Effect of the cortex-lytic enzyme SleC from non-food-borne Clostridium perfringens on the germination properties of SleC-lacking spores of a food poisoning isolate

2010 ◽  
Vol 56 (11) ◽  
pp. 952-958 ◽  
Author(s):  
Daniel Paredes-Sabja ◽  
Mahfuzur R. Sarker
Keyword(s):  
Clostridium Perfringens ◽  
Spore Germination ◽  
Type Strain ◽  
Wild Type Strain ◽  
Dipicolinic Acid ◽  
Food Poisoning ◽  
Lytic Enzyme ◽  
Wild Type ◽  
Lytic Enzymes ◽  
Food Borne

The hallmark of bacterial spore germination is peptidoglycan cortex hydrolysis by cortex-lytic enzymes. In spores of Clostridium perfringens wild-type strain SM101, which causes food poisoning, the sole essential cortex-lytic enzyme SleC is activated by a unique serine protease CspB. Interestingly, the non-food-borne wild-type strain F4969 encodes a significantly divergent SleC variant (SleCF4969) and 3 serine proteases (CspA, CspB, and CspC). Consequently, in this study we evaluated the functional compatibility of SleCF4969and SleCSM101by complementing the germination phenotypes of SM101ΔsleC spores with sleCF4969. Our results show that although pro-SleCF4969was processed into mature SleCF4969in the SM101ΔsleC spores, it partially restored spore germination with nutrient medium, with a mixture of l-asparagine and KCl, or with a 1:1 chelate of Ca2+and dipicolinic acid. While the amount of dipicolinic acid released was lower, the amount of hexosamine-containing material released during germination of SM101ΔsleC(sleCF4969) spores was similar to the amount released during germination of SM101 wild-type spores. The viability of SM101ΔsleC(sleCF4969) spores was 8- and 3-fold lower than that of SM101 and F4969 spores, respectively. Together, these data indicate that the peptidoglycan cortex hydrolysis machinery in the food poisoning isolate SM101 is functionally divergent than that in the non-food-borne isolate F4969.

scholarly journals Clostridium perfringens Spore Germination: Characterization of Germinants and Their Receptors

2007 ◽  
Vol 190 (4) ◽  
pp. 1190-1201 ◽  
Author(s):  
Daniel Paredes-Sabja ◽  
J. Antonio Torres ◽  
Peter Setlow ◽  
Mahfuzur R. Sarker
Keyword(s):  
Clostridium Perfringens ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Food Poisoning ◽  
Wild Type ◽  
Active Growth ◽  
Food Borne ◽  
High Concentrations ◽  
Auxiliary Role

ABSTRACT Clostridium perfringens food poisoning is caused by type A isolates carrying a chromosomal enterotoxin (cpe) gene (C-cpe), while C. perfringens-associated non-food-borne gastrointestinal (GI) diseases are caused by isolates carrying a plasmid-borne cpe gene (P-cpe). C. perfringens spores are thought to be the important infectious cell morphotype, and after inoculation into a suitable host, these spores must germinate and return to active growth to cause GI disease. We have found differences in the germination of spores of C-cpe and P-cpe isolates in that (i) while a mixture of l-asparagine and KCl was a good germinant for spores of C-cpe and P-cpe isolates, KCl and, to a lesser extent, l-asparagine triggered spore germination in C-cpe isolates only; and (ii) l-alanine or l-valine induced significant germination of spores of P-cpe but not C-cpe isolates. Spores of a gerK mutant of a C-cpe isolate in which two of the proteins of a spore nutrient germinant receptor were absent germinated slower than wild-type spores with KCl, did not germinate with l-asparagine, and germinated poorly compared to wild-type spores with the nonnutrient germinants dodecylamine and a 1:1 chelate of Ca2+ and dipicolinic acid. In contrast, spores of a gerAA mutant of a C-cpe isolate that lacked another component of a nutrient germinant receptor germinated at the same rate as that of wild-type spores with high concentrations of KCl, although they germinated slightly slower with a lower KCl concentration, suggesting an auxiliary role for GerAA in C. perfringens spore germination. In sum, this study identified nutrient germinants for spores of both C-cpe and P-cpe isolates of C. perfringens and provided evidence that proteins encoded by the gerK operon are required for both nutrient-induced and non-nutrient-induced spore germination.

