Induction of a Specific Humoral Immune Response by Nasal Delivery of Bcla2ctd of Clostridioides difficile

Abstract

Clostridioides difficile, formerly known as Clostridium difficile, is a spore-forming bacterium considered as the most common cause of nosocomial infections in developed countries. The spore of C. difficile is involved in the transmission of the pathogen and in its first interaction with the host; therefore, a therapeutic approach able to control C. difficile spores would improve the clearance of the infection. The C-terminal (CTD) end of BclA2, a spore surface protein of C. difficile responsible of the interaction with the host intestinal cells, was selected as a putative mucosal antigen. The BclA2 fragment, BclA2CTD, was purified and used to nasally immunize mice both as a free protein and after adsorption to the spore of Bacillus subtilis, a well-established mucosal delivery vehicle. While the adsorption to spores increased the in vitro stability of BclA2CTD, in vivo both free and spore-adsorbed BclA2CTD were able to induce a similar, specific humoral immune response in a murine model. Although in the experimental conditions utilized the immune response was not protective, the induction of specific IgG indicates that free or spore-bound BclA2CTD could act as a putative mucosal antigen targeting C. difficile spores.

Nasal Immunization with the C-Terminal Domain of Bcla3 Induced Specific IgG Production and Attenuated Disease Symptoms in Mice Infected with Clostridioides difficile Spores

“…Hence, His-tagged BclA3 CTD was overexpressed in Escherichia coli BL21(DE3) and purified by affinity chromatography with Ni-sepharose columns as described in the Methods section. Upon purification, the protein was loaded on a Sodium Dodecyl Sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) ( Figure 1 C) next to pure BclA2 CTD , an exosporium protein already purified by our group [ 23 ]. Whilst pure BclA2 CTD migrates on SDS-PAGE gel as a single band of approximately 14 kDa, as expected, pure BclA3 CTD consistently migrates in two bands, one with a molecular weight of about 20 kDa and the other of about 60 kDa that match with the predicted sizes of a monomer and a trimer, respectively, which may indicate protein aggregation.…”

“…The spore, form in which C. difficile persists inside the host, possibly modulating its immune system, is still not considered as a main target for therapies. We have recently demonstrated that the C-terminal domain of C. difficile exosporium protein BclA2 elicits an elevated humoral response after nasal immunization in mice [ 23 ]. Here, we have identified as a potential antigen to induce an anti-spore immune response, the C-terminal domain of the exosporium protein BclA3 (BclA3 CTD ) of the hyper-virulent strain R20291 of C. difficile .…”

“…Further investigation needs to be done in order to fully understand the role of BclA proteins in C. difficile spores; however, evidence suggest that these proteins are involved in the formation of the hair-like projections in most C. difficile strains such as the hyper-virulent strain R20291 [ 17 , 18 ] and their spore surface location propose them as potential antigens [ 19 ]. While the antigenicity of BclA1 and BclA2 has been recently tested [ 20 , 21 , 22 , 23 ], BclA3 has not been directly evaluated to date.…”

Nasal immunization with the C-terminal domain of BclA3 induced specific IgG production and attenuated disease symptoms in mice infected withClostridioides difficilespores

“…The spore, form in which C. difficile persists inside the host, possibly modulating its immune system, is still not considered as a main target for therapies. We have recently demonstrated that the C-terminal domain of BclA2 elicits an elevated humoral response [18]. Here, we have identified as a potential antigen to induce an anti-spore immune response, the C-terminal domain of the exosporium protein BclA3 (BclA3CTD) of the hyper-virulent strain R20291 of C. difficile.…”

“…Hence, His-tagged BclA3CTD was overexpressed in Escherichia coli BL21(DE3) and purified by affinity chromatography with Nisepharose columns as described in the Methods section. Upon purification, the protein was loaded on an SDS-Page gel ( Figure 1C) next to pure BclA2CTD, an exosporium protein already purified by our group [18]. Whilst pure BclA2CTD migrates on SDS-Page gel as a single band of approximately 14 kDa, as expected, pure BclA3CTD consistently migrates in two bands, one with about 20 kDa and other with approximately 60 kDa that match with the predicted sizes of a monomer and trimers, respectively, which may indicate protein aggregation.…”