SEC16A Variants Predispose to Chronic Pancreatitis by Impairing ER-to-Golgi Transport and Inducing ER Stress.

Chronic pancreatitis (CP) is a complex disease with genetic and environmental factors at play. Through trio exome sequencing, a de novo SEC16A frameshift variant in a Chinese teenage CP patient is identified. Subsequent targeted next-generation sequencing of the SEC16A gene in 1,061 Chinese CP patients and 1,196 controls reveals a higher allele frequency of rare nonsynonymous SEC16A variants in patients (4.90% vs 2.93%; odds ratio [OR], 1.71; 95% confidence interval [CI], 1.26-2.33). Similar enrichments are noted in a French cohort (OR, 2.74; 95% CI, 1.67-4.50) and in a biobank meta-analysis (OR, 1.16; 95% CI, 1.04-1.31). Notably, Chinese CP patients with SEC16A variants exhibit a median onset age 5 years earlier than those without (40.0 vs 45.0; p = 0.012). Functional studies using three CRISPR/Cas9-edited HEK293T cell lines show that loss-of-function SEC16A variants disrupt coat protein complex II (COPII) formation, impede secretory protein vesicles trafficking, and induce endoplasmic reticulum (ER) stress due to protein overload. Sec16a+/- mice, which demonstrate impaired zymogen secretion and exacerbated ER stress compared to Sec16a+/+, are further generated. In cerulein-stimulated pancreatitis models, Sec16a+/- mice display heightened pancreatic inflammation and fibrosis compared to wild-type mice. These findings implicate a novel pathogenic mechanism predisposing to CP.

Plasticity-induced actin polymerization in the dendritic shaft regulates intracellular AMPA receptor trafficking.

AMPA-type receptors (AMPARs) are rapidly inserted into synapses undergoing plasticity to increase synaptic transmission, but it is not fully understood if and how AMPAR-containing vesicles are selectively trafficked to these synapses. Here, we developed a strategy to label AMPAR GluA1 subunits expressed from their endogenous loci in cultured rat hippocampal neurons and characterized the motion of GluA1-containing vesicles using single-particle tracking and mathematical modeling. We find that GluA1-containing vesicles are confined and concentrated near sites of stimulation-induced structural plasticity. We show that confinement is mediated by actin polymerization, which hinders the active transport of GluA1-containing vesicles along the length of the dendritic shaft by modulating the rheological properties of the cytoplasm. Actin polymerization also facilitates myosin-mediated transport of GluA1-containing vesicles to exocytic sites. We conclude that neurons utilize F-actin to increase vesicular GluA1 reservoirs and promote exocytosis proximal to the sites of synaptic activity.

Rab10-CAV1 mediated intraluminal vesicle transport to migrasomes.

Migrasomes, vesicular organelles generated on the retraction fibers of migrating cells, play a crucial role in migracytosis, mediating intercellular communication. The cargoes determine the functional specificity of migrasomes. Migrasomes harbor numerous intraluminal vesicles, a pivotal component of their cargoes. The mechanism underlying the transportation of these intraluminal vesicles to the migrasomes remains enigmatic. In this study, we identified that Rab10 and Caveolin-1 (CAV1) mark the intraluminal vesicles in migrasomes. Transport of Rab10-CAV1 vesicles to migrasomes required the motor protein Myosin Va and adaptor proteins RILPL2. Notably, the phosphorylation of Rab10 by the kinase LRRK2 regulated this process. Moreover, CSF-1 can be transported to migrasomes through this mechanism, subsequently fostering monocyte-macrophage differentiation in skin wound healing, which served as a proof of the physiological importance of this transporting mechanism.

A role for synapsin tetramerization in synaptic vesicle clustering.

Although synapsins have long been proposed to be key regulators of synaptic vesicle (SV) clustering, their mechanism of action has remained mysterious and somewhat controversial. Here, we review synapsins and their associations with each other and with SVs. We highlight the recent hypothesis that synapsin tetramerization is a mechanism for SV clustering. This hypothesis, which aligns with numerous experimental results, suggests that the larger size of synapsin tetramers, in comparison to dimers, allows tetramers to form optimal bridges between SVs that overcome the repulsive force associated with the negatively charged membrane of SVs and allow synapsins to form a reserve pool of SVs within presynaptic terminals.

The WDR11 complex is a receptor for acidic-cluster-containing cargo proteins.

