Vesicle formation and cellulose degradation in Bacteroides succinogenes cultures: ultrastructural aspects

In 3-day-old cultures of Bacteroides succinogenes grown on filter paper, no cell division was observed. When grown on cellulosic substrate, bacteria exhibited vesicles clustered within cell wall pockets. In 2 day-old filter paper cultures, cells adhered tightly to the substrate. Twenty to 30% of them were dividing. There were cell wall pockets in about 25% of the bacteria, but no vesicles. Whether they adhered to the cellulosic substrate or not, and irrespective of the age of the bacteria, storage polysaccharides were found in the form of dense granules in the cytoplasm. It would appear that vesicles are not essential for cellulose degradation, but are rather a sign of ageing of the cells.     

Modulation of cellulosome composition in Clostridium cellulolyticum: Adaptation to the polysaccharide environment revealed by proteomic and carbohydrate‐active enzyme analyses

Clostridium cellulolyticum is a model mesophilic anaerobic bacterium that efficiently degrades plant cell walls. The recent genome release offers the opportunity to analyse its complete degradation system. A total of 148 putative carbohydrate‐active enzymes were identified, and their modular structures and activities were predicted. Among them, 62 dockerin‐containing proteins bear catalytic modules from numerous carbohydrate‐active enzymes' families and whose diversity reflects the chemical and structural complexity of the plant carbohydrate. The composition of the cellulosomes produced by C. cellulolyticum upon growth on different substrates (cellulose, xylan, and wheat straw) was investigated by LC MS/MS. The majority of the proteins encoded by the cip‐cel operon, essential for cellulose degradation, were detected in all cellulosome preparations. In the presence of wheat straw, the natural and most complex of the substrates studied, additional proteins predicted to be involved in hemicellulose degradation were produced. A 32‐kb gene cluster encodes the majority of these proteins, all harbouring carbohydrate‐binding module 6 or carbohydrate‐binding module 22 xylan‐binding modules along dockerins. This newly identified xyl‐doc gene cluster, specialised in hemicellulose degradation, comes in addition of the cip‐cel operon for plant cell wall degradation. Hydrolysis efficiencies determined on the different substrates corroborates the finding that cellulosome composition is adapted to the growth substrate.     

A comparative systems analysis of polysaccharide‐elicited responses in Neurospora crassa reveals carbon source‐specific cellular adaptations

Filamentous fungi are powerful producers of hydrolytic enzymes for the deconstruction of plant cell wall polysaccharides. However, the central question of how these sugars are perceived in the context of the complex cell wall matrix remains largely elusive. To address this question in a systematic fashion we performed an extensive comparative systems analysis of how the model filamentous fungus Neurospora crassa responds to the three main cell wall polysaccharides: pectin, hemicellulose and cellulose. We found the pectic response to be largely independent of the cellulolytic one with some overlap to hemicellulose, and in its extent surprisingly high, suggesting advantages for the fungus beyond being a mere carbon source. Our approach furthermore allowed us to identify carbon source‐specific adaptations, such as the induction of the unfolded protein response on cellulose, and a commonly induced set of 29 genes likely involved in carbon scouting. Moreover, by hierarchical clustering we generated a coexpression matrix useful for the discovery of new components involved in polysaccharide utilization. This is exemplified by the identification of lat‐1, which we demonstrate to encode for the physiologically relevant arabinose transporter in Neurospora. The analyses presented here are an important step towards understanding fungal degradation processes of complex biomass.     

Genetic modification in cellulose‐synthase reduces crystallinity and improves biochemical conversion to fermentable sugar

The cellulose synthase (CESA) membrane complex synthesizes microfibrils of cellulose that surround all plant cells. Cellulose is made of sugar (β,1‐4 glucan) and accessing the sugar in cellulose for biofuels is of critical importance to stem the use of fossil fuels and avoid competition with food crops and pristine lands associated with starch‐based biofuel production. The recalcitrance of cellulose to enzymatic conversion to a fermentable form of sugar is related to the degree of hydrogen bonding or crystallization of the glucan chain. Herein, we isolate the first viable low biomass‐crystallinity mutant by screening for altered cell wall structure using X‐ray scattering as well as screening for enzymatic conversion efficiency on a range of cell wall mutants in the model plant Arabidopsis thaliana (L.) Heynh. Through detailed analysis of the kinetics of bioconversion we identified a mutant that met both selection criteria. This mutant is ixr1‐2, which contains a mutation in a highly conserved consensus sequence among the C‐terminal transmembrane regions within CESA3. A 34% lower biomass crystallization index and 151% improvement in the efficiency of conversion from raw biomass to fermentable sugars was measured relative to that of wild type (Col‐0). Recognizing the inherent ambiguities with an insoluble complex substrate like cellulose and how little is still understood regarding the regulation of CESA we propose a general model for how to manipulate CESA enzymes to improve the recalcitrance of cellulose to enzymatic hydrolysis. This study also raises intriguing possibilities as to the functional importance of transmembrane anchoring in CESA complex and microfibril formation.     