scholarly journals Inorganic Phosphate and Sodium Ions Are Cogerminants for Spores of Clostridium perfringens Type A Food Poisoning-Related Isolates

2009 ◽  
Vol 75 (19) ◽  
pp. 6299-6305 ◽  
Author(s):  
Daniel Paredes-Sabja ◽  
Pathima Udompijitkul ◽  
Mahfuzur R. Sarker
Keyword(s):  
Clostridium Perfringens ◽  
Inorganic Phosphate ◽  
Dipicolinic Acid ◽  
Food Poisoning ◽  
Type A ◽  
Gastrointestinal Diseases ◽  
Wild Type ◽  
Similar Extent ◽  
Active Growth ◽  
Food Borne

ABSTRACT Clostridium perfringens type A isolates carrying a chromosomal copy of the enterotoxin (cpe) gene are involved in the majority of food poisoning (FP) outbreaks, while type A isolates carrying a plasmid-borne cpe gene are involved in C. perfringens-associated non-food-borne (NFB) gastrointestinal diseases. To cause diseases, C. perfringens spores must germinate and return to active growth. Previously, we showed that only spores of FP isolates were able to germinate with K+ ions. We now found that the spores of the majority of FP isolates, but none of the NFB isolates, germinated with the cogerminants Na+ and inorganic phosphate (NaPi) at a pH of ∼6.0. Spores of gerKA-KC and gerAA mutants germinated to a lesser extent and released less dipicolinic acid (DPA) than did wild-type spores with NaPi. Although gerKB spores germinated to a similar extent as wild-type spores with NaPi, their rate of germination was lower. Similarly, gerO and gerO gerQ mutant spores germinated slower and released less DPA than did wild-type spores with NaPi. In contrast, gerQ spores germinated to a slightly lesser extent than wild-type spores but released all of their DPA during NaPi germination. In sum, this study identified NaPi as a novel nutrient germinant for spores of most FP isolates and provided evidence that proteins encoded by the gerKA-KC operon, gerAA, and gerO are required for NaPi-induced spore germination.

scholarly journals The protease CspB is essential for initiation of cortex hydrolysis and dipicolinic acid (DPA) release during germination of spores of Clostridium perfringens type A food poisoning isolates

Microbiology ◽  
2009 ◽  
Vol 155 (10) ◽  
pp. 3464-3472 ◽  
Author(s):  
Daniel Paredes-Sabja ◽  
Peter Setlow ◽  
Mahfuzur R. Sarker
Keyword(s):  
Clostridium Perfringens ◽  
Spore Germination ◽  
Nutrient Medium ◽  
Deletion Mutant ◽  
Dipicolinic Acid ◽  
Food Poisoning ◽  
Wild Type ◽  
Cell Compartment ◽  
Gene Encoding

The genome of the Clostridium perfringens food poisoning isolate SM101 encodes a subtilisin-like protease, CspB, upstream of the sleC gene encoding the enzyme essential for degradation of the peptidoglycan cortex during spore germination. SleC is an inactive pro-SleC in dormant spores that is converted to active SleC during spore germination and Csp proteases convert pro-SleC to the active enzyme in vitro. In this work, the germination and viability of spores of a cspB deletion mutant of strain SM101, as well as cspB expression, were studied. The cspB gene was expressed only during sporulation, and only in the mother cell compartment. cspB spores were unable to germinate significantly with either a rich nutrient medium, KCl, or a 1 : 1 chelate of Ca2+ and dipicolinic acid (DPA); the viability of these spores was ∼104-fold lower than that of wild-type spores, although cspB and wild-type spores had similar viability on plates containing lysozyme, and cspB spores could not process inactive pro-SleC into active SleC during spore germination. Germination of cspB spores was blocked prior to DPA release and cortex hydrolysis, and germination and viability defects in these spores were complemented by an ectopic cspB. These results indicate that Csp proteases are essential to generate active SleC and allow cortex hydrolysis early in C. perfringens spore germination. However, Csp proteases likely play another role in spore germination, since cspB spores did not release DPA upon exposure to germinants, while sleC spores have been shown previously to release DPA, albeit slowly, upon exposure to germinants.

