C-reactive protein (CRP) is a component of innate immunity. The concentration of CRP in serum increases in microbial infections including Streptococcus pneumoniae infection. Employing a mouse model of pneumococcal infection, it has been shown that passively administered human wild-type CRP protects mice against infection, provided that CRP is injected into mice within two hours of administering pneumococci. Engineered CRP (E-CRP) molecules have been reported recently; unlike wild-type CRP, passively administered E-CRP protected mice against infection even when E-CRP was injected into mice after twelve hours of administering pneumococci. The current study was aimed at comparing the protective capacity of E-CRP with that of an antibiotic clarithromycin. We established a mouse model of pneumococcal infection in which both E-CRP and clarithromycin, when used alone, provided minimal but equal protection against infection. In this model, the combination of E-CRP and clarithromycin drastically reduced bacteremia and increased survival of mice when compared to the protective effects of either E-CRP or clarithromycin alone. E-CRP was more effective in reducing bacteremia in mice treated with clarithromycin than in untreated mice. Also, there was 90% reduction in antibiotic dosing by including E-CRP in the antibiotic-treatment for maximal protection of infected mice. These findings provide an example of cooperation between the innate immune system and molecules that prevent multiplication of bacteria, and that should be exploited to develop novel combination therapies for infections against multidrug-resistant pneumococci. The reduction in antibiotic dosing by including E-CRP in the combination therapy might also resolve the problem of developing antibiotic resistance.

There is a rise in prevalence of antibiotic resistance in Streptococcus pneumoniae, and its FDA-approved vaccines often do not mount effective immune responses in children, the elderly, or the immunocompromised. One reason these vaccines are generally less effective is because they do not utilize T-cell help. T-cell help can be accessed when di-, tri-, or tetra-saccharides positioned inside major histocompatibility complex (MHC) II are presented to T-cell receptors as a target antigen. Pairing MHC II-antigen complexes with T-cell receptors enables development of B and T lymphocytes that are highly specific to these antigens, granting an increase in antibody affinity and cell memory. One problem with today's vaccines against S. pneumoniae, in contrast, is that extracted, polymeric sugars cannot be presented to T-cells by MHC because they do not fit inside the MHC II complex due to their large molecular size. Thus, FDA-approved vaccines generate antibodies which have inadequate affinities and are largely nonspecific in their targets. This thesis covers the synthesis of a functionalized S. pneumoniae serotype 6A disaccharide and trisaccharide, which are core components of the repeating unit of natural capsular polysaccharides, and can be used to obtain necessary T-cell help in working vaccines and good monoclonal antibodies.

C-reactive protein (CRP) binds to several species of bacterial pathogens including Streptococcus pneumoniae. Experiments in mice have revealed that one of the functions of CRP is to protect against pneumococcal infection by binding to pneumococci and activating the complement system. For protection, however, CRP must be injected into mice within a few hours of administering pneumococci, that is, CRP is protective against early-stage infection but not against late-stage infection. It is assumed that CRP cannot protect if pneumococci got time to recruit complement inhibitor factor H on their surface to become complement attack-resistant. Since the conformation of CRP is altered under inflammatory conditions and altered CRP binds to immobilized factor H also, we hypothesized that in order to protect against late-stage infection, CRP needed to change its structure and that was not happening in mice. Accordingly, we engineered CRP molecules (E-CRP) which bind to factor H on pneumococci but do not bind to factor H on any host cell in the blood. We found that E-CRP, in cooperation with wild-type CRP, was protective regardless of the timing of administering E-CRP into mice. We conclude that CRP acts via two different conformations to execute its anti-pneumococcal function and a model for the mechanism of action of CRP is proposed. These results suggest that pre-modified CRP, such as E-CRP, is therapeutically beneficial to decrease bacteremia in pneumococcal infection. Our findings may also have implications for infections with antibiotic-resistant pneumococcal strains and for infections with other bacterial species that use host proteins to evade complement-mediated killing.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. One host defense function of C-reactive protein (CRP) is to protect against Streptococcus pneumoniae infection as shown by experiments employing murine models of pneumococcal infection. The protective effect of CRP is due to reduction in bacteremia. There is a distinct relationship between the structure of CRP and its anti-pneumococcal function. CRP is functional in both native and non-native pentameric structural conformations. In the native conformation, CRP binds to pneumococci through the phosphocholine molecules present on the C-polysaccharide of the pneumococcus and the anti-pneumococcal function probably involves the known ability of ligand-complexed CRP to activate the complement system. In the native structure-function relationship, CRP is protective only when given to mice within a few hours of the administration of pneumococci. The non-native pentameric conformation of CRP is created when CRP is exposed to conditions mimicking inflammatory microenvironments, such as acidic pH and redox conditions. In the non-native conformation, CRP binds to immobilized complement inhibitor factor H in addition to being able to bind to phosphocholine. Recent data using CRP mutants suggest that the factor H-binding function of non-native CRP is beneficial: in the non-native structure-function relationship, CRP can be given to mice any time after the administration of pneumococci irrespective of whether the pneumococci became complement-resistant or not. In conclusion, while native CRP is protective only against early stage infection, non-native CRP is protective against both early stage and late stage infections. Because non-native CRP displays phosphocholine-independent anti-pneumococcal activity, it is quite possible that CRP functions as a general anti-bacterial molecule.