Campylobacter jejuni outer membrane vesicle‐associated proteolytic activity promotes bacterial invasion by mediating cleavage of intestinal epithelial cell E‐cadherin and occludin

Outer membrane vesicles (OMVs) play an important role in the pathogenicity of Gram‐negative bacteria. Campylobacter jejuni produces OMVs that trigger IL‐8, IL‐6, hBD‐3 and TNF‐α responses from T84 intestinal epithelial cells and are cytotoxic to Caco‐2 IECs and Galleria mellonella larvae. Proteomic analysis of 11168H OMVs identified the presence of three proteases, HtrA, Cj0511 and Cj1365c. In this study, 11168H OMVs were shown to possess proteolytic activity that was reduced by pretreatment with specific serine protease inhibitors. OMVs isolated from 11168H htrA, Cj0511 or Cj1365c mutants possess significantly reduced proteolytic activity. 11168H OMVs are able to cleave both E‐cadherin and occludin, but this cleavage is reduced with OMVs pretreated with serine protease inhibitors and also with OMVs isolated from htrA or Cj1365c mutants. Co‐incubation of T84 monolayers with 11168H OMVs results in a visible reduction in both E‐cadherin and occludin. The addition of 11168H OMVs to the co‐culture of live 11168H bacteria with T84 cells results in enhanced levels of bacterial adhesion and invasion in a time‐dependent and dose‐dependent manner. Further investigation of the cleavage of host cell structural proteins by C. jejuni OMVs should enhance our understanding of the interactions of this important pathogen with intestinal epithelial cells.     

Hetero‐trans‐β‐glucanase,an enzyme unique to Equisetum plants,functionalizes cellulose

Cell walls are metabolically active components of plant cells. They contain diverse enzymes, including transglycanases (endotransglycosylases), enzymes that ‘cut and paste’ certain structural polysaccharide molecules and thus potentially remodel the wall during growth and development. Known transglycanase activities modify several cell‐wall polysaccharides (xyloglucan, mannans, mixed‐linkage β‐glucan and xylans); however, no transglycanases were known to act on cellulose, the principal polysaccharide of biomass. We now report the discovery and characterization of hetero‐trans‐β‐glucanase (HTG), a transglycanase that targets cellulose, in horsetails (Equisetum spp., an early‐diverging genus of monilophytes). HTG is also remarkable in predominantly catalysing hetero‐transglycosylation: its preferred donor substrates (cellulose or mixed‐linkage β‐glucan) differ qualitatively from its acceptor substrate (xyloglucan). HTG thus generates stable cellulose–xyloglucan and mixed‐linkage β‐glucan–xyloglucan covalent bonds, and may therefore strengthen ageing Equisetum tissues by inter‐linking different structural polysaccharides of the cell wall. 3D modelling suggests that only three key amino acid substitutions (Trp → Pro, Gly → Ser and Arg → Leu) are responsible for the evolution of HTG's unique specificity from the better‐known xyloglucan‐acting homo‐transglycanases (xyloglucan endotransglucosylase/hydrolases; XTH). Among land plants, HTG appears to be confined to Equisetum, but its target polysaccharides are widespread, potentially offering opportunities for enhancing crop mechanical properties, such as wind resistance. In addition, by linking cellulose to xyloglucan fragments previously tagged with compounds such as dyes or indicators, HTG may be useful biotechnologically for manufacturing stably functionalized celluloses, thereby potentially offering a commercially valuable ‘green’ technology for industrially manipulating biomass.     

