Polyamine transporter in Streptococcus pneumoniae is essential for evading early innate immune responses in pneumococcal pneumonia

Abstract

Streptococcus pneumoniae is the most common bacterial etiology of pneumococcal pneumonia in adults worldwide. Genomic plasticity, antibiotic resistance and extreme capsular antigenic variation complicates the design of effective therapeutic strategies. Polyamines are ubiquitous small cationic molecules necessary for full expression of pneumococcal virulence. Polyamine transport system is an attractive therapeutic target as it is highly conserved across pneumococcal serotypes. In this study, we compared an isogenic deletion strain of S. pneumoniae TIGR4 in polyamine transport operon (ΔpotABCD) with the wild type in a mouse model of pneumococcal pneumonia. Our results show that the wild type persists in mouse lung 24 h post infection while the mutant strain is cleared by host defense mechanisms. We show that intact potABCD is required for survival in the host by providing resistance to neutrophil killing. Comparative proteomics analysis of murine lungs infected with wild type and ΔpotABCD pneumococci identified expression of proteins that could confer protection to wild type strain and help establish infection. We identified ERM complex, PGLYRP1, PTPRC/CD45 and POSTN as new players in the pathogenesis of pneumococcal pneumonia. Additionally, we found that deficiency of polyamine transport leads to up regulation of the polyamine synthesis genes speE and cad in vitro.

Figure 1. Transition of S. pneumoniae TIGR4 deficient in polyamine transport operon (∆potABCD) from nasopharynx to lungs.

Recovery of S. pneumoniae TIGR4 or ∆potABCD (CFU/ml) from lung homogenates from C57BL/6 mice (n = 5) infected i.n. with 10⁷ CFU bacteria at [A] 4 h, [B] 12 h and [C] 24 h. Data are represented as mean ± SEM. Each datum point represents results from one animal. Mann-Whitney test was used to calculate statisical significance (**p-value = 0.008).

Figure 2. Evaluation of macrophage infiltration in lungs of C57BL/6 mice in response to infection with S. pneumoniae TIGR4 and ∆potABCD.

Expression of F4/80 and CD11b double positive cells from lung homogenates of C57BL/6 mice (n = 3) is shown. Data is expressed as percentage of F4/80, CD11b double positive cells compared to PBS at [A] 4 h and [B]12 h. Mice were challenged with 10⁷ S. pneumoniae TIGR4, ∆potABCD or PBS. Each datum point represents results from one animal. Data are represented as mean ± SEM. One-way ANOVA and Tukey’s multiple testing comparison was used to calculate the statistical significance (***p-value = 0.0001; ****p-value = <0.0001).

Figure 3. Evaluation of neutrophil infiltration in lungs of C57BL/6 mice in response to infection with S. pneumoniae TIGR4 and ∆potABCD.

Expression of Gr1 and CD11b double positive cells from lung homogenates of C57BL/6 mice (n = 3). Data is expressed as percentage of Gr1, CD11b double positive cells compared to PBS at [A] 4 h and [B]12 h. Mice were challenged with 10⁷ S. pneumoniae TIGR4, ∆potABCD or PBS. Each datum point represents results from one animal. Data are represented as mean ± SEM. One-way ANOVA and Tukey’s multiple testing comparison was used to calculate the statistical significance (**p-value = 0.003; ***p-value = 0.0001; ****p-value = <0.0001).

Figure 4. Opsonophagocytosis of S. pneumoniae TIGR4 and ΔpotABCD at two different bacteria: neutrophil ratios (1:10 and 1:100).

1 hr control represent the reaction mixture with neutrophils with no bacteria and T4 represents TIGR4. Data are represented as mean ± SEM. Two-way ANOVA and Sidak’s multiple comparison test were used to calculate the statistical significance (**p-value = 0.003; ****p-value = <0.0001).

Figure 5

[A] Deletion of polyamine transporter enhances the uptake of S. pneumoniae TIGR4 by murine alveolar macrophage (AMJ2.C8) cells. Cells were cultured in appropriate medium to 90% confluency, and were infected with TIGR4 or ΔpotABCD at MOI of 1:10 for 2 h, followed by 1 h incubation with penicillin and gentamicin at 37 °C in 5% CO₂. Cells were lysed in 0.0125% Triton X-100 for CFU enumeration. [B] Deletion of polyamine transporter enhances the invasion of S. pneumoniae TIGR4 in human BEAS.2B lung epithelial cells. Cells were cultured in appropriate medium to 90% confluency, and were infected with TIGR4 or ΔpotABCD at MOI of 1:10 for 2 h, followed by 1 h incubation with penicillin and gentamicin at 37 °C in 5% CO₂. Cells were lysed in 0.0125% Triton X-100 for CFU enumeration. [C,D] Deletion of polyamine transporter does not affect the ability of S. pneumoniae. TIGR4 to adhere to murine AMJ2.C8 [C] and human BEAS.2B [D] lung epithelial cells. Cells were cultured as mentioned in 5A, infected with TIGR4 or ΔpotABCD at MOI of 1:10 for 2 h at 37 °C in 5% CO₂, rinsed 3X in sterile PBS, and CFU enumerated. All assays were performed at least three times with two or more technical replicates. One-way ANOVA and Tukey’s multiple comparison tests were used to calculate the p-values. (****p-value = <0.0001, ns = p-value >0.05).

Figure 6. Predicted regulatory network effects identified from differential protein expression profile in lungs of C57BL/6 mice in response S. pneumoniae ∆potABCD 4 h p.i.

Based on significant up- (shown in pink and red) down- (shown in green) regulation of lung proteins (represented by diamond shape), Ingenuity pathways analysis (IPA) predicted activation of upstream regulators such as MYD88, PRKCA and TNFSF12 (shown in orange). IPA’s regulator effects algorithm connected upstream regulators, proteins in our dataset to downstream functions to generate regulator effects hypotheses with a consistency score. The predicted top regulatory network (15.7 consistency score) in response to ∆potABCD impacts inflammatory response, increase in quantity and movement of phagocytes which is consistent with the observed increase in neutrophils identified by FACS at 4 h p.i (Fig. 3).

Figure 7. Predicted regulatory network effects identified from differential protein expression profile in lungs of C57BL/6 mice in response S. pneumoniae TIGR4 12 h p.i.

Based on significant up- (shown in pink and red) down- (shown in green) regulation of lung proteins (represented by diamond shape), Ingenuity pathways analysis (IPA) predicted activation of upstream regulators such as EZH2, MYD88, IL-1β and PRDMI (shown in orange) and inhibition of INSIG1, MKL1 and 2 (shown in blue). IPA’s regulator effects algorithm connected the upstream regulators, proteins in our dataset to downstream functions to generate regulator effects hypotheses with a consistency score. The predicted top regulatory network (13.8 consistency score) in response to TIGR4 will impact chemotaxis of granulocytes, phagocytes and activation of neutrophils consistent with the establishment of infection, a delayed response when compared to ∆potABCD 4 h (Fig. 6).