Antisense-RNA-Mediated Decreased Synthesis of Small, Acid-Soluble Spore Proteins Leads to Decreased Resistance of Clostridium perfringens Spores to Moist Heat and UV Radiation

Abstract

Previous work has suggested that a group of ␣/␤-type small, acid-soluble spore proteins (SASP) is involved in the resistance of Clostridium perfringens spores to moist heat. However, this suggestion is based on the analysis of C. perfringens spores lacking only one of the three genes encoding ␣/␤-type SASP in this organism. We have now used antisense RNA to decrease levels of ␣/␤-type SASP in C. perfringens spores by ϳ90%. These spores had significantly reduced resistance to both moist heat and UV radiation but not to dry heat. These results clearly demonstrate the important role of ␣/␤-type SASP in the resistance of C. perfringens spores.Clostridium perfringens is a gram-positive, spore-forming, anaerobic bacterium that causes both histotoxic and gastrointestinal (GI) diseases in humans and animals (8, 10). C. perfringens isolates are classified into one of five types, types A through E (10, 11), based upon their ability to produce the four major lethal toxins, the alpha-, beta-, epsilon-and iota-toxins. The major lethal toxins, however, are not the only medically important toxins; some C. perfringens isolates (mostly those belonging to type A) produce C. perfringens enterotoxin (CPE). The CPE-producing type A isolates are important human GI pathogens, causing C. perfringens type A food poisoning as well as non-food-borne GI diseases (10,19). In addition to producing CPE, C. perfringens food poisoning isolates have the ability to form spores that are extremely resistant to heat and other environmental stress factors (20), facilitating spore survival in primary food vehicles (e.g., meat and poultry products) for C. perfringens type A food poisoning (10).The molecular basis for the resistance of C. perfringens spores to heat and other environmental stress factors remains unknown. However, the ␣/␤-type small, acid-soluble spore proteins (SASP) play a major role in the resistance of Bacillus subtilis spores to heat, UV radiation, and peroxides (24). Previous studies (1, 2) have shown that the genomes of Clostridium species contain multiple genes (termed ssp) encoding ␣/␤-type SASP, with three genes (ssp1, ssp2, and ssp3) in C. perfringens (13,25). However, in contrast to Bacillus species, Clostridium species do not contain genes encoding the ␥-type SASP (1). Evidence obtained recently has suggested that ␣/␤-type SASP plays a role in the resistance of C. perfringens spores to moist heat, as spores of a strain with an ssp3 deletion had slightly lower moist heat resistance than wild-type spores, and this defect was eliminated by complementing the ssp3 mutant with wild-type ssp3 (18). However, the link between ␣/␤-type SASP levels and spore heat resistance was tentative because only one of the three ssp genes in C. perfringens was deleted.In order to fully explore the relationship between levels of ␣/␤-type SASP and C. perfringens spore resistance, it would be necessary to generate a C. perfringens strain lacking all three ssp genes. However, since no technique is currently available to introduce multiple knockout muta...

Characterization of Clostridium perfringens Spores That Lack SpoVA Proteins and Dipicolinic Acid

“…Extraction of SASP from C. perfringens spores and their analysis by polyacrylamide gel electrophoresis at low pH were as described previously (49,50). Briefly, SASP were extracted from 40 mg (dry weight) of disrupted spores with dilute acid, and the extracts were processed and lyophilized.…”

“…(ii) SpoVA proteins and Ca-DPA release are not required for C. perfringens spore germination. (iii) A low spore core water content is essential for full resistance of C. perfringens spores to moist heat, UV radiation, and chemicals.Clostridium perfringens food poisoning is caused mainly by enterotoxigenic type A isolates that have the ability to form metabolically dormant spores that are extremely resistant to heat, radiation, and toxic chemicals (45,49,50,55). These highly resistant spores can survive traditional methods for cooking meat and poultry products as well as other processing treatments used in the food industry.…”

“…The factors involved in Bacillus subtilis spore resistance include the following: (i) relatively impermeable spore inner membrane; (ii) low water content of the spore core; (iii) high levels of pyridine-2,6-dicarboxylic acid (dipicolinic acid [DPA]) in the spore core and the type and amount of cations chelated by DPA; and (iv) the saturation of spore DNA with ␣/␤-type small, acid-soluble spore proteins (SASP) (63,64). Studies have shown that ␣/␤-type SASP are also an important factor in C. perfringens spore resistance to moist heat, UV radiation, and some chemicals (45,49,50), while small changes in the water content of the cores of these spores alter resistance to moist heat and nitrous acid (46). Spore germination has been well studied in B. subtilis (34,62) and can be initiated by a variety of compounds (termed germinants) including amino acids and some other nutrients and nutrient mixtures, a 1:1 chelate of Ca 2ϩ and DPA (Ca-DPA), cationic surfactants, and hydrostatic pressure (41,44).…”

Small acid-soluble spore proteins of Clostridium acetobutylicum are able to protect DNA in vitro and are specifically cleaved by germination protease GPR and spore protease YyaC

“…Three of them (Ssp1-3, 59-60 aa) are isoforms with about 90 % amino acid identity (Raju et al, 2007) and Ssp4 (90 aa) was detected later as it differs remarkably from Ssp1-3 in size and in amino acid identity (Li & McClane, 2008;Galperin et al, 2012). Two nearly identical a/b-type SASPs (w90 %) also exist in B. subtilis (SspA/ SspB), B. megaterium (SspA/SspC) and Clostridium bifermentans (SASP-a/SASP-b), and these SASPs are predominant in acidic protein extracts of dormant spores (Setlow, 1975b;Johnson & Tipper, 1981;Cabrera-Martinez et al, 1989).…”

“…In contrast, little is known about SASPs of strictly anaerobic sporeforming bacteria and recent studies have dealt only with the SASPs of the food poisoning pathogenic species Clostridium perfringens (Raju et al, 2006(Raju et al, , 2007Li & McClane, 2008;Li et al, 2009). With regard to Clostridium acetobutylicum, a Gram-positive, strictly anaerobic, endosporeforming bacterium, nothing was known about its SASPs on the biochemical level when we initiated this study.…”