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

ABSTRACTClostridium perfringenstype 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, ananInull 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 tonanIgene 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 incodYandccpAgene transcription levels in BMC202 versus wild-type CN3718. Similar to CodY, CcpA was found to positively control ETX production. A doublecodYccpAnull mutant produced even less ETX than acodYorccpAsingle null mutant. CcpA bound directly to sequences upstream of theetxorcodYstart codon, and bioinformatics identified putative CcpA-bindingcresites immediately upstream of both thecodYandetxstart codons, suggesting possible direct CcpA regulatory effects. AccpAmutation also decreasedcodYtranscription, suggesting that CcpA effects on ETX production can be both direct and indirect, including effects oncodYtranscription. 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 perfringensNanI 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 singleccpAorcodYnull mutants and accpAcodYdouble null mutant showed thatcodYandccpAregulate ETX production independently of one another but thatccpAalso affectscodYtranscription. Electrophoretic mobility shift assays and bioinformatic analyses suggest that both CodY and CcpA may directly regulateetxtranscription. Collectively, results of this study suggest that sialic acid generated by NanI from intestinal sources signals ETX-producingC. perfringensstrains, via CcpA and CodY, to upregulate ETX production and cause disease.

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CodY Is a Global Regulator of Virulence-Associated Properties for Clostridium perfringens Type D Strain CN3718

mBio ◽

10.1128/mbio.00770-13 ◽

2013 ◽

Vol 4 (5) ◽

Cited By ~ 27

Author(s):

Jihong Li ◽

Menglin Ma ◽

Mahfuzur R. Sarker ◽

Bruce A. McClane

Keyword(s):

Clostridium Perfringens ◽

Null Mutant ◽

Intestinal Infection ◽

Wild Type ◽

Global Regulator ◽

Content Type ◽

Gram Positive Bacteria ◽

Type D ◽

Epsilon Toxin ◽

Virulence Properties

ABSTRACT CodY is known to regulate various virulence properties in several Gram-positive bacteria but has not yet been studied in the important histotoxic and intestinal pathogen Clostridium perfringens. The present study prepared an isogenic codY-null mutant in C. perfringens type D strain CN3718 by insertional mutagenesis using the Targetron system. Western blot analysis indicated that, relative to wild-type CN3718 or a complementing strain, this isogenic codY mutant produces reduced levels of epsilon toxin (ETX). Using supernatants from cultures of the wild-type, codY-null mutant, and complementing strains, CodY regulation of ETX production was shown to have cytotoxic consequences for MDCK cells. The CodY regulatory effect on ETX production was specific, since the codY-null mutant still made wild-type levels of alpha-toxin and perfringolysin O. Sialidase activity measurements and sialidase Western blot analysis of supernatants from CN3718 and its isogenic derivatives showed that CodY represses overall exosialidase activity due to a reduced presence of NanH in culture supernatants. Inactivation of the codY gene significantly decreased the adherence of CN3718 vegetative cells or spores to host Caco-2 cells. Finally, the codY mutant showed increased spore formation under vegetative growth conditions, although germination of these spores was impaired. Overall, these results identify CodY as a global regulator of many C. perfringens virulence-associated properties. Furthermore, they establish that, via CodY, CN3718 coordinately regulates many virulence-associated properties likely needed for intestinal infection. IMPORTANCE Clostridium perfringens is a major human and livestock pathogen because it produces many potent toxins. C. perfringens type D strains cause intestinal infections by producing toxins, especially epsilon toxin (ETX). Previous studies identified CodY as a regulator of certain virulence properties in other Gram-positive bacteria. Our study now demonstrates that CodY is a global regulator of virulence-associated properties for type D strain CN3718. It promotes production of ETX, attachment of CN3718 vegetative cells or spores to host enterocyte-like Caco-2 cells, and spore germination; the last two effects may assist intestinal colonization. In contrast, CodY represses sporulation. These results provide the first evidence that CodY can function as a global regulator of C. perfringens virulence-associated properties and that this strain coordinately regulates its virulence-associated properties using CodY to increase ETX production, host cell attachment, and spore germination but to repress sporulation, as would be optimal during type D intestinal infection.

