The Role of the FOXO Pathway in the Regulation of Culex pipiens Diapause

Carbohydrate Metabolism and Energy Storage During Mosquito Diapause.  Culex pipiens, a major vector of West Nile virus and other human-pathogenic flaviviruses in North America, undergoes reproductive diapause in response to environmental cues, including shortened day length and lower temperatures. During this period, female mosquitoes alter their metabolism and accumulate glycogen and lipids to support survival through unfavorable winter conditions.

Using in vivo 13C solid-state nuclear magnetic resonance (SSNMR), we investigate how different dietary carbohydrates influence glycogen and lipid biosynthesis during diapause. Our findings provide insights into the metabolic pathways that govern energy storage and contribute to mosquito overwintering survival. This research also examines the role of the FOXO regulatory pathway in coordinating metabolic processes associated with diapause.

We are interested in characterizing insect lipid composition and understanding how lipid metabolism changes in response to physiological and environmental conditions. High-resolution mass spectrometry provides a powerful approach for identifying and characterizing complex lipid mixtures.

Selected publications

• RNA interference-mediated knockdown of FOXO-regulated genes reduces glycogen and lipid accumulation 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

Peptidoglycan Composition and Architecture of Bacterial Cell Walls

My research program combines solid-state nuclear magnetic resonance (SSNMR) and liquid chromatography–mass spectrometry (LC-MS) to characterize the composition, chemical modifications, and structural organization of bacterial cell walls. By investigating how bacterial cell walls respond to antibiotics and environmental stress, we seek to elucidate antibiotic mechanisms of action, understand antimicrobial resistance, and identify molecular determinants of biofilm formation.

Comprehensive Characterization of the Bacterial Peptidoglycome.  Peptidoglycan (PG) is a fundamental component of the bacterial cell wall that maintains cellular integrity and contributes to bacterial survival, pathogenicity, and adaptation. Its composition is highly heterogeneous because of the extensive chemical modifications and structural variations that occur in response to environmental conditions and antibiotic exposure. Conventional analytical approaches often remove or alter these modifications to simplify analysis, potentially obscuring structural information associated with bacterial adaptation. Comprehensive characterization of the native diversity of peptidoglycan therefore remains a significant analytical challenge. We have developed a mass spectrometry-based analytical platform designed to preserve and characterize peptidoglycan modifications.  Peptidoglycome represents the comprehensive profile of peptidoglycan structures present in a bacterial cell wall. Using this approach, we have characterized the peptidoglycomes of clinically important pathogens, including Staphylococcus aureus and Enterococcus faecalis. We have also established protocols for studying other organisms, including multidrug-resistant Pseudomonas aeruginosa and Mycobacterium smegmatis. These capabilities provide a foundation for comparative investigations of Mycobacterium tuberculosis and M. smegmatis.

Selected publications

• 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 Chemical Biology (2019). “L,D-transpeptidase specific probe reveals spatial activity of peptidoglycan crosslinking.”

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

• Biochemistry 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.”

Mechanisms of Action of Glycopeptide and Cyclic Peptide Antibiotics

We investigate the molecular mechanisms of disaccharide-modified glycopeptide antibiotics, including oritavancin and telavancin, and cyclic antimicrobial peptides, including plusbacin A3, tripropeptin C, daptomycin, and amphomycin.

Oritavancin and telavancin are semisynthetic antibiotics used against serious Gram-positive bacterial infections, including those caused by resistant pathogens. Cyclic antimicrobial peptides represent additional scaffolds for developing new antibiotics, although their clinical status varies by compound.

By integrating SSNMR and LC-MS, we examine antibiotic–cell-wall interactions and characterize changes in bacterial cell-wall biosynthesis following drug exposure. Our studies have identified drug-binding sites and revealed bactericidal mechanisms involving peptidoglycan and wall teichoic acid biosynthesis in Staphylococcus aureus, including methicillin-resistant S. aureus (MRSA). These findings provide molecular-level insights into antibiotic activity and inform the development of new antimicrobial strategies against resistant Gram-positive pathogens.

Selected publications

• 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 (2016). DOI: 10.1038/srep31757. “Solid-state NMR characterization of amphomycin effects on peptidoglycan and wall teichoic acid biosyntheses in Staphylococcus aureus.”

• Biochemistry 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.”

