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

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.

Polyamines are ubiquitous small cationic molecules that are important for growth and virulence of pneumococcus 14,15 . The most common intracellular polyamines, such as putrescine, spermidine and cadavarine, carry a net positive charge at physiological pH and form electrostatic bonds with negatively charged macromolecules, particularly nucleic acids, to maintain a stable conformation and regulate transcription among other processes 16 . In bacteria, polyamines are implicated in scavenging iron and free radicals, conferring acid resistance, promoting biofilm formation, helping escape from the phagolysosomes, and interactions with various components of cell envelopes 15,17 . Although the effect of polyamines on bacterial virulence has been studied in Neisseria gonorrhoeae, Escherichia coli, Vibrio cholerae, Helicobacter pylori, and Shigella flexneri 15 , the role of polyamines in pneumococcal pneumonia remains largely unexplored. Polyamine transport and synthesis genes are highly conserved among pneumococcal serotypes 18 and are potentially attractive therapeutic targets. In pneumococcus, the polyamine transport operon potABCD consists of a membrane associated cytosolic ATPase (PotA), trans-membrane channel forming proteins (PotB and PotC) and the extracellular polyamine recognition domain (PotD) 15 . Deletion of potD in a type 3 pneumococcus resulted in severe attenuation of virulence in systemic and pulmonary models of pneumococcal disease 19 . In mice, PotD vaccination confers protection against colonization, pneumococcal pneumonia and sepsis 20 . PotD, is a potential next generation vaccine candidate and its efficacy increases in combination with several other pneumococcal protein candidates such as sortase A and glutamyl tRNA synthetase 13,[19][20][21] . Recent studies showed delayed autolysis in a serotype 2 strain in which potD was deleted 22 . Although polyamines are implicated in a wide array of cellular processes, specific mechanistic roles for polyamines are yet to be assigned 17 .
It is known that pneumococci can invade lungs as early as a minute following intranasal challenge 23 . Our earlier findings showed that isogenic deletion of potABCD in TIGR4 (ΔpotABCD), a type 4 strain, led to attenuation in a mouse model of pneumococcal pneumonia 48 h following infection compared to the wild type (WT) strain, and active immunization with recombinant PotD affords protection against systemic infection 18,24,25 . The inability of polyamine transport mutants to survive in the host could be due to altered expression of pneumococcal genes regulated by polyamines, including the expression of virulence factors such as pneumolysin and capsular polysaccharides 18 . Differences in early host innate immune responses against the WT and polyamine transport deficient mutants could also explain the observed attenuation, but remain unknown.
In this study, we compared the early host immune responses to WT and polyamine transport deficient strain, in an intranasal challenge model of pneumococcal pneumonia in mice. Impaired polyamine transport in S. pneumoniae TIGR4 ∆potABCD resulted in its clearance from the lung and blood by 24 h. Concentration of several cytokines/chemokines were higher at 4 h and 12 h in mice infected with ∆potABCD. Consistent with this observation we found a significant increase in the infiltration of neutrophils in the lung. Comparative expression proteomics analysis of mouse lung tissue using 1D LC ESI MS/MS identified differential regulation of proteins involved in neutrophil killing and bacterial clearance such as PTPRC/CD45, PGLYRP1 and Ezrin-Radixin-Moesin, to name a few. In pneumococci, synthesis and transport mechanisms regulate intracellular polyamine concentrations 18,26 . We show upregulation of polyamine biosynthetic genes in vitro, in transport deficient ∆potABCD. We conclude that polyamine transport in the pneumococcus is essential to evade early innate immune responses. A thorough understanding of dedicated polyamine transport in the pneumococcus is an attractive avenue for developing small molecule based intervention strategies that do not depend on host immune status.

Clearance of a polyamine transporter mutant in a mouse model of pneumococcal pneumonia.
