Outer Membrane Research Articles

A dynamical systems model for the total fission rate in Drp1-dependent mitochondrial fission

Mitochondrial hyperfission in response to cellular insult is associated with reduced energy production and programmed cell death. Thus, there is a critical need to understand the molecular mechanisms coordinating and regulating the complex process of mitochondrial fission. We develop a nonlinear dynamical systems model of dynamin related protein one (Drp1)-dependent mitochondrial fission and use it to identify parameters which can regulate the total fission rate (TFR) as a function of time. The TFR defined from a nondimensionalization of the model undergoes a Hopf bifurcation with bifurcation parameter μ = k + M k - where M is the total concentration of mitochondrial fission factor (Mff) and k+ and k− are the association and dissociation rate constants between oligomers on the outer mitochondrial membrane. The variable μ can be thought of as the maximum build rate over the disassembling rate of oligomers. Though the nondimensionalization of the system results in four dimensionless parameters, we found the TFR and the cumulative total fission (TF) depend strongly on only one, μ. Interestingly, the cumulative TF does not monotonically increase as μ increases. Instead it increases with μ to a certain point and then begins to decrease as μ continues to increase. This non-monotone dependence on μ suggests interventions targeting k+, k−, or M may have a non-intuitive impact on the fission mechanism. Thus understanding the impact of regulatory parameters, such as μ, may assist future therapeutic target selection.

Abstract C003: Outer membrane vesicles from Bacteroides fragilis contain coding and non-coding small RNA species that modulate inflammatory signaling in intestinal epithelial cells

Abstract Alterations to the community structure and function of the microbiome are associated with changes to host physiology, including immune responses. However, the contribution of microbial-derived RNAs carried by outer membrane vesicles (OMVs) to host immune responses remains unclear. This paper investigates the role of OMVs and OMV-associated small RNA (sRNA) species from pathogenic and commensal Bacteroides fragilis (ETBF and NTBF respectively) in eliciting different immune responses in intestinal epithelial cells. To distinguish the differences in the sRNA profiles of both strains and their OMVs, small RNA-seq was conducted, and identified enrichment of discrete sRNA species in OMVs that were also differentially expressed between the two strains. To understand the effects of OMVs on pattern recognition receptors, Toll-like receptor (TLR) reporter cells were treated with OMVs, and activation of TLRs 2, 3, 4, and 7 were observed. Treatment of Caco-2 and HT29-MTX cells with OMVs demonstrated increased expression of IL-8. Surprisingly, we discovered that degradation of RNase-accessible RNAs within ETBF OMVs, but not NTBF OMVs, resulted in vesicles with enhanced capacity to stimulate IL-8 expression, indicating that these extravesicular RNAs exert an immunosuppressive effect. This suggests a dual role for OMV-associated RNAs in modulating host immune responses, with implications for both bacterial pathogenesis and therapeutic applications. Citation Format: Aadil Sheikh, Colin Scano, Julia Xu, Tolulope Ojo, Jessica M. Conforti, Kayla L. Haberman, Bryan King, James Lotter, Alysia Martinez, Harry Ojeas, Michelle Pujol, Juli Watkins, Emily Lin, Bernd Zechmann, Amanda Sevcik, Christie Sayes, Elyssia S Gallagher, Joshua Mell, Garth Ehrlich, Joseph H Taube, K. Leigh Greathouse. Outer membrane vesicles from Bacteroides fragilis contain coding and non-coding small RNA species that modulate inflammatory signaling in intestinal epithelial cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr C003.