Rice cellulose synthase‐like D4 is essential for normal cell‐wall biosynthesis and plant growth

Cellulose synthase‐like (CSL) proteins of glycosyltransferase family 2 (GT2) are believed to be involved in the biosynthesis of cell‐wall polymers. The CSL D sub‐family (CSLD) is common to all plants, but the functions of CSLDs remain to be elucidated. We report here an in‐depth characterization of a narrow leaf and dwarf1 (nd1) rice mutant that shows significant reduction in plant growth due to retarded cell division. Map‐based cloning revealed that ND1 encodes OsCSLD4, one of five members of the CSLD sub‐family in rice. OsCSLD4 is mainly expressed in tissues undergoing rapid growth. Expression of OsCSLD4 fluorescently tagged at the C‐ or N‐terminus in rice protoplast cells or Nicotiana benthamiana leaves showed that the protein is located in the endoplasmic reticulum or Golgi vesicles. Golgi localization was verified using phenotype‐rescued transgenic plants expressing OsCSLD4–GUS under the control of its own promoter. Two phenotype‐altered tissues, culms and root tips, were used to investigate the specific wall defects. Immunological studies and monosaccharide compositional and glycosyl linkage analyses explored several wall compositional effects caused by disruption of OsCSLD4, including alterations in the structure of arabinoxylan and the content of cellulose and homogalacturonan, which are distinct in the monocot grass species Oryza sativa (rice). The inconsistent alterations in the two tissues and the observable structural defects in primary walls indicate that OsCSLD4 plays important roles in cell‐wall formation and plant growth.     

Production of oligosaccharides and cellobionic acid by <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> S85 growing on sugars,cellulose and wheat straw

Extracellular culture fluid of Fibrobacter succinogenes S85 grown on glucose, cellobiose, cellulose or wheat straw was analysed by 2D-NMR spectroscopy. Cellodextrins did not accumulate in the culture medium of cells grown on cellulose or straw. Maltodextrins and maltodextrin-1P were identified in the culture medium of glucose, cellobiose and cellulose grown cells. New glucose derivatives were identified in the culture fluid under all the substrate conditions. In particular, a compound identified as cellobionic acid accumulated at high levels in the medium of F. succinogenes S85 cultures. The production of cellobionic acid (and cellobionolactone also identified) was very surprising in an anaerobic bacterium. The results suggest metabolic shifts when cells were growing on solid substrate cellulose or straw compared to soluble sugars.     

The F‐box protein Fbp1 functions in the invasive growth and cell wall integrity mitogen‐activated protein kinase (MAPK) pathways in Fusarium oxysporum

F‐box proteins determine substrate specificity of the ubiquitin–proteasome system. Previous work has demonstrated that the F‐box protein Fbp1, a component of the SCF^Fbp1 E3 ligase complex, is essential for invasive growth and virulence of the fungal plant pathogen Fusarium oxysporum. Here, we show that, in addition to invasive growth, Fbp1 also contributes to vegetative hyphal fusion and fungal adhesion to tomato roots. All of these functions have been shown previously to require the mitogen‐activated protein kinase (MAPK) Fmk1. We found that Fbp1 is required for full phosphorylation of Fmk1, indicating that Fbp1 regulates virulence and invasive growth via the Fmk1 pathway. Moreover, the Δfbp1 mutant is hypersensitive to sodium dodecylsulfate (SDS) and calcofluor white (CFW) and shows reduced phosphorylation levels of the cell wall integrity MAPK Mpk1 after SDS treatment. Collectively, these results suggest that Fbp1 contributes to both the invasive growth and cell wall integrity MAPK pathways of F. oxysporum.     

Three AtCesA6‐like members enhance biomass production by distinctively promoting cell growth in Arabidopsis

Cellulose is an abundant biopolymer and a prominent constituent of plant cell walls. Cellulose is also a central component to plant morphogenesis and contributes the bulk of a plant's biomass. While cellulose synthase (CesA) genes were identified over two decades ago, genetic manipulation of this family to enhance cellulose production has remained difficult. In this study, we show that increasing the expression levels of the three primary cell wall AtCesA6‐like genes (AtCesA2, AtCesA5, AtCesA6), but not AtCesA3, AtCesA9 or secondary cell wall AtCesA7, can promote the expression of major primary wall CesA genes to accelerate primary wall CesA complex (cellulose synthase complexes, CSCs) particle movement for acquiring long microfibrils and consequently increasing cellulose production in Arabidopsis transgenic lines, as compared with wild‐type. The overexpression transgenic lines displayed changes in expression of genes related to cell growth and proliferation, perhaps explaining the enhanced growth of the transgenic seedlings. Notably, overexpression of the three AtCesA6‐like genes also enhanced secondary cell wall deposition that led to improved mechanical strength and higher biomass production in transgenic mature plants. Hence, we propose that overexpression of certain AtCesA genes can provide a biotechnological approach to increase cellulose synthesis and biomass accumulation in transgenic plants.     