S. pneumoniae is a commensal of the nasopharynx and invasive disease requires transition to sterile sites such as lungs and blood. To test if polyamine transport modulates pneumococcal transition and adaption to the lung environment, we infected mice with TIGR4 or polyamine transporter mutant intranasally to produce pneumonia. Lung and blood were aseptically collected in PBS at 4 h, 12 h and 24 h post infection (p.i.). We found significant differences in the bacterial burden at all time points in the lung of TIGR4 and ∆ potABCD infected mice. Enumeration of bacteria from lungs showed a significantly higher number of ∆ potABCD compared to TIGR4 at 4 h (Fig. 1A) post infection (p.i.). In contrast, TIGR4 ultimately persists better in the lower respiratory tract, as there were significantly more WT bacteria at 12 h (Fig. 1B) and 24 h p.i. (Fig. 1C) compared to ∆ potABCD. The mutant strain was cleared by the host immune system by 24 h as demonstrated by the lack of bacteria recovered from lungs. Blood was sterile in all animals infected with WT and ∆ potABCD at 4 h, 12 h and 24 h p.i. suggesting that neither WT nor ∆ potABCD could invade the blood stream within 24 h of infection. Together, these results demonstrate that the deletion of the polyamine transporter in a capsular type 4 strain renders it more invasive yet susceptible to bacterial clearance mechanisms than the parent strain at early stages of infection.

Polyamine transport in S. pneumoniae effects cytokine/chemokine expression in mice.
Pneumococcal invasion of lungs results in complex early immune responses that can be characterized by cytokines and chemokines that induce pro-or anti-inflammatory responses necessary for bacterial clearance. The observed differences in bacteria in the lungs at 4 h and 12 h p.i. could result from differential cytokines/chemokine levels in the lung. To evaluate this, lung homogenates from animals challenged with TIGR4 and ∆ potABCD were evaluated for 32 unique cytokines and chemokines (See methods). Consistent with the observed increase in the bacterial burden, the concentrations of G-CSF, LIF, IP-10, KC, GM-CSF, IL-5, IL-17 and MCP-1 were significantly higher in lungs from mice infected with ∆potABCD at 4 h. p.i. (Table 1). Assessment of cytokine/chemokine levels at 12 h. p.i. showed elevated levels of IL-4, IFN-γ , MIG, GM-CSF, IL-17 and IL-7 relative to WT ( Table 2). Majority of these cytokines and chemokines are associated with neutrophil recruitment response. Increased IL-17 levels correlate with the recruitment and activation of neutrophils and pneumococcal clearance in murine models of pneumococcal colonization 27,28 . Together, the 4 h and 12 h cytokine/chemokine data suggests a model in which ∆ potABCD has decreased resistance to host immune response, potentially by decreased expression of virulence factors that facilitate evasion of early host cytokine responses and their effector functions.

Differences in immune cell infiltration in mice infected with TIGR4 WT and ∆potABCD strains.
We hypothesized that the comparatively higher bacterial burden at 4 h with ∆ potABCD and the observed increase in chemokines and cytokines could lead to significant differences in the recruitment of neutrophils and  macrophages to infected lungs. We tested this hypothesis using flow cytometry and determined changes in the distribution of neutrophils and macrophages in infected mice compared to control mice. Infiltrating non-alveolar macrophages in lung homogenates were quantified by counting cells expressing F4/80 and CD11b (Fig. 2) and neutrophils were quantitated by Gr-1 and CD11b expression (Fig. 3). We identified significantly higher number of neutrophils at 4 h p.i. with ∆ potABCD mutant when compared to TIGR4 (Fig. 3A). No differences were observed at 12 h (Fig. 3B). Unlike neutrophil infiltration, there were no significant differences in the recruitment of infiltrating macrophages to the lung tissue with WT or ∆ potABCD infection at 4 h or 12 h p.i. (Fig. 2A,B). Although we observed significant increase in the levels of MCP-1 in lung homogenates at 4 h (Table 1), a potential indication of macrophage and monocyte recruitment, previous studies have shown that these may not correlate with increased infiltration of macrophages 29 .
