About

Elizabeth D. Hay Professor of Cell Biology Suzanne Walker is a faculty member at Harvard Medical School, associated with the Walker Lab. Her research focuses on bacterial cell envelope biology and the enzyme O-GlcNAc transferase. The lab investigates the molecular mechanisms underlying bacterial cell envelope structure and function, contributing to the understanding of bacterial physiology and potential therapeutic targets. Her work is integral to advancing knowledge in cell biology, particularly in the context of bacterial pathogens and their interactions with host organisms.

Research topics

• Cell biology
• Sociology
• Social Science
• Biochemistry
• Biology
• Computer Science
• Engineering ethics
• Chemistry
• Genetics
• Pedagogy
• Microbiology
• Organic chemistry
• Engineering
• Engineering management

Selected publications

• SpbR controls lipoteichoic acid length by directly inhibiting signal peptidase SpsB in <i>Staphylococcus aureus</i>

  Proceedings of the National Academy of Sciences · 2025-06-30 · 3 citations

  articleOpen accessSenior authorCorresponding

  Staphylococcus aureus is a Gram-positive pathogen that causes life-threatening infections. Its cell envelope contains anionic polymers called teichoic acids that are required for cell viability. Teichoic acids come in two forms and are made by different biosynthetic pathways. One form, lipoteichoic acid (LTA), is anchored in the cell membrane; the other form, wall teichoic acid (WTA), is covalently linked to the peptidoglycan cell wall. Although the LTA and WTA biosynthetic pathways have been characterized, regulation of teichoic acid production is not well understood. Here, we identified SpbR ( SAOUHSC_00965 ), a polytopic membrane protein similar to a eukaryotic CAAX protease, as a factor that controls LTA levels in S. aureus cells. We show that loss of SpbR results in short LTAs and a synthetically sick phenotype when WTA biosynthesis is prevented, whereas overexpressing SpbR results in elongated LTAs. Mechanistically, we find that SpbR physically associates with the type I signal peptidase SpsB, which cleaves LtaS, the polymerase that assembles LTA on the extracellular side of the membrane, and we show that this physical interaction inhibits SpsB cleavage of LtaS both in vivo and in vitro. Although the phenotypes investigated here are dominated by SpbR’s effects on LtaS, it also inhibits cleavage of other SpsB substrates. Based on its role in regulating the activity of SpsB, we named this factor SpbR ( S ignal p eptidase b R egulator).

  PublisherDOI

• The role of lipoteichoic acid in Staphylococcus aureus cell wall integrity

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-01-16

  preprintSenior author

  In Staphylococcus aureus, lipoteichoic acid (LTA) is crucial for growth, cell division, osmoprotection, and beta-lactam resistance, yet its molecular mechanisms remain unclear. This study reveals that LTA binds to multiple proteins involved in cell wall processes and preserves cell wall integrity by regulating one of the LTA-binding proteins, ScaH. ScaH is a peptidoglycan hydrolase predicted to have N-acetylglucosaminidase and amidase/peptidase activities. LTA inhibits ScaH's enzymatic activities by direct binding and represses scaH transcription by an unknown mechanism. During early growth, LTA is highly expressed and sequesters ScaH at the cell membrane, preventing ScaH activity in the cell wall. However, LTA expression decreases during the late growth phase, leading to ScaH translocation into the cell wall. This reduction in LTA coincides with increased wall teichoic acid (WTA) expression and cleavage of the LTA synthase LtaS. In the LTA-null mutant, ScaH inactivation restored peptidoglycan crosslinking, osmoresistance, and beta-lactam resistance both in vitro and in vivo. These findings suggest that LTA protects the cell wall by suppressing ScaH expression and activity while sequestering it during active growth. Additionally, the reciprocal expression patterns of LTA and WTA indicate an interconnected regulation of teichoic acids in S. aureus, with their roles likely depending on the growth phase.

