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Streptococcus pneumoniae (S. pneumoniae, pneumococci) and Staphylococcus aureus (S. aureus) belong to the Gram-positive, facultative pathogenic bacteria. They are typical commensals of the human upper respiratory tract and most people get colonized at least once during their life. Nevertheless, these potentially pathogenic bacteria are able to spread from the site of colonization to invade into deeper tissues and the blood circulation. Thereby, severe local and invasive infections like bacteremia and life-threatening sepsis can be caused. Once reaching the bloodstream, bacteria get in contact with platelets. Platelets are small, anucleated cells and the second most abundant cell type in the circulation. The role of platelets in hemostasis is well known. Circulating resting platelets sense vessel injury independent of its cause. Platelets bind to injured endothelium and exposed molecules of the underlying extracellular matrix, get activated and release intracellular adhesion proteins and different modulatory molecules. This in turn initiates activation and binding of nearby platelets resulting in closure of vascular injury by formation of small thrombi. Despite being pivotal in maintenance of the endothelial barrier they got increasingly recognized as cells with important immune functions. Platelets excert functions of the immune response by either, i) interacting with immune cells of different pathways of the immune response, ii) releasing immunomodulatory molecules stored in their granules or iii) interacting with invading pathogens via direct or indirect binding.
The basis for this study were results demonstrating direct binding of different S. aureus proteins to platelets resulting in platelet activation. The identified proteins in the mentioned study are the S. aureus proteins Eap, AtlA-1, CHIPS and FlipR. Severe invasive infections with S. pneumoniae are quite often associated with development of thrombocytopenia or disseminated vascular dissemination. This frequent observation hints towards either a direct or indirect interplay of platelets with pneumococci. Hence, this study aims to analyze potential interactions and aims to decipher involved factors on both the platelet- and bacterial site.
A screening of recombinant pneumococcal surface proteins identified proteins belonging to the group of lipoproteins, sortase-anchored proteins and choline-binding proteins to directly activate human platelets. Besides these surface proteins also the intracellular pneumococcal pneumolysin (Ply) induced highly increased values for the platelet activation marker P-selectin. Since Ply is a major virulence factor of
S. pneumoniae the primary focus was set on involvement of this pore forming toxin on platelet activation. Surprisingly, our data revealed Ply induced platelet activation to be a false positive result based on formation of large Ply pores in the platelet membrane. In fact, it was clearly demonstrated that Ply lyses platelets even at low concentrations and thereby rendering them non-functional. Lysis of platelets could be inhibited by the addition of pharmaceutical immunoglobulin preparations as well as antibodies specifically targeting Ply. Inhibition of Ply also resulted in fully rescued platelet function either in washed platelets or in whole blood as shown by thrombus formation. Next to pneumococci also S. aureus expresses pore forming toxins, namely α-hemolysin (Hla) and different pairs of bicomponent pore forming leukocidins. Whereas the different tested leukocidins did not affect platelets, Hla acted in a two-step mechanism on human platelets. The results confirm previous data on Hla induced platelet activation via Hla resulting in e.g., reversible platelet aggregation or surface expression of activation markers. Nevertheless, platelet activation by Hla is followed by dose- and time-dependent lysis of platelets resulting in loss of platelet function and abrogated thrombus formation. Platelet lysis by Hla could neither be rescued with specific monoclonal anti-Hla antibodies nor with pharmaceutical IgG preparations containing anti-Hla IgGs. Taken together, the presented data reveal new pathomechanisms involving disturbance of platelets by bacterial pore forming toxins. Platelet lysis as well as impaired platelet function play an important role in development of severe complications during invasive infections. In life threatening infections caused by S. pneumoniae the usage of antibody formulations containing antibodies targeting Ply might be a promising approach for the prevention or even intervention and improvement of clinical outcome.
