scholarly journals Subtypes of the Plasmid-Encoded Serine Protease EspP in Shiga Toxin-Producing Escherichia coli: Distribution, Secretion, and Proteolytic Activity

2007 ◽  
Vol 73 (20) ◽  
pp. 6351-6359 ◽  
Author(s):  
Jens Brockmeyer ◽  
Martina Bielaszewska ◽  
Angelika Fruth ◽  
Marie Luise Bonn ◽  
Alexander Mellmann ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Serine Protease ◽  
Proteolytic Activity ◽  
Shiga Toxin ◽  
Point Mutations ◽  
Coagulation Factor ◽  
Factor V ◽  
Human Pathogens ◽  
E Coli ◽  
Bona Fide

ABSTRACT We investigated the prevalence, distribution, and structure of espP in Shiga toxin-producing Escherichia coli (STEC) and assessed the secretion and proteolytic activity of the encoded autotransporter protein EspP (extracellular serine protease, plasmid encoded). espP was identified in 56 of 107 different STEC serotypes. Sequencing of a 3,747-bp region of the 3,900-bp espP gene distinguished four alleles (espPα, espPβ, espPγ, and espPδ), with 99.9%, 99.2%, 95.3%, and 95.1% homology, respectively, to espP of E. coli O157:H7 strain EDL933. The espPβ, espPγ, and espPδ genes contained unique insertions and/or clustered point mutations that enabled allele-specific PCRs; these demonstrated the presence of espPα, espPβ, espPγ, and espPδ in STEC isolates belonging to 17, 16, 15, and 8 serotypes, respectively. Among four subtypes of EspP encoded by these alleles, EspPα (produced by enterohemorrhagic E. coli [EHEC] O157:H7 and the major non-O157 EHEC serotypes) and EspPγ cleaved pepsin A, human coagulation factor V, and an oligopeptide alanine-alanine-proline-leucine-para-nitroaniline, whereas EspPβ and EspPδ either were not secreted or were proteolytically inactive. The lack of proteolysis correlated with point mutations near the active serine protease site. We conclude that espP is widely distributed among STEC strains and displays genetic heterogeneity, which can be used for subtyping and which affects EspP activity. The presence of proteolytically active EspP in EHEC serogroups O157, O26, O111, and O145, which are bona fide human pathogens, suggests that EspP might play a role as an EHEC virulence factor.

scholarly journals Molecular Subtyping and Distribution of the Serine Protease from Shiga Toxin-Producing Escherichia coli among Atypical Enteropathogenic E. coli Strains

2009 ◽  
Vol 75 (7) ◽  
pp. 2246-2249 ◽  
Author(s):  
Adrian L. Cookson ◽  
Jenny Bennett ◽  
Carolyn Nicol ◽  
Fiona Thomson-Carter ◽  
Graeme T. Attwood
Keyword(s):  
Escherichia Coli ◽  
Serine Protease ◽  
Shiga Toxin ◽  
Molecular Subtyping ◽  
E Coli ◽  
Sequence Types

ABSTRACT Atypical enteropathogenic Escherichia coli (aEPEC) and Shiga toxin-producing E. coli (STEC) were examined to determine the prevalence and sequence of espP, which encodes a serine protease. These analyses indicated shared espP sequence types between the two E. coli pathotypes and thus provide further insights into the evolution of aEPEC and STEC.

EspP, An Extracellular Serine Protease From Enterohemorrhagic E. Coli, Induces Coagulopathy In Human Whole Blood and Plasma

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4416-4416
Author(s):  
Kevin H.M. Kuo ◽  
Shekeb Khan ◽  
Elena Brnjac ◽  
Emil F. Pai ◽  
Alden E. Chesney
Keyword(s):  
Serine Protease ◽  
Whole Blood ◽  
Ex Vivo ◽  
Coagulation Factor ◽  
Time Dependent ◽  
Coagulation Cascade ◽  
Factor V ◽  
Dependent Manner ◽  
E Coli ◽  
Negative Controls