Mitochondrial dynamics and metabolic remodeling in xenograft of IPSC-derived human neural precursors

It is well recognized that the regulation of mitochondrial functions affects the differentiation and maturation of neurons. The study of these processes is of both fundamental and practical importance for regenerative neurobiology. Aim of the study: to characterize the mitochondrial fission changes and their relation to the activation of oxidative phosphorylation (metabolic shift) during maturation of human IPSC-derived neural precursors grafted into rat striatum. Wistar rats (n = 15) were unilaterally injected into the caudate nucleus with neural precursors derived from human IPSCs. Changes in localization and expression of neuronal differentiation markers: nestin, NeuN, neuronal enolase, as well as mitochondrial outer membrane protein, ATP synthase and mitochondrial fission protein Drp1 were assessed by immunostaining. Measurements were performed on graft cells 2 weeks, 3 and 6 months after surgery. Maturation of grafted neurons was associated with fluctuations morphometric parameters of the mitochondrial fraction and Drp1 levels. Increased mitochondrial fission was detected 3 months after transplantation, before an increase in ATP synthase staining by 6th month and a switch of transplanted cells to oxidative phosphorylation. The conducted experiment demonstrated a link between mitochondrial dynamics and changes in the metabolic profile and maturation of transplanted neurons. The regulation of mitochondrial dynamics may have future implications for developing methods to improve the integration of transplanted neurons into recepient brain structures.

The Salmonella enterica EnvE is an Outer Membrane Lipoprotein and Its Gene Expression Leads to Transcriptional Repression of the Virulence Gene msgA.

The envE gene of Salmonella enterica serovar Typhimurium is encoded within Salmonella Pathogenicity Island-11 (SPI-11) and is located immediately upstream of the virulence gene msgA (macrophage survival gene A) in the same transcriptional orientation. To date, the characteristics and roles of envE remain largely unexplored. In this study, we show that EnvE, a predicted lipoprotein, is localized on the outer membrane using sucrose gradient ultracentrifugation. Under oxidative stress conditions, envE transcription is suppressed, while msgA transcription is induced, indicating an inverse correlation between the mRNA levels of the two neighboring genes. Importantly, inactivation of envE leads to constitutive transcription of msgA regardless of the presence of oxidative stress. Moreover, trans-complementation of the envE mutant with a plasmid-borne envE fails to prevent the induction of msgA transcription, suggesting that envE functions as a cis-regulatory element rather than a trans-acting factor. We further show that both inactivation and complementation of envE confer wild-type levels of resistance to oxidative stress by ensuring the expression of msgA. Our data suggest that the S. enterica envE gene encodes an outer membrane lipoprotein, and its transcription represses msgA expression in a cis-acting manner, probably by transcriptional interference, although the exact molecular details are yet unclear.

The impact of varying sizes of silver nanoparticles on the induction of cellular damage in Klebsiella pneumoniae involving diverse mechanisms

Abstract Silver nanoparticles (AgNPs) are extensively studied as potent antibacterial agents targeting antibiotic-resistant pathogens. Cellular damage induced through various mechanisms that can affect multiple cell components like the outer membrane, enzymes, and proteins is closely linked to their chemical and morphological characteristics. We investigated the impact of AgNPs’ size on their antibacterial effectiveness using two differently sized nanoparticles: silver nanoparticle-Citrus limon (AgCL) with an average size of 21 nm and silver nanoparticle-Citrus sinensis (AgCS) with an average size of 13 nm, derived from C. limon and C. sinensis through environmentally friendly methods. The study evaluated their antibacterial effects by assessing morphology changes via scanning electron microscopy, metabolic alterations using Fourier transform infrared (FT-IR) spectroscopy, and oxidative stress responses through biochemical markers in Klebsiella pneumoniae cells exposed to AgNPs. The results showed that both AgCL and AgCS exhibited remarkable antibacterial activity, evidenced by inhibition zones of 14 ± 1.5 and 16 ± 1.0 mm, respectively. Morphological changes in K. pneumoniae cells treated with AgNPs were size dependent, with notable alterations noted. FT-IR spectroscopy revealed size and concentration-dependent biochemical changes, particularly in shifts in functional groups involved in the fluidity of cell wall lipid, and protein structure. Exposure to AgNPs led to increased oxidative stress markers like lipid peroxides and reduced levels of enzymatic and non-enzymatic antioxidants, more prominently observed with smaller AgCS nanoparticles (13 nm). AgNPs induce oxidative stress and morphological changes in K. pneumoniae strains, with smaller nanoparticles demonstrating greater efficacy. These findings underscore the importance of nanoparticle size in optimizing the antibacterial properties against pathogens.

Tackling immunosuppression by Neisseria gonorrhoeae to facilitate vaccine design.