Vesicle trafficking is a fundamental process that allows for the sorting and transport of specific proteins (i.e., "cargoes") to different compartments of eukaryotic cells. Cargo recognition primarily occurs through coats and the associated proteins at the donor membrane. However, it remains unclear whether cargoes can also be selected at other stages of vesicle trafficking to further enhance the fidelity of the process. The WDR11-FAM91A1 complex functions downstream of the clathrin-associated AP-1 complex to facilitate protein transport from endosomes to the TGN. Here, we report the cryo-EM structure of human WDR11-FAM91A1 complex. WDR11 directly and specifically recognizes a subset of acidic clusters, which we term super acidic clusters (SACs). WDR11 complex assembly and its binding to SAC-containing proteins are indispensable for the trafficking of SAC-containing proteins and proper neuronal development in zebrafish. Our studies thus uncover that cargo proteins could be recognized in a sequence-specific manner downstream of a protein coat.

A RabGAP negatively regulates plant autophagy and immune trafficking.

Plants rely on autophagy and membrane trafficking to tolerate stress, combat infections, and maintain cellular homeostasis. However, the molecular interplay between autophagy and membrane trafficking is poorly understood. Using an AI-assisted approach, we identified Rab3GAP-like (Rab3GAPL) as a key membrane trafficking node that controls plant autophagy negatively. Rab3GAPL suppresses autophagy by binding to ATG8, the core autophagy adaptor, and deactivating Rab8a, a small GTPase essential for autophagosome formation and defense-related secretion. Rab3GAPL reduces autophagic flux in three model plant species, suggesting that its negative regulatory role in autophagy is conserved in land plants. Beyond autophagy regulation, Rab3GAPL modulates focal immunity against the oomycete pathogen Phytophthora infestans by preventing defense-related secretion. Altogether, our results suggest that Rab3GAPL acts as a molecular rheostat to coordinate autophagic flux and defense-related secretion by restraining Rab8a-mediated trafficking. This unprecedented interplay between a RabGAP-Rab pair and ATG8 sheds new light on the intricate membrane transport mechanisms underlying plant autophagy and immunity.

S-acylation of YKT61 modulates its unconventional participation in the formation of SNARE complexes in Arabidopsis.

Hetero-tetrameric soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) complexes are critical for vesicle-target membrane fusion within the endomembrane system of eukaryotic cells. SNARE assembly involves four different SNARE motifs, Qa, Qb, Qc, and R, provided by three or four SNARE proteins. YKT6 is an atypical R-SNARE that lacks a transmembrane domain and is involved in multiple vesicle-target membrane fusions. Although YKT6 is evolutionarily conserved and essential, its function and regulation in different phyla seem distinct. Arabidopsis YKT61, the yeast and metazoan YKT6 homologue, is essential for gametophytic development, plays a critical role in sporophytic cells, and mediates multiple vesicle-target membrane fusion. However, its molecular regulation is unclear. We report here that YKT61 is S-acylated. Abolishing its S-acylation by a C195S mutation dissociates YKT61 from endomembrane structures and causes its functional loss. Although interacting with various SNARE proteins, YKT61 functions not as a canonical R-SNARE but coordinates with other R-SNAREs to participate in the formation of SNARE complexes. Phylum-specific molecular regulation of YKT6 may be evolved to allow more efficient SNARE assembly in different eukaryotic cells.

Sucrose Treatment Enhances the Electrotransfer of DNA by Activating Phospholipase A2.

Our previous study discovered that sucrose and other non-reducing sugars (e.g., trehalose and raffinose) could be used to improve the electrotransfer (ET) of molecular cargo, including DNA, mRNA, and ribonucleoprotein in various cell lines and primary human cells in vitro and in vivo. To understand the molecular mechanisms of this improvement, we used RNA sequencing technology to analyze changes in the cell transcriptome after sucrose treatment. The results from our analysis demonstrated that the sucrose treatment upregulated phospholipase A2 and V-ATPase gene families, which could potentially influence the acidity of intracellular vesicles through augmenting vesicle fusion and the influx of proton, respectively. To determine how this upregulation affects ET efficiency, we treated cells with pharmaceutical inhibitors of phospholipase A2 and V-ATPase. The data demonstrated that the treatment with the phospholipase A2 inhibitor could reverse the ET improvement elicited by the sucrose treatment. The V-ATPase inhibitor treatment either had little influence or further enhanced the effect of the sucrose treatment on the ET efficiency. These observations provide a molecular explanation for our previous findings, demonstrating that the sucrose treatment primarily enhanced the ET efficiency by promoting vesicle trafficking and fusion through the activation of phospholipase A2.