In the grass species Brachypodium distachyon,the production of mixed‐linkage (1,3;1,4)‐β‐glucan (MLG) occurs in the Golgi apparatus

Mixed‐linkage (1,3;1,4)‐β‐glucan (MLG) is a glucose polymer with beneficial effects on human health and high potential for the agricultural industry. MLG is present predominantly in the cell wall of grasses and is synthesized by cellulose synthase‐like F or H families of proteins, with CSLF6 being the best‐characterized MLG synthase. Although the function of this enzyme in MLG production has been established, the site of MLG synthesis in the cell is debated. It has been proposed that MLG is synthesized at the plasma membrane, as occurs for cellulose and callose; in contrast, it has also been proposed that MLG is synthesized in the Golgi apparatus, as occurs for other matrix polysaccharides of the cell wall. Testing these conflicting possibilities is fundamentally important in the general understanding of the biosynthesis of the plant cell wall. Using immuno‐localization analyses with MLG‐specific antibody in Brachypodium and in barley, we found MLG present in the Golgi, in post‐Golgi structures and in the cell wall. Accordingly, analyses of a functional fluorescent protein fusion of CSLF6 stably expressed in Brachypodium demonstrated that the enzyme is localized in the Golgi. We also established that overproduction of MLG causes developmental and growth defects in Brachypodium as also occur in barley. Our results indicated that MLG production occurs in the Golgi similarly to other cell wall matrix polysaccharides, and supports the broadly applicable model in grasses that tight mechanisms control optimal MLG accumulation in the cell wall during development and growth. This work addresses the fundamental question of where mixed linkage (1,3;1,4)‐β‐glucan (MLG) is synthesized in plant cells. By analyzing the subcellular localization of MLG and MLG synthase in an endogenous system, we demonstrated that MLG synthesis occurs at the Golgi in Brachypodium and barley. A growth inhibition due to overproduced MLG in Brachypodium supports the general applicability of the model that a tight control of the cell wall polysaccharides accumulation is needed to maintain growth homeostasis during development.     

LPS targets host guanylate‐binding proteins to the bacterial outer membrane for non‐canonical inflammasome activation

Pathogenic and commensal Gram‐negative bacteria produce and release outer membrane vesicles (OMVs), which present several surface antigens and play an important role for bacterial pathogenesis. OMVs also modulate the host immune system, which makes them attractive as vaccine candidates. At the cellular level, OMVs are internalized by macrophages and deliver lipopolysaccharide (LPS) into the host cytosol, thus activating the caspase‐11 non‐canonical inflammasome. Here, we show that OMV‐induced inflammasome activation requires TLR4‐TRIF signaling, the production of type I interferons, and the action of guanylate‐binding proteins (GBPs), both in macrophages and in vivo. Mechanistically, we find that isoprenylated GBPs associate with the surface of OMVs or with transfected LPS, indicating that the key factor that determines GBP recruitment to the Gram‐negative bacterial outer membranes is LPS itself. Our findings provide new insights into the mechanism by which GBPs target foreign surfaces and reveal a novel function for GBPs in controlling the intracellular detection of LPS derived from extracellular bacteria in the form of OMVs, thus extending their function as a hub between cell‐autonomous immunity and innate immunity.     

Enterohaemorrhagic Escherichia coli produces outer membrane vesicles as an active defence system against antimicrobial peptide LL‐37

Antimicrobial peptides (AMPs) are important components of the innate immune system. Enterohaemorrhagic Escherichia coli (EHEC), a food‐borne pathogen causing serious diarrheal diseases, must overcome attack by AMPs. Here, we show that resistance of EHEC against human cathelicidin LL‐37, a primary AMP, was enhanced by butyrate, which has been shown to act as a stimulant for the expression of virulence genes. The increase of resistance depended on the activation of the ompT gene, which encodes the outer membrane protease OmpT for LL‐37. The expression of the ompT gene was enhanced through the activation system for virulence genes. The increase in ompT expression did not result in an increase in OmpT protease in bacteria but in enhancement of the production of OmpT‐loaded outer membrane vesicles (OMVs), which primarily contributed to the increase in LL‐37‐resistance. Furthermore, a sublethal dosage of LL‐37 stimulated the production of OMVs. Finally, we showed that OMVs produced by OmpT‐positive strains protect the OmpT‐negative strain, which is susceptible to LL‐37 by itself more efficiently than OMVs from the ompT mutant. These results indicate that EHEC enhances the secretion of OmpT‐loaded OMVs in coordination with the activation of virulence genes during infection and blocks bacterial cell attack by LL‐37.     