  PublisherDOI

• Acyltransferases that Modify Cell Surface Polymers Across the Membrane

  Biochemistry · 2025-04-02 · 2 citations

  reviewOpen accessSenior authorCorresponding

  Cell surface oligosaccharides and related polymers are commonly decorated with acyl esters that alter their structural properties and influence their interactions with other molecules. In many cases, these esters are added to polymers that are already positioned on the extracytoplasmic side of a membrane, presenting cells with a chemical challenge because the high-energy acyl donors used for these modifications are made in the cytoplasm. How activated acyl groups are passed from the cytoplasm to extra-cytoplasmic polymers has been a longstanding question. Recent mechanistic work has shown that many bacterial acyl transfer pathways operate by shuttling acyl groups through two covalent intermediates to their final destination on an extracellular polymer. Key to these and other pathways are cross-membrane acyltransferases─enzymes that catalyze transfer of acyl groups from a donor on one side of the membrane to a recipient on the other side. Here we review what has been learned recently about how cross-membrane acyltransferases in polymer acylation pathways function, highlighting the chemical and biosynthetic logic used by two key protein families, membrane-bound O-acyltransferases (MBOATs) and acyltransferase-3 (AT3) proteins. We also point out outstanding questions and avenues for further exploration.

  PublisherOA PDFDOI

• Loss of LafB activity reverses daptomycin resistance in <i>E. faecium</i>

  mBio · 2025-11-28 · 1 citations

  articleOpen access

  ABSTRACT Infections caused by multidrug-resistant enterococci, particularly vancomycin-resistant Enterococcus (VRE), present significant therapeutic challenges. Daptomycin, a last-line treatment for VRE, often loses efficacy due to the emergence of resistance. In this study, we revealed the critical role of the lafB gene as a key determinant of daptomycin susceptibility and resistance in E. faecium . We showed that in the absence of a functional lafB , daptomycin-resistant mutants did not emerge in vitro , and derivatives of clinical daptomycin-resistant strains engineered to lack functional lafB were rendered even more sensitive to daptomycin than wild-type daptomycin-susceptible strains. These findings indicated that functional lafB is critical for key known mechanisms of daptomycin resistance, and mutations in lafB have phenotypic dominance to those that otherwise confer resistance. Therefore, inhibiting the activity of the lafB gene product is predicted to prevent or reverse resistance, offering a promising new strategy for extending the efficacy of daptomycin for treating enterococcal infections. IMPORTANCE Daptomycin is one of the few remaining effective antibiotics for treating vancomycin-resistant enterococcal infections but is limited by the emergence of resistance during protracted therapy. Here, we show that without a functional lafB gene, daptomycin-resistant mutants do not arise under conditions where wild-type strains readily generate daptomycin-resistant mutants. Furthermore, we show that loss of function mutation of the lafB gene in daptomycin-resistant clinical isolates renders them more susceptible to daptomycin than wild-type Enterococcus faecium . This indicates that an effective small molecule inhibitor of LafB activity or lafB gene expression would be a useful adjunctive for extending and restoring the therapeutic utility of daptomycin.

  PublisherDOI

• The mechanism of peptidoglycan O-acetylation in Gram-negative bacteria typifies bacterial MBOAT-SGNH acyltransferases

  Journal of Biological Chemistry · 2025-04-23 · 3 citations

  articleOpen access

  Bacterial cell envelope polymers are commonly modified with acyl groups that provide fitness advantages. Many polymer acylation pathways involve pairs of membrane-bound O-acyltransferase (MBOAT) and SGNH family proteins. As an example, the MBOAT protein PatA and the SGNH protein PatB are required in Gram-negative bacteria for peptidoglycan O-acetylation. The mechanism for how MBOAT-SGNH transferases move acyl groups from acyl-CoA donors made in the cytoplasm to extracellular polymers is unclear. Using the peptidoglycan O-acetyltransferase proteins PatAB, we explore the mechanism of MBOAT-SGNH pairs. We find that the MBOAT protein PatA catalyzes auto-acetylation of an invariant Tyr residue in its conserved C-terminal hexapeptide motif. We also show that PatB can use a synthetic hexapeptide containing an acetylated tyrosine to donate an acetyl group to a peptidoglycan mimetic. Finally, we report the structure of PatB, finding that it has structural features that shape its activity as an O-acetyltransferase and distinguish it from other SGNH esterases and hydrolases. Taken together, our results support a model for peptidoglycan acylation in which a tyrosine-containing peptide at the MBOAT's C-terminus shuttles an acyl group from the MBOAT active site to the SGNH active site, where it is transferred to peptidoglycan. This model likely applies to other systems containing MBOAT-SGNH pairs, such as those that O-acetylate alginate, cellulose, and secondary cell wall polysaccharides.