scholarly journals SleC Is Essential for Cortex Peptidoglycan Hydrolysis during Germination of Spores of the Pathogenic Bacterium Clostridium perfringens

2009 ◽  
Vol 191 (8) ◽  
pp. 2711-2720 ◽  
Author(s):  
Daniel Paredes-Sabja ◽  
Peter Setlow ◽  
Mahfuzur R. Sarker
Keyword(s):  
Clostridium Perfringens ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Pathogenic Bacterium ◽  
Wild Type ◽  
Lytic Enzymes ◽  
Colony Forming Efficiency ◽  
Forming Efficiency ◽  
Peptidoglycan Hydrolysis

ABSTRACT Clostridial spore germination requires degradation of the spore's peptidoglycan (PG) cortex by cortex-lytic enzymes (CLEs), and two Clostridium perfringens CLEs, SleC and SleM, degrade cortex PG in vitro. We now find that only SleC is essential for cortex hydrolysis and viability of C. perfringens spores. C. perfringens sleC spores did not germinate completely with nutrients, KCl, or a 1:1 chelate of Ca2+ and dipicolinic acid (Ca-DPA), and the colony-forming efficiency of sleC spores was 103-fold lower than that of wild-type spores. However, sleC spores incubated with various germinants released most of their DPA, although slower than wild-type or sleM spores, and DPA release from sleC sleM spores was very slow. In contrast, germination and viability of sleM spores were similar to that of wild-type spores, although sleC sleM spores had 105-fold-lower viability. These results allow the following conclusions about C. perfringens spore germination: (i) SleC is essential for cortex hydrolysis; (ii) although SleM can degrade cortex PG in vitro, this enzyme is not essential; (iii) action of SleC alone or with SleM can accelerate DPA release; and (iv) Ca-DPA does not trigger spore germination by activation of CLEs.

scholarly journals The CcpA Protein Is Necessary for Efficient Sporulation and Enterotoxin Gene (cpe) Regulation in Clostridium perfringens

2004 ◽  
Vol 186 (16) ◽  
pp. 5221-5229 ◽  
Author(s):  
John Varga ◽  
Veronica L. Stirewalt ◽  
Stephen B. Melville
Keyword(s):  
Mutant Strain ◽  
Clostridium Perfringens ◽  
Exponential Growth ◽  
Catabolite Repression ◽  
Type Strain ◽  
Wild Type Strain ◽  
Food Poisoning ◽  
Wild Type ◽  
Antibiotic Associated Diarrhea ◽  
In The Wild

ABSTRACT Clostridium perfringens is the cause of several human diseases, including gas gangrene (clostridial myonecrosis), enteritis necroticans, antibiotic-associated diarrhea, and acute food poisoning. The symptoms of antibiotic-associated diarrhea and acute food poisoning are due to sporulation-dependent production of C. perfringens enterotoxin encoded by the cpe gene. Glucose is a catabolite repressor of sporulation by C. perfringens. In order to identify the mechanism of catabolite repression by glucose, a mutation was introduced into the ccpA gene of C. perfringens by conjugational transfer of a nonreplicating plasmid into C. perfringens, which led to inactivation of the ccpA gene by homologous recombination. CcpA is a transcriptional regulator known to mediate catabolite repression in a number of low-G+C-content gram-positive bacteria, of which C. perfringens is a member. The ccpA mutant strain sporulated at a 60-fold lower efficiency than the wild-type strain in the absence of glucose. In the presence of 5 mM glucose, sporulation was repressed about 2,000-fold in the wild-type strain and 800-fold in the ccpA mutant strain compared to sporulation levels for the same strains grown in the absence of glucose. Therefore, while CcpA is necessary for efficient sporulation in C. perfringens, glucose-mediated catabolite repression of sporulation is not due to the activity of CcpA. Transcription of the cpe gene was measured in the wild-type and ccpA mutant strains grown in sporulation medium by using a cpe-gusA fusion (gusA is an Escherichia coli gene encoding the enzyme β-glucuronidase). In the exponential growth phase, cpe transcription was two times higher in the ccpA mutant strain than in the wild-type strain. Transcription of cpe was highly induced during the entry into stationary phase in wild-type cells but was not induced in the ccpA mutant strain. Glucose repressed cpe transcription in both the wild-type and ccpA mutant strain. Therefore, CcpA appears to act as a repressor of cpe transcription in exponential growth but is required for efficient sporulation and cpe transcription upon entry into stationary phase. CcpA was also required for maximum synthesis of collagenase (kappa toxin) and acted as a repressor of polysaccharide capsule synthesis in the presence of glucose, but it did not regulate synthesis of the phospholipase PLC (alpha toxin).

