Molecular investigation of competence development by streptococcus pneumoniae during in vivo infection

Oh, Myung Whan

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https://hdl.handle.net/2142/124635 Copy

Description

Title

Molecular investigation of competence development by streptococcus pneumoniae during in vivo infection

Author(s)

Oh, Myung Whan

Issue Date

2024-03-28

Director of Research (if dissertation) or Advisor (if thesis)

Lau, Gee W

Doctoral Committee Chair(s)

Lau, Gee W

Committee Member(s)

• Weiping, Zhang
• Vanderpool, Carin
• Alam, Tauqeer

Department of Study

Pathobiology

Discipline

VMS - Pathobiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• streptococcus pneumoniae
• competence system
• competence regulon
• virulence
• homologous recombination
• pneumonia

Abstract

Streptococcus pneumoniae (pneumococcus) commonly inhabits the human nasopharyngeal tract as a commensal, but causes variety of serious opportunistic infections, ranging from mild respiratory disease to acute otitis media, invasive bacteremia, pneumonia-derived sepsis, and meningitis. S. pneumoniae is able to naturally enter a competent state upon colonization of nasopharyngeal space or during acute pneumonia and pneumonia-derived sepsis, during which the pathogen actively acquires novel genes for recombination resulting in genetic polymorphism. Polymorphism in certain genes, especially that are exposed to strong selective pressures, allow the pathogen to evolve and circumvent the modern preventive strategy including the pneumococcal conjugate vaccines, or antibiotic treatments. The pneumococcal competence regulon also serves as a major driver of virulence and disease pathogenesis wherein a wide range of virulence factors such as choline-binding proteins, bacteriocins, adhesins are expressed in a competent-state-specific manner. During the competence-dependent autolysis, also termed fratricide/allolysis, major virulence factors such as lipoteichoic acid (LTA)and cholesterol-dependent cytolysin pneumolysin (PLY) are also released. The pneumococcal regulon regulates the expression of four distinct subcategories of genes: “early”, “late”, “delayed” and “repressed” competence (com) genes. Competence develops in response to the accumulation of the competence-stimulating peptide (CSP) pheromone in the extracellular matrix. The comC gene encodes for the pre-CSP which is processed into 17 amino acid mature peptide and exported by ATP-binding cassette transporter ComAB. When the threshold concentration is achieved, CSP activates the two-component response regulator ComDE. The activated histidine kinase ComD phosphorylates the cognate response regulator ComE. The ComE~P dimers bind to promoters (ComE-box) of the early com genes and establish the positive transcriptional feedback loop of comABCDE, which drives the expression of CSP responsive genes. Beside comABCDE, comW, comM and comX are transcribed as early com genes. The identical comX1 and comX2 genes redundantly encode for alternative sigma factor ComX which is responsible for the transcription of “late” com genes that comprise of more than 80 genes. ComX binds to the “combox” containing promoters. The genes encoding for fratricins (or allolytic factors), such as lytA, lytC, cbpD, and cibAB are also ComX-regulated “late” com genes. During competent state, competent pneumococci release LytA, LytC, CbpD, and CibAB fratricins which attack and cause the lysis of the non-competent sister cells. This seemingly self-predatory event promotes the release of nutrients and genomic DNA (gDNA) presumable for genetic transformation. Important late com genes for genetic transformation include cglABCDEFG (also known as comGA, -GB, -GC, -GD, -GE, -GF, -GG) genes encoding the type IV pili that actively transports the donor DNA across the cell membrane for homologous recombination. The translocation apparatus is assembled by the double stranded DNA (dsDNA) receptor protein ComEA, an endonuclease EndA, a transmembrane channel for single stranded DNA (ssDNA) ComEC and ATP-binding protein ComFA. The DprA, encoded by the dprA gene, protects the incoming ssDNA and loads the RecA recombinase onto the ssDNA, after which the incoming ssDNA is incorporated into the genome via homologous recombination. The termination of competent state is facilitated by multiple layers of control. Dimerization of DprA with ComE~P promotes the dissociation of ComE~P from ComE-box of early com genes promoter, indicating the dual-role of DprA during competence. Competence