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Exogenous fatty acid (eFA) activation and utilization play key roles in bacterial physiology and confer growth advantages by bypassing the need to make fatty acids for lipid synthesis. In Gram-positive bacteria, eFA activation and utilization is generally carried out by the fatty acid kinase (FakAB) two-component system that converts eFA to acyl phosphate, and the acyl-ACP:phosphate transacylase (PlsX) that catalyzes the reversible conversion of acyl phosphate to acyl–acyl carrier protein. Acyl–acyl carrier protein is a soluble format of the fatty acid that is compatible with cellular metabolic enzymes and can feed multiple processes including the fatty acid biosynthesis pathway. The combination of FakAB and PlsX enables the bacteria to channel eFA nutrients. These key enzymes are peripheral membrane interfacial proteins that associate with the membrane through amphipathic helices and hydrophobic loops. In this review, we discuss the biochemical and biophysical advances that have established the structural features that drive FakB or PlsX association with the membrane, and how these protein–lipid interactions contribute to enzyme catalysis.

Commensal gut bacteria use oleate hydratase to release a spectrum of hydroxylated fatty acids using host-derived unsaturated fatty acids. These compounds are thought to attenuate the immune response, but the underlying signaling mechanism(s) remain to be established. The pathogen Staphylococcus aureus also expresses an oleate hydratase and 10-hydroxyoctadecanoic acid ( h 18:0) is the most abundant oleate hydratase metabolite found at Staphylococcal skin infection sites. Here, we show h 18:0 stimulates the transcription of a set of lipid metabolism genes associated with the activation of peroxisome proliferator activated receptor (PPAR) in the RAW 264.7 macrophage cell line and mouse primary bone marrow-derived macrophages. Cell-based transcriptional reporter assays show h 18:0 selectively activates PPARα. Radiolabeling experiments with bone marrow-derived macrophages show [1- 14 C] h 18:0 is not incorporated into cellular lipids, but is degraded by β-oxidation, and mass spectrometry detected shortened fragments of h 18:0 released into the media. The catabolism of h 18:0 was >10-fold lower in bone marrow-derived macrophages isolated from Ppara −/− knockout mice, and we recover 74-fold fewer S. aureus cells from the skin infection site of Ppara −/− knockout mice compared to wildtype mice. These data identify PPARα as a target for oleate hydratase-derived hydroxy fatty acids and support the existence of an oleate hydratase-PPARα signaling axis that functions to suppress the innate immune response to S. aureus .

Microgravity conditions have been used to improve protein crystallization from the early 1980s using advanced crystallization apparatuses and methods. Early microgravity crystallization experiments confirmed that minimal convection and a sedimentation-free environment is beneficial for growth of crystals with higher internal order and in some cases, larger volume. It was however realized that crystal growth in microgravity requires additional time due to slower growth rates. The progress in space research via the International Space Station (ISS) provides a laboratory-like environment to perform convection-free crystallization experiments for an extended time. To obtain detailed insights in macromolecular transport phenomena under microgravity and the assumed reduction of unfavorable impurity incorporation in growing crystals, microgravity and unit gravity control experiments for three different proteins were designed. To determine the quantity of impurity incorporated into crystals, fluorescence-tagged aggregates of the proteins (acting as impurities) were prepared. The recorded fluorescence intensities of the respective crystals reveal reduction in the incorporation of aggregates under microgravity for different aggregate quantities. The experiments and data obtained, provide insights about macromolecular transport in relation to molecular weight of the target proteins, as well as information about associated diffusion behavior and crystal lattice formation. Results suggest one explanation why microgravity-grown protein crystals often exhibit higher quality. Furthermore, results from these experiments can be used to predict which proteins may benefit more from microgravity crystallization.

The Toxin Complex (Tc) superfamily consists of toxin translocases that contribute to the targeting, delivery, and cytotoxicity of certain pathogenic Gram-negative bacteria. Membrane receptor targeting is driven by the A-subunit (TcA), which comprises IgG-like receptor binding domains (RBDs) at the surface. To better understand XptA2, an insect specific TcA secreted by the symbiont X. nematophilus from the intestine of entomopathogenic nematodes, we determined structures by X-ray crystallography and cryo-EM. Contrary to a previous report, XptA2 is pentameric. RBD-B exhibits an indentation from crystal packing that indicates loose association with the shell and a hotspot for possible receptor binding or a trigger for conformational dynamics. A two-fragment XptA2 lacking an intact linker achieved the folded pre-pore state like wild type (wt), revealing no requirement of the linker for protein folding. The linker is disordered in all structures, and we propose it plays a role in dynamics downstream of the initial pre-pore state.

