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Clostridium perfringens can produce up to three different sialidases, including NanI, its major exosialidase. The current study first showed that human intestinal strains of C. perfringens can grow by utilizing either glucose or sialic acids, such as N -acetylneuraminic acid (Neu5Ac), which are the end products of sialidase activity. For the human enteropathogenic strain F4969, it was then determined that culture supernatant sialidase activity and expression of exosialidase genes, particularly nanI , are influenced by the presence of Neu5Ac or glucose. Low Neu5Ac concentrations increased culture supernatant sialidase activity, largely by stimulating nanI transcription. In contrast, low glucose concentrations did not affect exosialidase activity or nanI transcription. However, either high Neu5Ac or high glucose concentrations repressed F4969 culture supernatant sialidase activity and nanI transcription levels. Furthermore, high glucose levels repressed F4969 culture sialidase activity and nanI expression even in the presence of low Neu5AC concentrations. To begin to evaluate the mechanistic basis for nanI expression, a nanR null mutant was used to demonstrate that NanR, a member of the RpiR family of regulatory proteins, decreases exosialidase activity and nanI transcription in the absence of sialic acid. The ability of C. perfringens to regulate its exosialidase activity, largely by controlling nanI expression, may affect intestinal pathogenesis by affecting the production of NanI, which may affect C. perfringens growth, adhesion, and toxin binding in vivo ., (Copyright © 2017 American Society for Microbiology.)

Clostridium perfringens is ubiquitous in nature and is often found as a commensal of the human and animal gastrointestinal tract. It is the primary etiological agent of clostridial myonecrosis, or gas gangrene, a serious infection that results in extensive tissue necrosis due to the action of one or more potent extracellular toxins. α-toxin and perfringolysin O are the major extracellular toxins involved in the pathogenesis of gas gangrene, but histotoxic strains of C. perfringens, such as strain 13, also produce many degradative enzymes such as collagenases, hyaluronidases, sialidases and the cysteine protease, α-clostripain. The production of many of these toxins is regulated either directly or indirectly by the global VirSR two-component signal transduction system. By isolating a chromosomal mutant and carrying out microarray analysis we have identified an orphan sensor histidine kinase, which we have named ReeS (regulator of extracellular enzymes sensor). Expression of the sialidase genes nanI and nanJ was down-regulated in a reeS mutant. Since complementation with the wild-type reeS gene restored nanI and nanJ expression to wild-type levels, as shown by quantitative reverse transcription-PCR and sialidase assays we concluded that ReeS positively regulates the expression of these sialidase genes. However, mutation of the reeS gene had no significant effect on virulence in the mouse myonecrosis model. Sialidase production in C. perfringens has been previously shown to be regulated by both the VirSR system and RevR. In this report, we have analyzed a previously unknown sensor histidine kinase, ReeS, and have shown that it also is involved in controlling the expression of sialidase genes, adding further complexity to the regulatory network that controls sialidase production in C. perfringens.

The inherent difficulty of expressing clostridial AT-rich genes in a heterologous host has limited their biotechnological application. We previously reported a plasmid for high-level expression of clostridial genes in Clostridium perfringens (Takamizawa et al., Protein Expr Purif 36:70-75, 2004). In this study, we examined the extracellular proteases of C. perfringens strain 13. Zymographic analysis and caseinase assaying of a culture supernatant showed that it contained a protease activated by dithiothreitol and Ca(2+), suggesting that clostripain-like protease (Clp) is the most likely candidate for the major extracellular protease. Disruption of the clp gene by homologous recombination markedly decreased the level of caseinase activity in the culture supernatant. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed that the Clp(-) mutant but not the wild type strain increased the levels of many polypeptides in the culture supernatant after the late exponential growth phase. Such polypeptides included both cytoplasmic and secretory proteins, suggesting proteins secreted or released into the medium were degraded by Clp. To assess the effects of Clp on the productivity and stability of recombinant proteins, 74-kDa NanI sialidase was expressed in the two strains. The mutant strain produced a higher level of NanI activity than the wild type strain. Furthermore, under the conditions where Clp was activated, NanI was degraded easily in the latter culture but not in the former one. These results indicate that the Clp(-) mutant could serve as a useful strain for efficiently expressing and preparing protease-free clostridial proteins.