Uptake of ∆potABCD by neutrophils does not require antibody opsonization. Increased influx of neutrophils and clearance of ∆ potABCD suggests a role for functioning polyamine transport in the WT for evading host neutrophil response. Increased uptake of ∆ potABCD by neutrophils when compared to the WT could result in enhanced clearance of the polyamine transport deficient strain (Fig. 4). Our results show that there is ~50% increase in the uptake of ∆ potABCD by murine neutrophils compared to WT. This uptake is dependent on phagocytosis and complement activation as addition of cytochalasin D and heat inactivation of serum had an inhibitory effect on bacterial uptake (See supplementary Fig. S1). However, we found that the uptake of ∆ potABCD by neutrophils was independent of antibody opsonization as serotype 4 specific antibody was not required for phagocytosis (See supplementary Fig. S2B). Although WT bacterial numbers were reduced in the presence of neutrophils, opsonization with serotype 4 specific antibody was required for its efficient uptake (see supplementary Figs S1 and S2A). Hence, we conclude that polyamine transport is critical for protecting S. pneumoniae from opsonophagocytosis by the host.  Table 2. Comparison of significantly altered cytokine/chemokine concentrations in lung of C57BL/6 mice infected with TIGR4 WT and ∆potABCD strains12 h p.i. Data shown represents the geometric means (SD) of concentration in pg/ml and represents values from 5 animals in each group with the significance level set at 0.05. One-way ANOVA and Tukey's multiple testing comparison was used to calculate the statistical significance (***p-value = 0.0001; ****p-value = < 0.0001).
Scientific RepoRts | 6:26964 | DOI: 10.1038/srep26964 Polyamine transport is required for invasion of phagocytic and non-phagocytic cells. Our flow cytometry analysis of phagocytic cell infiltration was specific for non-alveolar macrophages. Unlike interstitial macrophages, alveolar macrophages express lower levels of CD11b rendering them CD11b low or negative for flow cytometric analysis 30 . Clearance of pneumococci from the lower respiratory tract also requires functional tissue resident alveolar macrophages 31 . Based on the observed differential uptake of WT and mutant by neutrophils, we asked if a similar difference could be observed in alveolar macrophages. Results from our invasion assays of mouse alveolar macrophages (AMJ2.C8 cells) at a MOI of 1:10 (cell: bacteria) show that ∆potABCD is taken up more efficiently by macrophages (Fig. 5A) compared to WT. Our in vivo analysis showed differences in the transition of WT and ∆potABCD from nasopharynx to lungs. This could be the result of altered invasiveness of the mutant strain in epithelial cells. We tested this in BEAS.2B lung epithelial cells and found that ∆potABCD could invade lung epithelial cells better than WT (Fig. 5B). However, we did not see any differences in the ability of TIGR4 or transport mutant to adhere to either murine alveolar macrophages or lung epithelial cells (Fig. 5C,D respectively). These results suggest the requirement for a functional polyamine transporter for uptake of TIGR4 by phagocytic alveolar macrophages and invasion of the lung epithelial cell barrier.
Proteomic analyses of lung from mice infected with TIGR4 and ∆potABCD. The propensity of pneumococci to establish infection in a healthy host relies, in part, on its inherent capacity to evade innate host defense mechanisms. Our results demonstrate that an intact polyamine transporter is critical for evading  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). recruitment and uptake by host neutrophils. To identify specific changes in the lung proteome in response to TIGR4 and ∆potABCD infection, we conducted mass spectrometry-based expression proteomics 4 h and 12 h p.i.
[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% CO 2 . 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% CO 2 , 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).  (Table S2). Differentially expressed mouse lung proteins in response to WT and ∆potABCD strain were analyzed in the context of molecular function, signaling pathways and biological networks using Ingenuity Pathways analysis (IPA) software. Based on the expression pattern of proteins in a given dataset, IPA causal network analysis 32 predicts either the activation or inhibition of upstream regulators. Causal network analysis with WT and ∆potABCD at 4 h, predicted the activation of IL-6, TNF, MyD88 (activation Z-score ≥ 2.0). Predicted activation of interferon gamma was observed at 4 h and 12 h with ∆potABCD and only at 12 h with WT. At 12 h IL-1β , TNF, MyD88 were predicted to be activated while MAPK12 was inhibited with WT. Based on the observed differences in recovered bacteria, neutrophil infiltration and cytokine concentrations at 4 h and 12 h with WT and ∆potABCD, we expect to see a shift in host innate immune processes. In brief, a delay in early innate immune responses should be observed with WT. Regulator effects algorithm in IPA linked differentially expressed proteins, predicted activators/repressors to known impact/phenotype. Comparison of impact of observed differential expression on host immune responses with regulator effects algorithm identified that host response to ∆potABCD at 4 h (Fig. 6) is comparable to the response to WT at 12 h (Fig. 7).