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CodY Promotes Sporulation and Enterotoxin Production by Clostridium perfringens Type A Strain SM101

Infection and Immunity ◽

10.1128/iai.00855-16 ◽

2017 ◽

Vol 85 (3) ◽

Cited By ~ 9

Author(s):

Jihong Li ◽

John C. Freedman ◽

Daniel R. Evans ◽

Bruce A. McClane

Keyword(s):

Gene Expression ◽

Clostridium Perfringens ◽

Food Poisoning ◽

Type A ◽

Toxin Production ◽

Null Mutant ◽

Wild Type ◽

Content Type ◽

Type D ◽

Epsilon Toxin

ABSTRACT Clostridium perfringens type D strains cause enterotoxemia and enteritis in livestock via epsilon toxin production. In type D strain CN3718, CodY was previously shown to increase the level of epsilon toxin production and repress sporulation. C. perfringens type A strains producing C. perfringens enterotoxin (CPE) cause human food poisoning and antibiotic-associated diarrhea. Sporulation is critical for C. perfringens type A food poisoning since spores contribute to transmission and resistance in the harsh food environment and sporulation is essential for CPE production. Therefore, the current study asked whether CodY also regulates sporulation and CPE production in SM101, a derivative of C. perfringens type A food-poisoning strain NCTC8798. An isogenic codY-null mutant of SM101 showed decreased levels of spore formation, along with lower levels of CPE production. A complemented strain recovered wild-type levels of both sporulation and CPE production. When this result was coupled with the earlier results obtained with CN3718, it became apparent that CodY regulation of sporulation varies among different C. perfringens strains. Results from quantitative reverse transcriptase PCR analysis clearly demonstrated that, during sporulation, codY transcript levels remained high in SM101 but rapidly declined in CN3718. In addition, abrB gene expression patterns varied significantly between codY-null mutants of SM101 and CN3718. Compared to the levels in their wild-type parents, the level of abrB gene expression decreased in the CN3718 codY-null mutant strain but significantly increased in the SM101 codY-null mutant strain, demonstrating CodY-dependent regulation differences in abrB expression between these two strains. This difference appears to be important since overexpression of the abrB gene in SM101 reduced the levels of sporulation and enterotoxin production, supporting the involvement of AbrB repression in regulating C. perfringens sporulation.

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Epsilon Toxin Is Essential for the Virulence of Clostridium perfringens Type D Infection in Sheep, Goats, and Mice

Infection and Immunity ◽

10.1128/iai.00238-13 ◽

2013 ◽

Vol 81 (7) ◽

pp. 2405-2414 ◽

Cited By ~ 55

Author(s):

J. P. Garcia ◽

V. Adams ◽

J. Beingesser ◽

M. L. Hughes ◽

R. Poon ◽

...

Keyword(s):

Clostridium Perfringens ◽

Type Strain ◽

Wild Type Strain ◽

Null Mutant ◽

Wild Type ◽

Histological Changes ◽

Content Type ◽

Type D ◽

Necrotizing Colitis ◽

Epsilon Toxin

ABSTRACTClostridium perfringenstype D causes disease in sheep, goats, and other ruminants. Type D isolates produce, at minimum, alpha and epsilon (ETX) toxins, but some express up to five different toxins, raising questions about which toxins are necessary for the virulence of these bacteria. We evaluated the contribution of ETX toC. perfringenstype D pathogenicity in an intraduodenal challenge model in sheep, goats, and mice using a virulentC. perfringenstype D wild-type strain (WT), an isogenic ETX null mutant (etxmutant), and a strain where theetxmutation has been reversed (etxcomplemented). All sheep and goats, and most mice, challenged with the WT isolate developed acute clinical disease followed by death in most cases. Sheep developed various gross and/or histological changes that included edema of brain, lungs, and heart as well as hydropericardium. Goats developed various effects, including necrotizing colitis, pulmonary edema, and hydropericardium. No significant gross or histological abnormalities were observed in any mice infected with the WT strain. All sheep, goats, and mice challenged with the isogenicetxmutant remained clinically healthy for ≥24 h, and no gross or histological abnormalities were observed in those animals. Complementation ofetxknockout restored virulence; most goats, sheep, and mice receiving this complemented mutant developed clinical and pathological changes similar to those observed in WT-infected animals. These results indicate that ETX is necessary for type D isolates to induce disease, supporting a key role for this toxin in type D disease pathogenesis.

