Increased pathogenicity of pneumococcal serotype 1 is driven by rapid autolysis and release of pneumolysin

Streptococcus pneumoniae serotype 1 is the predominant cause of invasive pneumococcal disease in sub-Saharan Africa, but the mechanism behind its increased invasiveness is not well understood. Here, we use mouse models of lung infection to identify virulence factors associated with severe bacteraemic pneumonia during serotype-1 (ST217) infection. We use BALB/c mice, which are highly resistant to pneumococcal pneumonia when infected with other serotypes. However, we observe 100% mortality and high levels of bacteraemia within 24 hours when BALB/c mice are intranasally infected with ST217. Serotype 1 produces large quantities of pneumolysin, which is rapidly released due to high levels of bacterial autolysis. This leads to substantial levels of cellular cytotoxicity and breakdown of tight junctions between cells, allowing a route for rapid bacterial dissemination from the respiratory tract into the blood. Thus, our results offer an explanation for the increased invasiveness of serotype 1.


Statistics
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All studies must disclose on these points even when the disclosure is negative.  (10):1250-1259) and lungs and blood (Neill DR et al, PLoS Pathogens, 2012, e1002660). Initial experiments looking at lung and blood bacterial burdens in serotype 2 and serotype 1 infected mice, with 5 mice per group, suggested no difference between groups prior to 12 hours post-infection but a 129% difference thereafter (Log10 1.33 vs 3.05, std dev 0.69 vs 1.4). On this basis, we calculated that a total sample size of 9 mice per group would be needed for 90% power, alpha 0.05, using the methodology described above. We included an additional mouse at each time point to account for potential loss due to comorbidities or premature progression to licence severity endpoints.
No data was excluded Data in the following figures are from three independent experiments: Figure 1A, Figure 4C, Figure 5A, Figure 6, Figure 7A-D, Figure 9A Mice were randomized to cages on arrival at the University of Liverpool animal facility, by technical staff with no role in study design. At study onset, random allocation of unique cage I.D. numbers to experimental groups was performed by the research team. All mice used were female BALB/c of 6-10 weeks of age. Within each experiment, all mice used were within 2 weeks of age of each other.
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Flow Cytometry
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Methodology
Sample preparation Validation is shown above in "antibodies used" section but also in a BALB/c mouse lung tissue was harvested, weighed, placed into a petri dish and then cut into smaller pieces using a scalpel blade.
To help release immune cells via enzymatic digestion, lung tissue it was placed in 1.5ml Eppendorf tubes containing 1ml of PBS and 10mg per ml of Collagenase D (Roche). The Eppendorf tubes were then incubated at 37oC for 30 minutes. After digestion, tissue was passed through a 40m cell strainer (BD Biosciences) and washed with sterile PBS to create a single cell suspension. Cell suspensions were then centrifuged at 400 x g for 5 minutes. The cell pellet was re-suspended in 1x Red blood cell lysis buffer (Sigma) to lyse all red blood cells. The cell suspensions were then centrifuged at 400 x g for 5 minutes and cell pellet resuspended in cryopreservation media, for storage at -80oC. When needed, aliquots of cells were thawed quickly in the water bath.
BALB/c mouse nasopharyngeal tissue was harvested and placed into bijou tubes containing 3mls of sterile PBS. The tissue was then mechanically disrupted for~1 minute using a homogenizer (IKA T10). The homogenized tissue was then passed through a 40m pore cell strainer and centrifuged at 400 x g for 5 minutes. The cell pellet was either re-suspended in cryopreservation media or used for FACS analysis on the same day.
For staining and acquisition, samples were either thawed or used fresh from dissection. Cells were incubated with a 1 in 200