A "large" sialidase isozyme (NanI) from Clostridium perfringens is a representative microbial sialidase with broad substrate specificity, being used for the analysis of sialoglycoconjugates. It is also a possible virulence factor. However, purification of the native enzyme in a large quantity is not practical due to its low productivity. To obtain the enzyme in a satisfactory yield, a gene encoding the NanI was transcriptionally fused to the fdx gene promoter (P(fdx)) in a shuttle-vector, pFF, and transformed into C. perfringens 13. The resultant strain released the enzyme into the culture medium, as the original strain does. The enzyme activity increased during the first 6 h of culture and thereafter remained at maximal levels. The maximal activity was approximately 3000-fold compared with that of the original strain, and 15-fold compared with that of recombinant Escherichia coli, which possesses extra copies of the tRNA gene for selected rare codons. This suggests the usefulness of a P(fdx)-based plasmid for expressing AT-rich genes in C. perfringens. The enzyme was successfully purified by two-step procedure with a specific activity of 2860 U/mg using 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and a yield of 1.69 mg of NanI per 100 ml of culture. The method described here can facilitate purification of NanI in enough quality and quantity to analyze the role of sialoglycoconjugates in cells and the pathogenic importance of NanI sialidase.

The small nanH gene encoding a neuraminidase from Clostridium perfringens ATCC 10543 was cloned in JM109 using pUC19 as a vector. Sequence analysis revealed an ORF, nt 310-1455, encoding 382 amino acids that was proceeded by a typical Shine-Dalgarno sequence, GGACGAGA. The nt sequence in the 15-402 region had in vivo promoter activity in an Escherichia coli promoter probe plasmid pKK232-8, which suggested that the small nanH promoter is functional in E. coli. Four regions of amino acids demonstrated great similarity to the "Asp boxes" (-Ser-X-Asp-X-Gly-X-Thr-Trp-) of other bacterial nanH proteins. The small nanH expressing clone, pCPN-1, which was cultured under aerobic conditions resulted in NanH activity which was 203-fold in culture and 211-fold in intracellular fraction compared to that of C. perfringens which has to be cultured under anaerobic conditions. Production of small NanH was also induced by adding sialyllactose to the culture medium of JM109 [pCPN-1]. The enzyme activity could be detected in the periplasmic fraction and the culture medium of JM109 [pCPN-1] after culturing to the stationary phase. The molecular weight, K(m), and optimum pH and pI of the cloned enzyme are identical to those of the parent strain. The cloned, small nanH could be used to study the structure-functional relationship of nanH, while the pCPN-1 clone could be used in the aerobic production of neuraminidase.

A 1.4-kb gene encoding the "small" sialidase isoenzyme of Clostridium perfringens A99, including its own promoter, was previously cloned in and expressed by Escherichia coli JM 101. Since all attempts to purify this enzyme to homogeneity were unsuccessful, a new strategy was developed. The structural gene was amplified by means of a PCR technique and inserted into the plasmid vector pQE-10, transferring a six-histidine affinity tag (His6) to the N-terminus of the protein. In order to minimize proteolytic degradation of the sialidase protein, the gene was subcloned into the Escherichia coli strain BL21(DE3)pLys S with reduced protease activity. The sialidase production was increased about 2.5-fold when compared with that of the original clone. The enzyme, released by lysozyme treatment of the bacterial cells, was purified by metal chelate chromatography on Ni-nitrilo-triacetic acid agarose to apparent homogeneity in SDS-PAGE. The 42-kDa protein was enriched 62-fold with a yield of 82% and a specific activity of 280 U mg-1. A total amount of 1 mg sialidase was obtained from 1 liter of bacterial culture. For future studies, including crystallization experiments, the histidine affinity tag was removed from the sialidase enzyme by aminopeptidase K. The sialidase was then separated from aminopeptidase K by ion-exchange chromatography, resulting in an overall yield of 83% and a specific activity of 305 U mg-1 using 4-methylumbelliferyl- alpha-D-N-acetylneuraminic acid under standard conditions. The two forms (with or without the histidine tag) of sialidase exhibited similar kinetic properties when compared to the wild-type enzyme.

