Biology subjects

McCausland, J. W.

Publications and source records attributed to McCausland, J. W..

9 recordsLinked to original sources

bioRxiv2026

A lipid compendium of a metabolically compromised bacterium provides insights into lipid acquisition, biosynthesis, and metabolism

The Lyme disease agent Borrelia burgdorferi belongs to a class of metabolically compromised bacteria that cannot survive without host-derived lipids. Survival of the agent in tick and vertebrate hosts requires substantial nutrient acquisition and potential cell envelope remodeling. While prior studies identified cholesterol, cholesterol glycolipids, and phosphatidylcholines as membrane lipids in B. burgdorferi, the identity of many other membrane lipids, their origin, and their physiological relevance remain unknown. Here, we used a suite of untargeted and targeted high-resolution mass spectrometry methods to reveal a complex lipid profile of the pathogen and to identify the origin of its lipids. The analysis detected more than 500 lipids in B. burgdorferi, the majority of which are sourced from the environment. However, the bacterium selectively accumulates certain lipids while excluding others, suggesting discriminatory uptake. These include cholesteryl esters and triglycerides that are organized in foci within the pathogen. Intriguingly, the pathogen also synthesizes predominantly eukaryotic lipids such as the lysosomal bis(monoacylglycerol)phosphate and the plant glycolipid sulfoquinovosyl diacylglycerol (SQDG). The biosynthesis of the latter is carried out by enzymes that exhibit structural homology to plant oxidoreductases and galactosyltransferases, yet their closest orthologs are found in bacteria. This hints that the capability of SQDG synthesis is more widespread in spirochaetes and other bacteria. Together, the comprehensive lipid profiling we report here uncovers novel aspects of the physiology of the metabolically challenged B. burgdorferi and highlights lipid acquisition and synthesis pathways as potentially critical for pathogen survival.

biochemistry↗

bioRxiv2025

Bacterial and host enzymes modulate the inflammatory response produced by the peptidoglycan of the Lyme disease agent

The spirochete Borrelia burgdorferi causes Lyme disease. In some patients, an excessive, dysregulated proinflammatory immune response can develop in joints leading to persistent arthritis even after antibiotic therapy. In such patients, persistence of antigenic B. burgdorferi peptidoglycan (PGBb) fragments within joint tissues may contribute to immunopathogenesis pre- and post-antibiotic treatment. In live B. burgdorferi cells, the outer membrane shields the polymeric PGBb sacculus from exposure to the immune system. However, unlike most diderm bacteria, B. burgdorferi releases PGBb turnover products into its environment due to the absence of recycling activity. In this study, we identified the released PGBb fragments using a mass spectrometry-based approach. By characterizing the L,D-carboxypeptidase activity of B. burgdorferi protein BB0605 (renamed DacA), we found that PGBb turnover largely occurs at sites of PGBb synthesis. In parallel, we demonstrated that the lytic transglycosylase activity associated with BB0259 (renamed MltS) releases PGBb fragments with 1,6-anhydro bond on their N-acetylmuramyl residues. Stimulation of human cell lines with various synthetic PGBb fragments revealed that 1,6-anhydromuramyl-containing PGBb fragments are poor inducers of a NOD2-dependent immune response relative to their hydrated counterparts found in the polymeric PGBb isolated from dead bacteria. We also showed that the activity of the human N-acetylmuramyl-L-alanine amidase PGLYRP2, which reduces the immunogenicity of PGBb material, is low in joint (synovial) fluids relative to serum. Altogether, our findings suggest that MltS activity helps B. burgdorferi evade PG-based immune detection by NOD2 during growth despite shedding PGBb fragments and that PGBb-induced immunopathology likely results from host sensing of PGBb material from dead (lysed) spirochetes. Additionally, our results suggest the possibility that natural variation in PGLYRP2 activity may contribute to differences in susceptibility to PG-induced inflammation across tissues and individuals. Author summaryDuring bacterial infection, the presence of peptidoglycan- a polymeric element of bacterial cell walls-triggers a host inflammatory response. Although generally protective during acute phases, inflammation, when chronic, can contribute to disease development. Recent work has suggested that the persistence of pro-inflammatory peptidoglycan derived from the Lyme disease spirochete Borrelia burgdorferi in joints may contribute to persistent arthritis in some patients despite appropriate antibiotic therapy. Interestingly, B. burgdorferi sheds peptidoglycan turnover products into the environment during growth. Here, we show that these shed products from live spirochetes are poor effectors of an immune response by the human NOD2 immune receptor due to the formation of an anhydro bond on the N-acetyl-muramic residue during peptidoglycan hydrolysis by a B. burgdorferi lytic transglycosylase. We also show that human N-acetylmuramyl-L-alanine amidase activity, which abrogates the NOD2-dependent response to the immunogenic peptidoglycan isolated from lysed B. burgdorferi cells, is low in human joint fluids relative to serum. Based on our findings, we propose that immunopathogenesis by peptidoglycan material more likely derives from lysed spirochetes (killed by an immune attack or antibiotics) than live ones and that the level of human peptidoglycan hydrolytic enzymes across tissues and individuals influences susceptibility to chronic inflammation.

microbiology↗

bioRxiv2024

Borrelia burgdorferi loses essential genetic elements and cell proliferative potential during stationary phase in culture but not in the tick vector.

