Genetic dissection of desiccation tolerance, carbon catabolite repression, and protein vaccine candidates in Streptococcus pneumoniae
Matthews, Allison.
2021
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Thesis
(Ph.D.)--Tufts University, 2021.
Submitted to the Dept. of Molecular Microbiology.
Advisor: Andrew Camilli.
Committee: Wai-Leung Ng, Joan Mecsas, and Aimee Shen.
Keyword: Microbiology.
Streptococcus pneumoniae (the pneumococcus) is a Gram-positive bacterium that asymptomatically colonizes the nasopharynx. Dissemination to the lungs, ... read morebloodstream or central nervous system cause serious, often deadly infections worldwide. As colonization is considered a prerequisite for invasive disease, better understanding of transmission to susceptible hosts and subsequent growth on host-derived carbohydrates in the upper airway may inform the development of therapeutics or other control measures to inhibit this part of the lifecycle of S. pneumoniae. In addition, vaccination is an effective intervention in this pathogen's lifecycle which can prevent either the initial colonization event or progression to invasive disease. Investigation of these areas of pneumococcal biology and prevention is presented here as three independent manuscripts. Transmission of S. pneumoniae has classically been thought to occur through inhalation of respiratory droplets and direct contact with nasal secretions. However, recent work has demonstrated that S. pneumoniae is desiccation tolerant, which opens up the possibility that this pathogen is also transmitted via contaminated surfaces (fomites). To better understand the molecular mechanisms that enable S. pneumoniae to survive desiccation, we performed a high throughput mutant screen in search of genetic determinants of desiccation tolerance. We found that the nucleotide excision repair pathway, as well as other DNA repair pathways, are important for surviving desiccation. Deletion of the nucleotide excision repair gene uvrA resulted in decreased transmission efficiency between infant mice, suggesting a correlation between desiccation tolerance and pneumococcal transmission. Overall, we found that maintenance of genome integrity is key to surviving desiccation and may have important implications for fomite transmission of this pathogen. Once S. pneumoniae transmits to a susceptible host, it relies on scavenged carbohydrates for energy. In order to maximize metabolic efficiency in an environment with multiple carbohydrates, bacteria preferentially use readily metabolizable ones first until they are exhausted and then the less preferred ones, in a process called carbon catabolite repression (CCR). In S. pneumoniae CCR has been observed for a number of non-preferred carbohydrates. Although both global and specific regulatory mechanisms are known to mediate CCR in S. pneumoniae, knowledge of the components involved in incomplete. Here we investigate the impact of two previously identified small proteins, SP_0451 and SP_1473, on CCR. We used growth curves on non-preferred carbohydrates to characterize where these proteins fit into the metabolic regulatory network of S. pneumoniae. In addition, we created a functional epitope-tagged SP_1473 on the chromosome which can be used for future biochemical characterization of interacting partners of this regulatory protein. Characterization of metabolism will lead to a better understanding of how S. pneumoniae successfully grows in various niches in its human host. Due to increased incidence of invasive disease caused by serotypes that are not included in available capsular polysaccharide-based vaccines, there is a need for a broadly protective protein-based vaccine to complement the polysaccharide based ones. To limit the occurence of vaccine-escape mutants, ideally the target antigen should be essential for colonization and/or virulence. Although this is the case for the capsular polysaccharide, most or all surface proteins of S. pneumoniae are dispensable. To overcome this limitation, we performed a genetic interaction screen to identify functionally redundant surface protein pairs that could be used as bivalent protein vaccines, based on the observation that together, these protein pairs are essential for virulence. We identified four pairs of functionally redundant surface proteins that displayed a significant competitive disadvantage during murine pneumococcal pneumonia. Immunization with the most attenuated pair, CbpC and CbpJ, resulted in production of high titers of specific antibodies and increased median survival times of mice challenged with pneumococcal pneumonia. Thus, we have demonstrated a method to identify essential pairs of surface-associated virulence proteins that could be widely applied to many bacterial pathogens. Together these works improve our understanding of S. pneumoniae transmission and growth on host-derived carbohydrates and advance efforts towards developing an effective protein-based vaccine, all with the hope of developing interventions that will limit S. pneumoniae spread and the incidence of invasive disease.read less - ID:
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