scholarly journals Role Of Selected Fimbrial Adheins In Pathogenesis Of Acid-Induced Eneterohemorrhagic Escherichia Coli 0157:H7

2021 ◽  
Author(s):  
Shahnaz Haque
Keyword(s):  
Fatty Acid ◽  
Type Strain ◽  
Wild Type Strain ◽  
Short Chain Fatty Acid ◽  
Acid Stress ◽  
Chain Fatty Acid ◽  
Short Chain ◽  
Wild Type ◽  
Food Borne

Enterohemorrhagic Escherichia coli (EHEC) 0157:H7 is a food-borne pathogen that causes hemolytic uremic syndrome and hemorrhagic colitis. The mechanisms underlying the adhesion of EHEC 0157:H7 to intestinal epithelial cells are not well understood. Like other food-borne pathogens, ECEC 0157:H7 must survive the acid stress of the gastric juice in the stomach and short chain fatty acid in the intestine in order to colonize the large intestine. We have found that acid stress and short chain fatty acid stress significantly enhance host-adhesion of EHEC 0157:H7 and also upregulates expression of EHEC fimbrial genes, lpfA1, lpfA2 and yagZ, as demonstrated by our DNA microarray. We now report that disruption of the yagZ (also known as the E. coli common pilus A) gene results in loss of the acid-induced and short chain fatty acid-induced adhesion increase seen for the wild type strain. When the yagZ mutant is complemented with yagZ, the sress-induced and short chain fatty acid-induced adhesion increase seen for the wild type strain. When the yagZ mutant is complemented with yagZ, the stress-induced adhesion pehnotype is restored, confirming the role of yagZ in the acid as well as short chain fatty acid induced adhesion to HEp-2 cells. On the other hand, neither disruption in the long polar fimbria genes lpfA1 or lpfA2 in the wild type showed any effect in adherence to HEp-2 cells; rather displaying a hyperadherant phenotype to HEp-2 cells after acid-induced or short chain fatty acid-induced stress. The results also indicate that acid or short chain fatty acid stress, which is a part of the host's natural defense mechanism against pathogens, may regulate virulence factors resulting in enhanced bacteria-host attachment during colonization in the human or bovine host.

scholarly journals Characterization of Clostridium perfringens Spores That Lack SpoVA Proteins and Dipicolinic Acid

2008 ◽  
Vol 190 (13) ◽  
pp. 4648-4659 ◽  
Author(s):  
Daniel Paredes-Sabja ◽  
Barbara Setlow ◽  
Peter Setlow ◽  
Mahfuzur R. Sarker
Keyword(s):  
Water Content ◽  
Uv Radiation ◽  
Clostridium Perfringens ◽  
Spore Germination ◽  
Gastrointestinal Disease ◽  
Dipicolinic Acid ◽  
High Heat ◽  
Wild Type ◽  
Active Growth ◽  
Moist Heat