The production of neuraminidase by a classical strain of Clostridium welchii (C. perfringens) type A was studied. Good yields were produced in 5% Proteose Peptone-water medium (PPW5); the enzyme was essentially extracellular but some further neuraminidase could be released by ultrasonic disintegration of the cells. This also released N-acyl neuraminic acid-aldolase (NAN-aldolase) and the degree to which this interferes with the assay for neuraminidase was evaluated. Forty-one British reference food-poisoning strains of C. welchii type A were examined for extracellular neuraminidase production in PPW5. Twelve of 17 strains that produce so-called heat-sensitive spores were neuraminidase positive whereas 20 of 24 strains that are non-haemolytic and produce very heat-resistant sporeswere neuraminidase negative. Variation was found in the ability to produce neuraminidase among strains of a single Hobbs' serotype; four Hobbs' type-13 strains produced neuraminidase but a fifth did not. Disruption of the cells of a Hobbs' type-2 strain that did not produce any extracellular neuraminidase released NAN-aldolase but there was no evidence of cell-associated neuraminidase. British food-poisoning strains of C. welchii type A thus include some that are clearly neuraminidase positive and some that still cannot be shown to produce neuraminidase. There is no correlation between lack of neuraminidase production and the ability to cause food poisoning, although the majority of non-haemolytic heat-resistant strains do not produce neuraminidase. It remains possible that neuraminidase may play a part in C. welchii gas gangrene; it is suggested that the ability to define neuraminidase-negative strains may now be of value in investigating this possibility.

The production of neuraminidase (EC 3.2.1.18) by a range of clostridial species was investigated with techniques previously developed to distinguish neuraminidase-negative and neuraminidase-positive strains of Clostridium perfringens (welchii). Large amounts of extracellular neuraminidase were produced by representative strains of C. perfringens and C. septicum in the test media. Under similar conditions, two strains each of C. chauvoei and C. tertium were found to produce small amounts of the enzyme. All of 12 strains of C. sordellii were clearly shown to produce neuraminidase, often in large amounts, but none of five strains of the closely related but non-pathogenic C. bifermentans had demonstrable neuraminidase activity. No neuraminidase was produced by C. novyi (oedematiens) types A-D (10 strains), C. tetani (6), C. botulinum types A, B, C or E (4), C. sporogenes (4), C. histolyticum (4) or by single strains of five other clostridial species. Clostridial neuraminidase was predominantly extracellular and was not calcium-dependent. The investigation took account of variations in growth and enzyme production in different media. It was necessary to prolong the neuraminidase-assay reaction time to 24 h and to monitor for the presence of NAN-aldolase (EC 4.1.3.3) to define true negatives. It is suggested that neuraminidase production may be of value in taxonomic studies and that its production by several pathogenic species of clostridia may be of interest in studies of pathogenicity and virulence.

The relationship between the production of hemagglutinin and sialidase activities by Clostridium perfringens was investigated by screening for mutants producing reduced levels of hemagglutinin activity. Twelve mutants were isolated; all produced reduced levels of sialidase activity and several had other altered phenotypic markers. Revertants that regained the ability to produce active hemagglutinin were isolated. All of these revertants produced increased sialidase activity. These results show that the production of hemagglutinin activity is directly related to the production of sialidase activity. Evidence is also presented that the processes of sporulation and the production of extracellular proteins are interrelated.