Gonorrhoea, caused by Neisseria gonorrhoeae, is a common sexually transmitted infection. Increasing multi-drug resistance and the impact of asymptomatic infections on sexual and reproductive health underline the need for an effective gonococcal vaccine. Outer membrane vesicles (OMVs) from Neisseria meningitidis induce modest cross-protection against gonococcal infection. However, the presence of proteins in OMVs derived from N. gonorrhoeae that manipulate immune responses could hamper their success as a vaccine. Here we modified two key immunomodulatory proteins of the gonococcus; RmpM, which can elicit 'blocking antibodies', and PorB, an outer membrane porin which contributes to immunosuppression. As meningococcal PorB has adjuvant properties, we replaced gonococcal PorB with a meningococcal PorB. Immunisation with OMVs from N. gonorrhoeae lacking rmpM and expressing meningococcal porB elicited higher antibody titres against model antigens in mice compared to OMVs with native PorB. Further, a gonococcal protein microarray revealed stronger IgG antibody responses to a more diverse range of antigens in the Nm PorB OMV immunised group. Finally, meningococcal PorB OMVs resulted in a Th1-skewed response, exemplified by increased serum IgG2a antibody responses and increased IFNɣ production by splenocytes from immunised mice. In summary, we demonstrate that the replacement of PorB in gonococcal OMVs enhances immune responses and offers a strategy for gonococcal vaccine development.

Refolding dynamics and immunoinformatic insights into Vibrio cholerae OmpA, OmpK, and OmpV for vaccine applications.

OmpA, OmpK, and OmpV are crucial for the pathogenesis of Vibrio cholerae, functioning within the bacterium's outer membrane; they present significant potential as candidates for vaccine development. Due to their intrinsic β-sheet richness, these OMPs tend to form inclusion bodies whenever overexpression is attempted. To achieve a native-like structure, detergents can be utilized during the refolding of OMPs from inclusion bodies. The impact of different detergents is examined on the renaturation of these OMPs, specifically non-ionic and zwitterionic detergents. The findings provide valuable insights into detergent selection, with LDAO and DDM emerging as the best protein refolding agents, facilitating successful structural and functional studies of these OMPs. Furthermore, using immunoinformatics it is established that OmpA, OmpK, and OmpV carry B- and T-cell epitopes in their exposed extracellular regions. The presence of immunodominant regions makes it easier to employ these proteins as vaccine candidates as they are stable, non-allergenic, and likely to stimulate successful innate and active immune responses. Overall, with all three OMPs harboring numerous immunogenic epitopes, they can be employed in subunit vaccines against Vibrio spp. and contribute to the development of diagnostic tools for effective disease mitigation.

AKAP1/PKA-mediated GRP75 phosphorylation at mitochondria-associated endoplasmic reticulum membranes protects cancer cells against ferroptosis.

Emerging evidence suggests that signaling pathways can be spatially regulated to ensure rapid and efficient responses to dynamically changing local cues. Ferroptosis is a recently defined form of lipid peroxidation-driven cell death. Although the molecular mechanisms underlying ferroptosis are emerging, spatial aspects of its signaling remain largely unexplored. By analyzing a public database, we found that a mitochondrial chaperone protein, glucose-regulated protein 75 (GRP75), may have a previously undefined role in regulating ferroptosis. This was subsequently validated. Interestingly, under ferroptotic conditions, GRP75 translocated from mitochondria to mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) and the cytosol. Further mechanistic studies revealed a highly spatial regulation of GRP75-mediated antiferroptotic signaling. Under ferroptotic conditions, lipid peroxidation predominantly accumulated at the ER, which activated protein kinase A (PKA) in a cAMP-dependent manner. In particular, a signaling microdomain, the outer mitochondrial membrane protein A-kinase anchor protein 1 (AKAP1)-anchored PKA, phosphorylated GRP75 at S148 in MAMs. This caused GRP75 to be sequestered outside the mitochondria, where it competed with Nrf2 for Keap1 binding through a conserved high-affinity RGD-binding motif, ETGE. Nrf2 was then stabilized and activated, leading to the transcriptional activation of a panel of antiferroptotic genes. Blockade of the PKA/GRP75 axis dramatically increased the responses of cancer cells to ferroptosis both in vivo and in vitro. Our identification a localized signaling cascade involved in protecting cancer cells from ferroptosis broadens our understanding of cellular defense mechanisms against ferroptosis and also provides a new target axis (AKAP1/PKA/GRP75) to improve the responses of cancer cells to ferroptosis.

