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Title:Molecular dissection of competence regulated virulence in Streptococcus pneumoniae
Author(s):Lin, Jingjun
Director of Research:Lau, Gee W
Doctoral Committee Chair(s):Lau, Gee W
Doctoral Committee Member(s):Gaskins, H. Rex; Maddox, Carol W; Vanderpool, Cari
Department / Program:Pathobiology
Discipline:VMS - Pathobiology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Streptocococcus pneumoniae
competence
dprA
IVIS
scRNA
Abstract:Streptococcus pneumoniae is an important human pathogen capable of entering the competent state naturally and acquiring DNA from the environment for recombination. In addition to improving genome plasticity, a natural competence system directly contributes to pneumococcal virulence during pneumonia and bacteremia, independent of DNA uptake. Competence is activated when the competence stimulating peptide (CSP), encoded by the comC gene, is processed and exported through the ComAB transporter and stimulates the membrane localized two component system sensor kinase, ComD. Activated ComD autophosphorylates and activates its response regulator ComE, which in turn induces the expression of 24 “early” genes, including the comABCDE, comX1 and comX2 and ComM. Encoded by two identical redundant genes, ComX is an alternative sigma factor which activates the expression of 80 “late” genes, 16 of which are required for DNA uptake and recombination. Some of the proteins encoded by the “late” competence genes, including LytA, CbpD, and CibAB, are capable of attacking and lysing other non-competent pneumococcal cells to release DNA, in a process called allolysis or fratricide. One of these “late” genes, dprA, encodes a dual-functional protein called DNA processing protein A (DprA), which protects incoming single strand DNA for genetic transformation, as well as shutting off the activated competence system. We found that deletion of dprA resulted in prolonged and higher expression of competence genes in vitro in competent pneumococcus, and caused attenuation in mouse models of acute pneumonia and bacteremia. Also, when compared to the parental wild-type serotype 2 strain D39, ∆dprA entered competent state more easily, at lower concentrations of exogenously provided CSP. LytA, CbpD and CibAB were over-expressed in ∆dprA during competence induction in vitro. Deletion of the “combox”, the ComX binding site on the promoters of cbpD and cibAB genes abolished the attenuation ∆dprA genetic background, suggesting overexpression of the allolysis factors during competent state accounted for the virulence attenuation in ∆dprA. Additionally, deletion of the “early” competence gene comM in the ∆dprA genetic background also compensated for the virulence loss, suggesting that overexpression of ComM also contributed to virulence attenuation in ∆dprA. Lastly, because of its inability to turn off the competence regulon, overexpression of all the competence genes consumed excessive nutrients and energy that would otherwise diverted for normal cellular functions, which might also contribute to virulence attenuation. Even though the biology of pneumococcal competence development has been studied for nearly a century, the development of natural competence during pneumococcal diseases have not been examined. We generated a pneumococcal reporter strain using the wild-type serotype 2 strain D39 with the firefly luciferase gene (luc) fused to the promoter of a highly upregulated “late” competence gene ssbB. The resulting reporter strain D39 ssbB-luc was used to track the development of natural competence during pneumonia-derived sepsis (pneumonic sepsis). D39 ssbB-luc cells infecting the lungs of CD-1 mice were able to naturally enter the competent state during pneumonic sepsis. In contrast to the transient short burst of competent state in vitro (~2 hours) in liquid medium, the competent state during pneumonic sepsis was prolonged and sustained, beginning at approximately 20 hours post infection (hpi) and lasted for 12 hours to 40 hours. In mice where the competent state lasted for ~12 hours, the animals quickly progressed from acute pneumonia to sepsis, and the competence signal kept increasing until death. For mice with more prolonged course of the disease, the naturally-developed competent state might wane, but would increase again when the infection entered the sepsis state and lasted for >40 hours until the animal approached endpoint. Exogenously provided CSP had minimal impact on the development of natural competence during pneumonic sepsis. The amount of initial inoculum (1 x 106 – 5 x 107 CFU per mouse), as well as the subsequent bacterial burden in the lung after infection do not seem to be the key factors in determining the timing of natural competence development. DNA processing protein A (DprA), which is responsible for efficient competence shut off in vitro, was highly expressed during pneumonic sepsis, but failed to turn off the competent state in infected animals. Importantly, natural competence development was also detected in both serotypes 3 and 4 clinical pneumococcal strains. Lastly, during pneumonic sepsis, competent D39 bacteria appeared to transfer the competence signal by using a cell-cell contact mechanism rather than the quorum-sensing model based on freely diffusive CSP. Genetic transformation in pneumococcus involves both DNA uptake and homologous recombination. Previously, our lab has identified the small cytoplasmic RNA (scRNA) as essential for transformation. ScRNA is a component of the signal recognition particle (SRP) that forms a complex with both Ffh and FtsY, and together, delivers nascent peptide to the Sec translocon for membrane targeting or secretion. By marker-less deletion, we found that the first nucleotides 6-49 of the scRNA gene, which constitutes part of the helix IV structure, was required for genetic transformation. Considerable efforts were devoted for the deletion and analysis of both ffh and ftsY genes, because ∆ffh and ∆ftsY were unable to grow on commonly used Todd-Hewitt broth unless supplemented with free amino acids. Similar to ∆scRNA, deletion of ffh and ftsY abolished genetic transformation. Deletion of the insertase gene yidC2, which functional overlaps with SRP, did not affect transformation. Proteomic analysis revealed that three key proteins CglA, CglB, and CglC, which are required for the assembly of the indispensable a Type IV pilus-like DNA uptake apparatus, were found in reduced levels on the membrane of ∆scRNA than D39. Collectively, these results suggest that reduced efficiency in targeting and/or assembling DNA uptake apparatus impairs the genetic transformation in ∆scRNA.
Issue Date:2019-07-10
Type:Text
URI:http://hdl.handle.net/2142/105926
Rights Information:Copyright 2019 Jingjun Lin
Date Available in IDEALS:2019-11-26
Date Deposited:2019-08


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