Thrombozyten haben neben ihrer Funktion in der Hämostase eine wichtige Rolle in der Immunabwehr. Sie interagieren hierbei mit Komponenten des angeborenen und des adaptiven Immunsystems und sind in der Lage, direkte anti-mikrobielle Einflüsse zu vermitteln. Die Interaktion von Thrombozyten mit Gram-positiven Bakterien unterscheidet sich von jener mit Gram-negativen Erregern. Bei beiden Gruppen von Bakterien scheint die Aktivierung von Thrombozyten und Freisetzung anti-mikrobieller Peptiden aus den Granula ein wichtiger Bestandteil der direkten Pathogenabwehr durch Thrombozyten zu sein. Hierbei führt die Interaktion mit S. aureus direkt zu einer starken pathogen-induzierten Thrombozytenaktivierung, während bei Gram-negativen Organismen wie E. coli eine Verstärkung durch die Opsonierung mit PF4 und anti-PF4/H IgG notwendig scheint. Vermutlich ist die Bindung von PF4 und anti-PF4/H IgG an Gram-positive Bakterien von größerer Bedeutung für die Opsonierung für andere Immunzellen als für den direkten bakteriziden Effekt der Thrombozyten.
Der Gram-positive S. pneumoniae führt durch Funktionsstörung und Exposition von Phosphatidylserin zu einer Schädigung der Thrombozyten. Dieser schädigende Effekt auf Thrombozyten durch S. pneumoniae wird unter anderem durch Pneumolysin, ein porenbildendes Toxin der Pneumokokken, vermittelt. Dieses induziert bereits in geringen Konzentrationen die Porenbildung in der Thrombozytenmembran und führt zur Induktion von Apoptose.
In der Arbeit konnten die initialen Fragestellungen folgendermaßen beantwortet werden:
1.Thrombozyten können einen direkten schädigenden Effekt auf Gram-positive Bakterien vermitteln.
2.PF4 und anti-PF4/Polyanion IgG spielen in der direkten Thrombozyten-vermittelten Pathogenabwehr bei Gram-positiven Erregern, trotz der Bindung an Gram-positive Bakterien, eine untergeordnete Rolle. Sie verstärken weder die Thrombozyten-aktivierung noch den anti-bakteriellen Effekt.
3.Die Auswirkung der Co-Inkubation mit Bakterien auf die Thrombozyten ist heterogen und abhängig vom untersuchten Bakterienstamm. Es kommt zur Aktivierung der Thrombozyten durch S. aureus und zur Schädigung der Thrombozyten durch S. pneumoniae.
Infective endocarditis (IE) is a potentially life-threatening infection of the endocardial surfaces of the heart, most frequently the valves. It is typically caused by bacteria, less commonly by fungi. Over the past years, the morbidity and mortality of IE have gradually increased, and it is now the fourth most common life-threatening infection after sepsis, pneumonia, and intra-abdominal abscess. Despite advances in cardiac imaging and diagnostic techniques, the diagnosis of IE remains challenging. The lack of fast and reliable diagnosis of IE can lead to serious complications. Therefore, new diagnostic and therapeutic tools are urgently needed.
This study had two main aims: (i) to investigate whether a pathogen-specific antibody response in IE patients is mounted against different IE pathogens and whether analysis of such a response might be useful for complementing the classical blood culture diagnosis, and (ii) generate and characterize neutralizing monoclonal antibodies (mAbs) against three virulence factors of Staphylococcus aureus (S. aureus), which is the most common etiological agent in IE.
Our research group has recently established an xMAP® (Luminex®) technology-based serological assay that simultaneously quantifies the antibody response against 30 different pathogens. Within the research consortium Card-ii-Omics, we conducted a prospective, observational clinical discovery study involving 17 IE patients and 20 controls (i.e., patients with non-infectious heart-related conditions). Plasma samples were obtained on the day of IE diagnosis from all patients, while samples at later dates over the course of infection were available for only some patients. Invasive pathogens were identified by blood culture.