Epitope Analysis of Hypothetical Proteins in Leptospira interrogans Serovar Lai Reveals Potential Diagnostic Markers

Leptospirosis is a neglected zoonosis caused by a pathogenic spirochete, Leptospira interrogans. The mode of infection in humans is through an abrasion in human skin or the conjunctiva and mucous membrane. Infected patients usually show different symptoms resembling bacterial or viral infections such as the flu. Hence, diagnosing leptospirosis in the early stage is complex, and can be easily confused with other infections. A strategical pathway was developed to analyze the hypothetical proteins in L. interrogans and unveil their potential as diagnostic markers. Subcellular localization tools such as PSORTb, CELLO, SOSUI-GramN, and ProtCompB were used to segregate the outer membrane and surface proteins from the overall pool of hypothetical proteins. The shortlisted proteins were checked for their virulency, and antigenicity through tools such as VirulentPred, and VaxiJen, respectively. Proteins with the highest scores were fed into ElliPro which predicted both linear and discontinuous epitopes in each protein. Proteins with many epitopes were further analyzed with BepiPred 3.0, which provided the epitope probability for each protein’s amino acid. Epitope probability of the potential proteins was compared with the standard diagnostic marker, LipL32. The comparison revealed that a protein (UniProt ID D4YW28) has better immunogenic potential than the gold standard marker, LipL32. In conclusion, this protein can be used as a diagnostic marker for the detection of leptospirosis and it will also serve as a better vaccine candidate.

Overexpression of outer membrane protein A (OmpA) increases aminoglycoside sensitivity in mycobacteria.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb) complex infection, is a leading cause of death worldwide from a single infectious agent. The emergence of drug resistance Mtb clinical strains makes the situation more serious. The role of Mtb outer membrane protein A (OmpA) in antimicrobial resistance remains unclear. This study aimed to evaluate the effect of OmpA expression on mycobacterial drug resistance. In this study, a Mycobacterium smegmatis (Ms) strain overexpressing OmpA (Ms-OmpA) and a Mycobacterium bovis (Mb) strain overexpressing OmpA (Mb-OmpA) were constructed, and their susceptibility to anti-TB drugs was determined by performing the minimal inhibitory concentrations (MICs), the plate assay and the macrophage infection assays. The streptomycin MIC of the overexpressing strain was 2-fold lower than those of the wide-type (Ms) and empty plasmid strains (pMV-261) as well as amikacin and gentamicin. Moreover, both the plate and the macrophage infection assays indicate that overexpression of OmpA increases streptomycin sensitivity in Mycobacteria. The other aminoglycosides like amikacin and gentamicin have the same phenotypes as streptomycin on the plates for the virulent strain Mb-OmpA. The porin inhibitor spermidine can increase streptomycin tolerance in the overexpressing strain, and overexpressing OmpA can increase the intracellular accumulation of hydrophilic ethidium bromide, which indicates that porin protein OmpA contributes to aminoglycosides sensitivity in Mycobacteria. In this study, we have characterized the contribution of OmpA in the antimicrobial resistance phenotype of Mycobacteria, which may provide valuable insights for understanding antibiotic resistance and designing new strategies for TB treatment.

Tail-specific protease is an essential Chlamydia virulence factor that mediates the differentiation of elementary bodies into reticulate bodies.

