Peptidoglycan Composition of Bacterial Cell Wall

Students involved in the project: Tobi,  Leigh, and Nacorria.

Solid-state NMR analysis of Cell Wall.  

The host-cell attachment by Gram-positive pathogens, such as Staphylococcus aureus, Enterococcus faecalis, and Streptococcus pneumoniae are essential for understanding the mechanisms of microbial pathogenesis. In S. aureus, wall teichoic acid (WTA), a ribitol-phosphate polymer of cell wall, mediates the host binding. In E. faecalis and S. pneumoniae, pili are used to target the host extracellular matrices. Stable isotopes (13C, 15N, and 19F) are used to selectively label the extracellular matrices of bacteria and epithelial tissues. REDOR NMR is used to measure the heteronuclear dipolar coupling, hence internuclear distances between 19F-13C and 19F-15N to determine the mechanisms of bacterial attachment to host tissues. We also investigate the mechanisms biofilm formation by the Gram-positive pathogens. Biofilm-associated infections exhibit increased drug resistance and are difficult to eradicate, resulting in persistent infections. 

LC/MS analysis of Peptidoglycan 

Peptidoglycan is a dynamic component of the cell wall in bacteria that changes in response to growth conditions during the infection such as hypoxia, low pH, low nutrient, and antibiotic treatment.  The change in peptidoglycan composition (peptidoglycome) of microbial pathogens will be characterized using a combined liquid-chromatography/mass-spectrometry (LCMS) with the in silico muropeptide fragment library to provide an unprecedented level of peptidoglycan composition analysis.  The analysis will provide molecular and compositional insights into the mechanisms of pathogenesis, antibiotic resistance, increased virulence, persistence, and resuscitation, for the prevention and treatment of infections and the development of novel antimicrobial agents.

Recommended Background Reading for Peptidoglycan

• https://academic.oup.com/femsre/article/32/2/149/2683904

• https://academic.oup.com/femsre/article/32/2/287/2683968

• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC98950/

• https://academic.oup.com/femsre/article/43/5/548/5513445 

The Role of the Foxo Pathway in the Control of Culex pipiens Diapause

Students involved in the project: Tobi,  Alani, Mayana, Raiyan, Nnenna, and Rachelle.

Solid-state NMR analysis of Culex pipiens.
C. pipiens is the mosquito that vectors West Nile Virus and other human-pathogenic flavivruses in North America. In response to shortened day length and lower temperatures, female Cx. pipiense prepares for the diapause by actively feeding on carbohydrates to increase the biosynthesis of glycogen and lipid to store energy for overwintering. The effect of feeding different carbohydrates on glycogen and lipid biosynthesis in diapausing mosquitoes was investigated in vivo using 13C solid-state NMR. Our findings provide novel insights into the insect carbohydrate metabolism that governs glycogen and lipid biosynthesis during diapause, which is fundamental for the insect survival during inimical environments. 

• Dsi-RNA knockdown of genes regulated by Foxo reduces glycogen and lipid accumulations in diapausing Culex pipiens

• Suppression of glycogen synthase expression reduces glycogen and lipid storage during mosquito overwintering diapause

• Solid-state NMR reveals differential carbohydrate utilization in diapausing Culex pipiens

Lipidome of insects

• LIPID MAPS (https://www.lipidmaps.org/) 

• Exploris 480 www.planetorbitrap.com 

• Advances in Mass Spectrometry for Lipidomics

• High Resolution in Mass Spectrometry in Lipidomics

FTIR analysis of Culex pipiens.
PCA analysis with machine learning (Tobi and Raiyan)

 Collaborators

• Dr. Chad Weisbrod  (Ion Cyclotron Resonance (ICR) National High Magnetic Field Laboratory at FSU)

• Dr. Chulho Sim (Baylor University Group Lab)

• Dr. Yeona Kang (Howard University, Dept. of Mathematics)

Metabolome Analysis

Students involved in the project: Raiyan, Francis, Collin, Mayana, Nnenna, Nacorria, and Leigh.

LC/MS 

• LIPID MAPS (https://www.lipidmaps.org/) 

• Exploris 480 www.planetorbitrap.com 

• Advances in Mass Spectrometry for Lipidomics

• High Resolution in Mass Spectrometry in Lipidomics

 NMR 

• Chenomx 

Collaborators

• Dr. Joon Ha (Howard University, Dept. of Mathematics)

• Dr. Yeona Kang (Howard University, Dept. of Mathematics)

Solid and Lipid Nanoparticles

Central-carbon and nitrogen flux analysis in photosynthetic algae

Understanding the fundamental carbon metabolism in algae at the subcellular level is challenging because of complex carbon metabolic pathways that are governed by multiple organelles with built-in redundancy. Our goal is to provide the “global parameters” that can accommodate the “omics” approach and the systems analysis. We will achieve this by selective 13C, 12C, 15N, 2H, and 14N-isotope labeling strategies we have developed for Chlamydomonas reinhardtii (green alga).  The primary objective of this study is not for a mere understanding of the enhanced lipid production in algae, but to understand the fundamental central-carbon and nitrogen metabolisms in algae by using the C. reinhardtii strains cw15 and sta16 as a comparative model. To understand this different metabolic response, we use SSNMR for the analysis of intact whole cells to determine the total 13C and 15N fluxes. LC-MS and GC-MS analysis is used to determine the 13C and 15N-isotope incorporation efficiency (13C/12C and 15N/14N ratios) of metabolites and lipids from whole cells or isolated organelles. 

Department of Chemistry/ Howard University/ Chemistry Building (CHB)/ 525 College Street, N.W. Washington, D.C. 20059