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This dissertation is a compilation of published work and a manuscript that seeks to understand the role of polyamine metabolism in the regulation of pneumococcal physiology. Streptococcus pneumoniae (pneumococcus) is the major cause of community-acquired pneumonia, and otitis media worldwide. Genetic diversity and serotype replacement, and antibiotics resistance to confound existing therapeutic strategies and limit the effectiveness of the available capsule polysaccharide (CPS) based vaccines. Polyamines such as putrescine, spermidine and cadaverine are ubiquitous polycationic hydrocarbons that interact with negatively charged molecules and modulate important cellular processes. Intracellular polyamine concentrations are regulated by biosynthesis, degradation, and transport. This work investigated the impact of the deletion of polyamine biosynthesis gene, SP_0916 (cadA, lysine/arginine decarboxylase covered in the second, third and fourth chapters), on growth, Gram staining characteristics, capsule production, proteome and stress responses of virulent pneumococcal serotype 4 (TIGR4). We identified loss of capsular polysaccharide (CPS) in DELTA SP_0916 strain. Our proteome results showed a shift in metabolism towards the pentose phosphate pathway (PPP) that could reduce the availability of precursors for CPS and could explain the un-encapsulated phenotype of DELTA SP_0916. Since a shift towards the PPP is usually in response to stress, we compared the stress responses of DELTA SP_0916 to that of TIGR4. Our results show that the mutant was more susceptible to oxidative, nitrosative, and acid stress compared to the wild type. In the fifth chapter we compared the transcriptome, metabolome, stress responses and stress susceptibility of the polyamine transport deficient strain (DELTA potABCD) and S. pneumoniae TIGR4. Results in this chapter show that polyamine transport is essential for pneumococcal stress responses, and capsule biosynthesis. The impact of impaired polyamine synthesis (DELTA SP_0916), and transport (DELTA potABCD) on pneumococcal capsule is due to altered expression of Leloir pathway, reduced glycolysis, and increased PPP, possibly in response to impaired stress responses. These results demonstrate that alteration of polyamine pathways affects pneumococcal stress responses which in turn could limit the availability of precursors for capsule synthesis, and thus have an impact on virulence. Thus, polyamine metabolism is an attractive avenue for developing novel interventions for limiting the spread of S. pneumoniae, a versatile human pathogen.

C-reactive protein (CRP) binds to Streptococcus pneumoniae through the phosphocholine groups present in the cell wall and subsequently activates the complement system to kill the pathogen. To escape the attack of complement, pneumococci recruit a complement inhibitory protein, factor H, on their surface. It has been shown that CRP protects mice against pneumococcal infection only when injected within 2 hours after administering pneumococci. We hypothesized that CRP is not protective when injected at later times because, by then, factor H is recruited by pneumococci. In the current study, we evaluated the protective effects of an engineered CRP molecule (E-CRP) which does not bind to factor H in fluid phase but binds to factor H-coated pneumococci. We found that E-CRP, unlike native CRP, protected mice regardless of the timing of administering E-CRP by drastically reducing bacteremia and increasing survival of mice. Next, we established another murine model of pneumococcal infection using the antibiotic clarithromycin. We found that the combination of E-CRP and clarithromycin was more protective against infection when compared to the protective effects of either E-CRP alone or clarithromycin alone. These findings suggest that the structure of native CRP has to be altered to display its full anti-pneumococcal activity and that CRP and antibiotic act synergistically to protect against pneumococcal infection by decreasing bacteremia. These data also have implications for infections with other bacterial species that use factor H to evade the attack of complement. Additionally, the administration of E-CRP may be therapeutically beneficial to treat infections with antibiotic-resistant bacteria.

Background: The p53-up-regulated modulator of apoptosis (PUMA) protein is a pro-apoptotic, BH3-only member of the BCL2 family of effector proteins responsible for promoting organized cell death. PUMA is required for resolution of pneumococcal pneumonia in mice, as mice deficient of PUMA exhibit greater numbers of S. pneumoniae CFU within tissues and higher mortality rates than observed in Puma+/+ mice. Methods: Puma+/+ and Puma-/- mice were intranasally challenged with TIGR4 pneumococcus and sacrificed 24 h post-infection. Differences in cytokine levels from blood and whole lung tissue were detected by MILLIPLEX MAP Mouse Cytokine/Chemokine Magnetic Bead Panel. Lung transcriptomes from Puma+/+ and Puma-/- mice were prepared from total lung RNA using NEBNext Poly(A) mRNA Magnetic Isolation Module and NEBNext Ultra RNA Library Prep Kit for Illumina. Libraries were read by Illumina NovaSeq and transcript reads were referenced to Mus musculus. Results: Puma-/- mice exhibited significant differences in G-CSF, GM-CSF, IFN-gamma, IL-1-alpha and -beta, -6, -9, -10, -12 (p40 and p70), -13, and -17, IP-10, KC, MCP-1, MIP- iv 1alpha and -beta, MIP-2, RANTES, and TNF-alpha compared to Puma+/+ mice. Puma-/- lungs exhibited higher levels of IL-12, IFN-gamma, and IP-10. Loss of PUMA also resulted in expression of the pro-angiogenic genes Adam19 and Neurexin2. Additionally, Puma+/+ and Puma-/- mice displayed similar levels of colonization, but Puma-/- mice were more susceptible to subsequent dissemination to the lungs and blood. Conclusion: Polymorphonuclear cells (PMNs) were previously demonstrated to be one of the innate cell types responsible for Puma-dependent resolution of pneumococcal pneumonia in mice. Observations reported here suggest that this resolution is propelled by suppressing the inflammatory response via the inhibition of IL-12/IFN-gamma/IP-10 pro-inflammatory axis. Pulmonary tissue transcriptomic analysis also suggests PUMA-dependent positive regulation of homeostatic control of pulmonary vasculature, smooth muscle innervation, and maintenance of the interstitium. Gene ontological analysis further demonstrated Puma's modulatory role in Type I and II IFN signaling. For the first time, we report Puma's regulatory effects on pro-inflammatory cytokine signaling and gene expression during pneumococcal pneumonia.

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