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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.
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.