Short-distance vesicle transport via phase separation.

In addition to long-distance molecular motor-mediated transport, cellular vesicles also need to be moved at short distances with defined directions to meet functional needs in subcellular compartments but with unknown mechanisms. Such short-distance vesicle transport does not involve molecular motors. Here, we demonstrate, using synaptic vesicle (SV) transport as a paradigm, that phase separation of synaptic proteins with vesicles can facilitate regulated, directional vesicle transport between different presynaptic bouton sub-compartments. Specifically, a large coiled-coil scaffold protein Piccolo, in response to Ca2+ and via its C2A domain-mediated Ca2+ sensing, can extract SVs from the synapsin-clustered reserve pool condensate and deposit the extracted SVs onto the surface of the active zone protein condensate. We further show that the Trk-fused gene, TFG, also participates in COPII vesicle trafficking from ER to the ER-Golgi intermediate compartment via phase separation. Thus, phase separation may play a general role in short-distance, directional vesicle transport in cells.

Ehrlichia chaffeensis Etf-3 Induces Host RAB15 Upregulation for Bacterial Intracellular Growth.

Ehrlichia chaffeensis infects human monocytes or macrophages and causes human monocytic ehrlichiosis (HME), an emerging life-threatening zoonosis. After internalization, E. chaffeensis resides in membrane-bound inclusions, E. chaffeensis-containing vesicles (ECVs), which have early endosome-like characteristics and fuse with early autophagosomes but not lysosomes, to evade host innate immune microbicidal mechanisms and obtain nutrients for bacterial intracellular growth. The mechanisms exploited by E. chaffeensis to modulate intracellular vesicle trafficking in host cells have not been comprehensively studied. Here, we demonstrate that E. chaffeensis type IV secretion system (T4SS) effector Etf-3 induces RAB15 upregulation in host cells and that RAB15, which is localized on ECVs, inhibits ECV fusion with lysosomes and induces autophagy. We found that E. chaffeensis infection upregulated RAB15 expression using qRT-PCR, and RAB15 was colocalized with E. chaffeensis using confocal microscopy. Silence of RAB15 using siRNA enhanced ECV maturation to late endosomes and fusion with lysosomes, as well as inhibited host cell autophagy. Overexpression of Etf-3 in host cells specifically induced RAB15 upregulation and autophagy. Our findings deepen the understanding of E. chaffeensis pathogenesis and adaptation in hosts as well as the function of RAB15 and facilitate the development of new therapeutics for HME.

Hypersecretory production of glucose oxidase in Pichia pastoris through combinatorial engineering of protein properties, synthesis, and secretion.

Glucose oxidase (EC 1.1.3.4, GOD) is a widely used industrial enzyme. To construct a GOD-hyperproducing Pichia pastoris strain, combinatorial strategies have been applied to improve GOD activity, synthesis, and secretion. First, wild-type GOD was subjected to saturation mutagenesis to obtain an improved variant, MGOD1 (V20W/T30S), with 1.7-fold higher kcat /KM . Subsequently, efficient signal peptides were screened, and the copy number of MGOD1 was optimized to generate a high-producing strain, 8GM1, containing eight copies of AOX1 promoter-GAS1 signal peptide-MGOD1 expression cassette. Finally, the vesicle trafficking of 8GM1 was engineered to obtain the hyperproducing strain G1EeSe co-expressing the trafficking components EES and SEC. 22, and the EES gene (PAS_chr3_0685) was found to facilitate both protein secretion and production for the first time. Using these strategies, GOD secretion was enhanced 65.2-fold. In the 5-L bioreactor, conventional fed-batch fermentation without any process optimization resulted in up to 7223.0 U/mL extracellular GOD activity (3.3-fold higher than the highest level reported to date), with almost only GOD in the fermentation supernatant at a protein concentration of 30.7 g/L. Therefore, a GOD hyperproducing strain for industrial applications was developed, and this successful case can provide a valuable reference for the construction of high-producing strains for other industrial enzymes.

The Pseudomonas syringae pv. tomato DC3000 effector HopD1 interferes with cellular dynamics associated with the function of the plant immune protein AtNHR2B.

