The cloning of sialidase genes from Clostridium indolis and Clostridium cocleatum.

Abstract

Gut microbial microflora is composed of u10 bacteria per 24 hours. The DNA was partially digested with HindIII; 2to 10kb fragments were isolated from agarose gel. Two-tenths of a mg gram, representing u400 species. It comprises both endogenous (resident) and exogenous (transiting) microbiota, which in healthy of insert DNA was ligated with 1 mg of HindIII-digested and alcaline phosphatase–treated l Express vector (Stratagene, La humans is kept under tight equilibrium. Our laboratory has developed a simplified model in trixenic mouse representing minimal Jolla, CA). Following in vitro packaging (Gigapack, Stratagene), the titer resident flora capable of inhibiting implantation of Clostridium difficile, a pathogenic bacterium responsible for a disease spectrum of the genomic library thus obtained was determined by infecting Escherichia coli XL1-Blue MRF cells (Stratagene) with aliquots ranging from postantibiotic diarrhea to pseudomembranous colitis. This flora comprises three strains: Clostridium indolis, Clostridium of packaged phage; it was found to be 3 1 10 pfu/mL. Plating onto indicator plates containing isopropyl-b-D-thiogalactopyracocleatum, and Eubacterium species, which act in concert to impede colonization by C. difficile. However, the mechanism by noside (IPTG; Promega, Madison, WI) and 5-bromo-4-chloro-3indolyl-b-D-galactopyranoside (X-Gal; Promega) revealed that which this is accomplished remains unknown. To explain the barrier effect, we have focused our attention on 87% of lambda arms carried an insert. The library was amplified by reinfecting E. coli XL1-Blue MRF; its titer was 4.5 1 10 the interactions taking place at the level of the cecal mucus layer pfu/mL. between the three players implicated in the process: the host, the To identify potential sialidase-positive clones, the phage library barrier microbiota, and C. difficile. By scanning electron microswas converted into a plasmid one by in vivo excision of pBKcopy [1], we have observed that the three barrier strains are embedCMV phagemid (Stratagene) carried by l Express, as described ded in the mucus (figure 1); furthermore, they are able to degrade in Stratagene protocols. About 4,000 white colonies were picked mucins [2]. The latter is the result of bacterial osidase activity, from X-Gal/IPTG-containing plates and tested for sialidase activity which first removes blood group residues (fucose, a-galactose, by placement of a 3MM filter paper (Prolabo, Fontenay-sous-Bois, N-acetylgalactosamine) from the mucus surface, followed by the France) soaked with the synthetic sialidase substrate 4-methylumremoval of sialic acids, b-galactose, and N-acetylglucosamine. belliferyl-a-D-N-acetylneuraminic acid (Sigma, St. Louis) in conOur present studies have focused on the sialidase activities of tact with the colonies for 15 minutes at 377C. One clone expressing the barrier strains for two reasons. First, albeit weak, this is the sialidase activity was identified by its blue-white fluorescence unonly mucinolytic activity expressed by C. indolis. In contrast, der ultraviolet light (360 nm). This clone carries an insert of C. cocleatum displays much higher and varied mucin-degrading 2.7 kb. activity. Second, sialic acid residues usually occupy terminal posiThe restriction map is shown in figure 2. E. coli carrying the tions on sialoglycoconjugates such as mucins and thus represent recombinant plasmid expresses a sialidase of identical size (C70 the outermost surface available for interactions between host and kD), detectable in immunoblots by antiserum raised against the microbiota. large isoform of Clostridium perfringens. Two subclones of the The sialidase activity expressed by the barrier strains could play plasmid have been created by digesting the insert with Ssp I (figure a role in the barrier mechanism inasmuch as the removal of sialic 2); nucleotide sequencing is in progress. acids could remove an attachment site for C. difficile. We intend To clone the sialidase gene of C. cocleatum, a genomic library to study the potential role of the sialidase in the barrier function was constructed in l Zap II (Stratagene). The library was conby molecular approach, i.e., by constructing mutant strains devoid of sialidase activity that can be tested in the trixenic mouse model available in our laboratory, in order to elucidate the effect of this mutation on the barrier function. Isolation of the sialidase genes is the first step in this process. To clone the gene encoding the sialidase of C. indolis, a genomic library was constructed. High-molecular-weight DNA was isolated as described [3] from a strain grown in anaerobic conditions in TGY (Tryptone Glucose Yeast broth, Difco, Detroit) at 377C for