked to a Tn2 transposon on the mcr-1.26 IncX4 plasmids of three isolates, indicating progressive plasmid evolution. Overall, all described mcr-1.26-carrying plasmids contain a highly conserved core genome necessary for colistin resistance development, transmission, replication, and maintenance. Variations in the plasmid sequences are mainly caused by the acquisition of insertion sequences and alteration in intergenic sequences or genes of unknown function. IMPORTANCE Evolutionary events causing the emergence of new resistances/variants are usually rare and challenging to predict. Conversely, common transmission events of widespread resistance determinants are quantifiable and predictable. One such example is the transmissible plasmid-mediated colistin resistance. The main determinant, mcr-1, has been notified in 2016 but has successfully established itself in multiple plasmid backbones in diverse bacterial species across all One Health sectors. So far, 34 variants of mcr-1 are described, of which some can be used for epidemiological tracing-back analysis to identify the origin and transmission dynamics of these genes. Here, we report the presence of the rare mcr-1.26 gene in E. coli isolated from poultry since 2014. Based on the temporal occurrence and high similarity of the plasmids between poultry and human isolates, our study provides first indications for poultry husbandry as the primary source of mcr-1.26 and its transmission between different niches

Antimicrobial Resistance Surveillance: Data Harmonisation and Data Selection within Secondary Data Use

Resistance to last-resort antibiotics is a global threat to public health. Therefore, surveillance and monitoring systems for antimicrobial resistance should be established on a national and international scale. For the development of a One Health surveillance system, we collected exemplary data on carbapenem and colistin-resistant bacterial isolates from human, animal, food, and environmental sources. We pooled secondary data from routine screenings, hospital outbreak investigations, and studies on antimicrobial resistance. For a joint One Health evaluation, this study incorporates epidemiological metadata with phenotypic resistance information and molecular data on the isolate level. To harmonise the heterogeneous original information for the intended use, we developed a generic strategy. By defining and categorising variables, followed by plausibility checks, we created a catalogue for prospective data collections and applied it to our dataset, enabling us to perform preliminary descriptive statistical analyses. This study shows the complexity of data management using heterogeneous secondary data pools and gives an insight into the early stages of the development of an AMR surveillance programme using secondary data

Genomic Evidence of mcr-1.26 IncX4 Plasmid Transmission between Poultry and Humans

Colistin is still commonly used and misused in animal husbandry driving the evolution and dissemination of transmissible plasmid-mediated colistin resistance (mcr). mcr-1.26 is a rare variant and, so far, has only been detected in Escherichia coli obtained from a hospitalized patient in Germany in 2018. Recently, it was also notified in fecal samples from a pigeon in Lebanon. We report on the presence of 16 colistin-resistant, mcr-1.26-carrying extended-spectrum beta-lactamase (ESBL)-producing and commensal E. coli isolated from poultry samples in Germany, of which retail meat was the most common source. Short- and long-read genome sequencing and bioinformatic analyses revealed the location of mcr-1.26 exclusively on IncX4 plasmids. mcr-1.26 was identified on two different IncX4 plasmid types of 33 and 38 kb and was associated with an IS6-like element. Based on the genetic diversity of E. coli isolates, transmission of the mcr-1.26 resistance determinant is mediated by horizontal transfer of IncX4 plasmids, as confirmed by conjugation experiments. Notably, the 33-kb plasmid is highly similar to the plasmid reported for the human sample. Furthermore, we identified the acquisition of an additional beta-lactam resistance linked to a Tn2 transposon on the mcr-1.26 IncX4 plasmids of three isolates, indicating progressive plasmid evolution. Overall, all described mcr-1.26-carrying plasmids contain a highly conserved core genome necessary for colistin resistance development, transmission, replication, and maintenance. Variations in the plasmid sequences are mainly caused by the acquisition of insertion sequences and alteration in intergenic sequences or genes of unknown function.Colistin is still commonly used and misused in animal husbandry driving the evolution and dissemination of transmissible plasmid-mediated colistin resistance (mcr). mcr-1.26 is a rare variant and, so far, has only been detected in Escherichia coli obtained from a hospitalized patient in Germany in 2018. Recently, it was also notified in fecal samples from a pigeon in Lebanon. We report on the presence of 16 colistin-resistant, mcr-1.26-carrying extended-spectrum beta-lactamase (ESBL)-producing and commensal E. coli isolated from poultry samples in Germany, of which retail meat was the most common source. Short- and long-read genome sequencing and bioinformatic analyses revealed the location of mcr-1.26 exclusively on IncX4 plasmids. mcr-1.26 was identified on two different IncX4 plasmid types of 33 and 38 kb and was associated with an IS6-like element. Based on the genetic diversity of E. coli isolates, transmission of the mcr-1.26 resistance determinant is mediated by horizontal transfer of IncX4 plasmids, as confirmed by conjugation experiments. Notably, the 33-kb plasmid is highly similar to the plasmid reported for the human sample. Furthermore, we identified the acquisition of an additional beta-lactam resistance linked to a Tn2 transposon on the mcr-1.26 IncX4 plasmids of three isolates, indicating progressive plasmid evolution. Overall, all described mcr-1.26-carrying plasmids contain a highly conserved core genome necessary for colistin resistance development, transmission, replication, and maintenance. Variations in the plasmid sequences are mainly caused by the acquisition of insertion sequences and alteration in intergenic sequences or genes of unknown function

Klebsiella pneumoniae arms itself: poultry food chain drives spread and evolution of mcr-1.26-IncX4 plasmids

No abstract available