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

Li, Jihong; McClane, Bruce A.

doi:10.1128/aem.03440-13

Cited by 58 publications

(94 citation statements)

References 33 publications

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“…Both sialidase inhibitors reduced overall F4969 sialidase activity ( Fig. 4B and C), and from the inhibition curves, the IC 50 of NADNA was determined to be 11.86 M, while the IC 50 of SB was found to be 41 M. These IC 50 doses are similar to the IC 50 doses used previously to inhibit type D strain CN3718 sialidase activity in TH overnight culture supernatant (28).…”

Section: Fig 3 Creation and Characterization Of The Isogenic F4969 Nasupporting

confidence: 79%

“…The absorbance at 595 nm was then measured using a Bio-Rad microplate reader. Previous studies (21,28) showed that under these conditions, sialidase activity in the samples falls in the linear range of the assay.…”

Section: Methodsmentioning

confidence: 95%

“…Plasmids pJIR750nani and pJIR750nanicomp were prepared previously (21) Measurement of supernatant sialidase enzyme activity. Supernatant sialidase enzyme activity, which accounts for nearly all of the total sialidase activity present in overnight cultures of C. perfringens (21), was assayed as described previously (21,28). Briefly, a 0.2-ml aliquot of an FTG overnight (ϳ16-h) culture was transferred to 10 ml of fresh TH medium and cultured for 6 h at 37°C.…”

Section: Methodsmentioning

confidence: 99%

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Contributions of NanI Sialidase to Caco-2 Cell Adherence by Clostridium perfringens Type A and C Strains Causing Human Intestinal Disease

McClane

2014

Infect Immun

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Previous studies showed that Clostridium perfringens type D animal disease strain CN3718 uses NanI sialidase for adhering to enterocyte-like Caco-2 cells. The current study analyzed whether NanI is similarly important when type A and C human intestinal disease strains attach to Caco-2 cells. A PCR survey determined that the nanI gene was absent from typical type A food poisoning (FP) strains carrying a chromosomal enterotoxin (CPE) gene or the genetically related type C Darmbrand (Db) strains. However, the nanI gene was present in type A strains from healthy humans, type A strains causing CPE-associated antibioticassociated diarrhea (AAD) or sporadic diarrhea (SD), and type C Pig-Bel strains. Consistent with NanI sialidase being the major C. perfringens sialidase when produced, FP and Db strains had little supernatant sialidase activity compared to other type A or C human intestinal strains. All type A and C human intestinal strains bound to Caco-2 cells, but NanI-producing strains had higher attachment levels. When produced, NanI can contribute to host cell attachment of human intestinal disease strains, since a nanI null mutant constructed in type A SD strain F4969 had lower Caco-2 cell adhesion than wild-type F4969 or a complemented strain. Further supporting a role for NanI in host cell attachment, sialidase inhibitors reduced F4969 adhesion to Caco-2 cells. Collectively, these results suggest that NanI may contribute to the intestinal attachment and colonization needed for the chronic diarrhea of CPE-associated AAD and SD, but this sialidase appears to be dispensable for the acute pathogenesis of type A FP or type C enteritis necroticans.

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Section: Fig 3 Creation and Characterization Of The Isogenic F4969 Nasupporting

confidence: 79%

Section: Methodsmentioning

confidence: 95%

Section: Methodsmentioning

confidence: 99%

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Contributions of NanI Sialidase to Caco-2 Cell Adherence by Clostridium perfringens Type A and C Strains Causing Human Intestinal Disease

McClane

2014

Infect Immun

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“…CN3718, a C. perfringens type D livestock gastrointestinal (GI) disease strain, produces epsilon toxin (ETX) and all three sialidases (NanJ, NanI, and NanH) (32,33). Previously constructed mutants used in this study included the following: (i) BMC202, an isogenic CN3718 nanI null mutant strain; (ii) BMC207, an isogenic CN3718 nanJ, nanI, nanH, and etx quadruple null mutant strain; and (iii) BMC2072, a nanI complementing strain of BMC207 (32).…”

Section: Methodsmentioning

confidence: 99%

“…In addition to toxins, C. perfringens produces many enzymes, including three sialidases named NanJ, NanI, and NanH. The two larger sialidases, NanJ (129 kDa) and NanI (77 kDa), are secreted from C. perfringens, while the smallest enzyme, NanH (43 kDa), is located in the cytoplasm of log-phase cultures but can also appear extracellularly in older cultures (32,33). A recent study (32) found that type D strain CN3718 produces all three sialidases, with NanI being the major secreted sialidase of this strain, as it is for many other C. perfringens strains (34,35).…”

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confidence: 99%

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

Freedman

McClane

2015

J Bacteriol

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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...

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Impact of cell culture media additives on IgG glycosylation produced in Chinese hamster ovary cells

Ehret

Zimmermann

Eichhorn

et al. 2019

Biotech & Bioengineering

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Glycosylation is a key critical quality attribute for monoclonal antibodies and other recombinant proteins because of its impact on effector mechanisms and half‐life. In this study, a variety of compounds were evaluated for their ability to modulate glycosylation profiles of recombinant monoclonal antibodies produced in Chinese hamster ovary cells. Compounds were supplemented into the cell culture feed of fed‐batch experiments performed with a CHO K1 and a CHO DG44 cell line expressing a recombinant immunoglobulin G1 (IgG1). Experiments were performed in spin tubes or the ambr®15 controlled bioreactor system, and the impact of the compounds at various concentrations was determined by monitoring the glycosylation profile of the IgG and cell culture parameters, such as viable cell density, viability, and titer. Results indicate that the highest impact on mannosylation was achieved through 15 µM kifunensine supplementation leading to an 85.8% increase in high‐mannose containing species. Fucosylation was reduced by 76.1% through addition of 800 µM 2‐F‐peracetyl fucose. An increase of 40.9% in galactosylated species was achieved through the addition of 120 mM galactose in combination with 48 µM manganese and 24 µM uridine. Furthermore, 6.9% increased sialylation was detected through the addition of 30 µM dexamethasone in combination with the same manganese, uridine, and galactose mixture used to increase total galactosylation. Further compounds or combinations of additives were also efficient at achieving a smaller overall glycosylation modulation, required, for instance, during the development of biosimilars. To the best of our knowledge, no evaluation of the efficacy of such a variety of compounds in the same cell culture system has been described. The studied cell culture media additives are efficient modulators of glycosylation and are thus a valuable tool to produce recombinant glycoproteins.

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

Cited by 58 publications

References 33 publications

Contributions of NanI Sialidase to Caco-2 Cell Adherence by Clostridium perfringens Type A and C Strains Causing Human Intestinal Disease

Contributions of NanI Sialidase to Caco-2 Cell Adherence by Clostridium perfringens Type A and C Strains Causing Human Intestinal Disease

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

Impact of cell culture media additives on IgG glycosylation produced in Chinese hamster ovary cells

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