The Lyme disease agent Borrelia burgdorferi is a polyploid bacterium with a segmented genome in which both the chromosome and over 20 distinct plasmids are present in multiple copies per cell. This pathogen can survive at least nine months in its tick vector in an apparent dormant state between blood meals, without losing cell proliferative capability when re-exposed to nutrients. Cultivated B. burgdorferi cells grown to stationary phase or resuspended in nutrient-limited media are often used to study the effects of nutrient deprivation. However, a thorough assessment of the spirochetes ability to recover from nutrient depletion has been lacking. Our study shows that starved B. burgdorferi cultures rapidly lose cell proliferative. Loss of genetic elements essential for cell proliferation contributes to the observed proliferative defect in stationary phase. The gradual decline in copies of genetic elements is not perfectly synchronized between chromosomes and plasmids, generating cells that harbor one or more copies of the essential chromosome but lack all copies of one or more non-essential plasmids. This phenomenon likely contributes to the well-documented issue of plasmid loss during in vitro cultivation of B. burgdorferi. In contrast, B. burgdorferi cells from ticks starved for 14 months showed no evidence of reduced cell proliferative ability or plasmid loss. Beyond their practical implications for studying B. burgdorferi, these findings suggest that the midgut of the tick vector offers a unique environment that supports the maintenance of B. burgdorferis segmented genome and cell proliferative potential during periods of tick fasting. ImportanceBorrelia burgdorferi causes Lyme disease, a prevalent tick-borne illness. B. burgdorferi must survive long periods (months to a year) of apparent dormancy in the midgut of the tick vector between blood meals. Resilience to starvation is a common trait among bacteria. However, this study reveals that in laboratory cultures, B. burgdorferi poorly endures starvation and rapidly loses viability. This decline is linked to a gradual loss of genetic elements required for cell proliferation. These results suggest that the persistence of B. burgdorferi in nature is likely shaped more by unique environmental conditions in the midgut of the tick vector than by a general innate ability of this bacterium to endure nutrient deprivation.

microbiology↗

bioRxiv2024

The polyadenylase PAPI is required for virulence plasmid maintenance in pathogenic bacteria

Many species of pathogenic bacteria harbor critical plasmid-encoded virulence factors, and yet the regulation of plasmid replication is often poorly understood despite playing a critical role in plasmid-encoded gene expression. Human pathogenic Yersinia, including the plague agent Y. pestis and its close relative Y. pseudotuberculosis, require the type III secretion system (T3SS) virulence factor to subvert host defense mechanisms and colonize host tissues. The Yersinia T3SS is encoded on the IncFII plasmid for Yersinia virulence (pYV). Several layers of gene regulation enables a large increase in expression of Yersinia T3SS genes at mammalian body temperature. Surprisingly, T3SS expression is also controlled at the level of gene dosage. The number of pYV molecules relative to the number of chromosomes per cell, referred to as plasmid copy number, increases with temperature. The ability to increase and maintain elevated pYV plasmid copy number, and therefore T3SS gene dosage, at 37{degrees}C is important for Yersinia virulence. In addition, pYV is highly stable in Yersinia at all temperatures, despite being dispensable for growth outside the host. Yet how Yersinia reinforces elevated plasmid replication and plasmid stability remains unclear. In this study, we show that the chromosomal gene pcnB encoding the polyadenylase PAP I is required for regulation of pYV plasmid copy number (PCN), maintenance of pYV in the bacterial population outside the host, robust T3SS activity, and Yersinia virulence in a mouse infection model. Likewise, pcnB/PAP I is also required for robust expression of the Shigella flexneri virulence plasmid-encoded T3SS. Furthermore, Yersinia and Shigella pcnB/PAP I is required for maintaining normal PCN of model antimicrobial resistance (AMR) plasmids whose replication is regulated by sRNA, thereby increasing antibiotic resistance by ten-fold. These data suggest that pcnB/PAP I contributes to the spread and stabilization of virulence and AMR plasmids in bacterial pathogens, and is essential in maintaining the gene dosage required to mediate plasmid-encoded traits. Importantly pcnB/PAP I has been bioinformatically identified in many species of bacteria despite being studied in only a few species to date. Our work highlights the potential importance of pcnB/PAP I in antibiotic resistance, and shows for the first time that pcnB/PAP I reinforces PCN and virulence plasmid stability in natural pathogenic hosts with a direct impact on bacterial virulence. Author SummaryMany pathogens carry extrachromosomal DNA elements known as plasmids, which encode genes that confer bacterial virulence or antimicrobial resistance (AMR). Acquisition of these plasmids by bacteria can lead to the emergence of new pathogenic traits and the spread of AMR, yet the mechanisms by which plasmids are retained in bacterial populations particularly in the absence of selective pressure remain incompletely understood. Here we show that the major bacterial polyadenylase enzyme PAP I, encoded by the pcnB gene, is critical for the human pathogen Yersinia pseudotuberculosis to maintain its native virulence plasmid as well as AMR plasmids. Very little is known about the process of polyadenylation in bacteria, or the post-transcriptional addition of adenosine residues to the 3 end of transcripts. This study represents the first demonstration that PAP I-mediated polyadenylation contributes to bacterial pathogenesis.