The plant pathogenic bacterium Pseudomonas syringae pv tomato DC3000 (Pst DC3000) causes disease in tomato, in the model plant Arabidopsis thaliana, and conditionally in Nicotiana benthamiana. The pathogenicity of Pst DC3000 is mostly due to bacterial virulence proteins, known as effectors, that are translocated into the plant cytoplasm through the type III secretion system (T3SS). Bacterial type III secreted effectors (T3SEs) target plants physiological processes and suppress defense responses to enable and support bacterial proliferation. The Pst DC3000 T3SE HopD1 interferes with plant defense responses by targeting the transcription factor NTL9. This work shows that HopD1 also targets the immune protein AtNHR2B (Arabidopsis thaliana nonhost resistance 2B), a protein that localizes to dynamic vesicles of the plant endomembrane system. Live-cell imaging of Nicotiana benthamiana plants transiently co-expressing HopD1 fused to the epitope haemagglutinin (HopD1-HA) with AtNHR2B fused to the red fluorescent protein (AtNHR2B-RFP), revealed that HopD1-HA interferes with the abundance and cellular dynamics of AtNHR2B-RFP-containing vesicles. The results from this study shed light into an additional function of HopD1 while contributing to understanding how T3SEs also target vesicle trafficking-mediated processes in plants.

Multi-omics study identifies that PICK1 deficiency causes male infertility by inhibiting vesicle trafficking in Sertoli cells.

BACKGROUND: Infertility affects approximately 10-15% of reproductive-age men worldwide, and genetic causes play a role in one-third of cases. As a Bin-Amphiphysin-Rvs (BAR) domain protein, protein interacting with C-kinase 1 (PICK1) deficiency could lead to impairment of acrosome maturation. However, its effects on auxiliary germ cells such as Sertoli cells are unknown. PURPOSE: The present work was aimed to use multi-omics analysis to research the effects of PICK1 deficiency on Sertoli cells and to identify effective biomarkers to distinguish fertile males from infertile males caused by PICK1 deficiency. METHODS: Whole-exome sequencing (WES) was performed on 20 infertility patients with oligozoospermia to identify pathogenic PICK1 mutations. Multi-omics analysis of a PICK1 knockout (KO) mouse model was utilized to identify pathogenic mechanism. Animal and cell function experiments of Sertoli cell-specific PICK1 KO mouse were performed to verify the functional impairment of Sertoli cells. RESULTS: Two loss-of-function deletion mutations c.358delA and c.364delA in PICK1 resulting in transcription loss of BAR functional domain were identified in infertility patients with a specific decrease in serum inhibin B, indicating functional impairment of Sertoli cells. Multi-omics analysis of PICK1 KO mouse illustrated that targeted genes of differentially expressed microRNAs and mRNAs are significantly enriched in the negative regulatory role in the vesicle trafficking pathway, while metabolomics analysis showed that the metabolism of amino acids, lipids, cofactors, vitamins, and endocrine factors changed. The phenotype of PICK1 KO mouse showed a reduction in testis volume, a decreased number of mature spermatozoa and impaired secretory function of Sertoli cells. In vitro experiments confirmed that the expression of growth factors secreted by Sertoli cells in PICK1 conditional KO mouse such as Bone morphogenetic protein 4 (BMP4) and Fibroblast growth factor 2 (FGF2) were decreased. CONCLUSIONS: Our study attributed male infertility caused by PICK1 deficiency to impaired vesicle-related secretory function of Sertoli cells and identified a variety of significant candidate biomarkers for male infertility induced by PICK1 deficiency.

Nanoinjection of extracellular vesicles to single live cells by robotic fluidic force microscopy.

In the past decade, extracellular vesicles (EVs) have attracted substantial interest in biomedicine. With progress in the field, we have an increasing understanding of cellular responses to EVs. In this Technical Report, we describe the direct nanoinjection of EVs into the cytoplasm of single cells of different cell lines. By using robotic fluidic force microscopy (robotic FluidFM), nanoinjection of GFP positive EVs and EV-like particles into single live HeLa, H9c2, MDA-MB-231 and LCLC-103H cells proved to be feasible. This injection platform offered the advantage of high cell selectivity and efficiency. The nanoinjected EVs were initially localized in concentrated spot-like regions within the cytoplasm. Later, they were transported towards the periphery of the cells. Based on our proof-of-principle data, robotic FluidFM is suitable for targeting single living cells by EVs and may lead to information about intracellular EV cargo delivery at a single-cell level.