The infection array revealed antibodies against a broad range of pathogens in both controls and IE patients, suggesting a broad immune memory. Overall, antibody levels did not significantly differ between both groups, but we observed high antibody titers against those pathogens that were detected by blood culture. Whenever available (in the case of 13/17 IE patients), back-up and follow-up plasma samples (obtained before or after diagnosis, respectively) were included in the analyses that provided valuable information about the kinetics of antibody response during the course of infection. Notably, infection array data confirmed (and extended) the blood culture data in only 2/13 cases. In three cases, serology contradicted the microbiological diagnosis, and in three cases, the infection array was able to identify pathogens, while the microbiological diagnosis failed. In three cases, serology was negative while microbiological diagnosis was positive, and in two cases, both serology and microbiological diagnosis were negative. In 6 out of 8 cases with increases in antibody levels, this response was directed against gut microbes. This supports the leaky gut hypothesis, which assumes that breaching of the gut barrier causes translocation of gut microbes into the bloodstream, which then infect the heart valves. Moreover, we observed an increase in antibody titers in 4 patients against the yeast C. albicans, suggesting a secondary fungal infection. Finally, this study emphasized that the timing of plasma collection is crucial for studying antibody kinetics in IE.
After demonstrating that pathogen-specific antibodies are generated during IE, we aimed to generate mAbs against the prime IE pathogen S. aureus and study their functions on a molecular level. Using the hybridoma technology, our research group has recently generated mAbs against two S. aureus surface proteins/adhesion factors (clumping factor A (ClfA) and fibronectin-binding protein A (FnBPA)), both involved in biofilm formation, as well as an extracellular enzyme, the staphylococcal serine protease–like protein B (SplB), a virulence factor. In this work, the sequences of the mAbs were determined from hybridoma RNA. Then those mAbs were produced at a larger scale in order to determine their binding and neutralizing capacities using in vitro assays such as ELISA, Western blot, Dot blot, microscale thermophoresis, and in a mouse model.
The anti-SplB mAb specifically targeted SplB, with no cross-reactivity to other Spls or extracellular proteins (ECP) of S. aureus. Though anti-SplB mAb showed moderate binding to SplB with a Kd value of 2.54 μM and high sequence homology to the germline sequence, it neutralized the enzymatic activity of SplB up to 99% in 5-fold molar excess as showed in an in vitro substrate cleavage assay. Previous work showed that SplB facilitates the release of proinflammatory cytokines in endothelial cells and induces endothelial damage in mice. Here, we demonstrated that the anti-SplB mAb efficiently blocked the function of SplB in vivo, thus markedly reducing the damage to the endothelial barrier. In conclusion, we identified the strong neutralizing potential of a mAb against SplB, which merits further investigation as a candidate for the immunotherapy of SplB-induced S. aureus pathologies, including IE.
High antibody titers against S. aureus adhesins, including ClfA and FnBPA, have been reported in IE patients. Besides, ClfA is involved in serious S. aureus bloodstream and biofilm-related infections. Similarly, FnBPA facilitates biofilm formation and inhibits macrophage invasion. These important properties make the two bacterial adhesins ideal candidates for a passive vaccination strategy. We generated two murine ClfA-mAbs, ClfA-002 and ClfA-004, which showed strong specificity to ClfA. However, ClfA-004 showed reduced binding strength compared to ClfA-002 due to a single non-synonymous nucleotide change (Phe Tyr) at the CDR3 region. While the ClfA-002 mAb reduced the binding of ClfA to fibrinogen by around 60%, the ClfA-004 had no inhibitory capacity. We also generated two murine and twelve humanized anti-FnBPA mAbs, which showed similar and moderate binding to FnBPA. One murine mAb (anti-FnBPA D4) partially inhibited the binding of FnBPA to fibronectin. FnBPA contains 11 tandem repeats that can all bind to fibronectin. This redundancy could be the reason for the lack of complete inhibition. Hence, in this work, we characterized the properties of neutralizing mAbs against two adhesins of S. aureus. These mAbs should be tested in the future, alone and in combination with other mAbs and antibiotics, for their ability to reduce staphylococcal biofilm formation.