microbiology↗

bioRxiv2024

FtsN coordinates septal peptidoglycan synthesis and degradation through self-interaction of SPOR in E. coli

The E. coli cell division protein FtsN was proposed to coordinate septal peptidoglycan (sPG) synthesis and degradation to ensure robust cell wall constriction without lethal lesions. Although the precise mechanism remains unclear, previous work highlights the importance of two FtsN domains: the E domain, which interacts with and activates the sPG synthesis complex FtsWIQLB, and the SPOR domain, which binds to denuded glycan (dnG) strands, key intermediates in sPG degradation. Here, we used single-molecule tracking of FtsN and FtsW (a proxy for the sPG synthesis complex FtsWIQLB) to investigate how FtsN coordinates the two opposing processes. We observed dynamic behaviors indicating that FtsNs SPOR domain binds to dnGs cooperatively, which both sequesters the sPG synthesis complex on dnG (termed as the dnG-track) and protects dnGs from degradation by lytic transglycosylases (LTs). The release of the SPOR domain from dnGs leads to activating the sPG synthesis complex on the sPG-track and simultaneously exposing those same dnGs to degradation. Furthermore, FtsNs SPOR domain self-interacts and facilitates the formation of a multimeric sPG synthesis complex on both tracks. The cooperative self-interaction of the SPOR domain creates a sensitive switch to regulate the partitioning of FtsN between the dnG- and sPG-tracks, thereby controlling the balance between sequestered and active populations of the sPG synthesis complex. As such, FtsN coordinates sPG synthesis and degradation in space and time.

microbiology↗

bioRxiv2022

Integration of cell wall synthesis activation and chromosome segregation during cell division in Caulobacter

To divide, bacteria must synthesize and remodel their peptidoglycan (PG) cell wall, a protective meshwork that maintains cell shape. FtsZ, a tubulin homolog, dynamically assembles into a midcell band, recruiting division proteins including the PG synthases FtsW and FtsI. FtsWI are activated to synthesize PG and drive constriction at the appropriate time and place, however their activation pathway remains unresolved. In Caulobacter crescentus, FtsWI activity requires FzlA, an essential FtsZ-binding protein. Through time-lapse imaging and single-molecule tracking of C. crescentus FtsW and FzlA in perturbed genetic backgrounds, we demonstrate that FzlA is a limiting constriction activation factor that converts inactive, fast-moving FtsW to an active, slow-moving state. We find that FzlA interacts with the DNA translocase FtsK, and place FtsK genetically in a pathway with FzlA and FtsWI. Misregulation of the FzlA-FtsK-FtsWI pathway leads to heightened DNA damage and cell death. We propose that FzlA integrates the FtsZ ring, chromosome segregation, and PG synthesis to ensure robust and timely constriction during Caulobacter division.

microbiology↗

bioRxiv2021

FtsN activates septal cell wall synthesis by forming a processive complex with the septum-specific peptidoglycan synthase in E. coli

