# Rickettsia cell envelope glycoconjugates are derived from the host cell amino sugar biosynthesis pathway

> **NIH NIH R21** · UNIVERSITY OF MARYLAND BALTIMORE · 2020 · $231,750

## Abstract

Project Summary
The global impact of rickettsial diseases is highlighted by historical records, reemergence of fatal arthropod-borne
spotted and typhus fever rickettsioses, and emergence of new pathogens. The intracellular lifestyle of Rickettsia spp.
poses immense challenges to research; nevertheless, a transdisciplinary approach alleviates the near intractability of
these bacteria to genetic manipulation. This application employs such an approach, targeting one of the most
understudied aspects of rickettsial virulence, host-dependent metabolic parasitism. Our recent reconstruction of the
Rickettsia metabolic and transport network identified 51 host-acquired metabolites needed to compensate for
degraded biosynthesis pathways. Without glycolysis and the pentose phosphate pathway, peptidoglycan (PGN)
and lipopolysaccharide (LPS) must be synthesized using host sugars. N-acetylglucosamine 1-P (NAG-1-P), a
precursor of both PGN and lipid A disaccharide backbones (as well as other LPS sugars), is usually synthesized by
bacteria using the bifunctional enzyme GlmU: the C-terminal acetyltransferase domain generates NAG-1-P from
glucosamine-1-P (GlcN-1-P), while the N-terminal uridyltransferase domain converts NAG-1-P to UDP-NAG.
Curiously, Rickettsia spp. contain a “halfling” GlmU enzyme with only the uridyltransferase domain, indicating
rickettsiae do not synthesize GlcN-1-P. Given the eukaryotic pathway (synthesizing sialic acid or chitin) generates
NAG-1-P from NAG-6-P (not GlcN-1-P), we speculate that rickettsiae import host NAG-1-P and convert it to UDP-
NAG using a streamlined enzyme (GlmU-N) tailored to eukaryotic metabolism. Specifically, we hypothesize that
rickettsiae uniquely utilize host-derived NAG-1-P for biosynthesis of cell envelope glycoconjugates, and that such
metabolite thievery from the host amino sugar biosynthesis pathway is essential for rickettsial intracellular
replication and survival. To test our hypothesis, we will characterize rickettsial uptake and metabolism of host NAG-
1-P using sophisticated biochemical techniques (AIM 1), and determine the essentiality of host NAG-1-P pilfering
using genetic tools for silencing host and pathogen metabolic genes (AIM 2). Under AIM 1, we will augment host cell
amino sugar metabolism (using chemical engineering and isotope supplementation) to monitor synthetic NAG-1-P
incorporation into the rickettsial cell envelope. Specifically, we’ll measure azido sugars incorporated into PGN and
LPS (via click chemistry and IFA), and 15N-labeled NAG-1-P-derived metabolites incorporated into PGN (via mass
spectrometry). Under AIM 2, we will show that rickettsiae must acquire host NAG-1-P for synthesis of
glycoconjugates (and thus replication) by using siRNAs to assess the impact of silencing host amino sugar
synthesis genes on R. typhi infection, as well as peptide nucleic acid-mediated knockdown of protein expression to
determine the functional significance of GlmU-N during early host infection. Colle...

## Key facts

- **NIH application ID:** 9935029
- **Project number:** 5R21AI146773-02
- **Recipient organization:** UNIVERSITY OF MARYLAND BALTIMORE
- **Principal Investigator:** Joseph J Gillespie
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $231,750
- **Award type:** 5
- **Project period:** 2019-06-01 → 2022-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9935029

## Citation

> US National Institutes of Health, RePORTER application 9935029, Rickettsia cell envelope glycoconjugates are derived from the host cell amino sugar biosynthesis pathway (5R21AI146773-02). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/9935029. Licensed CC0.

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