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Muszyński, Artur

Publications and source records attributed to Muszyński, Artur.

10 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

Rhizobial Secretion of Truncated Exopolysaccharides Severely Impairs the Mesorhizobium-Lotus Symbiosis

The symbiosis between Mesorhizobium japonicum R7A and Lotus japonicus Gifu is an important model system for investigating the role of bacterial exopolysaccharides (EPS) in plant-microbe interactions. Previously, we showed that R7A exoB mutants that are affected at an early stage of EPS synthesis and in lipopolysaccharide (LPS) synthesis induce effective nodules on L. japonicus Gifu after a delay, whereas exoU mutants affected in the biosynthesis of the EPS side chain induce small uninfected nodule primordia and are impaired in infection. The presence of a halo around the exoU mutant when grown on Calcofluor-containing media suggested the mutant secreted a truncated version of R7A EPS. A nonpolar ΔexoA mutant defective in the addition of the first glucose residue to the EPS backbone was also severely impaired symbiotically. Here, we used a suppressor screen to show that the severe symbiotic phenotype of the exoU mutant was due to the secretion of an acetylated pentasaccharide, as both monomers and oligomers, by the same Wzx/Wzy system that transports wild-type exopolysaccharide. We also present evidence that the ΔexoA mutant secretes an oligosaccharide by the same transport system, contributing to its symbiotic phenotype. In contrast, ΔexoYF and polar exoA and exoL mutants have a similar phenotype to exoB mutants, forming effective nodules after a delay. These studies provide substantial evidence that secreted incompatible EPS is perceived by the plant, leading to abrogation of the infection process.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

A phage tail–like bacteriocin suppresses competitors in metapopulations of pathogenic bacteria

Bacteria can repurpose their own bacteriophage viruses (phage) to kill competing bacteria. Phage-derived elements are frequently strain specific in their killing activity, although there is limited evidence that this specificity drives bacterial population dynamics. Here, in this study, we identified intact phage and their derived elements in a metapopulation of wild plant–associated Pseudomonas genomes. We discovered that the most abundant viral cluster encodes a phage remnant resembling a phage tail called a tailocin, which bacteria have co-opted to kill bacterial competitors. Each pathogenic Pseudomonas strain carries one of a few distinct tailocin variants that target the variable polysaccharides in the outer membrane of co-occurring pathogenic Pseudomonas strains. Analysis of herbarium samples from the past 170 years revealed that the same tailocin and bacterial receptor variants have persisted in Pseudomonas populations. These results suggest that tailocin genetic diversity can be mined to develop targeted “tailocin cocktails” for microbial control.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

The multifaceted role of c-di-AMP signaling in the regulation of Porphyromonas gingivalis lipopolysaccharide structure and function

This study unveils the intricate functional association between cyclic di-3’,5’-adenylic acid (c-di-AMP) signaling, cellular bioenergetics, and the regulation of lipopolysaccharide (LPS) profile in Porphyromonas gingivalis, a Gram-negative obligate anaerobe considered as a keystone pathogen involved in the pathogenesis of chronic periodontitis. Previous research has identified variations in P. gingivalis LPS profile as a major virulence factor, yet the underlying mechanism of its modulation has remained elusive. We employed a comprehensive methodological approach, combining two mutants exhibiting varying levels of c-di-AMP compared to the wild type, alongside an optimized analytical methodology that combines conventional mass spectrometry techniques with a novel approach known as FLAT n . We demonstrate that c-di-AMP acts as a metabolic nexus, connecting bioenergetic status to nuanced shifts in fatty acid and glycosyl profiles within P. gingivalis LPS. Notably, the predicted regulator gene cdaR, serving as a potent regulator of c-di-AMP synthesis, was found essential for producing N-acetylgalactosamine and an unidentified glycolipid class associated with the LPS profile. The multifaceted roles of c-di-AMP in bacterial physiology are underscored, emphasizing its significance in orchestrating adaptive responses to stimuli. Furthermore, our findings illuminate the significance of LPS variations and c-di-AMP signaling in determining the biological activities and immunostimulatory potential of P. gingivalis LPS, promoting a pathoadaptive strategy. The study expands the understanding of c-di-AMP pathways in Gram-negative species, laying a foundation for future investigations into the mechanisms governing variations in LPS structure at the molecular level and their implications for host-pathogen interactions.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Low-level resource partitioning supports coexistence among functionally redundant bacteria during successional dynamics

