Chemokines are small chemotactic cytokines that control the migration and positioning of immune cells during immune system development and in the innate and adaptive immune responses. Notably, chemokines share structural features with the defensin-class of antimicrobial peptides (AMPs), and many types of chemokines show bactericidal activity comparable to AMPs in vitro. There is also evidence suggesting that chemokine bactericidal activity plays roles in innate immune responses. Yet, relatively little is known about the basic mechanisms by which chemokines kill bacteria or the mechanisms by which resistance develops to chemokine killing. The goal of this application is to fill in these major gaps in knowledge by elucidating the molecular mechanisms of chemokine CXCL10 killing and resistance of Streptococcus pneumoniae, which is a major human pathogen that also serves as a highly tractable genetic and cell biological bacterial model. This application is based on a large body of unpublished data showing that S. pneumoniae is sensitive to killing by CXCL10 and related chemokines. Resistance of S. pneumoniae to CXCL10 killing is imparted by amino acid changes in extracellular loop domains of the transmembrane FtsX division protein. FtsX forms a complex with cytoplasmic ATPase FtsE and extracellular peptidoglycan (PG) hydrolase PcsB, and the FtsEX:PcsB complex functions as a regulated PG hydrolase in cell division. The locations of the amino acid changes in the FtsX loop domains are consistent with decreased CXCL10 binding or impaired PcsB activation as mechanisms of CXCL10 resistance. An NMR solution structure of the large FtsX loop domain (ECL1) is near completion and will allow direct testing of these mechanisms. Together, these data support the central hypothesis of this application that CXCL10 binding to FtsX aberrantly activates PcsB PG hydrolase, thereby cleaving cell walls and killing cells. This central hypothesis and alternate hypotheses will be tested by three specific aims.
Aim 1 will identify and characterize new classes of mechanistically informative CXCL10-resistant mutations and determine the mode of CXCL10 killing and sensitivity of S. pneumoniae cells in culture and in a host-relevant model of biofilm formation.
Aim 2 will use NMR methods and biochemical assays to determine the structure of the FtsX loops and their interactions with CXCL10 and whether CXCL10 stimulates PcsB hydrolysis activity.
Aim 3 will use microscopic methods to examine where CXCL10 binds relative to FtsEX:PcsB on pneumococcal cells and will also determine amino acids and regions in CXCL10 required for binding and killing of pneumococcal cells. Results from this application will provide the first detailed study of the physiological and biochemical mechanisms of chemokine killing of S. pneumoniae and the structural basis for CXCL10 resistance by amino acid changes in loop domains of FtsX. Besides filling in major gaps in knowledge about bactericidal chemokines compared to AMPs, this work will shed light on the function and regulation of the FtsEX:PcsB PG hydrolase in cell division and possibly provide a prototype for a new class of antibiotics.

Public Health Relevance

Besides their roles as chemotactic cytokines, chemokines share structural features with antimicrobial peptides and exhibit bactericidal activity to which resistance can sometimes develop. The goals of this project are to determine the molecular and structural mechanisms of chemokine killing and resistance using a model developed for the experimentally tractable pathogenic bacterium, Streptococcus pneumoniae. Results from this project will fill in major knowledge gaps about how chemokines kill bacteria and how resistance develops to bactericidal chemokines, and this work will possibly point the way to an entirely new mechanistic class of antibiotics.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM114315-03
Application #
9211339
Study Section
Bacterial Pathogenesis Study Section (BACP)
Program Officer
Reddy, Michael K
Project Start
2015-04-10
Project End
2019-01-31
Budget Start
2017-02-01
Budget End
2018-01-31
Support Year
3
Fiscal Year
2017
Total Cost
$258,069
Indirect Cost
$84,819
Name
Indiana University Bloomington
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
006046700
City
Bloomington
State
IN
Country
United States
Zip Code
47401
Bruce, Kevin E; Rued, Britta E; Tsui, Ho-Ching Tiffany et al. (2018) The Opp (AmiACDEF) Oligopeptide Transporter Mediates Resistance of Serotype 2 Streptococcus pneumoniae D39 to Killing by Chemokine CXCL10 and Other Antimicrobial Peptides. J Bacteriol 200:
Mura, Andrea; Fadda, Daniela; Perez, Amilcar J et al. (2017) Roles of the Essential Protein FtsA in Cell Growth and Division in Streptococcus pneumoniae. J Bacteriol 199:
Lisher, John P; Tsui, Ho-Ching Tiffany; Ramos-MontaƱez, Smirla et al. (2017) Biological and Chemical Adaptation to Endogenous Hydrogen Peroxide Production in Streptococcus pneumoniae D39. mSphere 2:
Martin, Julia E; Lisher, John P; Winkler, Malcolm E et al. (2017) Perturbation of manganese metabolism disrupts cell division in Streptococcus pneumoniae. Mol Microbiol 104:334-348
Martin, Julia E; Edmonds, Katherine A; Bruce, Kevin E et al. (2017) The zinc efflux activator SczA protects Streptococcus pneumoniae serotype 2 D39 from intracellular zinc toxicity. Mol Microbiol 104:636-651
Rued, Britta E; Zheng, Jiaqi J; Mura, Andrea et al. (2017) Suppression and synthetic-lethal genetic relationships of ?gpsB mutations indicate that GpsB mediates protein phosphorylation and penicillin-binding protein interactions in Streptococcus pneumoniae D39. Mol Microbiol 103:931-957
Sharifzadeh, Shabnam; Boersma, Michael J; Kocaoglu, Ozden et al. (2017) Novel Electrophilic Scaffold for Imaging of Essential Penicillin-Binding Proteins in Streptococcus pneumoniae. ACS Chem Biol 12:2849-2857
Zheng, Jiaqi J; Perez, Amilcar J; Tsui, Ho-Ching Tiffany et al. (2017) Absence of the KhpA and KhpB (JAG/EloR) RNA-binding proteins suppresses the requirement for PBP2b by overproduction of FtsA in Streptococcus pneumoniae D39. Mol Microbiol 106:793-814
Zheng, Jiaqi J; Sinha, Dhriti; Wayne, Kyle J et al. (2016) Physiological Roles of the Dual Phosphate Transporter Systems in Low and High Phosphate Conditions and in Capsule Maintenance of Streptococcus pneumoniae D39. Front Cell Infect Microbiol 6:63
Bajaj, Ruchika; Bruce, Kevin E; Davidson, Amy L et al. (2016) Biochemical characterization of essential cell division proteins FtsX and FtsE that mediate peptidoglycan hydrolysis by PcsB in Streptococcus pneumoniae. Microbiologyopen 5:738-752

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