Search Results (9)

Bile acids, the primary components of bile, are cholesterol-derived molecules synthesized in the liver and secreted to the small intestine. Besides their primary digestive roles, bile acids have antimicrobial properties and serve as an environmental cue for intestinal pathogens, modulating the expression of virulence factors, e.g., toxins and effector proteins. Whereas timely recognition and neutralization of pathogenic toxin effectors by the host is critical, our understanding of the effects of bile on their structure and function is limited. In this work, we found that bile effectively protected cultured IEC-18 enterocytes from the mixture of Aeromonas hydrophila secreted toxins, containing hemolysin, aerolysin, and RtxA (MARTX). To explore whether these effects have broad specificity, we employed biochemical and biophysical techniques to test the in vitro effects of bile and bile acids on several effector domains of MARTX and VgrG toxins from Vibrio cholerae and Aeromonas hydrophila, and catalytic domains of TcdA and TcdB toxins from Clostridioides difficile. Bile compromised the structural integrity of the tested effectors to various degrees in a protein charge-dependent manner. Bile and bile acids promoted exposure of hydrophobic residues and the unfolding of most, but not all, of the tested effectors, facilitating their precipitation and cleavage by chymotrypsin. Bile also inhibited specific activities of the tested effector enzymes, partially due to imposed oxidation of their catalytic residues. To summarize, this work validated bile as a non-proteinaceous factor of innate immunity, capable of compromising the structural integrity and function of the effector domains of various bacterial toxins. Full article

Competition between bacterial species is a major factor shaping microbial communities. It is possible but remains largely unexplored that competition between bacterial pathogens can be mediated through antagonistic effects of bacterial effector proteins on host systems, particularly the actin cytoskeleton. Using Salmonella Typhimurium invasion into cells as a model, we demonstrate that invasion is inhibited if the host actin cytoskeleton is disturbed by actin-specific toxins, namely, Vibrio cholerae MARTX actin crosslinking (ACD) and Rho GTPase inactivation (RID) domains, Photorhabdus luminescens TccC3, and Salmonella’s own SpvB. We noticed that ACD, being an effective inhibitor of tandem G-actin-binding assembly factors, is likely to inhibit the activity of another Vibrio effector, VopF. In reconstituted actin polymerization assays and by live-cell microscopy, we confirmed that ACD potently halted the actin nucleation and pointed-end elongation activities of VopF, revealing competition between these two V. cholerae effectors. These results suggest that bacterial effectors from different species that target the same host machinery or proteins may represent an effective but largely overlooked mechanism of indirect bacterial competition in host-associated microbial communities. Whether the proposed inhibition mechanism involves the actin cytoskeleton or other host cell compartments, such inhibition deserves investigation and may contribute to a documented scarcity of human enteric co-infections by different pathogenic bacteria. Full article

Actin is an essential element of both innate and adaptive immune systems and can aid in motility and translocation of bacterial pathogens, making it an attractive target for bacterial toxins. Pathogenic Vibrio and Aeromonas genera deliver actin cross-linking domain (ACD) toxin into the cytoplasm of the host cell to poison actin regulation and promptly induce cell rounding. At early stages of toxicity, ACD covalently cross-links actin monomers into oligomers (AOs) that bind through multivalent interactions and potently inhibit several families of actin assembly proteins. At advanced toxicity stages, we found that the terminal protomers of linear AOs can get linked together by ACD to produce cyclic AOs. When tested against formins and Ena/VASP, linear and cyclic AOs exhibit similar inhibitory potential, which for the cyclic AOs is reduced in the presence of profilin. In coarse-grained molecular dynamics simulations, profilin and WH2-motif binding sites on actin subunits remain exposed in modeled AOs of both geometries. We speculate, therefore, that the reduced toxicity of cyclic AOs is due to their reduced configurational entropy. A characteristic feature of cyclic AOs is that, in contrast to the linear forms, they cannot be straightened to form filaments (e.g., through stabilization by cofilin), which makes them less susceptible to neutralization by the host cell. Full article

After invading a host, bacterial pathogens secrete diverse protein toxins to disrupt host defense systems. To ensure successful infection, however, pathogens must precisely regulate the expression of those exotoxins because uncontrolled toxin production squanders energy. Furthermore, inappropriate toxin secretion can trigger host immune responses that are detrimental to the invading pathogens. Therefore, bacterial pathogens use diverse transcriptional regulators to accurately regulate multiple exotoxin genes based on spatiotemporal conditions. This review covers three major exotoxins in pathogenic Vibrio species and their transcriptional regulation systems. When Vibrio encounters a host, genes encoding cytolysin/hemolysin, multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin, and secreted phospholipases are coordinately regulated by the transcriptional regulator HlyU. At the same time, however, they are distinctly controlled by a variety of other transcriptional regulators. How this coordinated but distinct regulation of exotoxins makes Vibrio species successful pathogens? In addition, anti-virulence strategies that target the coordinating master regulator HlyU and related future research directions are discussed. Full article

