Molecular Charaterization of Streptococcus Pneumoniae Surface Protein A (PspA) in Pakistan Isolate

Abstract

Streptococcus pneumoniae (pneumococcus) has a major role in morbidity and mortality of children as well as of the elder population worldwide. Pneumococcus causes pneumonia, otitis media in addition to invasive diseases such as bacteremia and meningitis, which are collectively called pneumococcal diseases or infections. Pakistan, a country located in South Asia, is the third largest contributor in pneumococcal infections, specially ‘pneumonia’ in the world (IVAC/PATH Pneumonia and Diarrhea Progress Reports 2013-2017), it makes an ideal scenario to begin with preliminary exploring of pneumococcus characterization. Above in view, first part of the present study was designed to determine the antibiotic susceptibility and serotype distribution of the pneumococcal isolates. Further studies were undertaken for pneumococcal surface protein A gene (pspA) family/clade distribution, in-silico determination of its antigenic sites, their fusion, cloning and recombinant pilot expression study in E. coli. Thirty-eight pneumococcal strains from patients with pneumococcal disease (PD) were collected from a local hospital. Antibiotic susceptibility tests indicated that all strains were susceptible to chloramphenicol and linezolid, while 80% were resistant to gentamycin. Serotype distribution revealed that 19F was the most prevalent serotype (23.6%), while ∼80% of the strains carried pspA gene belonging to PspA family 2 and clade 3. The in-silico determined PspA fusion protein was found antigenic in the immunoblotting assay. Recent epidemiological studies reveal that biofilm support and develop most human chronic infections. Keeping this, the second part of the thesis is focused on the characteristics of pneumococcal biofilm, grown on the biotic and abiotic substrate. Antibiotic susceptibility (erythromycin, 2μg/mL) and growth pattern of pneumococcal biofilm grown on biotic (Detroit-562 cells) and abiotic substrate (Thermonox coverslips) were evaluated. Results indicated that pneumococcal biofilm grown on immobilized biotic substrate challenge erythromycin more significantly than abiotic substrate. The similar results were obtained when the study was done using a bioreactor model, more xii than ∼90% increase in biofilm formation was noted when biotic substrate was provided for growth as compared to the abiotic substrate. The epithelial cells as well as the intercellular tight junction play an important role and are involved in the control of paracellular permeability. The S. pneumoniae colonize with epithelial cell layer, disrupted epithelial cells as well as tight junction, and translocate to other body parts, that promotes the development of pneumococcal invasive diseases. The third part of the study is concentrated on the analysis of invasive property of pneumococcus, using Trans Epithelial Electrical Resistance (TEER). In this study, two different types of epithelial cells, Detroit 562 cells (pharyngeal cells) and Calu-3 cells (lung cells) were used; substantial decline in TEER was observed in the pneumococcal infected epithelial cells. Our study presented that pneumococcus invades the epithelial cells and causes damage to epithelial cell barrier with cytopathic effects. It was also observed that the invasion of pneumococcus is independent of its biofilm mass density. Previous research findings have shown an adverse association between S. pneumoniae and S. aureus, present in nasopharyngeal carriage. But these epidemiological observations have no conclusive findings. The fourth part of the study was designed to demonstrate, how S. pneumoniae opposes and eradicate S. aureus. We have confirmed in this study that TIGR4, rapidly eradicated, preformed S. aureus biofilm, including biofilm produced by MRSA strain ‘USA300’. The eradication requires physical contact between S. pneumoniae and S. aureus. The requirement of physical contact was confirmed through confocal microscopy, colocalization experiments, and experiments utilizing a transwell system to separate the two bacterial species. The physical contact-mediated eradication was found very efficient as it completely eradicated a viable lawn of S. aureus biofilm with in 2 hours.