Vesicle trafficking in rice: too little is known.

The vesicle trafficking apparatus is a fundamental machinery to maintain the homeostasis of membrane-enclosed organelles in eukaryotic cells. Thus, it is broadly conserved in eukaryotes including plants. Intensive studies in the model organisms have produced a comprehensive picture of vesicle trafficking in yeast and human. However, with respect to the vesicle trafficking of plants including rice, our understanding of the components and their coordinated regulation is very limited. At present, several vesicle trafficking apparatus components and cargo proteins have been identified and characterized in rice, but there still remain large unknowns concerning the organization and function of the rice vesicle trafficking system. In this review, we outline the main vesicle trafficking pathways of rice based on knowledge obtained in model organisms, and summarize current advances of rice vesicle trafficking. We also propose to develop methodologies applicable to rice and even other crops for further exploring the mysteries of vesicle trafficking in plants.

Sec8 specifically interacts with the PDZ2 domain of synapse associated protein 102 (SAP102).

The exocyst is an evolutionarily conserved protein complex tethering secretory vesicles before their docking and fusion with the plasma membrane. The complex also plays important roles in cell migration, synaptogenesis, and neurite outgrowth. One of its subunits, Sec8, was reported to interact with two major synaptic scaffolding proteins SAP102 and PSD-95 that share high sequence homology and contain three PDZ domains at their N-terminal region. The interaction is via the binding of the C-terminal ITTV motif in Sec8 to the PDZ domains of the two synaptic proteins. However, it remains elusive to which PDZ domain(s) Sec8 binds and how their interaction occurs. Here we reported a 2.5 Å resolution crystal structure of the C-terminal half of rat Sec8 containing the ITTV motif. The structure shows that Sec8 contains an enormously long helix at its C-terminus, which bears a unique long "spacer" of 14 residues to bridge the ITTV motif to the compact core of Sec8. We found that Sec8 preferentially binds PDZ2 over PDZ1 and PDZ3 of SAP102. Deletion of the spacer completely abolished the binding of Sec8 to SAP102. Overall, our structural studies, biochemical data and modeling analyses altogether provide an explanation for how Sec8 interacts with SAP102.

Identification of CD133+ intercellsomes in intercellular communication to offset intracellular signal deficit.

CD133 (prominin 1) is widely viewed as a cancer stem cell marker in association with drug resistance and cancer recurrence. Herein, we report that with impaired RTK-Shp2-Ras-Erk signaling, heterogenous hepatocytes form clusters that manage to divide during mouse liver regeneration. These hepatocytes are characterized by upregulated CD133 while negative for other progenitor cell markers. Pharmaceutical inhibition of proliferative signaling also induced CD133 expression in various cancer cell types from multiple animal species, suggesting an inherent and common mechanism of stress response. Super-resolution and electron microscopy localize CD133 on intracellular vesicles that apparently migrate between cells, which we name 'intercellsome.' Isolated CD133+ intercellsomes are enriched with mRNAs rather than miRNAs. Single-cell RNA sequencing reveals lower intracellular diversity (entropy) of mitogenic mRNAs in Shp2-deficient cells, which may be remedied by intercellular mRNA exchanges between CD133+ cells. CD133-deficient cells are more sensitive to proliferative signal inhibition in livers and intestinal organoids. These data suggest a mechanism of intercellular communication to compensate for intracellular signal deficit in various cell types.

The liver is an important metabolic organ that is responsible for digesting nutrients. Over time, it can become damaged by the toxins it receives from food and drink, as well as during infections. Thankfully, cells in the liver can divide and replace the parts that have become harmed allowing the organ to continue carrying out its vital role in the body. Experiments in mice have identified various genes and proteins involved in regenerating the liver. This includes the protein Shp2 which instructs liver cells to divide. However, scientists have found mice lacking the gene for Shp2 could still repair their livers. But how exactly these genetically modified mice were able to do this remained unclear. To investigate, Kaneko et al. examined the shape and size of cells in the livers of mice lacking Shp2. This revealed clusters of dividing cells that could still repair the liver that contained abundant amounts of a protein called CD133. The CD133 molecules resided in very small vesicles about 50 to 150 nm in width which Kaneko et al. named 'intercellsomes' because they could move from one liver cell to the next. Further experiments revealed that the intercellsomes contained important materials essential for cell division, making them distinct from other well-known vesicles. These newly discovered structures may allow liver cells to share replication signals with other cells that may be struggling to divide during liver regeneration. CD133 is also present in cancer cells that are resistant to treatment and can multiply under stress. Kaneko et al. found that treating various types of tumor cells with drugs that inhibit proliferation led to an increase in CD133. This suggests that some cancer cells may use the intercellsome mechanism to keep dividing following treatment, potentially resulting in a relapse of the malignant disease. Taken together, this study hints at the existence of a previously unknown communication system that helps cells to divide when their replication is inhibited. Further experiments are needed to see if this mechanism is widely employed by various cell types, how exactly the CD133 vesicles migrate between cells, and if intercellsomes carry out any other roles.