The FtsN protein of Escherichia coli and other proteobacteria is an essential and highly conserved bitopic membrane protein that triggers the inward synthesis of septal peptidoglycan (sPG) during cell division. Previous work has shown that the activation of sPG synthesis by FtsN involves a series of interactions of FtsN with other divisome proteins and the cell wall. Precisely how FtsN achieves this role is unclear, but a recent study has shown that FtsN promotes the relocation of the essential sPG synthase FtsWI from an FtsZ-associated track (where FtsWI is inactive) to an sPG-track (where FtsWI engages in sPG synthesis). Whether FtsN works by displacing FtsWI from the Z-track or capturing/retaining FtsWI on the sPG-track is not known. Here we use single-molecule imaging and genetic manipulation to investigate the organization and dynamics of FtsN at the septum and how they are coupled to sPG synthesis activity. We found that FtsN exhibits a spatial organization and dynamics distinct from those of the FtsZ-ring. Single FtsN molecules move processively as a single population with a speed of [~] 9 nm s-1, similar to the speed of active FtsWI molecules on the sPG-track, but significantly different from the [~] 30 nm s-1 speed of inactive FtsWI molecules on the FtsZ-track. Furthermore, the processive movement of FtsN is independent of FtsZs treadmilling dynamics but driven exclusively by active sPG synthesis. Importantly, only the essential domain of FtsN, a three-helix bundle in the periplasm, is required to maintain the processive complex containing both FtsWI and FtsN on the sPG-track. We conclude that FtsN activates sPG synthesis by forming a processive synthesis complex with FtsWI exclusively on the sPG-track. These findings favor a model in which FtsN captures or retains FtsWI on the sPG-track rather than one in which FtsN actively displaces FtsWI from the Z-track.

microbiology↗

bioRxiv2019

Treadmilling FtsZ polymers drive the directional movement of sPG-synthesis enzymes via a Brownian ratchet mechanism

FtsZ, a highly conserved bacterial tubulin GTPase homolog, is a central component of the cell division machinery in nearly all walled bacteria. FtsZ polymerizes at the future division site and recruits greater than 30 proteins to assemble into a macromolecular complex termed the divisome. Many of these divisome proteins are involved in septal cell wall peptidoglycan (sPG) synthesis. Recent studies found that FtsZ polymers undergo GTP hydrolysis-coupled treadmilling dynamics along the circumference the division site, driving the processive movement of sPG synthesis enzymes. How FtsZs treadmilling drives the directional transport of sPG enzymes and what its precise role is in bacterial cell division are unknown. Combining theoretical modeling and experimental testing, we show that FtsZs treadmilling drives the directional movement of sPG-synthesis enzymes via a Brownian ratchet mechanism, where the shrinking end of FtsZ polymers introduces an asymmetry to rectify diffusions of single sPG enzymes into persistent end-tracking movement. Furthermore, we show that the processivity of this directional movement is dependent on the binding potential between FtsZ and the enzyme, and hinges on the balance between the enzymes diffusion and FtsZs treadmilling speed. This interplay could provide a mechanism to control the level of available enzymes for active sPG synthesis both in time and space, explaining the distinct roles of FtsZ treadmilling in modulating cell wall constriction rate observed in different bacterial species.

biophysics↗

bioRxiv2019

FtsW exhibits distinct processive movements driven by either septal cell wall synthesis or FtsZ treadmilling in E. coli

During bacterial cell division, synthesis of new septal peptidoglycan (sPG) is crucial for successful cytokinesis and cell pole morphogenesis. FtsW, a SEDS (Shape, Elongation, Division and Sporulation) family protein and an indispensable component of the cell division machinery in all walled bacterial species, was recently identified in vitro as a new monofunctional peptidoglycan glycosyltransferase (PGTase). FtsW and its cognate monofunctional transpeptidase (TPase) class B penicillin binding protein (PBP3 or FtsI in E. coli) may constitute the essential, bifunctional sPG synthase specific for new sPG synthesis. Despite its importance, the septal PGTase activity of FtsW has not been documented in vivo. How its activity is spatiotemporally regulated in vivo has also remained unknown. Here we investigated the septal PGTase activity and dynamics of FtsW in E. coli cells using a combination of single-molecule imaging and genetic manipulations. We show that FtsW exhibits robust activity to incorporate an N-acetylmuramic acid analog at septa in the absence of other known PGTases, confirming FtsW as the essential septum-specific PGTase in vivo. Notably, we identified two populations of processive moving FtsW molecules at septa. A fast-moving population is driven by the treadmilling dynamics of FtsZ and independent of sPG synthesis. A slow-moving population is driven by active sPG synthesis and independent of FtsZs treadmilling dynamics. We further identified that FtsN, a potential sPG synthesis activator, plays an important role in promoting the slow-moving, sPG synthesis-dependent population. Our results support a two-track model, in which inactive sPG synthase molecules follow the fast treadmilling "Z-track" to be distributed along the septum; FtsN promotes their release from the "Z-track" to become active in sPG synthesis on the slow "sPG-track". This model explains how the spatial information is integrated into the regulation of sPG synthesis activity and suggests a new mechanistic framework for the spatiotemporal coordination of bacterial cell wall constriction.

biophysics↗