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NanR Regulates Sporulation and Enterotoxin Production byClostridium perfringensType F Strain F4969

Infection and Immunity ◽

10.1128/iai.00416-18 ◽

2018 ◽

Vol 86 (10) ◽

Cited By ~ 4

Author(s):

Eric Mi ◽

Jihong Li ◽

Bruce A. McClane

Keyword(s):

Sialic Acid ◽

Clostridium Perfringens ◽

Vegetative Growth ◽

Foodborne Illness ◽

Null Mutant ◽

Wild Type ◽

Gastrointestinal Infections ◽

Content Type ◽

Antibiotic Associated Diarrhea ◽

Uptake And Metabolism

ABSTRACTClostridium perfringenstype F strains, which produceC. perfringensenterotoxin (CPE), are a major cause of gastrointestinal infections, including the second most prevalent bacterial foodborne illness and 5 to 10% cases of antibiotic-associated diarrhea. Virulence of type F strains is primarily ascribable to CPE, which is synthesized only during sporulation. Many type F strains also produce NanI sialidase and carry ananoperon that likely facilitates uptake and metabolism of sialic acid liberated from glycoconjugates by NanI. During vegetative growth of type F strain F4969, NanR can regulate expression ofnanI. Given their importance for type F disease, the current study investigated whether NanR can also influence sporulation and CPE production when F4969 or isogenic derivatives are cultured in modified Duncan-Strong sporulation (MDS) medium. An isogenic F4969nanRnull mutant displayed much less sporulation and CPE production but more NanI production than wild-type F4969, indicating that NanR positively regulates sporulation and CPE production but represses NanI production in MDS. Results for thenanRmutant also demonstrated that NanR regulates expression of thenanoperon. AnanI nanRdouble null mutant mirrored the outcome of thenanRnull mutant strain but with a stronger inhibition of sporulation and CPE production, even after overnight incubation. Coupled with results using ananInull mutant, which had no impairment of sporulation or CPE production, NanR appears to carefully modulate the availability of NanI,nanoperon-encoded proteins and sialic acid to provide sufficient nutrients to sustain sporulation and CPE production when F4969 is cultured in MDS medium.

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Epsilon-Toxin Production by Clostridium perfringens Type D Strain CN3718 Is Dependent upon the agr Operon but Not the VirS/VirR Two-Component Regulatory System

mBio ◽

10.1128/mbio.00275-11 ◽

2011 ◽

Vol 2 (6) ◽

Cited By ~ 28

Author(s):

Jianming Chen ◽

Julian I. Rood ◽

Bruce A. McClane

Keyword(s):

Quorum Sensing ◽

Clostridium Perfringens ◽

Regulatory System ◽

Toxin Production ◽

Broth Culture ◽

Type B ◽

Content Type ◽

Type D ◽

Two Component ◽

Epsilon Toxin

ABSTRACT Clostridium perfringens type B and D strains cause enterotoxemias and enteritis in livestock after proliferating in the intestines and producing epsilon-toxin (ETX), alpha-toxin (CPA), and, usually, perfringolysin O (PFO). Although ETX is one of the most potent bacterial toxins, the regulation of ETX production by type B or D strains remains poorly understood. The present work determined that the type D strain CN3718 upregulates production of ETX upon close contact with enterocyte-like Caco-2 cells. This host cell-induced upregulation of ETX expression was mediated at the transcriptional level. Using an isogenic agrB null mutant and complemented strain, the agr operon was shown to be required when CN3718 produces ETX in broth culture or, via a secreted signal consistent with a quorum-sensing (QS) effect, upregulates ETX production upon contact with host cells. These findings provide the first insights into the regulation of ETX production, as well as additional evidence that the Agr-like QS system functions as a global regulator of C. perfringens toxin production. Since it was proposed previously that the Agr-like QS system regulates C. perfringens gene expression via the VirS/VirR two-component regulatory system, an isogenic virR null mutant of CN3718 was constructed to evaluate the importance of VirS/VirR for CN3718 toxin production. This mutation affected production of CPA and PFO, but not ETX, by CN3718. These results provide the first indication that C. perfringens toxin expression regulation by the Agr-like quorum-sensing system may not always act via the VirS/VirR two-component system. IMPORTANCE Mechanisms by which Clostridium perfringens type B and D strains regulate production of epsilon-toxin (ETX), a CDC class B select toxin, are poorly understood. Production of several other toxins expressed by C. perfringens is wholly or partially regulated by both the Agr-like quorum-sensing (QS) system and the VirS/VirR two-component regulatory system, so the present study tested whether ETX expression by type D strain CN3718 also requires these regulatory systems. The agr operon was shown to be essential for signaling CN3718 to produce ETX in broth culture or to upregulate ETX production upon close contact with enterocyte-like Caco-2 cells, which may have pathogenic relevance since ETX is produced intestinally. However, ETX production remained at wild-type levels after inactivation of the VirS/VirR system in CN3718. These findings provide the first information regarding regulation of ETX production and suggest Agr-like QS toxin production regulation in C. perfringens does not always require the VirS/VirR system.