In vitro analysis of speE and cad gene expression in ∆potABCD. Polyamines regulate a wide array of cellular processes; consequently, their intracellular concentrations are tightly regulated 18 , by balancing extracellular transport with de novo synthesis of intracellular polyamines. Similar to E. coli, pneumococcus has SpeE that converts putrescine to spermidine, and Cad, which catalyzes the conversion of L-lysine to cadaverine. To address if lack of transport is compensated by synthesis, we evaluated the expression of speE and cad genes by qRT-PCR and found that compared to TIGR4, speE and cad levels were up regulated by 14.4 and 17.0 fold respectively in ∆ potABCD (Table 5). Although these changes were found in cells grown in vitro we posit that cellular synthesis of polyamines by pneumococci growing in a host may wholly or partially compensate for diminished extracellular transport.

Discussion
Despite intensive investigation into the natural history and pathogenesis of pneumococcal pneumonia, the ability of the pneumococcus to interact and adapt to the host milieu to establish infection is still confounding. Although much experimental focus has been placed on capsule, components of the cell wall, toxins, sensing systems and membrane transporters critical for adaption to host environments are just starting to gain attention in pneumococcal physiology and virulence 18 . Pneumococci can subvert innate immune responses by several mechanisms such as impeding the recruitment of phagocytic cells, circumventing intake by phagocytic cells, inducing the expression of host proteins that can block antimicrobial properties and inhibit/reduce complement activation and antibody-mediated opsonization 33 . Here we show that significantly reducing polyamine transport by deletion of a polyamine membrane transporter dampens several of these resistance mechanisms. In the present study, we used a polyamine transporter-deficient mutant to identify host innate immune effectors that normally serve to protect against pneumococcal pneumonia.
Our previous findings indicated that deletion of polyamine transport led to attenuation at 48 h 18 , in an intranasal challenge model of pneumococcal pneumonia in mice. In this study, we studied the kinetics of bacterial clearance in the lung in an intranasal challenge model and identified a significant difference in the bacterial load between the WT and ΔpotABCD as early as 4 h p.i. While the WT can persist in the lungs 24 h p.i., the mutant is cleared by host early innate immune responses. The larger number of mutant bacteria at 4 h reflects either a differential suppression of growth by the host or inherent differences in the two bacterial strains to replicate in vivo, early after entering the lower respiratory tract. Also, pneumococcal derived factors could produce a net growth advantage for the mutant bacteria early in the course of infection. The observed increase in the expression of specific cytokines/chemokines suggests that the host innate immune cells are differentially recruited to the sites of infection in pneumococci lacking potABCD. G-CSF coordinates the maturation and release of neutrophils from bone marrow 34 . Binding of KC to CXCR2 is required for releasing neutrophils into the circulation and G-CSF is known to facilitate this release by KC 35 . The up regulation of these neutrophil attractants suggest increased recruitment of neutrophils by polyamine transporter-deficient pneumococci. Although MCP-1 is shown to be involved in the recruitment of neutrophils and bacterial clearance in Gram-negative bacterial infections, studies on serotype 3 strains showed that MCP-1 is not required for recruitment of innate immune cells in pneumococcal pneumonia 36 . At 4 h and 12 h p.i. with ∆potABCD we observed increased IL-17. Levels of IL-17 correlate with the recruitment and activation of neutrophils and pneumococcal clearance in murine models of pneumococcal colonization 27,28 . This finding suggests that intracellular polyamine levels in pneumococci are critical to bacterial defenses against host innate