Tail-specific proteases (Tsp) are members of a widely distributed family of serine proteases that commonly target and process periplasmic proteins in Gram-negative bacteria. The obligately intracellular, Gram-negative Chlamydia encode a highly conserved Tsp homolog whose target and function are unclear. We identified a Chlamydia muridarum mutant with a nonsense mutation in tsp. Differentiation of the tsp mutant elementary bodies into vegetative reticulate bodies was delayed at 37°C and completely blocked at 40°C. Tsp localized to C. muridarum cells but was not detected outside the inclusion, suggesting that it targets chlamydial rather than host proteins. The abundance of key chlamydia outer membrane complex and virulence-related proteins differed in wild-type and tsp mutant elementary bodies, consistent with the possibility that Tsp regulates developmental cycle progression. The altered abundances of chlamydial structural and virulence factors could explain why the mutant, but not an isogenic recombinant with wild-type tsp, was highly attenuated in a mouse intravaginal infection model. Thus, chlamydial Tsp is required for timely differentiation of elementary bodies into reticulate bodies in vitro and is an essential virulence factor in vivo.

Calcium bridges built by mitochondria-associated endoplasmic reticulum membranes: potential targets for neural repair in neurological diseases

The exchange of information and materials between organelles plays a crucial role in regulating cellular physiological functions and metabolic levels. Mitochondria-associated endoplasmic reticulum membranes serve as physical contact channels between the endoplasmic reticulum membrane and the mitochondrial outer membrane, formed by various proteins and protein complexes. This microstructural domain mediates several specialized functions, including calcium (Ca2+) signaling, autophagy, mitochondrial morphology, oxidative stress response, and apoptosis. Notably, the dysregulation of Ca2+ signaling mediated by mitochondria-associated endoplasmic reticulum membranes is a critical factor in the pathogenesis of neurological diseases. Certain proteins or protein complexes within these membranes directly or indirectly regulate the distance between the endoplasmic reticulum and mitochondria, as well as the transduction of Ca2+ signaling. Conversely, Ca2+ signaling mediated by mitochondria-associated endoplasmic reticulum membranes influences other mitochondria-associated endoplasmic reticulum membrane-associated functions. These functions can vary significantly across different neurological diseases–such as ischemic stroke, traumatic brain injury, Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and Huntington's disease–and their respective stages of progression. Targeted modulation of these disease-related pathways and functional proteins can enhance neurological function and promote the regeneration and repair of damaged neurons. Therefore, mitochondria-associated endoplasmic reticulum membranes-mediated Ca2+ signaling plays a pivotal role in the pathological progression of neurological diseases and represents a significant potential therapeutic target. This review focuses on the effects of protein complexes in mitochondria-associated endoplasmic reticulum membranes and the distinct roles of mitochondria-associated endoplasmic reticulum membranes-mediated Ca2+ signaling in neurological diseases, specifically highlighting the early protective effects and neuronal damage that can result from prolonged mitochondrial Ca2+ overload or deficiency. This article provides a comprehensive analysis of the various mechanisms of Ca2+ signaling mediated by mitochondria-associated endoplasmic reticulum membranes in neurological diseases, contributing to the exploration of potential therapeutic targets for promoting neuroprotection and nerve repair.

Inserting Omp22 into the flagellin protein, replacing its hypervariable region, results in stronger protection against lethal Acinetobacter baumannii infection.

Acinetobacter baumannii, a common nosocomial pathogen, is known for its rapid acquisition of antimicrobial resistance, underscoring the urgent need to develop an effective vaccine against this pathogen. Outer membrane protein 22 (Omp22) regulates the biogenesis of outer membrane vesicles to transport virulence-promoting factors into the host cells and facilitates the progression of A. baumannii infection. In this study, we used a mouse model to assess a vaccine's immunogenicity and protective efficacy using recombinant Omp22 protein within the hypervariable region of flagellin (FliC-Omp22). FliC-Omp22 demonstrated superior protection following challenge with a lethal dose of multidrug-resistant (MDR) A. baumannii strain 58ST compared to Omp22 alone. In addition, it elicited increased IgG1/IgG2a and IL-4/IFN-γ ratios, indicating a predominant Th2 immune response. Furthermore, the FliC-Omp22 vaccination elicited strong specific antibodies that inhibited the adhesion and invasion of A. baumannii 58ST and enhanced the opsonic killing activity against the pathogen. FliC-Omp22 immunization significantly reduced bacterial loads in infected mice's spleen, lungs, and liver, thereby improving their survival against the lethal infection caused by MDR A. baumannii 58ST. This study suggests that integrating Omp22 into the hypervariable domain of flagellin holds promise for developing an effective vaccine against A. baumannii infections.