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The Agr-Like Quorum-Sensing System Is Important for Clostridium perfringens Type A Strain ATCC 3624 To Cause Gas Gangrene in a Mouse Model

mSphere ◽

10.1128/msphere.00500-20 ◽

2020 ◽

Vol 5 (3) ◽

Author(s):

Mauricio A. Navarro ◽

Jihong Li ◽

Juliann Beingesser ◽

Bruce A. McClane ◽

Francisco A. Uzal

Keyword(s):

Quorum Sensing ◽

Mouse Model ◽

Clostridium Perfringens ◽

Regulatory System ◽

Type A ◽

Gas Gangrene ◽

Null Mutant ◽

Wild Type ◽

Content Type

ABSTRACT Clostridium perfringens type A is involved in gas gangrene in humans and animals. Following a traumatic injury, rapid bacterial proliferation and exotoxin production result in severe myonecrosis. C. perfringens alpha toxin (CPA) and perfringolysin (PFO) are the main virulence factors responsible for the disease. Recent in vitro studies have identified an Agr-like quorum-sensing (QS) system in C. perfringens that regulates the production of both toxins. The system is composed of an AgrB membrane transporter and an AgrD peptide that interacts with a two-component regulatory system in response to fluctuations in the cell population density. In addition, a synthetic peptide named 6-R has been shown to interfere with this signaling mechanism, affecting the function of the Agr-like QS system in vitro. In the present study, C. perfringens type A strain ATCC 3624 and an isogenic agrB-null mutant were tested in a mouse model of gas gangrene. When mice were intramuscularly challenged with 106 CFU of wild-type ATCC 3624, severe myonecrosis and leukocyte aggregation occurred by 4 h. Similar numbers of an agrB-null mutant strain produced significantly less severe changes in the skeletal muscle of challenged mice. Complementation of the mutant to regain agrB expression restored virulence to wild-type levels. The burdens of all three C. perfringens strains in infected muscle were similar. In addition, animals injected intramuscularly with wild-type ATCC 3624 coincubated with the 6-R peptide developed less severe microscopic changes. This study provides the first in vivo evidence that the Agr-like QS system is important for C. perfringens type A-mediated gas gangrene. IMPORTANCE Clostridium perfringens type A strains produce toxins that are responsible for clostridial myonecrosis, also known as gas gangrene. Toxin production is regulated by an Agr-like quorum-sensing (QS) system that responds to changes in cell population density. In this study, we investigated the importance of this QS system in a mouse model of gas gangrene. Mice challenged with a C. perfringens strain with a nonfunctional regulatory system developed less severe changes in the injected skeletal muscle compared to animals receiving the wild-type strain. In addition, a synthetic peptide was able to decrease the effects of the QS in this disease model. These studies provide new understanding of the pathogenesis of gas gangrene and identified a potential therapeutic target to prevent the disease.