immune responses. Other physiological pathways in the pneumococcus will undoubtedly be shown to have similar roles in bacterial adaption and response to various host environments. The clearance of bacteria in pneumococcal pneumonia is primarily mediated by neutrophils 37,38 . Increased early infiltration of neutrophils into the lungs and clearance of bacteria seen in transporter mutant infected mice suggested neutrophil killing as a potential mechanism of bacterial clearance. S. pneumoniae has evolved mechanisms to escape neutrophil extracellular traps (NETs) by degradation of the DNA scaffold through the endonuclease, endA 39 . Although we did not test neutrophil killing, our opsonophagocytosis assay clearly demonstrates that unlike WT, transporter-deficient mutants are taken up by neutrophils independent of opsonization. We show that transporter mutants are cleared by phagocytosis, as treating the neutrophils with cytochalsin D effectively inhibited the uptake of both WT and transporter mutant. Similarly, heat-inactivated serum reduced uptake of WT and transporter mutants by neutrophils, suggesting complement-mediated killing mechanisms. Our results demonstrate a role for ABC transporters such as PotABCD to alter the bacterial physiology to adapt to different host niches as infection is established. These adaptations can significantly alter the interface of pathogen and host immune responses. In lungs infected with WT pneumococci, we show increased expression of SerpinA1 (alpha-1-antitrypsin precursor), a known inhibitor of neutrophil serine proteases involved in bacterial killing (Table S2). This significant up regulation of a host resistance mechanism is lost with ΔpotABCD, which could in part explain its susceptibility to neutrophil killing. Further, increased expression of Leukotriene A4 hydrolase (Table 3) in ΔpotABCD infected animals could lead to increase in leukotriene mediated neutrophil ROS. This observation suggests that ΔpotABCD is possibly cleared by ROS mediated neutrophil killing mechanisms, which TIGR4 is resistant to 37 .
Proteome analysis of the lung showed significant up regulation of complement component C3 with both WT and mutant at 4 h, with a subsequent reduction at 12 h, which supports our neutrophil uptake data. C3 is critical for clearance of pneumococci 40 . Here we have shown that WT and polyamine transporter-deficient pneumococci both require complement for uptake by neutrophils, although the transport mutant succumbed quickly to phagocytosis (Fig. 4). This suggests that despite the differences in bacterial load, TIGR4 and ∆potABCD elicit similar host responses. However, lack of polyamine transport could alter pneumococcal surface properties rendering it more susceptible to complement activation. This is further substantiated by the fact that the expression of ELANE, a neutrophil elastase, was significantly elevated in both WT and transport mutant at 4 h. Neutrophil elastase is a serine protease required for neutrophil killing 37 . Similarly, MMP9 is crucial for phagocytosis of S. pneumoniae by murine PMNS and intracellular bacterial killing 41 . The expression of MMP9 was comparable in WT and the mutant at 4 h, despite the differences observed in the early immune responses.
The proteome of mouse lungs challenged with ∆potABCD parallels observations with immunoassays. Our proteomic screen revealed unique proteins that were differentially regulated in ∆potABCD at 4 h. We found    . 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). up regulation of PTPRC/CD45 in ∆potABCD relative to WT at 4 h (Table 3). PTPRC/CD45 participates in the clearance of Staphylococcus aureus in animal models 42 . Ptprc −/− mice display impaired neutrophil recruitment, weaker host defense and die prematurely 43 . The observed increase in the levels of PTPRC/CD45 may correlate with increased recruitment of neutrophils and clearance of ∆potABCD by 24 h. Similarly, the expression of PGLYRP1 was up-regulated in ∆potABCD relative to WT at 4 h. PGLYRP1 is an antimicrobial innate immunity protein commonly implicated in