Dysregulated haemolysin promotes bacterial outer membrane vesicles‐induced pyroptotic‐like cell death in zebrafish

Inflammasomes are important innate immune components in mammals. However, the bacterial factors modulating inflammasome activation in fish, and the mechanisms by which they alter fish immune defences, remain to be investigated. In this work, a mutant of the fish pathogen Edwardsiella piscicida (E. piscicida), called 0909I, was shown to overexpress haemolysin, which could induce a robust pyroptotic‐like cell death dependent on caspase‐5‐like activity during infection in fish nonphagocyte cells. E. piscicida haemolysin was found to mainly associate with bacterial outer membrane vesicles (OMVs), which were internalised into the fish cells via a dynamin‐dependent endocytosis and induced pyroptotic‐like cell death. Importantly, bacterial immersion infection of both larvae and adult zebrafish suggested that dysregulated expression of haemolysin alerts the innate immune system and induces intestinal inflammation to restrict bacterial colonisation in vivo. Taken together, these results suggest a critical role of zebrafish innate immunity in monitoring invaded pathogens via detecting the bacterial haemolysin‐associated OMVs and initiating pyroptotic‐like cell death. These new additions to the understanding of haemolysin‐mediated pathogenesis in vivo provide evidence for the existence of noncanonical inflammasome signalling in lower vertebrates.     

Degradation of Wheat Straw by Fibrobacter succinogenes S85: a Liquid- and Solid-State Nuclear Magnetic Resonance Study

Wheat straw degradation by Fibrobacter succinogenes was monitored by nuclear magnetic resonance (NMR) spectroscopy and chemolytic methods to investigate the activity of an entire fibrolytic system on an intact complex substrate. In situ solid-state NMR with ¹³C cross-polarization magic angle spinning was used to monitor the modification of the composition and structure of lignocellulosic fibers (of ¹³C-enriched wheat straw) during the growth of bacteria on this substrate. There was no preferential degradation either of amorphous regions of cellulose versus crystalline regions or of cellulose versus hemicelluloses in wheat straw. This suggests either a simultaneous degradation of the amorphous and crystalline parts of cellulose and of cellulose and hemicelluloses by the enzymes or degradation at the surface at a molecular scale that cannot be detected by NMR. Liquid-state two-dimensional NMR experiments and chemolytic methods were used to analyze in detail the various sugars released into the culture medium. An integration of NMR signals enabled the quantification of oligosaccharides produced from wheat straw at various times of culture and showed the sequential activities of some of the fibrolytic enzymes of F. succinogenes S85 on wheat straw. In particular, acetylxylan esterase appeared to be more active than arabinofuranosidase, which was more active than α-glucuronidase. Finally, cellodextrins did not accumulate to a great extent in the culture medium.     

The Effect of Cellobiose,Glucose, and Cellulose on the Survival of <Emphasis Type="Italic">Fibrobacter succinogenes</Emphasis> A3C Cultures Grown Under Ammonia Limitation

The ruminal, cellulolytic bacterium, Fibrobacter succinogenes A3C, grew rapidly on cellulose, cellobiose, or glucose, but it could not withstand long periods of energy source starvation. If ammonia was limiting and either cellobiose or glucose was in excess, the viability declined even faster. The carbohydrate-excess, ammonia-limited cultures did not spill energy, but they accumulated large amounts of cellular polysaccharide. Cultures that were carbohydrate-limited had approximately 4 nmol ATP mg cell protein^–1, but ATP could not be detected in cultures that had an excess of soluble carbohydrates. However, if F. succinogenes A3C was provided with excess cellulose and ammonia was limiting, ATP did not decline, and the cultures digested the cellulose soon after additional nitrogen sources were added. From these results, it appears that excess soluble carbohydrates can promote the death of F. succinogenes, but cellulose does not.     

