Extracellular Pneumococcal Serine Proteases Affect Nasopharyngeal Colonization

Abstract

Streptococcus pneumoniae has evolved versatile strategies to colonize the nasopharynx of humans. Colonization is facilitated by direct interactions with host cell receptors or via binding to components of the extracellular matrix. In addition, pneumococci hijack host-derived extracellular proteases such as the serine protease plasmin(ogen) for ECM and mucus degradation as well as colonization. S. pneumoniae expresses strain-dependent up to four serine proteases. In this study, we assessed the role of secreted or cell-bound serine proteases HtrA, PrtA, SFP, and CbpG, in adherence assays and in a mouse colonization model. We hypothesized that the redundancy of serine proteases compensates for the deficiency of a single enzyme. Therefore, double and triple mutants were generated in serotype 19F strain EF3030 and serotype 4 strain TIGR4. Strain EF3030 produces only three serine proteases and lacks the SFP encoding gene. In adherence studies using Detroit-562 epithelial cells, we demonstrated that both TIGR4Δcps and 19F mutants without serine proteases or expressing only CbpG, HtrA, or PrtA have a reduced ability to adhere to Detroit-562 cells. Consistent with these results, we show that the mutants of strain 19F, which preferentially colonizes mice, abrogate nasopharyngeal colonization in CD-1 mice after intranasal infection. The bacterial load in the nasopharynx was monitored for 14 days. Importantly, mutants showed significantly lower bacterial numbers in the nasopharynx two days after infection. Similarly, we detected a significantly reduced pneumococcal colonization on days 3, 7, and 14 post-inoculations. To assess the impact of pneumococcal serine proteases on acute infection, we infected mice intranasally with bioluminescent and invasive TIGR4 or isogenic triple mutants expressing only CbpG, HtrA, PrtA, or SFP. We imaged the acute lung infection in real-time and determined the survival of the mice. The TIGR4lux mutant expressing only PrtA showed a significant attenuation and was less virulent in the acute pneumonia model. In conclusion, our results showed that pneumococcal serine proteases contributed significantly to pneumococcal colonization but played only a minor role in pneumonia and invasive diseases. Because colonization is a prerequisite for invasive diseases and transmission, these enzymes could be promising candidates for the development of antimicrobials to reduce pneumococcal transmission.

Keywords: adherence; colonization; pneumococci; pneumonia; serine proteases.

Figure 1

Schematic models and in silico analysis of pneumococcal serine proteases. High-temperature requirement A belongs to the family of trypsin-like proteases (enzymatic domain shown in yellow). PrtA (pneumococcal protease A) is a cell wall-associated serine protease (enzymatic domain in gray), which belongs to the subtilisin-like proteases with an N-terminal signal peptide (SP) and a C-terminal LPKTG sortase A anchoring motif. Subtilase family protein with the peptidase domain (purple) but lacking an anchoring motif. Choline binding protein G (enzymatic domain orange), which contains in the C-terminal part a short choline-binding module consisting of three CW-repeats.

Figure 2

Growth behavior of serine protease deficient pneumococci. Wild-type and isogenic mutants were cultured at 37°C and 5% CO₂ in THY and chemically defined medium (RPMI_modi). Pneumococcal growth was monitored at OD₆₀₀. The mean of four individual growth experiments is shown for 19F_EF3030 (A, B), TIGR4Δcps (C, D), and TIGR4lux (E, F). Error bars represent SD (n = 4). The symbol “g” indicates the generation time, calculated from four biological replicates. The data were statistically analyzed using a two-way ANOVA analysis *P < 0.05.

Figure 3

Impact of pneumococcal serine proteases on pneumococcal adherence to host epithelial cells. Human Detroit-562 cells were infected for 4 h with an MOI of 50 of S. pneumoniae EF3030, TIGR4Δcps wild-type or mutant strain. (A, B) Colony-forming units were determined post-infection by plating host cell adherent pneumococci on blood agar plates. Results were presented as the mean ± SD for at least four independent experiments performed in triplicates. n.s. *P < 0.05, and **P ≤ 0.01 relative to the parental 19F pneumococcal strain. (C) Immunofluorescence microscopy of pneumococci attached to Detroit-562 cells after 4 h infection. Adherent pneumococci were stained with anti-pneumococcal antiserum followed by secondary Alexa-488 conjugated anti-IgG antibody (green). The epithelial F-actin was stained with Phalloidin-iFlour-594 conjugate (red).

Figure 4

Nasopharyngeal colonization in a murine infection model. Eight to ten-week-old female CD-1 outbred mice (n = 7) were infected intranasally with a CFU of 1 × 10⁷ pneumococci of serotype 19F (EF3030) or isogenic serine protease mutants: 19F_EF3030ΔhtrAΔcbpG (prtA+), 19F_EF3030ΔprtAΔcbpG (htrA+), 19F_EF3030ΔhtrAΔprtA (cbpG+), or 19F_EF3030ΔhtrAΔprtAΔcbpG. Mice were sacrificed at day 2, 3, 7, or 14 post-infections, and pneumococci recovered by a nasopharyngeal (A) or bronchoalveolar lavage (B) and plated on blood agar plates for quantification. Results are shown as scatter plots, where each dot represents one individual mouse. The data were statistically analyzed using a Kruskal-Wallis test *p < 0.05; **p < 0.01 and ***p < 0.001. The dashed line represents the limit of detection.

Figure 5

Acute infections in an experimental pneumonia model. Eight to ten-week-old female CD-1 mice (n = 14) were intranasally infected with S. pneumoniae TIGR4lux wild-type or isogenic triple serine protease mutants using an infection dose of 9×10⁷ CFU. (A, B) The course of infection was monitored in real-time using the IVIS^®-Spectrum in vivo bioimaging system. (C, D) The multiplication and dissemination of bioluminescent pneumococci in infected mice were quantified at indicated time points by measuring the luminescence intensity (photons/second). Data are shown as a Box-Whisker graph showing the values for each mouse. Kruskal-Wallis test was used for statistical analysis. (E, F) Kaplan-Meier survival curves of mice infected with S. pneumoniae TIGR4lux or serine protease deficient mutants. The log-rank (Mantel-Cox) test was used for statistical analysis. *p < 0.05; **p < 0.01 and ***p < 0.001.