Delivery of Biologically Active Anti-Inflammatory Cytokines IL-10 and IL-1ra In Vivo by the Shigella Type III Secretion Apparatus

Abstract

Pathogenicity of many Gram-negative bacteria relies on a type III secretion (T3S) apparatus, which is used for delivery of bacterial effectors into the host cell cytoplasm allowing the bacteria to manipulate host cell cytoskeleton network as well as to interfere with intracellular signaling pathways. In this study, we investigated the potential of the Shigella flexneri T3SA as an in vivo delivery system for biologically active molecules such as cytokines. The anti-inflammatory cytokines IL-10 and IL-1 receptor antagonist (IL-1ra) were genetically fused to the first 30 or 60 residues of the Shigella T3S effector IpaH9.8 or to the first 50 residues of the Yersinia enterocolitica effector YopE and the recombinant fusion proteins were expressed in S. flexneri. YopE50-IL-10, IpaH60-IL-10, and IpaH60-IL-1ra were efficiently secreted via the T3S apparatus of Shigella. Moreover, these recombinant proteins did not impair the invasive ability of the bacteria in vitro. In a murine model, Shigella strains expressing YopE50-IL-10, IpaH60-IL-10, and IpaH60-IL-1ra induced a lower mortality in mice that was associated with reduced inflammation and a restricted localization of bacteria within the lung tissues as compared with wild-type Shigella. Moreover, the level of TNF-α and IL-1β mRNA were reduced in the lungs following infection by IL-10- and IL-1ra-secreting Shigella, respectively. These findings demonstrate that the Shigella T3S apparatus can deliver biologically active cytokines in vivo, thus opening new avenues for the use of attenuated bacteria to deliver proteins for immunomodulation or gene therapy purposes.

Tobacco Upregulates P. gingivalis Fimbrial Proteins Which Induce TLR2 Hyposensitivity

“…This inflammatory suppression is reflected in lower concentrations of pro-inflammatory mediators (IL-1β, IL-6 and, perhaps, TNF-α) in the gingival crevicular fluid and periodontal tissues of smokers, compared to non-smokers [53], [54]. Inefficient TLR stimulation and the promotion of a less vigorous inflammatory response is an established means of immune evasion for several pathogens, including Bordetella Shigella and pathogenic Escherichia [55], [56], [57], [58].…”

Type III secretion systems: the bacterial flagellum and the injectisome

“…Similarly, the flagellar T3SS was shown to be able to export a variety of different protein types [191] and subsequently optimized for protein secretion [263]. The injectisome, a system evolutionarily optimized for protein translocation, has also been used to directly transport proteins into host cells, especially to deliver vaccines [264][265][266][267], but also for immunomodulatory cytokines [268]. Traditionally, attenuated strains were used for this purpose, but recently, achromosomal, but T3SS-active minicells were also shown to be functional for translocation [269].…”

Periplasmic Delivery of Biologically Active Human Interleukin-10 in <i>Escherichia coli</i> via a Sec-Dependent Signal Peptide

“…Alternatively, genetically modified microorganisms provide a possible means for the in situ delivery of therapeutic proteins [Beninati et al, 2000;Krüger et al, 2002;Steidler et al, 1998]. Lactococcus lactis and Shigella flexneri strains have been engineered to secrete biologically active IL-10 [Chamekh et al, 2008;Steidler et al, 2000]. However, the good colonization properties and the lack of virulence determinants may render nonpathogenic E. coli strains such as probiotic E. coli Nissle 1917 a more ideal carrier chassis for intestinal drug delivery.…”