Host serum factor triggers germination of Clostridium perfringens spores lacking the cortex hydrolysis machinery

Abstract

Clostridium perfringens type A is the causative agent of a variety of histotoxic and enteric diseases. The ability of C. perfringens spores to germinate in vivo might be due to the presence of nutrient germinants in the host tissue and blood. In the current study, we investigated the ability of spores of C. perfringens wild-type and mutation strains to germinate in blood. Results indicate that spores of all three surveyed C. perfringens wild-type isolates germinated better in blood than in brain heart infusion (BHI) broth. However, as expected, spores lacking germinant receptor (GR) protein GerAA or GerKB germinated like wild-type spores in BHI broth and blood. Strikingly, while spores lacking GR proteins GerKA and GerKC showed significantly decreased germination in BHI broth, these spores germinated well in blood, suggesting that blood factor(s) can trigger spore germination through a GR-independent pathway. Using C. perfringens spores lacking cortex lytic enzymes (DcspB or DsleC DsleM), we were able to identify a host serum germination factor with peptidoglycan hydrolysing activity that (i) restored the colony-forming efficiencies of DcspB and DsleC DsleM spores up to~5-20 % of that of total colony-forming spores; (ii) increased the number of c.f.u. of decoated DcspB and DsleC DsleM spores to~99 % of that of colony-forming spores; (iii) and finally lost enzymic activity after heat inactivation, consistent with serum germination factor being an enzyme. Further characterization demonstrated that serum germination factor is very likely lysozyme, which can form a stable high molecular mass complex of 120 kDa in serum.In conclusion, the current study indicates that a host serum germination factor with peptidoglycan hydrolysing activity is capable of triggering germination of C. perfringens spores by directly degrading the spore peptidoglycan cortex. Collectively, this study contributes to our understanding of the mechanism of in vivo germination of spores of C. perfringens. INTRODUCTIONClostridium perfringens is a Gram-positive, spore forming, anaerobic bacterium, which is highly associated with a wide array of gastrointestinal (GI) and histotoxic diseases in both humans and animals (Amimoto et al., 2007; Keyburn et al., 2008;McClane, 2007). However, the most common cause of C. perfringens-associated food poisoning in humans is the consumption of C. perfringens vegetative cells followed by sporulation in the gut (McClane, 2007;Paredes-Sabja & Sarker, 2009). The early steps in the development of C. perfringens-associated gas gangrene and other non-food-borne GI illnesses, such as antibioticassociated diarrhoea, occur when C. perfringens spores ubiquitously found in the environment come in contact with the host, where spores undergo germination followed by outgrowth, cell proliferation and toxin secretion. Therefore, C. perfringens spore germination can be considered as the most essential and earliest step in the progression of gas gangrene and antibiotic-associated diarrhoea.Bacterial spores break their dormancy wh...

Unique Regulatory Mechanism of Sporulation and Enterotoxin Production in Clostridium perfringens

“…Since a previous report (21) indicated that C. perfringens strain 13 has a frameshift mutation in the gene of the master regulator, spo0A (22,23), we examined the ability of spore formation of strain 13 under the same conditions as those used for SM101. As expected, strain 13 did not form spores (data not shown).…”

Clostridium perfringens Sporulation and Sporulation-Associated Toxin Production

“…The composition and function of the spore coat in C. perfringens has not yet been carefully studied; however, in other Gram-positive spore formers, the spore coat is thought to be comprised of >50 spore-specific proteins and provides protection to the spore from reactive chemicals and lytic enzymes (12-14). In C. perfringens , a small fraction of the spore population (∼5%) is defective in spore coats (15). Those spores are permeable to lysozyme (15) so they can germinate inside the host under specific conditions.…”

“…In C. perfringens , a small fraction of the spore population (∼5%) is defective in spore coats (15). Those spores are permeable to lysozyme (15) so they can germinate inside the host under specific conditions.…”

“…Among these are lysozyme, which is released by Peyer patches in the small intestine (54), present in the serum (55), and comprises part of the antibacterial arsenal of phagocytic cells. Lysozyme can trigger germination of spores of C. perfringens strain SM101 by directly degrading the spore peptidoglycan cortex (15). This germination pathway might have implications for the pathogenesis of C. perfringens (15), especially for superdormant spores that are extremely slow to germinate (56).…”

Pesquisa de Clostridium perfringens em carnes bovinas embaladas a vácuo comercializadas no Distrito Federal e entorno

“…(11)(12)(13) Os meios de cultivo mais usados para o isolamento de C. perfringens são o ágar Sulfito Polimixina Sulfadiazina (SPS) e o ágar Triptose Sulfito Cicloserina (TSC). (14) De acordo com a Agência Nacional de Vigilância Sanitária, a toxinfecção alimentar por C. perfringens é a sexta mais frequente no Brasil, entretanto, deve-se notar que em 51,34% de todos os casos de toxinfecção alimentar o agente causador é ignorado.…”