Requirements for Germination ofClostridium sordelliiSporesIn Vitro

Abstract

Clostridium sordellii is a spore-forming, obligately anaerobic, Gram-positive bacterium that can cause toxic shock syndrome after gynecological procedures. Although the incidence of C. sordellii infection is low, it is fatal in most cases. Since spore germination is believed to be the first step in the establishment of Bacilli and Clostridia infections, we analyzed the requirements for C. sordellii spore germination in vitro. Our data showed that C. sordellii spores require three structurally different amino acids and bicarbonate for maximum germination. Unlike the case for Bacilli species, D-alanine had no effect on C. sordellii spore germination. C. sordellii spores germinated only in a narrow pH range between 5.7 and 6.5. In contrast, C. sordellii spore germination was significantly less sensitive to temperature changes than that of the Bacilli. The analysis of the kinetics of C. sordellii spore germination showed strong allosteric behavior in the binding of L-phenylalanine and L-alanine but not in that of bicarbonate or L-arginine. By comparing germinant apparent binding affinities to their known in vivo concentrations, we postulated a mechanism for differential C. sordellii spore activation in the female reproductive tract.

The Ger Receptor Family from Sporulating Bacteria

“…This is similar to our recent findings that showed that C. sordellii, a close relative of C. bifermentans, needs a combination of a small amino acid, a basic amino acid, an aromatic amino acid, and bicarbonate to germinate (Ramirez and Abel-Santos, 2010). Even though germination rates are highest when spores are incubated with a mixture of L-alanine, L-phenylalanine, and L-arginine, germination also occurs when each amino acid is substituted with compounds with similar physical and chemical properties.…”

“…Even though germination rates are highest when spores are incubated with a mixture of L-alanine, L-phenylalanine, and L-arginine, germination also occurs when each amino acid is substituted with compounds with similar physical and chemical properties. Thus, C. sordellii spores seem to be quite promiscuous in the recognition of germinants (Ramirez and Abel-Santos, 2010).…”

“…sordellii is an opportunistic pathogen that causes toxic shock syndrome after gynecological procedures (Aldape et al, 2006). In this context, the fact that C. sordellii spores requires bicarbonate and prefer slightly acidic pH for germination is intriguing (Ramirez and Abel-Santos, 2010). Maximal germination is observed at a pH range where both carbonic acid and bicarbonate are present in equilibrium.…”

“…L-alanine-mediated germination has been mostly characterized in B. subtilis spores (McCann et al, 1996). However, L-alanine can also induce germination in B. cereus (Barlass et al, 2002), B. anthracis (Fisher and Hanna, 2005), B. megaterium (Christie and Lowe, 2007), C. botulinum (Broussolle et al, 2002), C. sporogenes (Broussolle et al, 2002) C. perfringens (Paredes-Sabja et al, 2008), and C. sordellii (Ramirez and Abel-Santos, 2010). Indeed, L-alanine seems to be the most ubiquitous germinant for bacterial spores.…”

“…Furthermore, by comparing apparent K m values to germinant concentrations in vivo, models for spore-germinant complex distribution can be proposed, and rate-limiting steps for the germination process can be inferred. Thus, kinetic analysis can yield information on spore activation even if the identities of the germination receptors are not known (Ramirez and Abel-Santos, 2010). …”