Proteomic Adaptation of Streptococcus pneumoniae to the Human Antimicrobial Peptide LL-37

Abstract

Secreted antimicrobial peptides (AMPs) are an important part of the human innate immune system and prevent local and systemic infections by inhibiting bacterial growth in a concentration-dependent manner. In the respiratory tract, the cationic peptide LL-37 is one of the most abundant AMPs and capable of building pore complexes in usually negatively charged bacterial membranes, leading to the destruction of bacteria. However, the adaptation mechanisms of several pathogens to LL-37 are already described and are known to weaken the antimicrobial effect of the AMP, for instance, by repulsion, export or degradation of the peptide. This study examines proteome-wide changes in Streptococcus pneumoniae D39, the leading cause of bacterial pneumonia, in response to physiological concentrations of LL-37 by high-resolution mass spectrometry. Our data indicate that pneumococci may use some of the known adaptation mechanisms to reduce the effect of LL-37 on their physiology, too. Additionally, several proteins seem to be involved in resistance to AMPs which have not been related to this process before, such as the teichoic acid flippase TacF (SPD_1128). Understanding colonization- and infection-relevant adaptations of the pneumococcus to AMPs, especially LL-37, could finally uncover new drug targets to weaken the burden of this widespread pathogen.

Keywords: LL-37; Streptococcus pneumoniae; adaptation; antimicrobial peptide; proteomics.

Figure 1

Established workflow for proteomic investigation of LL-37 stress on pneumococci.

Figure 2

(A) Effect of 2.5 μg/mL LL-37 on the growth of S. pneumoniae D39. The red arrow indicates the application of the stress after two hours of cultivation. Error bars represent the standard deviation. n = 6. (B) Colony-forming units (CFU) per ml OD₆₀₀ = 1 bacterial suspension. Samples were taken every hour after the addition of 2.5 μg/mL LL-37. Means are shown and error bars represent the standard deviation. n = 3.

Figure 3

Graphical representation of proteome coverage and correlation of replicates.

Figure 4

Graphical representation of significantly# affected proteins after LL-37 treatment via Volcano plots. Proteins with an increase in abundance after application of the stress are labeled in red and proteins with a decrease in protein abundance in blue, respectively. # Student’s t-test (p-value= 0.01, min. fold change = 1.5).

Figure 5

Graphical representation of significantly# affected proteins and their function after LL-37 treatment. The arrows indicate an increase or a decrease in protein abundance after application of the stress, respectively. # Student’s t-test (p-value = 0.01, min. fold change = 1.5).

Figure 6

Summary of selected proteomic changes of S. pneumoniae D39 upon LL-37 exposure after comparison of significant# proteome changes with published transcriptome and genome data. The arrows indicate an increase or a decrease in protein abundance after application of the stress, respectively. # Student’s t-test (p-value= 0.01, min. fold change= 1.5).

Figure 7

Effect of 2.5 μg/mL LL-37 on the growth of S. pneumoniae D39 wildtype (wt) in comparison to stressed D39ΔdltD and D39ΔlicD2 mutants. The red arrow indicates the application of the stress after two hours of cultivation. The numbers represent the growth inhibition of stressed pneumococci compared to corresponding untreated D39 strains in percentage. * Significant difference between mutant and wt inhibition using one-tailed t-tests assuming unequal variance, p < 0.05. The error bars represent the standard deviation. n = 3.