Members of microbial communities can substantially overlap in substrate use. However, what enables functionally redundant microorganisms to coassemble or even stably coexist remains poorly understood. Here, we show that during unstable successional dynamics on complex, natural organic matter, functionally redundant bacteria can coexist by partitioning low-concentration substrates even though they compete for one simple, dominant substrate. We allowed ocean microbial communities to self-assemble on leachates of the brown seaweed Fucus vesiculosus and then analyzed the competition among 10 taxonomically diverse isolates representing two distinct stages of the succession. All, but two isolates, exhibited an average of 90% ± 6% pairwise overlap in resource use, and functional redundancy of isolates from the same assembly stage was higher than that from between assembly stages, leading us to construct a simpler four-isolate community with two isolates from each of the early and late stages. We found that, although the short-term dynamics of the four-isolate communities in F. vesiculosus leachate was dependent on initial isolate ratios, in the long term, the four isolates stably coexist in F. vesiculosus leachate, albeit with some strains at low abundance. We therefore explored the potential for nonredundant substrate use by genomic content analysis and RNA expression patterns. This analysis revealed that the four isolates mainly differed in peripheral metabolic pathways, such as the ability to degrade pyrimidine, leucine, and tyrosine, as well as aromatic substrates. These results highlight the importance of fine-scale differences in metabolic strategies for supporting the frequently observed coexistence of large numbers of rare organisms in natural microbiomes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Impact of Polymicrobial Infection on Fitness of Streptococcus gordonii In Vivo

Pathogenic microbial ecosystems are often polymicrobial, and interbacterial interactions drive emergent properties of these communities. In the oral cavity, Streptococcus gordonii is a foundational species in the development of plaque biofilms, which can contribute to periodontal disease and, after gaining access to the bloodstream, target remote sites such as heart valves. Here, we used a transposon sequencing (Tn-Seq) library of S. gordonii to identify genes that influence fitness in a murine abscess model, both as a monoinfection and as a coinfection with an oral partner species, Porphyromonas gingivalis. In the context of a monoinfection, conditionally essential genes were widely distributed among functional pathways. Coinfection with P. gingivalis almost completely changed the nature of in vivo gene essentiality. Community-dependent essential (CoDE) genes under the coinfection condition were primarily related to DNA replication, transcription, and translation, indicating that robust growth and replication are required to survive with P. gingivalis in vivo. Interestingly, a group of genes in an operon encoding streptococcal receptor polysaccharide (RPS) were associated with decreased fitness of S. gordonii in a coinfection with P. gingivalis. Individual deletion of two of these genes (SGO_2020 and SGO_2024) resulted in the loss of RPS production by S. gordonii and increased susceptibility to killing by neutrophils. P. gingivalis protected the RPS mutants by inhibiting neutrophil recruitment, degranulation, and neutrophil extracellular trap (NET) formation. These results provide insight into genes and functions that are important for S. gordonii survival in vivo and the nature of polymicrobial synergy with P. gingivalis. Furthermore, we show that RPS-mediated immune protection in S. gordonii is dispensable and detrimental in the presence of a synergistic partner species that can interfere with neutrophil killing mechanisms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Molecular basis for polysaccharide recognition and modulated ATP hydrolysis by the O antigen ABC transporter