Identification of genes required for Plasmodium gametocyte-to-sporozoite development in the mosquito vector.

Malaria remains one of the most devastating infectious diseases. Reverse genetic screens offer a powerful approach to identify genes and molecular processes governing malaria parasite biology. However, the complex regulation of gene expression and genotype-phenotype associations in the mosquito vector, along with sexual reproduction, have hindered the development of screens in this critical part of the parasite life cycle. To address this, we developed a genetic approach in the rodent parasite Plasmodium berghei that, in combination with barcode sequencing, circumvents the fertilization roadblock and enables screening for gametocyte-expressed genes required for parasite infection of the mosquito Anopheles coluzzii. Our results confirm previous findings, validating our approach for scaling up, and identify genes necessary for mosquito midgut infection, oocyst development, and salivary gland infection. These findings can aid efforts to study malaria transmission biology and to develop interventions for controlling disease transmission.

A tyrosine phospho-switch within the Longin domain of VAMP721 modulates SNARE functionality.

The final step in secretion is membrane fusion facilitated by SNARE proteins that reside in opposite membranes. The formation of a trans-SNARE complex between one R and three Q coiled-coiled SNARE domains drives the final approach of the membranes providing the mechanical energy for fusion. Biological control of this mechanism is exerted by additional domains within some SNAREs. For example, the N-terminal Longin domain (LD) of R-SNAREs (also called Vesicle-associated membrane proteins, VAMPs) can fold back onto the SNARE domain blocking interaction with other cognate SNAREs. The LD may also determine the subcellular localization via interaction with other trafficking-related proteins. Here, we provide cell-biological and genetic evidence that phosphorylation of the Tyrosine57 residue regulates the functionality of VAMP721. We found that an aspartate mutation mimics phosphorylation, leading to protein instability and subsequent degradation in lytic vacuoles. The mutant SNARE also fails to rescue the defects of vamp721vamp722 loss-of-function lines in spite of its wildtype-like localization within the secretory pathway and the ability to interact with cognate SNARE partners. Most importantly, it imposes a dominant negative phenotype interfering with root growth, normal secretion and cytokinesis in wildtype plants generating large aggregates that mainly contain secretory vesicles. Non-phosphorylatable VAMP721Y57F needs higher gene dosage to rescue double mutants in comparison to native VAMP721 underpinning that phosphorylation modulates SNARE function. We propose a model where short-lived phosphorylation of Y57 serves as a regulatory step to control VAMP721 activity, favoring its open state and interaction with cognate partners to ultimately drive membrane fusion.

Gaining New Insights into Fundamental Biological Pathways by Bacterial Toxin-Based Genetic Screens.

Genetic screen technology has been applied to study the mechanism of action of bacterial toxins-a special class of virulence factors that contribute to the pathogenesis caused by bacterial infections. These screens aim to identify host factors that directly or indirectly facilitate toxin intoxication. Additionally, specific properties of certain toxins, such as membrane interaction, retrograde trafficking, and carbohydrate binding, provide robust probes to comprehensively investigate the lipid biosynthesis, membrane vesicle transport, and glycosylation pathways, respectively. This review specifically focuses on recent representative toxin-based genetic screens that have identified new players involved in and provided new insights into fundamental biological pathways, such as glycosphingolipid biosynthesis, protein glycosylation, and membrane vesicle trafficking pathways. Functionally characterizing these newly identified factors not only expands our current understanding of toxin biology but also enables a deeper comprehension of fundamental biological questions. Consequently, it stimulates the development of new therapeutic approaches targeting both bacterial infectious diseases and genetic disorders with defects in these factors and pathways.