state is also shut off by the competition for binding for ComW stabilizing factor and RNA polymerase between ComX and RpoD sigma-factors. The CiaRH two-component signal transduction system (TCSTS) of the delayed com genes also negatively regulate competence induction through HtrA-mediated competence inhibition via digestion of extracellular CSP. Despite the accumulating knowledge in pneumococcal competence development in vitro, natural induction of pneumococcal competence during infection remains largely unknown. The in vitro pneumococcal competence induction manifests a short burst of transient state in an early log phase and requires artificial stimulation by synthetic CSP to activate the comABCDE positive feedback loop. However, during host infection (e.g., acute pneumonia), not only the competence induction is spontaneous but the exogenously provided CSP has limiting effects in competence induction. Further, previous studies indicate neither the initial bacterial inoculum dose (106 -108 colony forming units) nor the bacterial loads in lungs during infection have appreciable impact in the kinetics of competence induction during pneumonia-derived sepsis model in mice. These findings indicate that in vivo pneumococcal competence induction may be limited by various unknown host factors that strongly influence the development of the competent state. We created a pneumococcal reporter strain by incorporating the firefly luciferase gene (luc) into the promoter of the highly activated late com gene ssbB, using the wild-type serotype 2 strain D39. This reporter strain, named D39-ssbB-luc, was employed to monitor natural competence at both protein- and transcript-level during pneumonic sepsis in mice by using an IVIS SpectrumCT imaging system and RNA-sequencing (RNA-seq), respectively. Transcriptomic analysis of murine lungs at 0-, 12-, 24- and >40-hours post infection (hpi) after intranasal challenge revealed upregulation of early, late and delayed com genes as early as 12-hpi, indicating the association of competence regulon to adaptation in the lungs. Furthermore, genes of the histidine triad (pht) gene family experienced a significant upregulation at 12-hpi following steep decline during the remaining infection cycle, indicating that Pht proteins are involved in the early adaptation within the lung microcompartment. Further analyses demonstrated that Pht proteins play a role in competence regulation through divalent metal homeostasis. A total of 19 genes, organized into 9 operons, have been characterized as delayed com genes based on the observation of gene expression peaking after the preceding waves of early- and late-com genes in vitro. Four of these operons encode chaperones or proteases, along with dnaK heat-shock operon, and have led to the speculation that delayed com genes likely function in pneumoccocal stress response. Notable members of the delayed com genes encoding the two-component regulatory system CiaRH (SP0798 and SP0799), previously reported as key participant in competence regulation and virulence. Further, the virulence role of ClpL protease (SP0338) and HtrA serine protease (SP2239) are well-documented. By gene deletion, we reveal that the GntR (SP1714) transcriptional regulator and multidrug ABC transporter (SP1715) confer virulence in a pneumonia-derived sepsis model. Transcriptomic profiling by RNA-seq revealed upregulations of htrA, parB, spd_1524 (gntR), spd_1525, spd_0913, and spd_1213 at 12-hpi during pneumonia-derived sepsis in murine lungs at which competence genes are actively transcribed. The ciaRH genes were only moderately upregulated until 24-hpi followed by downregulation onward. Additionally, the ∆spd_1524 (gntR) mutant demonstrated attenuated competence induction in vitro. The ∆spd_1524 (gntR) mutant also exhibited attenuated adherence to A549 cells and susceptibility to various stresses including elevated pH, osmotic and metal toxicity, and oxidative stress. Interestingly, the hypothetical protein SP1027 (SPD_0913) was revealed as a putative extracellular protein that contributes to virulence with a role in cell adherence. Collectively, these results suggest the role of GntR in competence induction, and characterizes the previously unknown SP1027 as the putative virulence factor that contributes to cell adherence.

Graduation Semester

2024-05

Type of Resource

Thesis

Copyright and License Information

Copyright 2022 American Chemical Society, Copyright 2024 Oh et al.

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