Expression of recombinant proteins in a lipid A mutant of Escherichia coli BL21 with a strongly reduced capacity to induce dendritic cell activation and maturation

Cognet, Isabelle; Coignac, Amélie Benoit de; Magistrelli, Giovanni; Jeannin, Pascale; Aubry, Jean‐Pierre; Maisnier-Patin, Karine; Caron, Gersende; Chevalier, Sylvie; Humbert, F; Nguyen, Thien Ngoc; Beck, Alain; Velin, Dominique; Delneste, Yves; Malissard, Martine; Gauchat, Jean‐François

doi:10.1016/s0022-1759(02)00506-9

Cited by 37 publications

(38 citation statements)

References 54 publications

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“…The secondary acyl chains have been demonstrated to be involved in the LPS recognition mechanism by the TLR4/MD-2 complex (35). Especially, the penta-acylated E. coli LPS produced by LpxM mutant lost the reactivity almost completely (26). This may be explained by the loss of the binding activity for the penta-acylated LPS to MD-2.…”

Section: Discussionmentioning

confidence: 99%

“…Further incorporation of single myristate and laurate moieties in the final step of biosynthesis occurs in E. coli. Human TLR4/MD-2 complexes do not respond to LPS produced in E. coli that possess a disrupted lpxM gene (26). The lpxM gene product promotes acylation of the (R)-3-hydroxymyristoyl chain located at the 3Ј position of the glucosamine disaccharide backbone leading to addition of the secondary myristoyl chain.…”

Section: Penta-acylated Lps Produced In Lpxmϫ Strain Does Not Bind Tomentioning

confidence: 99%

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The Functional and Structural Properties of MD-2 Required for Lipopolysaccharide Binding Are Absent in MD-1

Tsuneyoshi

Fukudome

Kohara

et al. 2005

The Journal of Immunology

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MD-1 and MD-2 are secretory glycoproteins that exist on the cell surface in complexes with transmembrane proteins. MD-1 is anchored by radioprotective 105 (RP105), and MD-2 is associated with TLR4. In vivo studies revealed that MD-1 and MD-2 have roles in responses to LPS. Although the direct binding function of MD-2 to LPS has been observed, the physiological function of MD-1 remains unknown. In this study, we compared the LPS-binding functions of MD-1 and MD-2. LPS binding to cell surface complexes was detected for cells transfected with TLR4/MD-2. In contrast, binding was not observed for RP105/MD-1-transfected cells. When rMD-2 protein was expressed in Escherichia coli, it was purified in complexes containing LPS. In contrast, preparations of MD-1 did not contain LPS. When rMD-2 protein was prepared in a mutant strain lacking the lpxM gene, LPS binding disappeared. Therefore, the secondary myristoyl chain attached to the (R)-3-hydroxymyristoyl chain added by LpxM is required for LPS recognition by MD-2, under these conditions. An amphipathic cluster composed of basic and hydrophobic residues in MD-2 has been suggested to be the LPS-binding site. We specifically focused on two Phe residues (119 and 121), which can associate with fatty acids. A mutation at Phe191 or Phe121 strongly reduced binding activity, and a double mutation at these residues prevented any binding from occurring. The Phe residues are present in MD-2 and absent in MD-1. Therefore, the LPS recognition mechanism by RP105/MD-1 is distinct from that of TLR4/MD-2.

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Section: Discussionmentioning

confidence: 99%

Section: Penta-acylated Lps Produced In Lpxmϫ Strain Does Not Bind Tomentioning

confidence: 99%

The Functional and Structural Properties of MD-2 Required for Lipopolysaccharide Binding Are Absent in MD-1

Tsuneyoshi

Fukudome

Kohara

et al. 2005

The Journal of Immunology

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“…Because the E. coli BL21(DE3) lpxM-host strain produces a penta-rather than hexa-acylated (nonmyristoylated) LPS, it has very low LPS activity as described by Cognet et al, (13). SubA A272 B, a nonfunctional variant of SubAB that has a point mutation in the active site serine residue of SubA subunit and has lost its protease (and also UPR-inducing) activity, was developed by site-directed mutagenesis of SubA as previously described (1).…”

Section: Reagentsmentioning

confidence: 99%

A Subcytotoxic Dose of Subtilase Cytotoxin Prevents Lipopolysaccharide-Induced Inflammatory Responses, Depending on its Capacity to Induce the Unfolded Protein Response

Harama

Koyama

Mukai

et al. 2009

The Journal of Immunology

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Subtilase cytotoxin (SubAB) is the prototype of a newly identified family of AB5 cytotoxins produced by Shiga toxigenic Escherichia coli. SubAB specifically cleaves the essential endoplasmic reticulum (ER) chaperone BiP (GRP78), resulting in the activation of ER stress-induced unfolded protein response (UPR). We have recently shown that the UPR following ER stress can suppress cellular responses to inflammatory stimuli during the later phase, in association with inhibition of NF-κB activation. These findings prompted us to hypothesize that SubAB, as a selective UPR inducer, might have beneficial effects on inflammation-associated pathology via a UPR-dependent inhibition of NF-κB activation. The pretreatment of a mouse macrophage cell line, RAW264.7, with a subcytotoxic dose of SubAB-triggered UPR and inhibited LPS-induced MCP-1 and TNF-α production associated with inhibition of NF-κB activation. SubAA272B, a SubAB active site mutant that cannot induce UPR, did not show such effects. In addition, pretreatment with a sublethal dose of SubAB, but not SubAA272B, protected the mice from LPS-induced endotoxic lethality associated with reduced serum MCP-1 and TNF-α levels and also prevented the development of experimental arthritis induced by LPS in mice. Collectively, although SubAB has been identified originally as a toxin associated with the pathogenesis of hemolytic uremic syndrome, the unique ability of SubAB to selectively induce the UPR may have the potential to prevent LPS-associated inflammatory pathology under subcytotoxic conditions.