In conclusion, we showed that antibody profiling of IE patients can provide valuable insights into the causative agent(s), and can help in guiding the antibiotic therapy. However, sampling is crucial in IE, which often dwells for many weeks before being clinically diagnosed. Because of the severity of IE, which can be life-threatening, I suggest to establish biobanks to store patient samples upon hospital admission that will provide a baseline in case of a later microbial infection. Moreover, our results suggest that C. albicans plays an important and so far underestimated role in IE. In the second part of the thesis, we characterized several mAbs against an S. aureus protease and two adhesins. Of high interest is a neutralizing mAb against SplB, which shows promising results in vitro and in vivo. Further in vitro and in vivo tests need to be conducted to study the anti-biofilm activity of the anti-FnBPA- and anti-ClfA-mAbs and explore their utility as therapeutic agents.
Functional characterization of a novel protease isolated from a mouse-adapted S. aureus strain
(2018)
Background: The high incidence of methicillin-resistant Staphylococcus aureus
(MRSA) strengthens the need for new effective antibiotics and a protective vaccine. Up till now, mainly human-adapted Staphylococcus aureus strains were used to study S. aureus pathogenicity in mouse models. However, it is known that S. aureus is highly host-specific. Recently, a mouse-adapted S. aureus strain, JSNZ, was identified. This strain could be a promising tool in developing more appropriate infection models. JSNZ produces high amounts of a putative extracellular protease, named JSNZ extracellular protease (Jep). Since the jep gene was only detected in S. aureus isolates from laboratory mice and wild small rodents and shrews, we hypothesize that Jep is important for colonization and infection in mice. The jep deletion mutant previously created by our collaborators from the University of Auckland, New Zealand, intriguingly showed a reduced survival and growth fitness in murine serum and whole blood as compared to the JSNZ wild type (WT) strain.
Objective: To elucidate the role of Jep in the interaction between S. aureus and its
host by comparing the impact of JSNZ WT with a mutant and a complement strain on the murine immune system. In addition, the elucidation of possible genetic factors behind host-adaptation of S. aureus strains isolated from wild rodents and shrews.
Methods: A jep complemented strain was generated by chromosomal replacement.
JSNZ WT, the jep mutant and the complement strain were subjected to functional
assays (whole blood survival assay, coagulation assay). In addition, the genetic
background that might confer host specificity was tested by staph array genotyping.
Results: The mutant strain JSNZDjep was successfully complemented with the jep
gene using a chromosomal integration approach. The WT strain and the
complemented strain produced the Jep protein in comparable amounts.
Unexpectedly, the complemented strains did not behave like the WT strain but rather like the mutant in a series of in vitro assays. Firstly, the growth of both the deletion mutant and the complemented strains was slightly reduced in TSB as compared to the WT strain. Secondly, the jep knockout strain showed a strongly reduced survival in murine whole blood compared to its wild type counterpart, but so did the complemented strain. Finally, the coagulation of murine plasma was less pronounced for the jep deletion mutant and the complemented strain as compared to the JSNZ WT. To exclude a defect in jep gene expression, we compared the amount of Jep expressed during growth in TSB medium for the three strains. The complemented strain produced Jep in a manner similar to the WT strain in a growth-phase dependent manner, suggesting that Jep expression was not affected during the creation of the complemented strain.
The array data showed some differences in the genetic makeup between animal
isolated strains and matched human strains. For example, while all animal isolates of the CC88 lacked the resistance mecA gene it was found in some human isolates of the same strain.
Conclusion: In conclusion, our unidentified mutation created during the generation
of the jep knock-out strain rather than the jep gene itself manipulated the murine
immune response. The responsible gene and the underlying mechanisms remain to
be clarified. Genetic profiling of S. aureus strains allowed us to obtain some valuable information including data about CC49, the most frequently isolated lineage in wild rodents and shrews where compared to the human isolates the murine strains showed clear signs of host adaptation. However, the analysis had several limitations including the small sample size.