O antigens are ubiquitous protective extensions of lipopolysaccharides in the extracellular leaflet of the Gram-negative outer membrane. Following biosynthesis in the cytosol, the lipid-linked polysaccharide is transported to the periplasm by the WzmWzt ABC transporter. Often, O antigen secretion requires the chemical modification of its elongating terminus, which the transporter recognizes via a carbohydrate-binding domain (CBD). Here, using components from A. aeolicus, we identify the O antigen structure with methylated mannose or rhamnose as its cap. Crystal and cryo electron microscopy structures reveal how WzmWzt recognizes this cap between its carbohydrate and nucleotide-binding domains in a nucleotide-free state. ATP binding induces drastic conformational changes of its CBD, terminating interactions with the O antigen. ATPase assays and site directed mutagenesis reveal reduced hydrolytic activity upon O antigen binding, likely to facilitate polymer loading into the ABC transporter. Our results elucidate critical steps in the recognition and translocation of polysaccharides by ABC transporters.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Differentiation and classification of bacterial endotoxins based on surface enhanced Raman scattering and advanced machine learning

Bacterial endotoxin, a major component of the Gram-negative bacterial outer membrane leaflet, is a lipopolysaccharide shed from bacteria during their growth and infection and can be utilized as a biomarker for bacterial detection. Here, the surface enhanced Raman scattering (SERS) spectra of eleven bacterial endotoxins with an average detection amount of 8.75 pg per measurement have been obtained based on silver nanorod array substrates, and the characteristic SERS peaks have been identified. With appropriate spectral pre-processing procedures, different classical machine learning algorithms, including support vector machine, k-nearest neighbor, random forest, etc., and a modified deep learning algorithm, RamanNet, have been applied to differentiate and classify these endotoxins. It has been found that most conventional machine learning algorithms can attain a differentiation accuracy of >99%, while RamanNet can achieve 100% accuracy. Such an approach has the potential for precise classification of endotoxins and could be used for rapid medical diagnoses and therapeutic decisions for pathogenic infections.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

Kinetic proofreading of lipochitooligosaccharides determines signal activation of symbiotic plant receptors

Plant cell surface receptors perceive carbohydrate signaling molecules and hereby establish communication with surrounding microbes. Genetic studies have identified two different classes of lysin motif receptor kinases as gatekeepers that together trigger the symbiotic pathway in plants; however, no structural or functional data of the perception mechanisms switching these receptors from resting state into activation is known. In this study, we use structural biology, biochemical, and genetic approaches to demonstrate how the NFP/NFR5 class of lipochitooligosaccharide (LCO) receptors discriminate bacterial symbionts based on a kinetic proofreading mechanism that controls receptor activation and signaling specificity. We show that the LCO binding site can be engineered to support symbiotic functions, which greatly advance future opportunities for receptor engineering in legumes and nonlegumes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Structure of Lipopolysaccharide from Liberibacter crescens Is Low Molecular Weight and Offers Insight into Candidatus Liberibacter Biology

Huanglongbing (HLB) disease, also known as citrus greening disease, was first reported in the US in 2005. Since then, the disease has decimated the citrus industry in Florida, resulting in billions of dollars in crop losses and the destruction of thousands of acres of citrus groves. The causative agent of citrus greening disease is the phloem limited pathogen Candidatus Liberibacter asiaticus. As it has not been cultured, very little is known about the structural biology of the organism. Liberibacter are part of the Rhizobiaceae family, which includes nitrogen-fixing symbionts of legumes as well as the Agrobacterium plant pathogens. To better understand the Liberibacter genus, a closely related culturable bacterium (Liberibacter crescens or Lcr) has attracted attention as a model organism for structural and functional genomics of Liberibacters. Given that the structure of lipopolysaccharides (LPS) from Gram-negative bacteria plays a crucial role in mediating host-pathogen interactions, we sought to characterize the LPS from Lcr. We found that the major lipid A component of the LPS consisted of a pentaacylated molecule with a β-6-GlcN disaccharide backbone lacking phosphate. The polysaccharide portion of the LPS was unusual compared to previously described members of the Rhizobiaceae family in that it contained ribofuranosyl residues. The LPS structure presented here allows us to extrapolate known LPS structure/function relationships to members of the Liberibacter genus which cannot yet be cultured. It also offers insights into the biology of the organism and how they manage to effectively attack citrus trees.

59 BASIC BIOLOGICAL SCIENCES↗