Vibrio cholerae non-O1, non-O139 bacteria are natural inhabitants of aquatic ecosystems and have been sporadically associated with human infections. They mostly lack the two major virulence factors of toxigenic V. cholerae serogroups O1 and O139 strains, which are the causative agent of cholera. Non-O1, non-O139 strains are found in water bodies, sediments, and in association with other aquatic organisms. Occurrence of these bacteria in fecal specimens of waterfowl were reported, and migratory birds likely contribute to the long-distance transfer of strains. We investigated four V. cholerae non-O1, non-O139 isolates for phenotypic traits and by whole genome sequencing (WGS). The isolates were recovered from organs of domestic ducks with serious disease symptoms. WGS data revealed only a distant genetic relationship between all isolates. The isolates harbored a number of virulence factors found in most V. cholerae strains. Specific virulence factors of non-O1, non-O139 strains, such as the type III secretion system (TTSS) or cholix toxin, were observed. An interesting observation is that all isolates possess multifunctional autoprocessing repeats-in-toxin toxins (MARTX) closely related to the MARTX of toxigenic El Tor O1 strains. Different primary sequences of the abundant OmpU proteins could indicate a significant role of this virulence factor. Phenotypic characteristics such as hemolysis and antimicrobial resistance (AMR) were studied. Three isolates showed susceptibility to a number of tested antimicrobials, and one strain possessed AMR genes located in an integron. Knowledge of the environmental occurrence of V. cholerae non-O1, non-O139 in Germany is limited. The source of the infection of the ducks is currently unknown. In the context of the ‘One Health’ concept, it is desirable to study the ecology of V. cholerae non-O1, non-O139, as it cannot be excluded that the isolates possess zoonotic potential and could cause infections in humans. Full article

Many Gram-negative bacterial pathogens directly deliver numerous effector proteins from the bacterium to the host cell, thereby altering the target cell physiology. The already well-characterized effector delivery systems are type III, type IV, and type VI secretion systems. Multifunctional autoprocessing repeats-in-toxin (MARTX) toxins are another effector delivery platform employed by some genera of Gram-negative bacteria. These single polypeptide exotoxins possess up to five effector domains in a modular fashion in their central regions. Upon binding to the host cell plasma membrane, MARTX toxins form a pore using amino- and carboxyl-terminal repeat-containing arms and translocate the effector domains into the cells. Consequently, MARTX toxins affect the integrity of the host cells and often induce cell death. Thus, they have been characterized as crucial virulence factors of certain human pathogens. This review covers how each of the MARTX toxin effector domains exhibits cytopathic and/or cytotoxic activities in cells, with their structural features revealed recently. In addition, future directions for the comprehensive understanding of MARTX toxin-mediated pathogenesis are discussed. Full article

Martentoxin (MarTX), a 37-residue peptide purified from the venom of East-Asian scorpion (Buthus martensi Karsch), was capable of blocking large-conductance Ca²⁺-activated K⁺ (BK) channels. Here, we report an effective expression and purification approach for this toxin. The cDNA encoding martentoxin was expressed by the prokaryotic expression system pGEX-4T-3 which was added an enterokinase cleavage site by PCR. The fusion protein (GST-rMarTX) was digested by enterokinase to release hetero-expressed toxin and further purified via reverse-phase HPLC. The molecular weight of the hetero-expressed rMarTX was 4059.06 Da, which is identical to that of the natural peptide isolated from scorpion venom. Functional characterization through whole-cell patch clamp showed that rMarTX selectively and potently inhibited the currents of neuronal BK channels (α + β4) (IC₅₀ = 186 nM), partly inhibited mKv1.3, but hardly having any significant effect on hKv4.2 and hKv3.1a even at 10 μM. Successful expression of martentoxin lays basis for further studies of structure-function relationship underlying martentoxin or other potassium-channel specific blockers. Full article

Protease domains within toxins typically act as the primary effector domain within target cells. By contrast, the primary function of the cysteine protease domain (CPD) in Multifunctional Autoprocessing RTX-like (MARTX) and Clostridium sp. glucosylating toxin families is to proteolytically cleave the toxin and release its cognate effector domains. The CPD becomes activated upon binding to the eukaryotic-specific small molecule, inositol hexakisphosphate (InsP₆), which is found abundantly in the eukaryotic cytosol. This property allows the CPD to spatially and temporally regulate toxin activation, making it a prime candidate for developing anti-toxin therapeutics. In this review, we summarize recent findings related to defining the regulation of toxin function by the CPD and the development of inhibitors to prevent CPD-mediated activation of bacterial toxins. Full article

Actin crosslinking toxins produced by Gram-negative bacteria represent a small but unique class of bacterial protein toxins. For each of these toxins, a discrete actin crosslinking domain (ACD) that is a distant member of the ATP-dependent glutamine synthetase family of protein ligases is translocated to the eukaryotic cell cytosol. This domain then incorporates a glutamate-lysine crosslink between actin monomers, resulting in destruction of the actin cytoskeleton. Recent studies argue that the function of these toxins during infection is not destruction of epithelial layers, but rather may specifically target phagocytic cells to promote survival of bacteria after the onset of innate immune defenses. This review will summarize key experiments performed over the past 10 years to reveal the function of these toxins. Full article