Immunogenicity and cross-protective efficacy induced by delayed attenuated Salmonella with regulated length of lipopolysaccharide in mice

ABSTRACT Non-typhoidal Salmonella enterica (NTS) is a major global foodborne pathogen that poses a major public health concern worldwide, and no vaccines are available for protecting against infection of multiple Salmonella serotypes, therefore, the development of Salmonella vaccines to provide broad protection is valuable. In this work, we aimed to regulate lipopolysaccharide (LPS) synthesis of live Salmonella in vivo for exposing conserved protein antigens on the outer membrane while maintaining smooth LPS patterns in vitro to keep their original ability to invade host cells for inducing cross-protection against infection of multiple Salmonella serotypes. We generated a series of mutants defective in genes to affect the length of LPS. These mutants exhibit in vivo regulated-delayed attenuation and altered length of LPS, and all these mutants were derived from SW067 (ΔpagL7 ΔpagP81::Plpp lpxE ΔlpxR9 Δfur9) containing ∆pagP81::Plpp lpxE mutation to reduce their endotoxic activity. Animal experiments demonstrated that all regulated delayed attenuated mutants exhibited reduced ability to colonize the organs of the mice, and SW114 (waaI), SW116 (waaJ), SW118 (waaL), and SW120 (wbaP) induced a significant production of IgG and IgA against OMPs isolated from S. Typhimurium, S. Enteritidis, and S. Choleraesuis. SW114 (waaI), SW116 (waaJ), and SW118 (waaL) were capable of conferring significant protection against infection of wild-type S. Enteritidis and S. Choleraesuis, with SW118 (waaL) triggering significant CD4+ T-cell responses as well as the B220low IgG+ BM cell. In conclusion, regulated delayed attenuated Salmonella vaccines with the whole core oligosaccharides of LPS showed a goo.d ability to expose conserved outer antigens and to trigger strong cross-immune responses against both homologous and heterologous Salmonella infections. These results give new insight into the development of the Salmonella vaccine against multiple serotypes of Salmonella.

Development of a starch-fermenting Zymomonas mobilis strain for bioethanol production

BackgroundBiorefinery using microorganisms to produce biofuels and value-added biochemicals derived from renewable biomass offers a promising alternative to meet our sustainable energy and environmental goals. The ethanologenic strain Zymomonas mobilis is considered as an excellent chassis for constructing microbial cell factories for diverse biochemicals due to its outstanding industrial characteristics in ethanol production, high specific productivity, and Generally Recognized as Safe (GRAS) status. Nonetheless, the restricted substrate range constrains its application.ResultsThe truncated ice nucleation protein InaK from Pseudomonas syringae was used as an autotransporter passenger, and α-amylase was fused to the C- terminal of InaK to equip the ethanol-producing bacterium with the capability to ferment renewable biomass. Western blot and flow cytometry analysis confirmed that the amylase was situated on the outer membrane. Whole-cell activity assays demonstrated that the amylase maintained its activity on the cell surface. The recombinant Z. mobilis facilitated the hydrolysis of starch into oligosaccharides and enabled the streamlining of simultaneous saccharification and fermentation (SSF) processes. In a 5% starch medium under SSF, recombinant strains containing Peno reached a maximum titer of 13.61 ± 0.12 g/L within 48 h. This represents an increase of 111.0% compared to the control strain's titer of titer of 6.45 ± 0.25 g/L.ConclusionsBy fusing the truncated ice nucleation protein InaK with α-amylase, we achieved efficient expression and surface display of the enzyme on Z. mobilis. This fusion protein exhibited remarkable enzymatic activity. Its presence enabled a cost-effective bioproduction process using starch as the sole carbon source, and it significantly reduced the required cycle time for SSF. This study not only provides an excellent Z. mobilis chassis for sustainable bioproduction from starch but also highlights the potential of Z. mobilis to function as an effective cellular factory for producing high-value products from renewable biomass.