Profiles of human nasal colonization consistently demonstrate that and can co-exist in the nasopharynx. Several studies have demonstrated the antagonist relationship between the two organisms via several molecular mechanisms including competition for nutrients as well as via direct killing by hydrogen peroxide. During nasal colonization, the pneumococcus is in direct contact with the fatty acid 18:0, which is released into the extracellular environment by . We report that 18:0 is specifically toxic to the pneumococcus amongst the pathogenic streptococci, providing a unique mechanism for interspecies competition during colonization. Exposure of cells to 18:0 revealed that could rapidly adapt to and overcome the observed toxicity. Whole genome analysis revealed the mechanism underlying this resistance being linked to a truncation of a glycosyltransferase in the capsule biosynthesis locus and a genomic inversion in the phase variation locus, leading to altered cell surface charge and membrane lipid composition. These physiological differences in the resistant isolates may aid in repelling toxic, charged fatty acids such as 18:0 from the cell membrane. The pneumococcus and are two of the most well-characterized residents of the human nasopharynx; yet much remains unknown regarding how the two bacteria interact. Here, we describe the potential of produced 18:0, whose function and biological impact are still being described, to act as an inter-species competition molecule against , and how the pneumococcus can adapt to overcome its toxicity.

In Staphylococcus aureus, the branched-chain amino acid biosynthetic pathway provides essential intermediates for membrane biosynthesis. Threonine deaminase (IlvA) is the first enzyme in the pathway, and isoleucine feedback-regulates the enzyme in Escherichia coli. These studies on E. coli IlvA (EcIlvA) introduced the concept of allosteric regulation. To investigate the regulation of S. aureus IlvA (SaIlvA), we first conducted additional studies on EcIlvA. The previously determined crystal structure of EcIlvA revealed a tetrameric assembly of protomers, each with catalytic and regulatory domains, but the structural basis of isoleucine regulation was not characterized. Here, we present the crystal structure of the EcIlvA regulatory domain bound to isoleucine, which reveals the isoleucine binding site and conformational changes that initiate at Phe352 and propagate 23 Angstrom across the domain. This suggests an allosteric pathway that extends to the active site of the adjacent protomer, mediating regulation across the protomer-protomer interface. The EcIlvA(F352A) mutant binds isoleucine but is feedback-resistant due to the absence of the initiating Phe352. In contrast, SaIlvA is not feedback-regulated by isoleucine and does not bind it. The structure of the SaIlvA regulatory domain reveals a different organization that lacks the isoleucine binding site. Other potential allosteric inhibitors of SaIlvA, including phospholipid intermediates, do not affect enzyme activity. We propose that the absence of feedback inhibition in SaIlvA is due to its role in membrane biosynthesis. These findings enhance our understanding of IlvA's allosteric regulation and offer opportunities for engineering feedback-resistant IlvA variants for biotechnological use.

Bacterial vaginosis (BV), a common syndrome characterized by -deficient vaginal microbiota, is associated with adverse health outcomes. BV often recurs after standard antibiotic therapy in part because antibiotics promote microbiota dominance by instead of , which has more beneficial health associations. Strategies to promote and inhibit are thus needed. We show that oleic acid (OA) and similar long-chain fatty acids simultaneously inhibit and enhance growth. These phenotypes require OA-inducible genes conserved in and related species, including an oleate hydratase ( ) and putative fatty acid efflux pump ( ). FarE mediates OA resistance, while OhyA is robustly active in the human vaginal microbiota and sequesters OA in a derivative form that only -harboring organisms can exploit. Finally, OA promotes dominance more effectively than antibiotics in an model of BV, suggesting a novel approach for treatment.

The largest market sector of the global antibiotics industry is on the verge of becoming obsolete because the incidence of ?-lactam antibiotic resistance in the clinic continues to rise. Therefore, we are in dire need of new therapeutics to address the increasing threat of antibiotic resistance. Novel targets that could lead to a new drug class are ABC (ATP-binding cassette) importers, which are only found in bacteria. The substrate-binding protein (SBP) components of these transporters present an intriguing subject of study because of their abundance in the cell and potential roles in infection. As a contribution to the scholarship of SBP structural biology, this dissertation presents atomic structures of two SBPs involved in iron transport as well as an innovative method for obtaining the apo (substrate-free) forms of SBPs that directly bind metal atoms. Using a biophysical approach including X-ray fluorescence and anomalous X-ray scattering, I demonstrate how YfeA, a polyspecific SBP and Yersinia pestis virulence factor, can use a single canonical substrate-binding site to bind iron, manganese, or zinc atoms. Using a combination of structural analyses and substrate docking simulations, I argue that apo YiuA, a Y. pestis SBP with unknown substrate, is designed to bind a chelate complex and may bind multiple xenosiderophores. In addition, I present evidence that cell fractionation can successfully extract apo YfeA from the periplasm of Escherichia coli when YfeA is exposed to the Yfe transporter. Time-resolved crystallographic experiments reveal that apo YfeA can spontaneously revert to the holo (substrate-bound) YfeA form while still crystallized. Based on the structural changes that occur between apo and holo forms, I argue that YfeA uses a trap door mechanism for substrate transfer to the Yfe transporter. This study simultaneously elucidates a new fundamental mechanism (trap door mechanism) that is distinct from the standard (Venus flytrap mechanism) that currently describes SBP substrate transfer, a method for obtaining apo Cluster A-1 SBPs without artificial intervention such as mutagenesis, partial denaturation, or chelation, and expansion of the SBP structural biology knowledge base.