  PublisherDOI

• The mycomembrane proteins PorH and ProtX are inserted at polar growth zones and linked to the cell wall

  bioRxiv (Cold Spring Harbor Laboratory) · 2025-10-14

  preprintOpen access

  ABSTRACT The Mycobacteriales order of bacteria includes important pathogens such as Mycobacterium tuberculosis . These organisms are surrounded by a unique cell envelope architecture that includes a two-layered cell wall composed of peptidoglycan (PG) and arabinogalactan. They also build an outer membrane called the mycomembrane that is made of mycolic acids. Mycolate outer membrane proteins (MOMPs) reside within the mycomembrane and a subset are thought to form pores that allow essential nutrients to permeate the envelope. However, little is known about the structure of these proteins or the mechanism by which they are assembled. Here, we investigate MOMP assembly in the model organism Corynebacterium glutamicum ( Cglu ) using PorH as a model MOMP. PorH is encoded in an operon with the MOMP PorA, and the two small, alpha-helical proteins have been proposed to form hetero-oligomeric pores in the mycomembrane. Consistent with this proposal, AlphaFold2 predicts a high confidence structure of a hetero-oligomeric pore formed by five copies each of PorH and its partner PorA, and we show that PorA is required for the surface assembly of PorH. Using a fluorescence assay for detection of surface-exposed PorH or another MOMP called ProtX, we found that MOMP assembly occurs within zones of active PG synthesis at the cell poles. We also discovered that PorH and ProtX are linked to the cell wall. Thus, like Gram-negative bacteria, Cglu and potentially other members of Mycobacteriales order, coordinate outer membrane protein assembly with PG biogenesis and use proteins to connect the mycomembrane and the cell wall. SIGNIFICANCE Diderm bacteria in the Mycobacteriales order have a distinctive outer layer called the mycomembrane. Proteins that reside within the mycomembrane play critical roles in virulence and cell viability. However, how proteins are assembled into the mycomembrane has remained an outstanding question in the field. Here, we investigate the biogenesis of mycomembrane proteins in the model organism Corynebacterium glutamicum . We show that these proteins are inserted into the mycomembrane in a manner that correlates with polar growth and are attached to the cell wall. Many features of these mycomembrane proteins are shared between species in the Mycobacteriales, suggesting that our findings may be conserved in other species within this order.

  PublisherOA PDFDOI

• AuxB interacts directly with GpsB and PknB to coordinate cell envelope processes that contribute to intrinsic antibiotic resistance in <i>Staphylococcus aureus</i>