Radical-Mediated Nucleophilic Peptide Cross-Linking in Dynobactin Biosynthesis.

Dynobactins are recently discovered ribosomally synthesized and post-translationally modified peptide (RiPP) antibiotics that selectively kill Gram-negative pathogens by inhibiting the β-barrel assembly machinery (Bam) located on their outer membranes. Such activity of dynobactins derives from their unique cross-links between Trp1-Asn4 and His6-Tyr8. In particular, the His6-Tyr8 cross-link is formed between Nτ of His6 and Cβ of Tyr8, an unprecedented type of cross-link in RiPP natural products. The mechanism of the C-N cross-link formation remains elusive. In this work, using in vitro characterizations, we demonstrate that both cross-links in dynobactins are biosynthesized by the radical S-adenosylmethionine (SAM) enzyme DynA. Subsequent mechanistic studies using deuterium-labeled DynB precursor peptides suggested that the C-N cross-linking proceeds through the Tyr8-Hβ atom abstraction by 5'-deoxyadenosyl radical. The absence of solvent exchange of Tyr8-Hα suggested that the mechanism unlikely involves α,β-desaturation of Tyr8. Furthermore, DynA catalyzed covalent modification of Tyr8 of H6A-DynB with small-molecule nucleophiles, suggesting the presence of a highly electrophilic Tyr-derived intermediate. Based on all these observations, we propose that DynA catalyzes Tyr8-Hβ atom abstraction to generate Tyr8-Cβ radical followed by its oxidation to a p-quinone methide intermediate, to which His6-Nτ attacks to form the C-N cross-link. This quinone methide-dependent mechanism of RiPPs cross-linking is distinct from the previously reported RiPPs cross-linking mechanisms and represents a novel mechanism in RiPPs biosynthesis. We will also discuss the functional, mechanistic, and evolutional relationships of DynA with other peptide-modifying radical SAM enzymes.

Development of High-Production Bacterial Biomimetic Vesicles for Inducing Mucosal Immunity Against Avian Pathogenic Escherichia coli

To evaluate the immunoprotective effect of bacterial biomimetic vesicles (BBVs) against avian pathogenic Escherichia coli (APEC), a ΔtolA J11 mutant strain was generated by deleting the tolA gene in the low pathogenic O78 serotype J11 strain. The total protein content of outer membrane vesicles (OMVs) derived from the ΔtolA J11 strain exhibited a sevenfold increase compared to the wild-type strain. Additionally, high-pressure homogenization technology was employed to produce BBVs, resulting in a sixfold increase in total protein content compared to spontaneously secreted OMVs from ΔtolA J11. The immunogenicity of both OMVs and BBVs was assessed through intranasal or intramuscular immunization in specific pathogen-free (SPF) chickens. Results demonstrated that intranasal immunization with OMVs or BBVs in chickens elicited specific IgY antibodies against APEC outer membrane proteins and specific sIgA antibodies in the nasal cavity and trachea, as well as a significant increase in the proliferation response of chicken peripheral blood lymphocytes. The bacterial load in the blood and various organs of the challenged chickens were significantly reduced, resulting in a 66.67% and 58.30% survival rate against a high pathogenic serotype O78 strain challenge, while the control group exhibited only a 16.67% survival rate. The intramuscular immunization with OMVs or BBVs in chickens only induced specific IgY antibodies, with a survival rate of only 33.33% for challenged chickens during the same period. Therefore, intranasal vaccination of the highly productive BBVs is capable of eliciting an immune response similar to that of OMVs and providing protection against APEC infection, thus offering innovative insights for the advancement of APEC vaccines.

