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

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

 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)

Peptidoglycan Composition of Bacterial Cell Wall

My research program focuses on the use of solid-state nuclear magnetic resonance (SSNMR) and liquid chromatography-mass spectrometry (LC-MS) to characterize the structure, composition, and organization of bacterial cell walls. This work aims to elucidate the antibiotic mode of action and to provide fundamental insights into antibiotic resistance and biofilm formation.

“Peptidoglycome” of Bacteria. 

Peptidoglycan (PG) is a key bacterial cell wall component.  Determining its chemical composition is crucial for understanding microbial pathogenesis, persistence during infection, and adaptive responses to external stimuli such as antibiotic exposure and nutrient restriction. However, analysing PG is challenging due to the wide range of chemical modifications that create a complex and heterogeneous mixture. Most laboratories address this complexity by removing chemical modifications, simplifying the analysis by eliminating the rich muropeptide diversity that encodes bacterial responses to environmental stimuli. As a result, only a few labs worldwide have performed comprehensive peptidoglycan analyses with limited success.

Our method is unique in that it preserves peptidoglycan modifications while employing a novel analytical approach developed in my lab. This methodology enables precise measurement and quantification of peptidoglycan composition in the bacterial cell wall. Our innovative strategy includes: i) developing a new method for cell wall isolation and enzymatic digestion that retains chemical modifications, ii) creating an in silico muropeptide-fragment mass library of all possible peptidoglycan fragments with known modifications, generated in-house using MATLAB for rapid identification, and iii) implementing a new approach for the accurate quantification of muropeptide fragments.

This approach has provided unprecedented insights into PG composition. We coined the term “peptidoglycome” to describe the comprehensive profile of PG structures. Using this method, we successfully characterized the peptidoglycome of two clinically significant pathogens, Staphylococcus aureus and Enterococcus faecalis. 

Additionally, we developed protocols to investigate the peptidoglycome of other clinically important pathogens, including: multi-drug-resistant Pseudomonas aeruginosa, and Mycobacterium smegmatis. As my lab has successfully characterized the peptidoglycome of M. smegmatis, we are prepared to perform comparative analyses of M. tuberculosis and M. smegmatis PG composition. Representative publications from this output are listed below.

• Scientific Reports 13, 12227 (2023) "Daptomycin inhibits the transpeptidation step of peptidoglycan biosynthesis in Enterococcus faecalis"

• Scientific Reports 12, 11061 (2022) "Peptidoglycan compositional analysis of Mycobacterium smegmatis using high-resolution LC-MS"

• ACS Chem. Bio. (2019) "L,D-transpeptidase specific probe reveals spatial activity of peptidoglycan crosslinking"

• Journal of Bacteriology, DOI: 10.1128/JB.00249-17 (2017) “Inhibition of Staphylococcus aureus cell wall biosynthesis by desleucyl-oritavancin: a quantitative peptidoglycan composition analysis by mass spectrometry”

• Biochemistry, Vol. 56 (10): 1529–1535 (2017) “Quantification of the d-Ala-d-Lac-terminated peptidoglycan structure in vancomycin-resistant Enterococcus faecalis using a combined solid-state nuclear magnetic resonance and mass spectrometry analysis”

Mode of Action of Glycopeptides and Cyclic Peptide Antibiotics Against Gram-Positive Pathogens

We investigate the in situ mode of action of disaccharide-modified glycopeptides (oritavancin and telavancin) and cyclic antimicrobial peptides (plusbacin A3, tripropeptin C, daptomycin, and amphomycin). Oritavancin and telavancin are semisynthetic antibiotics effective against multidrug-resistant Gram-positive pathogens. While most cyclic antimicrobial peptides are not yet available for clinical use (except daptomycin), they represent promising scaffolds for developing novel antibiotics through chemical modifications. Using a combination of solid-state NMR and LC-MS, we examined the mode of action of these antibiotic classes. Our study identified in situ drug-binding sites and elucidated their bactericidal mechanisms against methicillin-resistant Staphylococcus aureus (MRSA), a highly drug-resistant pathogen often referred to as the "Super Bug." These findings provide critical structural and molecular insights into the mechanisms of action of these antimicrobial agents, forming the basis for future antibiotic development. Representative publications from this research are listed below.

• Scientific Reports 12, 7087 (2022) "Molecular dynamics simulation of the secondary-binding site in disaccharide-modified glycopeptide antibiotics"

• The Journal of Physical Chemistry B, 121 (16): 3925-3932 (2017)  “A hidden mode of action for glycopeptide antibiotics: Inhibition of wall teichoic acid biosynthesis”

• The Journal of Physical Chemistry B, 121 (7): 1499-1505 (2017)  “Dual mode of action for plusbacin A3 in Staphylococcus aureus”

• Chemical Communications, 53, 5649-5652 (2017)  “Inhibition of D-Ala incorporation into wall teichoic acid biosynthesis in Staphylococcus aureus by desleucyl-oritavancin”

• Scientific Reports 6, 31757, doi:10.1038/srep31757 (2016)  “Solid-state NMR characterization of amphomycin effects on peptidoglycan and wall teichoic acid biosyntheses in Staphylococcus aureus”

• Biochemistry, Vol. 55 (24): 3383-3391 (2016)  “The carboxyl-terminus of eremomycin facilitates binding to the non-d-Ala-d-Ala segment of the peptidoglycan pentapeptide stem”

Characterization of Staphylococcus aureus Biofilm: Composition and Structural Analysis. 