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“…4, control), reinforcing the cytotoxic properties of ''heat labile'' rCARDS TX and negating the possible contribution of E. coli endotoxin in recombinant protein preparations. In the latter case, all recombinant proteins were expressed and purified from lpxM-inactivated E. coli BL21 (DE3) (24), which produces a nonmyristylated lipopolysaccharide (nmLPS) with markedly reduced endotoxicity. Also, purified recombinant proteins were passed through sequential polymixin columns to reduce remaining endotoxin contamination before use, and we performed Limulus assays to determine endotoxin concentrations in each recombinant preparation.…”

Section: Cytopathic Effects (Cpes) Of Rcards Tx On Mammalian Monolayementioning

confidence: 99%

ADP-ribosylating and vacuolating cytotoxin of Mycoplasma pneumoniae represents unique virulence determinant among bacterial pathogens

Kannan

Baseman

2006

Proc. Natl. Acad. Sci. U.S.A.

190

278

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Unlike many bacterial pathogens, Mycoplasma pneumoniae is not known to produce classical toxins, and precisely how M. pneumoniae injures the respiratory epithelium has remained a mystery for >50 years. Here, we report the identification of a virulence factor (MPN372) possibly responsible for airway cellular damage and other sequelae associated with M. pneumoniae infections in humans. We show that M. pneumoniae MPN372 encodes a 68-kDa protein that possesses ADP-ribosyltransferase (ART) activity. Within its N terminus, MPN372 contains key amino acids associated with NAD binding and ADP-ribosylating activity, similar to pertussis toxin (PTX) S1 subunit (PTX-S1). Interestingly, MPN372 ADP ribosylates both identical and distinct mammalian proteins when compared with PTX-S1. Remarkably, MPN372 elicits extensive vacuolization and ultimate cell death of mammalian cells, including distinct and progressive patterns of cytopathology in tracheal rings in organ culture that had been previously ascribed to infection with WT virulent M. pneumoniae. We observed dramatic seroconversion to MPN372 in patients diagnosed with M. pneumoniae-associated pneumonia, indicating that this toxin is synthesized in vivo and possesses highly immunogenic epitopes.ADP ribosylation ͉ community-acquired respiratory distress syndrome toxin ͉ vacuolization T he earliest reports of mycoplasmas as infectious agents in humans appeared in the 1940s (1). Definitive studies in the early 1960s established Mycoplasma pneumoniae as the singular cause of cold agglutinin-associated primary atypical pneumonia (2, 3). Today, M. pneumoniae is the best known of the human mycoplasmas (4). These bacteria are most unusual, lacking typical cell walls possessed by other prokaryotes, using UGA to encode tryptophan, and requiring cholesterol for growth and maintenance of membrane function and integrity. Much has been learned about the role of M. pneumoniae as a respiratory tract pathogen (5). M. pneumoniae infections constitute 20-40% of all community-acquired pneumonia and are frequently associated with other airway disorders, such as tracheobronchitis and pharyngitis. Extrapulmonary manifestations, such as hematopoietic, dermatologic, joint, central nervous system, liver, pancreas, kidney, and cardiovascular syndromes are considered sequelae of primary M. pneumoniae infections. Also, M. pneumoniae has been linked to fulminant disease, with multiorgan involvement (6). Therefore, M. pneumoniae causes a wide spectrum of pathologies, with more extensive complications than previously recognized (6), yet no single virulence determinant has been associated with these clinical signs and symptoms. In addition, definitive diagnosis and therapeutic decisions relative to M. pneumoniae infections are often delayed or lacking because of the long incubation period (average 1-2 weeks) before clinical symptoms can be observed. Further, direct isolation of M. pneumoniae from patients frequently fails, and, when successful, broth or colony growth requires 10-21 days.The early stages of t...

show abstract

Expression of recombinant proteins in a lipid A mutant of Escherichia coli BL21 with a strongly reduced capacity to induce dendritic cell activation and maturation

Cited by 37 publications

References 54 publications

The Functional and Structural Properties of MD-2 Required for Lipopolysaccharide Binding Are Absent in MD-1

The Functional and Structural Properties of MD-2 Required for Lipopolysaccharide Binding Are Absent in MD-1

A Subcytotoxic Dose of Subtilase Cytotoxin Prevents Lipopolysaccharide-Induced Inflammatory Responses, Depending on its Capacity to Induce the Unfolded Protein Response

ADP-ribosylating and vacuolating cytotoxin of Mycoplasma pneumoniae represents unique virulence determinant among bacterial pathogens

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