Helicobacter pylori outer membrane vesicles directly promote Aβ aggregation and enhance Aβ toxicity in APP/PS1 mice

Helicobacter pylori (H. pylori) infection has been found associated with Alzheimer’s disease (AD) with unclear mechanisms. Outer Membrane Vesicles (OMVs) are spherical particles secreted by Gram-negative bacteria. Here we explore the effect of H. pylori OMVs on Aβ aggregation and toxicity. We show intraperitoneally-injected H. pylori OMVs enter the brain and co-localize with Aβ plaques in APP/PS1 mice, accompanied by aggravated Aβ pathology, exacerbated cognitive deficits and synaptic impairment, indicating that H. pylori OMVs promote β-amyloidosis and AD development. The in vitro results further identify that H. pylori OMVs significantly accelerate Aβ aggregation and increase Aβ-induced neurotoxicity. Through lipidomic analysis, we reveal that lipid components, particularly LPC 18:0 in H. pylori OMVs accelerate Aβ aggregation and enhance Aβ neurotoxicity. Moreover, H. pylori OMVs-enhanced Aβ neurotoxicity is mediated by Ca2+. These findings reveal a mechanism of H. pylori OMVs in accelerating AD development in which the bacterial OMVs-originated lipid components play a key role in promoting Aβ aggregation and neurotoxicity.

Förster Resonance Energy Transfer Measurements in Living Bacteria for Interaction Studies of BamA with BamD and Inhibitor Identification

The β-barrel assembly machinery (BAM) is a multimeric protein complex responsible for the folding of outer membrane proteins in gram-negative bacteria. It is essential for cell survival and outer membrane integrity. Therefore, it is of impact in the context of antibiotic resistance and can serve as a target for the development of new antibiotics. The interaction between two of its subunits, BamA and BamD, is essential for its function. Here, a FRET-based assay to quantify the affinity between these two proteins in living bacterial cells is presented. The method was applied to identify two interaction hotspots at the binding interface. BamDY184 was identified to significantly contribute to the binding between both proteins through hydrophobic interactions and hydrogen bonding. Additionally, two salt bridges formed between BamDR94, BamDR97, and BamAE127 contributed substantially to the binding of BamA to BamD as well. Two peptides (RFIRLN and VAEYYTER) that mimic the amino acid sequence of BamD around the identified hotspots were shown to inhibit the interaction between BamA and BamD in a dose-dependent manner in the upper micromolar range. These two peptides can potentially act as antibiotic enhancers. This shows that the BamA–BamD interaction site can be addressed for the design of protein–protein interaction inhibitors. Additionally, the method, as presented in this study, can be used for further functional studies on interactions within the BAM complex.

Alzheimer's Disease-Derived Outer Membrane Vesicles Exacerbate Cognitive Dysfunction, Modulate the Gut Microbiome, and Increase Neuroinflammation and Amyloid-β Production.

Although our understanding of the molecular biology of Alzheimer's disease (AD) continues to improve, the etiology of the disease, particularly the involvement of gut microbiota disturbances, remains a challenge. Outer membrane vesicles (OMVs) play a key role in central nervous system diseases, but the impact of OMVs on AD progression remains unclear. In this study, we hypothesized that AD-derived OMVs (OMVsAD) were a risk factor in AD pathology. To test our hypothesis, young APP/PS1 mice (AD mice) were given OMVsAD by gavage. Young AD mice were euthanized 120days after gavage to assess the intestinal barrier, gut microbiota diversity, mediators of neuroinflammation, glial markers, amyloid burden, and short-chain fatty acid (SCFA) levels. Our results showed that OMVsAD accelerated cognitive dysfunction after 120days of intragastric administration. Morris water maze experiment and new object recognition test showed that OMVsAD caused significantly poorer spatial ability learning and memory of the AD mice. We observed the OMVsAD-treated APP/PS1 mice display OMVs disrupting the intestinal barrier compared with controls of normal human-derived OMVs. Compared with the OMVsHC group, claudin-5 and ZO-1 related to the intestinal barrier were significantly downregulated in the OMVsAD group. The OMVsAD activate microglia in the cerebral cortex and hippocampus of AD mice, and the levels of IL-1β, IL-6, TNF-α, and NF-Κb were upregulated. We also found that OMVsAD increased Aβ production. 16S rRNA sequencing showed that OMVsAD negatively regulated the α- and β-diversity index of intestinal flora and reduced the levels of SCFA. OMVsAD may change the intestinal flora of young AD, damage the intestinal mucosa and blood-brain barrier, and accelerate AD neuropathological damage.