  mBio · 2025-08-25

  articleOpen accessSenior author

  ABSTRACT Staphylococcus aureus , a leading cause of serious infections, produces various factors important for intrinsic resistance to antibiotics. Understanding what intrinsic resistance factors do may enable strategies to potentiate existing antibiotics. The membrane protein AuxB is an intrinsic resistance factor that helps S. aureus withstand diverse compounds that target the cell envelope, but its cellular functions are unknown. We show here that AuxB is a four-pass transmembrane protein with an intracellular C-terminus that interacts directly with the cytosolic cell cycle regulator GpsB. We also show AuxB’s membrane domain forms a homodimer that exists in equilibrium with a heterodimer of AuxB and PknB, a eukaryotic-like serine/threonine kinase that has been implicated in cell envelope processes. Shifting the equilibrium to favor AuxB-bound PknB impairs growth on tunicamycin, a condition where PknB is essential, which suggests that AuxB binding antagonizes a PknB function. To link PknB’s domains to compound susceptibility phenotypes, we assessed the fitness of PknB variants under several conditions. We find that PknB’s extracellular and kinase domains are not functionally interdependent but instead play distinct roles in withstanding cell envelope stress. AuxB evidently antagonizes functions of PknB’s extracellular PASTA ( p enicillin-binding protein a nd S er/ T hr kinase- a ssociated) domain, the presence of which is beneficial under tunicamycin treatment regardless of whether the kinase domain is active. On compounds where the PASTA domain is deleterious, increasing the amount of AuxB-bound PknB can also ameliorate sensitivity. Collectively, our data suggest that AuxB, as a homodimer and through its interactions with GpsB and PknB, modulates cell envelope processes during cell growth and division. IMPORTANCE Staphylococcus aureus is a leading cause of fatal infections worldwide. It encodes diverse genes that contribute to the organism's high intrinsic resistance to antibiotics. Understanding the biological roles of these genes and how their features contribute to intrinsic resistance may enable better antibiotic therapies. Here, we investigate AuxB, an intrinsic resistance factor to compounds that target the cell envelope. We find that AuxB interacts directly with the cell cycle regulator GpsB and the eukaryotic-like serine/threonine kinase PknB, another intrinsic resistance factor that is proposed to sense and respond to cell wall status. Based on our findings, we propose that AuxB impacts cell physiology through three mechanisms: (i) by antagonizing PknB's p enicillin-binding protein a nd S er/ T hr kinase- a ssociated domain function; (ii) by coordinating the phosphorylation of cell division proteins; and (iii) by forming a homodimer that interacts with GpsB hexamers to enable the formation of extended GpsB interaction networks.

  PublisherDOI

• Environmental cues in different host niches shape the survival fitness of Staphylococcus aureus

  Nature Communications · 2025-07-28 · 10 citations

  articleOpen access

  The ability of Staphylococcus aureus to adapt and thrive in diverse host niches adds to the challenge in combating this ubiquitous pathogen. While extensive research has been pursued on the adaptive mechanisms of methicillin-resistant S. aureus (MRSA) in various infection models, a comprehensive analysis of its fitness across different host niches is lacking. In this study, we employ transposon sequencing to analyze the adaptive strategies of MRSA in various infection niches. Our analysis encompasses a cell model that mimics an intracellular niche, human blood, which represents a major extracellular environment as well as a major intermediary route encountered by bacteria during systemic infection, and a male murine sepsis model that recapitulates intra-organ environments. Our findings reveal substantial differences in the genetic determinants essential for bacterial survival in intracellular and blood environments. Moreover, we show that each organ imposes unique growth constraints, thus fostering heterogeneity within the mutant population that can enter and survive in each organ of the mouse. By comparing genes important for survival across all examined host environments, we identify 27 core genes that represent potential therapeutic targets for treating S. aureus infections. Additionally, our findings aid in understanding how bacteria adapt to diverse host environments.

  PublisherOA PDFDOI

• <i>Staphylococcus aureus</i> uses a GGDEF protein to recruit diacylglycerol kinase to the membrane for lipid recycling