allergic asthma. Interestingly, pglyrp1 −/− mice are compromised in the recruitment of neutrophils, lymphocytes, eosinophils and macrophages to lung 44 , thereby substantiating increased recruitment of neutrophils by ∆potABCD at 4 h p.i. Though not detected above baseline in TIGR4 after 4 h, we observed elevated expression of PGLYRP1at 12 h. The majority of differentially regulated proteins unique to mice challenged with ∆potABCD were down regulated at 4 h ( Table 3). For example, periostin (POSTN) is a secreted extracellular matrix protein commonly associated with bronchial hyperresponsiveness and subepithelial fibrosis in asthmatic patients 45,46 and its concentration is thought to correlate with disease severity 45 . Postn −/− mice exhibit reduced inflammation of the airways in response to allergens such as house dust mites 45 . The observation that POSTN expression is significantly reduced in animals challenged with ∆potABCD suggests its requirement in establishing infection by WT TIGR4 in lungs. We found all three components of the Ezrin-Radixin-Moesin (ERM) complex significantly down regulated in ∆potABCD challenged animals 4 h p.i. Ezrin connects apical membranes to the cytoskeleton by crosslinking to actin filaments. Listeria monocytogenes effectively use the ERM complex to link the actin in their tail to membrane cytoskeleton enabling cell-cell spread 47 . Impairing the ERM complex results in inefficient establishment of infection 47 . Consequently, Listeria monocytogenes requires the ERM complex to evade host immune responses. Ezrin and radixin were significantly down regulated by 10-and 5-fold respectively (Table 3) and moesin levels down significantly by 1.7-fold (data not included, as it did not meet the ≥2-fold cut off). Neisseria meningitidis recruit ERM proteins that inhibit the formation of the endothelial docking structures critical for leukocyte diapedesis. When these docking sites with endothelial cells were eliminated by a dominant negative approach, leukocyte diapedesis was inhibited 48 . Taken together, the down regulation of Ezrin, Radixin and Moesin with ∆potABCD suggest a novel mechanism by which pneumococci can escape host innate immunity and spread infection in the lung by regulating the expression of ERM complex. Although ERM complex seems to be critical for TIGR4 to establish infection, the pathogen-directed differential expression of this and other host factors warrants further investigation.
Previous studies showed that Δ speE and Δ cad were severely attenuated in a manner similar to Δ potABCD, in a mouse model of pneumococcal pneumonia 18 . Polyamine levels are stringently controlled by intracellular synthesis and transport mechanisms in pneumococci 18,26 . We found increased expression of speE and cad genes in ΔpotABCD in vitro. We previously reported proteomics of ΔpotABCD, and showed the increased expression of capsular polysaccharide biosynthesis proteins, pneumolysin and pneumococcal surface protein A 18 . The contribution of these proteins to the observed differences in the host innate immune response remains open for investigation.
In summary, deficiencies in polyamine transport cause attenuation of virulence in murine models of pneumonia 18 , and genes involved in polyamine transport appear to be conserved within the species, providing a potential new class of broad-based vaccine candidates or therapeutic targets. However, to leverage this knowledge for vaccine development, a better understanding of how extracellular polyamine transport promotes invasive infection is necessary. This will require an understanding of polyamine dependent expression of pneumococcal genes, and host immune mechanisms in pneumonia. This study focused on host response during infection and identified reduced resistance to neutrophil killing in polyamine transport deficient pneumococci. Comprehensive description of host-pneumococcal interactions responsive to altered polyamine metabolism will be critical for therapies that reduce the global disease burden in public health domain due to this important human pathogen.