The chemical composition and local structure of S. aureus biofilms were determined using SSNMR.  While S. aureus is the leading cause of biofilm-related infections, the composition and mechanisms of its biofilm formation remain poorly understood. To address this, we used SSNMR to analyse a series of ¹³C- and ¹⁵N-labeled biofilms, enabling the precise quantification of sortase-mediated surface proteins attached to bacterial cell walls. Our findings provided critical insights into S. aureus biofilm formation. 

Additionally, we initiated the analysis of biofilm-induced change in PG composition using LC-MS. By integrating SSNMR and LC-MS techniques, we aim to uncover new insights into the mode of action of novel antibacterials targeting biofilms.  This work is essential for addressing persistent infection, eradication of biofilm, and enhancing our understanding of biofilm formation mechanisms. Representative publications from this research are listed below.

• ACS Omega 9, 36, 37610–37620 (2024) "Scanning electron microscopy and energy dispersive X-ray spectroscopic of Staphylococcus aureus biofilm"

The Effects of Induced Antibiotic Resistance on Pathogen Fitness. 

We are investigating the underlying mechanisms behind a fascinating clinical observation where the treatment of infections caused by multidrug-resistant pathogens with antibiotics can increase pathogen virulence and lead to poorer patient outcomes. For instance, treating methicillin-resistant S aureus (MRSA) infections with methicillin has been shown to worsen patient conditions. Using combined SSNMR and LC-MS, we have discovered that antibiotic-resistant pathogens respond to antibiotic therapy by altering their bacterial cell wall. These modifications hamper the innate immune system's ability to detect and clear the infection, contributing to increased pathogen fitness and disease severity. One published manuscript and 2 manuscripts in preparation are listed below.

• Scientific Reports 7, 46500; doi:10.1038/srep46500 (2017) “Peptidoglycan O-acetylation increases in response to vancomycin treatment in vancomycin-resistant Enterococcus faecalis”

Solid-state NMR characterization of Staphylococcus aureus biofilm to determine the composition and structural organization.  

The goal of this project is to gain structural and compositional insight into the bacterial biofilm using solid-state NMR and mass spectrometric approaches.  Our specific aims are: i) determine the composition and structural organization of mature biofilm in S. aureus, ii) determine the mode of action of oritavancin’s bactericidal activity against S. aureus biofilm, and iii) determine the mode of action of novel anti-biofilm agents. 

• Biochimica et Biophysica Acta Biomembrane, 1860 (3): 749-756 (2018)  “Surface proteins and the formation of biofilms by Staphylococcus aureus”

Solid-State NMR Characterization of Peptidoglycan Tertiary Structure in Bacterial Cell Walls. 

The goal of this project is to elucidate the 3D architecture of bacterial PG using SSNMR.  Our studies revealed that PG in bacterial cell walls exhibits a surprising order structure, and its tertiary organization undergoes significant changes when the inter-bridge chain length is altered.  These structural modifications have critical implications for cell wall assembly, integrity, drug resistance, and bacterial physiology.  Representative publications from this research are listed below.

• Biochimica et Biophysica Acta Biomembrane Vol. 1859 (11): 2171-2180 (2017) “Characterization of the tertiary structure of the peptidoglycan of Enterococcus faecalis”

• Biochimica et Biophysica Acta Biomembrane, Vol. 1848 (1): 350-362 (2015)  “Peptidoglycan architecture of Gram-positive bacteria by solid-state NMR”

• Biochimica et Biophysica Acta Biomembrane, Vol. 1848 (1): 363-368 (2015)  “REDOR constraints on the peptidoglycan lattice architecture of Staphylococcus aureus and its FemA mutant”

• Biochemistry, Vol. 53 (9): 1420-1427 (2014)  “Cross-link formation and peptidoglycan lattice assembly in the FemA mutant of Staphylococcus aureus”

Development of Stable Isotope Labeling by Amino Acids in Bacterial Culture (SILAB) for Accurate Quantification of Peptidoglycan Composition Changes During Planktonic-to-Biofilm Transition Using LC-MS. 

We have developed SILAB, a novel adaptation of Stable Isotope Labelling by Amino Acids in Cell Culture (SILAC), for isotope labeling in autotrophic and bacterial systems. This method uses heavy Lys (L-[13C6, 2D9, 15N2]Lys) to label  PG, and we established an analytical approach to accurately measure the isotopic enrichment and isotopic incorporation efficiency. Using SILAB, we analyzed 47 selected muropeptide pairs from isolated cell walls of planktonic and biofilm samples.  Our results demonstrated that PG in biofilm shows an increase in cross-linking and N-deacetylation of MurNAc, while a decrease in O-acetylation of GlcNAc.  The resulting publication from this effort is shown below.

• Biochemistry, 57, 7, 1274-1283. (2018) "Peptidoglycan compositional analysis of Enterococcus faecalis biofilm by stable isotope labeling by amino acids in a bacterial culture" 

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