Conservation of the "Outside-in" Germination Pathway in Paraclostridium bifermentans.

Disha Bhattacharjee,Joseph A Sorg

doi:10.3389/fmicb.2018.02487

Abstract

Clostridium difficile spore germination is initiated in response to certain bile acids and amino acids (e.g., glycine). Though the amino acid-recognizing germinant receptor is unknown, the bile acid germinant receptor is the germination-specific, subtilisin-like pseudoprotease, CspC. In C. difficile the CspB, CspA, and CspC proteins are involved in spore germination. Of these, only CspB is predicted to have catalytic activity because the residues important for catalysis are mutated in the cspA and cspC sequence. The CspB, CspA, and CspC proteins are likely localized to the outer layers of the spore (e.g., the cortex or the coat layers) and not the inner membrane where the Ger-type germinant receptors are located. In C. difficile, germination proceeds in an “outside-in” direction, instead of the “‘inside-out” direction observed during the germination of Bacillus subtilis spores. During C. difficile spore germination, cortex fragments are released prior to the release of 2,4-dipicolinic acid (DPA) from the spore core. This is opposite to what occurs during B. subtilis spore germination. To understand if the mechanism C. difficile spore germination is unique or if spores from other organisms germinate in a similar fashion, we analyzed the germination of Paraclostridium bifermentans spores. We find that P. bifermentans spores release cortex fragments prior to DPA during germination and the DPA release from the P. bifermentans spore core can be blocked by high concentrations of osmolytes. Moreover, we find that P. bifermentans spores do not respond to steroid-like compounds (unlike the related C. difficile and P. sordellii organisms), indicating that the mere presence of the Csp proteins does permit germination in response to steroid compounds. Our findings indicate that the “outside in” mechanism of spore germination observed in C. difficile can be found in other bacteria suggesting that this mechanism is a novel pathway for endospore germination.

Highlights

The endospore-forming Paraclostridium bifermentans belongs to the Clostridia family and P. bifermentans subsp. malaysia is the only known anaerobic larvicidal toxin producer whose toxins target Anopheles and Aedes mosquitoes (Qureshi et al, 2014)
This indicates that the P. bifermentans CspBA protein sequence is intact and has the potential to function to what is observed in C. difficile
Triton X-100 did not increase the rate of germination by P. bifermentans spores either by OD (Figure 2G) or dipicolinic acid (DPA) release (Figure 2H). These results suggest that, though taurocholic acid (TA) and deoxycholic acid (DCA) increase the rate of OD change during germination, DPA release is unaffected by bile acids suggesting that they do not influence P. bifermentans spore germination and the observed effects on OD are likely an artifact

Summary

INTRODUCTION

The endospore-forming Paraclostridium bifermentans belongs to the Clostridia family and P. bifermentans subsp. malaysia is the only known anaerobic larvicidal toxin producer whose toxins target Anopheles and Aedes mosquitoes (Qureshi et al, 2014). In C. perfringens, the CspA, CspB, and CspC proteases cleave the inactive SCLE, pro-SleC, to its active form resulting in cortex degradation (Shimamoto et al, 2001). Based upon the predicted location of CspB, CspA, CspC, and SleC (near the cortex layer and not in/on the inner spore membrane) (Miyata et al, 1997), we hypothesized that C. difficile spore germination may be initiated differently than what had been described in other endospore-forming bacteria (Francis et al, 2015). We hypothesized that bile acids and glycine would stimulate cortex degradation prior to the release of DPA from the spore core (a process opposite to what is observed during B. subtilis spore germination). Like C. difficile, P. bifermentans spores germinate through an “outsidein” mechanism and add to the list of organisms that germinate through this novel pathway of spore germination

RESULTS

DISCUSSION

MATERIALS AND METHODS