NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718

Abstract

Clostridium perfringens type D strains are usually associated with diseases of livestock, and their virulence requires the production of epsilon toxin (ETX). We previously showed (J. Li, S. Sayeed, S. Robertson, J. Chen, and B. A. McClane, PLoS Pathog 7:e1002429, 2011, http://dx.doi.org/10.1371/journal.ppat.1002429) that BMC202, a nanI null mutant of type D strain CN3718, produces less ETX than wild-type CN3718 does. The current study proved that the lower ETX production by strain BMC202 is due to nanI gene disruption, since both genetic and physical (NanI or sialic acid) complementation increased ETX production by BMC202. Furthermore, a sialidase inhibitor that interfered with NanI activity also reduced ETX production by wild-type CN3718. The NanI effect on ETX production was shown to involve reductions in codY and ccpA gene transcription levels in BMC202 versus wild-type CN3718. Similar to CodY, CcpA was found to positively control ETX production. A double codY ccpA null mutant produced even less ETX than a codY or ccpA single null mutant. CcpA bound directly to sequences upstream of the etx or codY start codon, and bioinformatics identified putative CcpA-binding cre sites immediately upstream of both the codY and etx start codons, suggesting possible direct CcpA regulatory effects. A ccpA mutation also decreased codY transcription, suggesting that CcpA effects on ETX production can be both direct and indirect, including effects on codY transcription. Collectively, these results suggest that NanI, CcpA, and CodY work together to regulate ETX production, with NanI-generated sialic acid from the intestines possibly signaling type D strains to upregulate their ETX production and induce disease. IMPORTANCEClostridium perfringens NanI was previously shown to increase ETX binding to, and cytotoxicity for, MDCK host cells. The current study demonstrates that NanI also regulates ETX production via increased transcription of genes encoding the CodY and CcpA global regulators. Results obtained using single ccpA or codY null mutants and a ccpA codY double null mutant showed that codY and ccpA regulate ETX production independently of one another but that ccpA also affects codY transcription. Electrophoretic mobility shift assays and bioinformatic analyses suggest that both CodY and CcpA may directly regulate etx transcription. Collectively, results of this study suggest that sialic acid generated by NanI from intestinal sources signals ETX-producing C. perfringens strains, via CcpA and CodY, to upregulate ETX production and cause disease. C lostridium perfringens is an important cause of human and livestock infections, including many diseases originating in the intestines (1, 2). The virulence of C. perfringens is largely attributable to its prolific toxin-producing ability, with different toxins involved in specific diseases (1,3,4). By definition, type D strains must produce both C. perfringens alpha toxin (CPA) and epsilon toxin (ETX). ETX is a class B NIAID priority toxin, since it is one of the most potent ba...

Clostridium perfringens Sialidases: Potential Contributors to Intestinal Pathogenesis and Therapeutic Targets

“…However, the NADNA sialidase inhibitor did not inhibit sialidase activity in CN3718 cultures, even at very high doses of the inhibitor. In the presence of NADNA, CN3718 cultures still possessed strong sialidase activity and also made the same amounts of ETX as wild-type CN3718 grown in the absence of any sialidase inhibitor [57]. Collectively, these results suggest that at least some sialidase inhibitors may be potentially useful therapeutics against C. perfringens infections.…”

“…Earlier studies demonstrated that NanI production also impacts the production of several C. perfringens toxins [34,37,57]. Specifically, inactivating the nanI gene in C. perfringens type A strain 13 caused a slight increase in supernatant activities of alpha toxin and perfringolysin O (PFO), which are contributors to gas gangrene [37], but their role (if any) in most intestinal infections, particularly human infections, is unsettled [3].…”

“…Specifically, inactivating the nanI gene in C. perfringens type A strain 13 caused a slight increase in supernatant activities of alpha toxin and perfringolysin O (PFO), which are contributors to gas gangrene [37], but their role (if any) in most intestinal infections, particularly human infections, is unsettled [3]. However, it was later demonstrated that inactivating the nanI gene in type D strain CN3718 reduced ETX toxin production in vitro and that either genetic or physical complementation could recover this ETX production [57]. NanI effects on ETX production involved reductions in both codY and ccpA transcript levels, suggesting a model whereby NanI generates sialic acid release in the intestines and that free sialic acid then alerts a type D strain of its presence in the intestines, making it worthwhile for this bacterium to upregulate ETX production to induce disease.…”

“…Two classic sialidase inhibitors, i.e., Siastatin B (SB) and N-acetyl-2,3-dehydro-2-deoxyneuraminic acid (NADNA), have been tested against C. perfringens [33,36,57,69,70]. SB, a broad-spectrum sialidase inhibitor isolated from a Streptomyces spp.…”

“…Recent in vitro studies suggest that sialidases, particularly NanI, may be important contributors to C. perfringens intestinal infections. Those contributions could include upregulated production of some toxins relevant to intestinal infections, enhanced binding and activity of some of those toxins, increased adherence of C. perfringens to host cells, and possibly generation of substrates for growth and metabolism [33,34,36,57]. There is a clear need to move these promising findings into appropriate animal models to firmly evaluate sialidase contributions to C. perfringens virulence.…”