Chemical Composition and Structural Organization of Staphylococcus aureus Biofilms

Biofilms are complex microbial communities whose structural organization and chemical composition contribute to persistent infections and reduced susceptibility to antimicrobial treatment. Despite the clinical importance of S. aureus biofilms, important aspects of their molecular composition and formation remain incompletely understood.

We use SSNMR to characterize intact, isotopically labeled biofilms and quantify the composition and organization of their molecular components. Our studies have examined sortase-mediated surface proteins attached to bacterial cell walls, providing insights into the molecular interactions that contribute to biofilm formation. We are extending this work through LC-MS-based analysis of biofilm-associated changes in peptidoglycan composition. By integrating structural and compositional analyses, we aim to understand how bacterial cell walls adapt during biofilm development and how these changes influence the activity of novel antibiofilm agents. This research provides a foundation for investigating persistent bacterial infections and developing strategies to disrupt biofilm formation and improve antimicrobial effectiveness.

Selected publication

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

Antibiotic-Induced Cell-Wall Remodeling and Pathogen Fitness

Antibiotic exposure can induce adaptive responses that alter bacterial physiology and may influence pathogen survival, immune recognition, and disease outcomes. We investigate the molecular basis of these responses, focusing on changes in bacterial cell-wall composition associated with antibiotic resistance.

Using complementary SSNMR and LC-MS approaches, we have shown that antibiotic exposure can alter peptidoglycan chemistry in resistant bacteria. Such modifications may affect interactions between bacterial cells and the host innate immune system, potentially contributing to bacterial persistence and altered pathogen fitness.

Our work seeks to clarify how antibiotic-induced cell-wall remodeling influences bacterial survival and resistance-associated phenotypes, with the broader goal of identifying new vulnerabilities that can be exploited therapeutically.

Selected publication

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

Structural Organization of Peptidoglycan in Bacterial Cell Walls

The mechanical integrity and biological function of bacterial cell walls depend on the three-dimensional organization of their peptidoglycan networks. We use SSNMR to investigate the tertiary structure of peptidoglycan and determine how variations in cross-linking and interpeptide bridge length affect cell-wall architecture.

Our studies have revealed that bacterial peptidoglycan exhibits an unexpectedly ordered structural organization and that changes in inter-bridge chain length can substantially alter its architecture. These findings provide insight into the relationship between cell-wall structure, bacterial physiology, cell-wall assembly, and antimicrobial resistance.

Selected publications

• Biochimica et Biophysica Acta (BBA) – Biomembranes 1859 (11), 2171–2180 (2017). “Characterization of the tertiary structure of the peptidoglycan of Enterococcus faecalis.”

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

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

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

Stable-Isotope Labeling for Quantitative Analysis of Peptidoglycan Remodeling

We developed Stable Isotope Labeling by Amino Acids in Bacterial Culture (SILAB), an adaptation of Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) for bacterial systems. The method uses heavy lysine (L-[^13C₆,^2H₉,^15N₂]lysine) to label peptidoglycan and enables the measurement of isotopic enrichment and incorporation efficiency.

Using SILAB and LC-MS, we analyzed 47 selected pairs of muropeptides from isolated cell walls of planktonic and biofilm-associated Enterococcus faecalis. Our results revealed increased peptidoglycan cross-linking and MurNAc N-deacetylation in biofilm-associated cells, accompanied by decreased GlcNAc O-acetylation.

This approach provides a quantitative framework for investigating cell-wall remodeling during the transition from planktonic growth to biofilm formation.

Selected publication

• 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 Metabolism in Photosynthetic Algae

Understanding carbon and nitrogen metabolism in photosynthetic algae is challenging because metabolic pathways are distributed across multiple organelles and involve overlapping biochemical functions. Our research aims to establish quantitative, system-level parameters that can support integrated metabolomics and systems biology analyses.

We developed selective isotope-labeling strategies using ^13C, ^12C, ^15N, ^2H, and ^14N for the green alga Chlamydomonas reinhardtii. Using the cw15 and sta16 strains as comparative models, we investigate fundamental differences in central carbon and nitrogen metabolism rather than focusing exclusively on enhanced lipid production.

Our analytical approach combines SSNMR of intact cells to characterize overall ^13C and ^15N incorporation with LC-MS and GC-MS to quantify isotope incorporation into metabolites and lipids from whole cells or isolated organelles. This integrated strategy provides a framework for investigating metabolic fluxes and the organization of carbon and nitrogen metabolism in photosynthetic microorganisms.

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