ABSTRACT Spores of Clostridium perfringens possess high heat resistance, and when these spores germinate and return to active growth, they can cause gastrointestinal disease. Work with Bacillus subtilis has shown that the spore's dipicolinic acid (DPA) level can markedly influence both spore germination and resistance and that the proteins encoded by the spoVA operon are essential for DPA uptake by the developing spore during sporulation. We now find that proteins encoded by the spoVA operon are also essential for the uptake of Ca2+ and DPA into the developing spore during C. perfringens sporulation. Spores of a spoVA mutant had little, if any, Ca2+ and DPA, and their core water content was approximately twofold higher than that of wild-type spores. These DPA-less spores did not germinate spontaneously, as DPA-less B. subtilis spores do. Indeed, wild-type and spoVA C. perfringens spores germinated similarly with a mixture of l-asparagine and KCl (AK), KCl alone, or a 1:1 chelate of Ca2+ and DPA (Ca-DPA). However, the viability of C. perfringens spoVA spores was 20-fold lower than the viability of wild-type spores. Decoated wild-type and spoVA spores exhibited little, if any, germination with AK, KCl, or exogenous Ca-DPA, and their colony-forming efficiency was 103- to 104-fold lower than that of intact spores. However, lysozyme treatment rescued these decoated spores. Although the levels of DNA-protective α/β-type, small, acid-soluble spore proteins in spoVA spores were similar to those in wild-type spores, spoVA spores exhibited markedly lower resistance to moist heat, formaldehyde, HCl, hydrogen peroxide, nitrous acid, and UV radiation than wild-type spores did. In sum, these results suggest the following. (i) SpoVA proteins are essential for Ca-DPA uptake by developing spores during C. perfringens sporulation. (ii) SpoVA proteins and Ca-DPA release are not required for C. perfringens spore germination. (iii) A low spore core water content is essential for full resistance of C. perfringens spores to moist heat, UV radiation, and chemicals.

scholarly journals GerO, a Putative Na+/H+-K+ Antiporter, Is Essential for Normal Germination of Spores of the Pathogenic Bacterium Clostridium perfringens

2009 ◽  
Vol 191 (12) ◽  
pp. 3822-3831 ◽  
Author(s):  
Daniel Paredes-Sabja ◽  
Peter Setlow ◽  
Mahfuzur R. Sarker
Keyword(s):  
Clostridium Perfringens ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Ectopic Expression ◽  
Monovalent Cation ◽  
Wild Type ◽  
Cell Compartment ◽  
E Coli ◽  
Modeled Structure ◽  
Cation Transporters

ABSTRACT The genome of the pathogen Clostridium perfringens encodes two proteins, GerO and GerQ, homologous to monovalent cation transporters suggested to have roles in the germination of spores of some Bacillus species. GerO and GerQ were able to transport monovalent cations (K+ and/or Na+) in Escherichia coli, and gerO and gerQ were expressed only in the mother cell compartment during C. perfringens sporulation. C. perfringens spores lacking GerO were defective in germination with a rich medium, KCl, l-asparagine, and a 1:1 chelate of Ca2+ and dipicolinic acid (DPA), but not with dodecylamine, and the defect was prior to DPA release in germination. All defects in gerO spores were complemented by ectopic expression of wild-type gerO. Loss of GerQ had much smaller effects on spore germination, and these effects were most evident in spores also lacking GerO. A modeled structure of GerO was similar to that of the E. coli Na+/H+ antiporter NhaA, and GerO, but not GerQ contained two adjacent Asp residues thought to be important in the function of this group of cation transporters. Replacement of these adjacent Asp residues in GerO with Asn reduced the protein's ability to complement the germination defect in gerO spores but not the ability to restore cation transport to E. coli cells defective in K+ uptake. Together, these data suggest that monovalent cation transporters play some role in C. perfringens spore germination. However, it is not clear whether this role is directly in germination or perhaps in spore formation.

scholarly journals Evaluating the Involvement of Alternative Sigma Factors SigF and SigG in Clostridium perfringens Sporulation and Enterotoxin Synthesis

