Lactococcus lactis as a vehicle for the heterologous expression of fungal ribotoxin variants with reduced IgE-binding affinity

Álvarez-García, Elisa; Alegre-Cebollada, Jorge; Batanero, Eva; Monedero, Vicente; Pérez-Martı́nez, Gaspar; García‐Fernández, Rosa A.; Gavilanes, José G.; Pozo, Álvaro Martı́nez del

doi:10.1016/j.jbiotec.2007.12.011

Cited by 5 publications

(4 citation statements)

References 45 publications

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“…These non-cytotoxic and hypoallergenic mutant variants of Aspf1 and α-sarcin might be suitable for their use in immunomodulating therapies against Aspergillus hypersensitivity and its diagnosis. Indeed, they have been produced in Lactococcus lactis , a GRAS (generally regarded as safe) microorganism that could be used in immunotherapeutic protocols for Aspf1-related diseases as a vaccination vehicle [ 92 ]. Furthermore, a mouse model of A. fumigatus sensitization has been established, that could be suitable for preclinical testing of recombinant allergens and their derivatives [ 93 ].…”

Section: The Potential Biotechnological Uses Of Fungal Ribotoxinsmentioning

confidence: 99%

Fungal Ribotoxins: A Review of Potential Biotechnological Applications

Olombrada

Lázaro-Gorines

López-Rodríguez

et al. 2017

Toxins

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Fungi establish a complex network of biological interactions with other organisms in nature. In many cases, these involve the production of toxins for survival or colonization purposes. Among these toxins, ribotoxins stand out as promising candidates for their use in biotechnological applications. They constitute a group of highly specific extracellular ribonucleases that target a universally conserved sequence of RNA in the ribosome, the sarcin-ricin loop. The detailed molecular study of this family of toxic proteins over the past decades has highlighted their potential in applied research. Remarkable examples would be the recent studies in the field of cancer research with promising results involving ribotoxin-based immunotoxins. On the other hand, some ribotoxin-producer fungi have already been studied in the control of insect pests. The recent role of ribotoxins as insecticides could allow their employment in formulas and even as baculovirus-based biopesticides. Moreover, considering the important role of their target in the ribosome, they can be used as tools to study how ribosome biogenesis is regulated and, eventually, may contribute to a better understanding of some ribosomopathies.

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Section: The Potential Biotechnological Uses Of Fungal Ribotoxinsmentioning

confidence: 99%

Fungal Ribotoxins: A Review of Potential Biotechnological Applications

Olombrada

Lázaro-Gorines

López-Rodríguez

et al. 2017

Toxins

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“…ssl7 was inserted into pDONR221 using Gateway BP Clonase II and transferred into the shuttle vector pOri23T using Gateway LP Clonase II. The completed plasmid was electroporated into L. lactis as described previously 37 . Expression of soluble SSL7 was confirmed by western blot analysis of culture supernatant.…”

Section: Methodsmentioning

confidence: 99%

“…The completed plasmid was electroporated into L. lactis as described previously. 37 Expression of soluble SSL7 was confirmed by western blot analysis of culture supernatant. Samples were separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane (PALL Life Sciences, Pensacola, FL, USA) in Towbin western transfer buffer (0.192 M glycine/25 mM Tris:Hcl, pH 8.3/1.3 mM sodium dodecyl sulfate/20% methanol) and protein expression confirmed with rabbit polyclonal anti-SSL7 (in-house).…”

Section: Protein Expression and Purificationmentioning

confidence: 97%

Full functional activity of SSL7 requires binding of both complement C5 and IgA

et al. 2013

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Staphylococcus aureus is an opportunistic bacterial pathogen responsible for a range of diseases, from local skin infections through to life-threatening illnesses such as toxic shock syndrome. S. aureus produces an assortment of molecules designed to evade or subvert the host immune system. One example is the 23 kDa staphylococcal superantigen-like protein 7 (SSL7) that simultaneously binds immunoglobulin A (IgA) and complement C5 to inhibit complement-mediated hemolytic and bactericidal activity. The avirulent bacterium Lactococcus lactis was engineered to express SSL7 so that its role in bacterial survival could be assessed without interference from other virulence factors. Expression of SSL7 by L. lactis led to significantly enhanced bacterial survival in whole human blood and prevented the membrane attack complex (C5b-9) forming on the cell wall. To further understand the mechanism of action of SSL7, the activity of wild-type SSL7 protein was compared with a panel of mutant proteins lacking the capacity to bind IgA, C5, or both IgA and C5. SSL7 potently inhibited in vitro chemotaxis of inflammatory myeloid cells in response to a pathogenic stimulus and when injected into mice, SSL7 blocked the migration of neutrophils into the peritoneum in response to an inoculum of heat-killed S. aureus. Mutagenesis of the C5-binding site on SSL7 abolished all inhibitory activity, while mutation of the IgA-binding site had only partial effects, indicating that while IgA binding enhances activity it is not essential. SSL7 is an important staphylococcal virulence factor with potent anti-inflammatory properties, which are mediated by targeting complement C5 and IgA.

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“…Within this idea, it must be remarked how these potentially hypoallergenic ribotoxin molecules have been recently cloned and produced in Lactococcus lactis [98], a microorganism 'generally regarded as safe' (GRAS). This GRAS status of L. lactis, altogether with the fact that the hypoallergenic variants of Asp f 1 are secreted by the engineered bacteria, confers this system with the features required to try immunotherapeutic protocols for Asp f 1-related allergic diseases.…”

Section: The a Fumigatus Allergen Asp Fmentioning

confidence: 99%

The Therapeutic Potential of Fungal Ribotoxins

Carreras-Sangrà

Álvarez-García²,

Herrero-Galán³

et al. 2008

CPB

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Ribotoxins constitute a family of toxic extracellular fungal RNases that exert a highly specific activity on a conserved region of the larger molecule of rRNA, known as the sarcin-ricin loop. This cleavage of a single phosphodiester bond inactivates the ribosome and leads to protein synthesis inhibition and cell death. In addition to this ribonucleolytic activity, ribotoxins can cross lipid membranes in the absence of any known protein receptor. This ability is due to their capacity to interact with acid phospholipid-containing membranes. Both activities together explain their cytotoxic character, being rather specific when assayed against some transformed cell lines. The determination of highresolution structures of some ribotoxins, the characterization of a large number of mutants, and the use of lipid model vesicles and transformed cell lines have been the tools used for the study of their mechanism of action at the molecular level. The present knowledge suggests that wild-type ribotoxins or some modified variants might be used in human therapies. Production of hypoallergenic mutants and immunotoxins designed against specific tumors stand out as feasible alternatives to treat some human pathology in the midterm future.3

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Lactococcus lactis as a vehicle for the heterologous expression of fungal ribotoxin variants with reduced IgE-binding affinity

Cited by 5 publications

References 45 publications

Fungal Ribotoxins: A Review of Potential Biotechnological Applications

Fungal Ribotoxins: A Review of Potential Biotechnological Applications

Full functional activity of SSL7 requires binding of both complement C5 and IgA

The Therapeutic Potential of Fungal Ribotoxins

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