Genome-wide association studies (GWAS) and fine-mapping efforts to date have identified more than 100 prostate cancer (PrCa)-susceptibility loci. We meta-analyzed genotype data from a custom high-density array of 46,939 PrCa cases and 27,910 controls of European ancestry with previously genotyped data of 32,255 PrCa cases and 33,202 controls of European ancestry. Our analysis identified 62 novel loci associated ( P  < 5.0 × 10 −8 ) with PrCa and one locus significantly associated with early-onset PrCa (≤55 years). Our findings include missense variants rs1800057 (odds ratio (OR) = 1.16; P  = 8.2 × 10 −9 ; G>C, p.Pro1054Arg) in ATM and rs2066827 (OR = 1.06; P  = 2.3 × 10 −9 ; T>G, p.Val109Gly) in CDKN1B . The combination of all loci captured 28.4% of the PrCa familial relative risk, and a polygenic risk score conferred an elevated PrCa risk for men in the ninetieth to ninety-ninth percentiles (relative risk = 2.69; 95% confidence interval (CI): 2.55–2.82) and first percentile (relative risk = 5.71; 95% CI: 5.04–6.48) risk stratum compared with the population average. These findings improve risk prediction, enhance fine-mapping, and provide insight into the underlying biology of PrCa 1 . A large meta-analysis combining genome-wide and custom high-density genotyping array data identifies 63 new susceptibility loci for prostate cancer, enhancing fine-mapping efforts and providing insights into the underlying biology.

The reasons why some individuals have severe neuropathy following an infection are not known. Through the agnostic screening of children with acute axonal neuropathy after an infection, we identified several families with biallelic variants in RCC1. We aimed to describe the clinical phenotype of these patients, and the molecular and cellular pathology associated with the genetic variants identified in these families. For this case series, we identified children affected by a severe, acute-onset axonal neuropathy following infection through an international research consortium of paediatric neurologists and clinical geneticists from nine countries (Canada, Cyprus, Czechia, Germany, Iran, Saudi Arabia, Slovakia, Türkiye, and the UK). Clinical assessments included nerve conduction studies and neuroimaging. We did exome or genome sequencing in DNA samples from all patients. We characterised the proteins encoded by the genetic variants by use of thermal stability and enzymatic assays, using recombinantly expressed proteins. We assessed cellular protein transport under heat or oxidative stress by use of immunofluorescence in primary fibroblasts, obtained from patients. We generated a humanised Drosophila knock-in model to assess the effects of stress on the in vivo function of RCC1. Between Nov 2, 2011, and July 10, 2024, we identified 24 individuals from 12 families who had severe, acute-onset axonal neuropathy following infection (13 female and 11 male patients, with a mean age at diagnosis of 1 year 10 months [SD 2·27]). Eight biallelic missense variants in RCC1 were identified in affected individuals with autosomal recessive inheritance. Patients had variable phenotypes, ranging from rapidly progressive fatal axonal neuropathy to mild motor neuropathy with impaired walking. Neurological presentation was often secondary to an infection, resulting in initial misdiagnoses of Guillain-Barré syndrome in several patients. 15 children had disease recurrence. The disease was fatal in 15 patients. The RCC1 variants in these patients code for proteins that alter GDP-to-GTP exchange activity and have reduced thermal stability in vitro. In primary fibroblasts, heat shock or oxidative stress revealed defects in Ran nuclear localisation and impaired nucleocytoplasmic transport. A Drosophila model of the disease revealed a fatal intolerance to oxidative stress. We describe an autosomal recessive, acute-onset paediatric axonal neuropathy, seemingly triggered by infection, that affects individuals with biallelic RCC1 variants. In these children, the disease can mimic Guillain-Barré syndrome. The pathological mechanisms underlying this novel axonal neuropathy might overlap with those of amyotrophic lateral sclerosis. Cellular studies indicate that RCC1 variants affect nucleocytoplasmic transport, which is crucial for healthy axonal function. Future studies should be directed at pre-symptomatic treatment by exploring ways to maintain nucleocytoplasmic transport. National Institute for Health and Care Research, LifeArc, and Wellcome Trust.