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ExpR Coordinates the Expression of Symbiotically Important, Bundle-Forming Flp Pili with Quorum Sensing in Sinorhizobium meliloti

Applied and Environmental Microbiology ◽

10.1128/aem.04088-13 ◽

2014 ◽

Vol 80 (8) ◽

pp. 2429-2439 ◽

Cited By ~ 12

Author(s):

Hardik M. Zatakia ◽

Cassandra E. Nelson ◽

Umair J. Syed ◽

Birgit E. Scharf

Keyword(s):

Quorum Sensing ◽

Sinorhizobium Meliloti ◽

Consensus Sequence ◽

Homoserine Lactone ◽

Host Cells ◽

Wild Type ◽

Mobility Shift ◽

Plant Host ◽

Symbiotic Interaction ◽

Content Type

ABSTRACTType IVb pili in enteropathogenic bacteria function as a host colonization factor by mediating tight adherence to host cells, but their role in bacterium-plant symbiosis is currently unknown. The genome of the symbiotic soil bacteriumSinorhizobium meliloticontains two clusters encoding proteins for type IVb pili of the Flp (fimbrial low-molecular-weight protein) subfamily. To establish the role of Flp pili in the symbiotic interaction ofS. melilotiand its host,Medicago sativa, we deletedpilA1, which encodes the putative pilin subunit in the chromosomalflp-1cluster and conducted competitive nodulation assays. ThepilA1deletion strain formed 27% fewer nodules than the wild type. Transmission electron microscopy revealed the presence of bundle-forming pili protruding from the polar and lateral region ofS. melilotiwild-type cells. The putative pilus assembly ATPase CpaE1 fused to mCherry showed a predominantly unilateral localization. Transcriptional reporter gene assays demonstrated that expression ofpilA1peaks in early stationary phase and is repressed by the quorum-sensing regulator ExpR, which also controls production of exopolysaccharides and motility. Binding of acyl homoserine lactone-activated ExpR to thepilA1promoter was confirmed with electrophoretic mobility shift assays. A 17-bp consensus sequence for ExpR binding was identified within the 28-bp protected region by DNase I footprinting analyses. Our results show that Flp pili are important for efficient symbiosis ofS. melilotiwith its plant host. The temporal inverse regulation of exopolysaccharides and pili by ExpR enablesS. melilotito achieve a coordinated expression of cellular processes during early stages of host interaction.

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The Sialidases of Clostridium perfringens Type D Strain CN3718 Differ in Their Properties and Sensitivities to Inhibitors

Applied and Environmental Microbiology ◽

10.1128/aem.03440-13 ◽

2013 ◽

Vol 80 (5) ◽

pp. 1701-1709 ◽

Cited By ~ 28

Author(s):

Jihong Li ◽

Bruce A. McClane

Keyword(s):

Sialic Acid ◽

Clostridium Perfringens ◽

Intracellular Location ◽

Content Type ◽

Sialidase Activity ◽

Type D ◽

Genes Encoding ◽

Isogenic Mutants ◽

Culture Supernatants ◽

Sialic Acid Linkage

ABSTRACTClostridium perfringenscauses histotoxic infections and diseases originating in animal or human intestines. A prolific toxin producer, this bacterium also produces numerous enzymes, including sialidases, that may facilitate infection.C. perfringenstype D strain CN3718 carries genes encoding three sialidases, including two large secreted sialidases (named NanI and NanJ) and one small sialidase (named NanH) that has an intracellular location in log-phase cultures but is present in supernatants of death phase cultures. Using isogenic mutants of CN3718 that are capable of expressing only NanJ, NanI, or NanH, the current study characterized the properties and activities of each sialidase. The optimal temperature determined for NanJ or NanH enzymatic activity was 37°C or 43°C, respectively, while NanI activity increased until temperature reached 48°C. NanI activity was also the most resistant against higher temperatures. All three sialidases showed optimal activities at pH 5.5. Compared to NanJ or NanH, NanI contributed most to the sialidase activity in CN3718 culture supernatants, regardless of the substrate sialic acid linkage; NanI also released the most sialic acid from Caco-2 cells. Only NanI activity was enhanced by trypsin pretreatment and then only for substrates with an α-2,3- or α-2,6-sialic acid linkage. NanJ and NanI activities were more sensitive than NanH activity to two sialidase inhibitors (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid and siastatin B). The activities of the three sialidases were affected differently by several metal ions. These results indicated that eachC. perfringenssialidase has distinct properties, which may allow these enzymes to play different roles depending upon environmental conditions.