Materials and Methods
Bacterial strains and growth conditions. Streptococcus pneumoniae serotype 4 strain TIGR4 was used in this study 49 . All strains were grown in Todd-Hewitt broth supplemented with 0.5% yeast extract (THY) or on 5% sheep blood agar (BA) plates. An isogenic mutant of TIGR4 deficient in the polyamine transport operon potABCD was generated by PCR-ligation mutagenesis as described previously 50 . Briefly, PCR primers were designed to amplify chromosomal DNA 600 nt 5′ to the start codon and 600 nt 3′ from the transcription termination site of potABCD. Erythromycin resistance gene (ermB) was amplified from pJY4163 51 . Genomic pieces were joined by gene splicing by overlap extension (SOEing) PCR 52 using a forward primer of the upstream flanking region and the reverse primer of the downstream flanking region. The recombinant product was transformed into TIGR4 as described previously 53 . Transformants were selected on BA plates with erythromycin (0.5 μ g/ml) by an overnight incubation at 37 °C with 5% CO 2 . The identity of mutant bacterial colonies were confirmed by PCR and sequencing. There was no significant difference between the WT and ∆potABCD growth in THY as reported previously 18 .    . Sequest was set up to match mass spectra and tandem mass spectra (see supplementary information) against a non-redundant mouse protein database appended with S. pneumoniae proteins and common laboratory contaminants. A reversed decoy database was utilized to assess false discovery rate for peptide identification. Scaffold 4 (Proteome Software, Portland, OR) was used to filter Discoverer output. Peptide identifications were accepted if they could be established at greater than 92.0% probability to achieve an FDR less than 0.5% by the Scaffold Local FDR algorithm (see supplementary tables S5B-8B). Protein identifications were accepted if they could be established at greater than 99.0% probability and contained at least 2 identified peptides (see supplementary tables S5A-8A). Significant changes in protein expression between uninfected control versus TIGR4 and uninfected control versus ΔpotABCD at 4 h and 12 h were identified by Fisher's exact test at a p-value of ≤ 0.05 and fold change in protein expression was calculated using weighted normalized spectra with 0.5 imputation value. The PRoteomics IDEntifications (PRIDE) database is a centralized, standards compliant, public data repository for proteomics data. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium 55 via the PRIDE partner repository with the dataset identifier PXD002300 and 10.6019/PXD002300. Significantly altered lung proteins were analyzed using Ingenuity Pathways Analysis (IPA) as was done earlier 56 (see supplementary methods). Based on the distinct up-and down regulation pattern of protein expression, IPA predicted activation of upstream and downstream regulators and utilized the regulator effects algorithm to connect identified upstream regulators with proteins in a dataset and downstream functions to generate a hypothesis with a consistency score. The predicted regulatory networks helped interpret the impact of observed protein expression changes on host response.
Isolation of murine neutrophils. Murine neutrophils were isolated as previously described 37 . In brief, mice were intraperitoneally injected with 1 ml of 10% casein in PBS. A second dose was administered after 24 h and neutrophils were harvested 2 h following the second dose. Murine polymorphonuclear leukocytes (PMNs) were collected by lavage of the peritoneal cavity with Hanks buffer (no Ca ++ and Mg ++ ) supplemented with 0.1% gelatin. Neutrophils were enriched by Ficoll density gradient, washed with 5 ml of Hanks buffer (no Ca ++ and Mg ++ ) with 0.1% gelatin and resuspended in Hanks buffer (Ca ++ and Mg ++ ) and 0.1% gelatin.
In brief, 10 3 bacterial cells (in 10 μ l) were preopsonized with Type 4 specific (Hyp4M3) monoclonal antibody 57 unless mentioned otherwise (gift from Dr. Moon H. Nahm, The University of Alabama, at Birmingham) for 30 min at 37 °C. Neutrophils were then added at 1:10 and 1:100 (bacteria:neutrophils) and incubated for 1 h at 37 °C with rotation. Percent survival was determined relative to control without neutrophils. Cytochalasin D (20 μ M) was used to pretreat neutrophils (30 min at 37 °C), and serum was heated at 56 °C for 30 min to inactivate complement.
Quantitative Real Time PCR. The primers used for quantitative PCR (qRT-PCR) are listed in Table S9.
In brief, total RNA was purified from mid-log phase cultures of TIGR4 and ΔpotABCD grown in THY media using the RNeasy Midi kit and QIAcube (QIAGEN, Valencia, CA). Purified total RNA (7.5 ng/reaction) was transcribed into cDNA and qRT-PCR was performed using the SuperScript ™ III Platinum ® SYBR ® Green One-Step qRT-PCR Kit (Fisher Scientific, Pittsburgh, PA) according to manufacturer's instructions. Relative quantification of gene expression were obtained using the Stratagene Mx3005P qPCR system (Agilent, Santa Clara, CA). Expression of target genes, speE (SP_0918) and cad (SP_0916) were normalized to the expression of gdh (glucose-6-phosphate 1-dehydrogenase, SP_1243) housekeeping gene and fold change determined by the comparative Ct method.
Statistical Analysis. GraphPad Prism (version 5.02 for Windows, GraphPad Software, USA) was used for all statistical analysis performed unless mentioned otherwise. Statistical significance (p-values) were calculated as described in the figure legends.