Inhibition of fucosylation of cell wall components by 2‐fluoro 2‐deoxy‐l‐fucose induces defects in root cell elongation

Screening of commercially available fluoro monosaccharides as putative growth inhibitors in Arabidopsis thaliana revealed that 2‐fluoro 2‐l ‐fucose (2F‐Fuc) reduces root growth at micromolar concentrations. The inability of 2F‐Fuc to affect an Atfkgp mutant that is defective in the fucose salvage pathway indicates that 2F‐Fuc must be converted to its cognate GDP nucleotide sugar in order to inhibit root growth. Chemical analysis of cell wall polysaccharides and glycoproteins demonstrated that fucosylation of xyloglucans and of N‐linked glycans is fully inhibited by 10 μm 2F‐Fuc in Arabidopsis seedling roots, but genetic evidence indicates that these alterations are not responsible for the inhibition of root development by 2F‐Fuc. Inhibition of fucosylation of cell wall polysaccharides also affected pectic rhamnogalacturonan‐II (RG‐II). At low concentrations, 2F‐Fuc induced a decrease in RG‐II dimerization. Both RG‐II dimerization and root growth were partially restored in 2F‐Fuc‐treated seedlings by addition of boric acid, suggesting that the growth phenotype caused by 2F‐Fuc was due to a deficiency of RG‐II dimerization. Closer investigation of the 2F‐Fuc‐induced growth phenotype demonstrated that cell division is not affected by 2F‐Fuc treatments. In contrast, the inhibitor suppressed elongation of root cells and promoted the emergence of adventitious roots. This study further emphasizes the importance of RG‐II in cell elongation and the utility of glycosyltransferase inhibitors as new tools for studying the functions of cell wall polysaccharides in plant development. Moreover, supplementation experiments with borate suggest that the function of boron in plants might not be restricted to RG‐II cross‐linking, but that it might also be a signal molecule in the cell wall integrity‐sensing mechanism.     

Salmonella enterica delivers its genotoxin through outer membrane vesicles secreted from infected cells

Cytolethal‐distending toxins (CDTs) belong to a family of DNA damage inducing exotoxins that are produced by several Gram‐negative bacteria. Salmonella enterica serovar Typhi expresses its CDT (named as Typhoid toxin) only in the Salmonella‐containing vacuole (SCV) of infected cells, which requires its export for cell intoxication. The mechanisms of secretion, release in the extracellular space and uptake by bystander cells are poorly understood. We have addressed these issues using a recombinant S. Typhimurium strain, MC71‐CDT, where the genes encoding for the PltA, PltB and CdtB subunits of the Typhoid toxin are expressed under control of the endogenous promoters. MC71‐CDT grown under conditions that mimic the SCV secreted the holotoxin in outer membrane vesicles (OMVs). Epithelial cells infected with MC71‐CDT also secreted OMVs‐like vesicles. The release of these extracellular vesicles required an intact SCV and relied on anterograde transport towards the cellular cortex on microtubule and actin tracks. Paracrine internalization of Typhoid toxin‐loaded OMVs by bystander cells was dependent on dynamin‐1, indicating active endocytosis. The subsequent induction of DNA damage required retrograde transport of the toxin through the Golgi complex. These data provide new insights on the mode of secretion of exotoxins by cells infected with intracellular bacteria.     

Association of Vibrio cholerae 569B outer membrane vesicles with host cells occurs in a GM1‐independent manner

The primary virulence factor of Vibrio cholerae, cholera toxin (CT), initiates a pathway in epithelial cells that leads to the severe diarrhoea characteristic of cholera. Secreted CT binds to GM1 on the surface of host cells to facilitate internalisation. Many bacterial toxins, including CT, have been shown to be additionally delivered via outer membrane vesicles (OMVs). A fraction of the closely related heat labile toxin produced by enterotoxigenic Escherichia coli has been demonstrated to reside on the surface of OMVs, where it binds GM1 to facilitate OMV internalisation by host cells. In this work, we investigated whether OMV‐associated CT is likewise trafficked to host cells in a GM1‐dependent mechanism. We demonstrated that a majority of CT is secreted in its OMV‐associated form and is located exclusively inside the vesicle. Therefore, the toxin is unable to bind GM1 on the host cell surface, and the OMVs are trafficked to the host cells in a GM1‐independent mechanism. These findings point to a secondary, noncompeting mechanism for secretion and delivery of CT, beyond its well‐studied secretion via a Type II secretion system and underscore the importance of focusing future studies on understanding this GM1‐independent delivery mechanism to fully understand Vibrio cholerae pathogenesis.