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NanI Sialidase Can Support the Growth and Survival of Clostridium perfringens Strain F4969 in the Presence of Sialyated Host Macromolecules (Mucin) or Caco-2 Cells

Infection and Immunity ◽

10.1128/iai.00547-17 ◽

2017 ◽

Vol 86 (2) ◽

Cited By ~ 4

Author(s):

Jihong Li ◽

Bruce A. McClane

Keyword(s):

Sialic Acid ◽

Clostridium Perfringens ◽

Growth Promotion ◽

Food Poisoning ◽

Host Cells ◽

Content Type ◽

Growth And Survival ◽

Gi Infections ◽

Human Enterocyte ◽

Free Sialic Acid

ABSTRACT Enterotoxin-producing Clostridium perfringens type A strains cause human gastrointestinal (GI) infections, including a very common food poisoning and 5 to 10% of all cases of antibiotic-associated diarrhea. This bacterium can utilize free sialic acid for growth, but most sialic acids in the GI tract are sequestered on macromolecules, such as the mucin proteins of mucus or glycoconjugates in host cells. However, many C. perfringens strains produce sialidases that might promote growth and survival by generating free sialic acid from those sialyated host macromolecules or by exposing underlying carbohydrates or proteins for digestion by other enzymes. The current study tested that possibility and found that the C. perfringens nonfoodborne human GI disease strain F4969 can use either a mucin preparation or Caco-2 cells, which are human enterocyte-like cells, to support its growth and survival. An isogenic nanI null mutant and complemented strain were used to show that this enhanced growth and survival using mucin or Caco-2 cells involved NanI, which is the major exosialidase of F4969 and many other C. perfringens strains. Experiments also suggested that, at least in part, this growth promotion involves utilization of NanI-generated sialic acid. In addition, a sialidase inhibitor named siastatin B reduced the growth and survival of F4969 growing with either the mucin preparation or Caco-2 cells. These findings suggest that, when produced, NanI may be a significant contributor to C. perfringens human GI infections by promoting the intestinal growth and survival of this bacterium. They also suggest the possibility that sialidase inhibitors might inhibit C. perfringens infections.

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Native or Proteolytically Activated NanI Sialidase Enhances the Binding and Cytotoxic Activity of Clostridium perfringens Enterotoxin and Beta Toxin

Infection and Immunity ◽

10.1128/iai.00730-17 ◽

2017 ◽

Vol 86 (1) ◽

Cited By ~ 9

Author(s):

James R. Theoret ◽

Jihong Li ◽

Mauricio A. Navarro ◽

Jorge P. Garcia ◽

Francisco A. Uzal ◽

...

Keyword(s):

Cytotoxic Activity ◽

Clostridium Perfringens ◽

Full Length ◽

Host Cells ◽

Proteolytic Activation ◽

Clostridium Perfringens Enterotoxin ◽

Content Type ◽

Sialidase Activity ◽

Epsilon Toxin ◽

Beta Toxin

ABSTRACTManyClostridium perfringensstrains produce NanI as their major sialidase. Previous studies showed that NanI could potentiateC. perfringensepsilon toxin cytotoxicity by enhancing the binding of this toxin to host cells. The present study first determined that NanI exerts similar cytotoxicity-enhancing effects onC. perfringensenterotoxin and beta toxin, which are also important toxins forC. perfringensdiseases (enteritis and enterotoxemia) originating in the gastrointestinal (GI) tract. Building upon previous work demonstrating that purified trypsin can activate NanI activity, this study next determined that purified chymotrypsin or mouse intestinal fluids can also activate NanI activity. Amino acid sequencing then showed that this effect involves the N-terminal processing of the NanI protein. Recombinant NanI (rNanI) species corresponding to major chymotrypsin- or small intestinal fluid-generated NanI fragments possessed more sialidase activity than did full-length rNanI, further supporting the proteolytic activation of NanI activity. rNanI species corresponding to proteolysis products also promoted the cytotoxic activity and binding of enterotoxin and beta toxin more strongly than did full-length rNanI. Since enterotoxin and beta toxin are produced in the intestines during human and animal disease, these findings suggest that intestinal proteases may enhance NanI activity, which in turn could further potentiate the activity of intestinally active toxins during disease. Coupling these new results with previous findings demonstrating that NanI is important for the adherence ofC. perfringensto enterocyte-like cells, NanI sialidase is now emerging as a potential auxiliary virulence factor forC. perfringensenteritis and enterotoxemia.

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