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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.
Streptococcus pneumoniae is a commensal of the human upper respiratory tract and
the etiological agent of several life-threatening diseases. This pathogen is the model bacterium
for natural competence. Furthermore, the pneumococci played an important role in the
identification of DNA as the main molecule involved in bacterial transformation. As a result,
studies on the pneumococcal genome provided an initial overview of the genetic potential of
this pathogen. The pneumococcus is a highly versatile bacterium possessing a high rate of
uptake and recombination of exogenous DNA from neighboring bacteria. As such, a significant
diversity in the genome content among the different pneumococcal strains has been reported.
The capsular polysaccharide, an important pneumococcal virulence factor, is the best example
on the pneumococcal diversity. There are over 98 serotypes characterized to date presenting
differences in their capsule (cps) locus. Additional to the cps locus, the pneumococcus also
presents 13 genomic islets annotated as regions of diversity (RD) encoded in the auxiliary
genome. Remarkably, 8 of the pneumococcal RD studied so far have been associated with
virulence. Furthermore, the ongoing sequencing of over 4000 pneumococcal genomes have
shed light on the conservation level of well-known pneumococcal virulence factors.
Interestingly, important pneumococcal virulence determinants show variations in the gene and
protein sequence among the different strains. Prototypes are for example the pneumococcal
surface protein C (PspC) and pneumococcal adherence and virulence factor B (PavB).
Conversely, gene regulation in S. pneumoniae is carried out by highly conserved and genome-
wide distributed transcriptional factors. Overall, the pneumococci interplays with its
environment with 4 major regulatory systems: quorum sensing (QS), stand-alone
transcriptional regulators, small RNAs (sRNAs) and two-component regulatory systems (TCS).
Some of these systems are multifaceted and share more than one feature. Furthermore, there
is crosstalk among the different systems, requiring the activation of a signaling cascade to
function properly.
A comprehensive analysis of the distribution and conservation of pneumococcal
virulence factors and TCS was obtained in this study. The results are summarized as a
simplified variome in which 25 pneumococcal strains with a complete sequenced genome were
analyzed. Interestingly, the genes encoding the glycolytic protein enolase and the toxin
pneumolysin were the most conserved virulence determinants. Additionally, the high level of
conservation was confirmed for the pneumococcal TCS regulators, especially for WalKR,
CiaRH and TCS08.
The main focus of this study was on the regulatory functions of pneumococcal TCS.
With this in mind, an extensive and detailed systematic review of the 13 pneumococcal TCS
and its orphan RR was undertaken. For this purpose, every pneumococcal TCS was analyzed
for its reported functional and structural information along with its contribution to the main
pathophysiology of the pneumococci. In brief, S. pneumoniae can utilize its TCS for the
regulation of important cellular processes and the sensing of detectable signals in the
environment. Additionally, the role of TCS in pneumococcal processes and signal sensing can
be divided further. In the first place, pneumococcal TCS regulate competence and fratricide,
the production of bacteriocins and host-pathogen interaction processes, while the detectable
signals include cell-wall perturbations, environmental stress, and nutrients. As a conclusion
from this section, it is possible to analyze the pneumococcal TCS in a comprehensive manner.
There is a complex network among the different pneumococcal regulators and the TCS play
an important role. Moreover, these systems are highly conserved and essential for the proper
functioning of the pneumococcus as a pathogen.
Following up on pneumococcal TCS, this study focused especially on the TCS08.
Interestingly, the pneumococcal TCS08 has been previously associated with the regulation of the cellobiose metabolism. Furthermore, this system has also been reported to regulate the
expression of genes encoded in the RD4 (Pilus-1). Remarkably, the pneumococcal TCS08
was shown to be highly homologous to the SaeRS system of Staphylococcus aureus. Initially,
mutant strains lacking a single (Δrr08 or Δhk08) or both components (Δtcs08) of the TCS08
were generated in pneumococcal D39 and TIGR4 strains. Transcriptomics and functional
assays showed a downregulation of the PI-1 in the absence of the complete tcs08, while PavB
presented an upregulation in the Δhk08 knockout. Moreover, an important number of genes
coding for intermediary metabolism proteins were also found to be differentially expressed by
microarray analysis. As such, the TIGR4Δhk08 strain presented a downregulation for the
cellobiose operon (cel). In contrast, an upregulation was reported for the fatty acid biosynthesis
(fab) and arginine catabolism (arc) operons. Conversely, a decrease in gene expression was
seen in the TIGR4Δrr08 strain for the arc operon. Finally, in vivo murine pneumonia and sepsis
models highlighted an involvement of TCS08 in pneumococcal virulence. Remarkably, the
different TCS08 mutants presented a strain dependent effect on their virulence severity. The
TIGR4Δrr08, and all TCS08 mutants in D39 showed a decrease in virulence in the pneumonia
model, with no changes in sepsis. Conversely, the absence of HK08 in TIGR4 presented a
highly virulent phenotype in both pneumonia and sepsis models. To sum up, the pneumococcal
TCS08 influenced the expression of genes involved in fitness and colonization. Specifically,
those coding for the adhesins PavB and PI-1 and fitness proteins from the cel, arc and fab
operons. Remarkably, the highest changes in expression were observed in the strains lacking
the HK08. Additionally, TCS08 has a strain dependent impact on pneumococcal virulence as
showed by murine pneumonia and sepsis models when comparing the effects in D39 and
TIGR4.