Airway proteolytic control of pneumococcal competence

Streptococcus pneumoniae is an opportunistic pathogen that colonizes the upper respiratory tract asymptomatically and, upon invasion, can lead to severe diseases including otitis media, sinusitis, meningitis, bacteremia, and pneumonia. One of the first lines of defense against pneumococcal invasive disease is inflammation, including the recruitment of neutrophils to the site of infection. The invasive pneumococcus can be cleared through the action of serine proteases generated by neutrophils. It is less clear how serine proteases impact non-invasive pneumococcal colonization, which is the key first step to invasion and transmission. One significant aspect of pneumococcal biology and adaptation in the respiratory tract is its natural competence, which is triggered by a small peptide CSP. In this study, we investigate if serine proteases are capable of degrading CSP and the impact this has on pneumococcal competence. We found that CSP has several potential sites for trypsin-like serine protease degradation and that there were preferential cleavage sites recognized by the proteases. Digestion of CSP with two different trypsin-like serine proteases dramatically reduced competence in a dose-dependent manner. Incubation of CSP with mouse lung homogenate also reduced recombination frequency of the pneumococcus. These ex vivo experiments suggested that serine proteases in the lower respiratory tract reduce pneumococcal competence. This was subsequently confirmed measuring in vivo recombination frequencies after induction of protease production via poly (I:C) stimulation and via co-infection with influenza A virus, which dramatically lowered recombination events. These data shed light on a new mechanism by which the host can modulate pneumococcal behavior and genetic exchange via direct degradation of the competence signaling peptide. Author summaryStreptococcus pneumoniae is a major human pathogen that usually colonizes the nasopharynx asymptomatically, but can progress to disease causing meningitis, bacteremia, otitis media, and sinusitis. In response to invasive pneumococci, the host produces an inflammatory response, including the influx of serine proteases. Invasive S. pneumoniae can be cleared through the action of serine proteases, however little is known on how serine proteases impact S. pneumoniae processes during colonization. One key modulator of pneumococcal survival in the host environment is its innate competence to uptake DNA. Here, we investigate how serine proteases impact pneumococcal competence through modulation of a key signaling peptide, CSP. We show that two different recombinant serine proteases degrade CSPs and this degradation has a direct impact on pneumococcal competence. We further demonstrate a dose-dependent reduction in pneumococcal competence upon CSP incubation with mouse lung homogenates, which suggests that serine proteases produced in mouse lungs similarly degrade CSP. These data reveal a novel mechanism by which the host can modulate pneumococcal behavior and genetic exchange, including that required for the spread of antibiotic resistance, a growing threat as the number of cases involving multi-drug resistant pneumococci have increased worldwide.