2010 ◽  
Vol 78 (10) ◽  
pp. 4286-4293 ◽  
Author(s):  
Jihong Li ◽  
Bruce A. McClane
Keyword(s):  
Bacillus Subtilis ◽  
Clostridium Perfringens ◽  
Food Poisoning ◽  
Sigma Factors ◽  
Wild Type ◽  
Alternative Sigma Factors ◽  
Food Borne ◽  
Food Borne Illness ◽  
Bacterial Food ◽  
Null Mutants

ABSTRACT Clostridium perfringens type A food poisoning is the second most commonly identified bacterial food-borne illness. Sporulation contributes to this disease in two ways: (i) most food-poisoning strains form exceptionally resistant spores to facilitate their survival of food-associated stresses, and (ii) the enterotoxin (CPE) responsible for the symptoms of this food poisoning is synthesized only during sporulation. In Bacillus subtilis, four alternative sigma factors mediate sporulation. The same four sigma factors are encoded by C. perfringens genomes, and two (SigE and SigK) have previously been shown to be necessary for sporulation and CPE production by SM101, a transformable derivative of a C. perfringens food-poisoning strain (K. H. Harry, R. Zhou, L. Kroos, and S. B. Melville, J. Bacteriol. 2009, 191:2728-2742). However, the importance of SigF and SigG for C. perfringens sporulation or CPE production had not yet been assessed. In the current study, after confirming that sporulating wild-type SM101 cultures produce SigF (from a tricistronic operon) and SigG, we prepared isogenic sigF- or sigG-null mutants. Whereas SM101 formed heat-resistant, phase-refractile spores, spore formation was blocked in the sigF- and sigG-null mutants. Complementation fully restored sporulation by both mutants. By use of these mutants and complementing strains, CPE production was shown to be SigF dependent but SigG independent. This finding apparently involved regulation of the production of SigE and SigK, which Harry et al. showed to be necessary for CPE synthesis, by SigF. By combining these findings with those previous results, it is now apparent that all four alternative sigma factors are necessary for C. perfringens sporulation, but only SigE, SigF, and SigK are needed for CPE synthesis.

scholarly journals Regulation and Characterization of a Newly Deduced Cell Wall Hydrolase Gene (cwlJ) Which Affects Germination of Bacillus subtilis Spores

1998 ◽  
Vol 180 (6) ◽  
pp. 1375-1380 ◽  
Author(s):  
Shu Ishikawa ◽  
Kunio Yamane ◽  
Junichi Sekiguchi
Keyword(s):  
Bacillus Subtilis ◽  
Type Strain ◽  
Wild Type Strain ◽  
Catalytic Domain ◽  
Slow Release ◽  
Dipicolinic Acid ◽  
Northern Hybridization ◽  
Predicted Amino Acid Sequence ◽  
Wild Type

ABSTRACT The predicted amino acid sequence of Bacillus subtilis ycbQ (renamed cwlJ) exhibits high similarity to those of the deduced C-terminal catalytic domain of SleBs, the specific cortex-hydrolyzing enzyme of B. cereus and the deduced one of B. subtilis. We constructed acwlJ::lacZ fusion in the B. subtilischromosome. The β-galactosidase activity and results of Northern hybridization and primer extension analyses of the cwlJgene indicated that it is transcribed by EςE RNA polymerase. cwlJ-deficient spores responded to bothl-alanine and AGFK, the A 580 values of spore suspensions decreased more slowly than in the case of the wild-type strain, and the mutant spores released less dipicolinic acid than did those of the wild-type strain during germination. However, the mutant spores released only slightly less hexosamine than did the wild-type spores. In contrast, B. subtilis sleB spores did not release hexosamine at a significant level. While cwlJand sleB spores were able to germinate, CJSB (cwlJ sleB) spores could not germinate but exhibited initial germination reactions, e.g., partial decrease inA 580 and slow release of dipicolinic acid. CJSB spores became slightly gray after 6 h in the germinant, but their refractility was much greater than that of sleB mutant spores. The roles of the sleB and cwlJmutations in germination and spore maturation are also discussed.

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