  Proceedings of the National Academy of Sciences · 2025-03-18 · 5 citations

  articleOpen accessSenior authorCorresponding

  Staphylococcus aureus is a Gram-positive pathogen responsible for numerous antibiotic-resistant infections. Identifying vulnerabilities in S. aureus is crucial for developing new antibiotics to treat these infections. With this in mind, we probed the function of GdpS, a conserved Staphylococcal membrane protein containing a cytoplasmic GGDEF domain. These domains are canonically involved in cyclic-di-GMP signaling processes, but S. aureus is not known to make cyclic-di-GMP. Using a transposon screen, we found that loss of GdpS is lethal when combined with disruption in synthesis of the glycolipid anchor of a cell surface polymer called lipoteichoic acid (LTA) or with deletion of genes important in cell division. Taking advantage of a small molecule that inhibits LTA glycolipid anchor synthesis, we selected for suppressors of Δ gdpS lethality. The most prevalent suppressors were hypermorphic alleles of dgkB , which encodes a soluble diacylglycerol (DAG) kinase required to recycle DAG to phosphatidylglycerol. By following up on these suppressors, we found that the GGDEF domain of GdpS interacts directly with DgkB, orienting its active site at the membrane to promote DAG recycling. DAG kinase hypermorphs also suppressed the lethality caused by combined loss of gdpS and cell division factors, highlighting the importance of lipid homeostasis for cell division. GdpS’ positive regulation of DAG kinase function was dependent on the GGDEF domain but not its catalytic residues. As the sole conserved GGDEF-domain protein in Staphylococci, GdpS promotes an enzymatic process independent of cyclic-di-GMP signaling, revealing a new function for the ubiquitously conserved GGDEF domain.

  PublisherOA PDFDOI

• A diverse single-stranded DNA–annealing protein library enables efficient genome editing across bacterial phyla

  Proceedings of the National Academy of Sciences · 2025-04-21 · 5 citations

  articleOpen access

  Genome modification is essential for studying and engineering bacteria, yet making efficient modifications to most species remains challenging. Bacteriophage-encoded single-stranded DNA–annealing proteins (SSAPs) can facilitate efficient genome editing by homologous recombination, but their typically narrow host range limits broad application. Here, we demonstrate that a single library of 227 SSAPs enables efficient genome-editing across six diverse bacteria from three divergent classes: Actinomycetia ( Mycobacterium smegmatis and Corynebacterium glutamicum ), Alphaproteobacteria ( Agrobacterium tumefaciens and Caulobacter crescentus ), and Bacilli ( Lactococcus lactis and Staphylococcus aureus ). Surprisingly, the most effective SSAPs frequently originated from phyla distinct from their bacterial hosts, challenging the assumption that phylogenetic relatedness is necessary for recombination efficiency, and supporting the value of a large unbiased library. Across these hosts, the identified SSAPs enable genome modifications requiring efficient homologous recombination, demonstrated through three examples. First, we use SSAPs with Cas9 in C. crescentus to introduce single amino acid mutations with &gt;70% efficiency. Second, we adapt SSAPs for dsDNA editing in C. glutamicum and S. aureus , enabling one-step gene knockouts using PCR products. Finally, we apply SSAPs for multiplexed editing in S. aureus to precisely map the interaction between a conserved protein and a small-molecule inhibitor. Overall, this library-based SSAP screen expands engineering capabilities across diverse, previously recalcitrant microbes, enabling efficient genetic manipulation for both fundamental research and biotechnological applications.

  PublisherDOI

Recent grants

• NIH Grant R03TW009424

  NIH · $135k · 2016

• NIH Grant R01AI099144

  NIH · $4.3M · 2019

• NIH Grant R01AI044854

  NIH · $2.3M · 2008

• Defining OGT's Essential Functions to Guide Therapeutic Approaches

  NIH · $5.8M · 2012–2024

• Discovery and characterization of new bacterial cell wall targets and inhibitors to treat resistant infections

  NIH · $5.5M · 2020–2029

Frequent coauthors

• Larry Bryant

  International Rescue Committee

  109 shared

• James Madara

  St. Louis County Missouri

  109 shared

• Annette Flanagin

  109 shared

• Paul Ruich

  University of Washington

  109 shared

• Karen Bucher

  109 shared

• Michael Berkwits

  109 shared

• Phil Fontanarosa

  109 shared

• Emily Ling

  Boston Children's Hospital

  105 shared

Labs

• Walker LabPI

Education

• Ph.D., Chemistry

  Harvard University

  1995

• B.S., Chemistry

  University of California, Berkeley

  1989