Abstract Abstract 4416 EspP (E. coli secreted serine protease, large plasmid encoded) is an extracellular serine protease produced by enterohemorrhagic E. coli (EHEC) O157:H7. Brunder et al. (Mol Microbiol 1997, 24:767–78) have shown that EspP cleaves, amongst other proteins, human coagulation factor V, and the authors hypothesized that it may contribute to the mucosal hemorrhage in patients with EHEC infection. We have since shown that EspP also cleaves factor VIII. Since the mechanism by which EHEC induces diarrhea-associated Hemolytic Uremic Syndrome (D+HUS) has not been fully elucidated, and EspP has been cited as a putative virulence factor in D+HUS, we investigated the role of EspP in primary and secondary hemostasis in the pathogenesis of D+HUS. Wild type EspP (EspPwt) and EspPS263A, where the serine at the active site was mutated to an alanine thereby abolishing its proteolytic activity, were expressed in the non-pathogenic E. coli host BL21(DE3) and purified by hydrophobic interaction and size-exclusion chromatography. EspPwt at 1.0 mg/mL was incubated for 0.5, 2.0 and 4.0 hours ex vivo with citrated plasma from 6 healthy adults. EspPS263A, bovine serum albumin (BSA) and phosphate buffer saline-glycerol (PBS-G) served as negative controls. PT, aPTT and TT were found to be significantly prolonged and activity of factors V, VII, VIII and XII were reduced in a time- and concentration-dependent manner (Figures 1 and Figure 2). When citrated plasma was incubated with 1 mg/mL EspPwt at 37°C for 4 hours, PT was prolonged by 23.2 +/− 3.8 s, aPTT by 41.6 +/− 8.3 s and TT by 6.1 +/− 0.6 s, relative to the negative controls. Factor V activity decreased by 0.82 +/− 0.14 U/mL, factor VII by 0.72 +/− 0.28 U/mL, factor VIII by 0.69 +/− 0.31 U/mL and factor XII by 0.36 +/− 0.09 U/mL, relative to the negative controls. Prothrombin activity was significantly reduced (0.16 +/− 0.08 U/mL) compared to all negative controls but remained above 0.75 U/mL. Factors IX, × and XI activity, and fibrinogen concentration were not significantly different from the controls. To determine whether any cellular components in whole blood contribute to EspP's effect on the coagulation cascade, the experiment was repeated using citrated whole blood in place of plasma during the incubation phase. Plasma was then recovered and analyzed. Similar results were observed. The results suggest that EspP has proteolytic activity against specific coagulation factors at least in an ex vivo setting. In patients with EHEC infection, EspP may contribute to the hemorrhagic diarrhea by impairing the coagulation cascade. Further studies are needed to determine whether EspP is able to induce coagulopathy in vivo and if so, whether induction of such a coagulopathic state may favour the entry of Shiga toxin into systemic circulation in patients with D+HUS. Figure 1 EspP prolongs PT, aPTT and TT in a time-dependent manner. Figure 1. EspP prolongs PT, aPTT and TT in a time-dependent manner. Figure 2 EspP reduces coagulation factor activity in a time-dependent manner. Figure 2. EspP reduces coagulation factor activity in a time-dependent manner. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Presence of Escherichia coli O157 and O157:H7 in raw milk and Van herby cheese

2015 ◽  
Vol 59 (4) ◽  
pp. 511-514 ◽  
Author(s):  
Yakup Can Sancak ◽  
Hakan Sancak ◽  
Ozgur Isleyici
Keyword(s):  
Public Health ◽  
Escherichia Coli ◽  
Drug Administration ◽  
Shiga Toxin ◽  
Raw Milk ◽  
Escherichia Coli O157 ◽  
Human Pathogens ◽  
Emerging Pathogens ◽  
E Coli ◽  
Milk Samples

Abstract The Shiga toxin-producing Escherichia coli (STEC) strains are currently considered important emerging pathogens threatening public health. Among Shiga toxin-producing Escherichia coli, E. coli O157:H7 strains have emerged as important human pathogens. This study was conducted to determine the presence of Escherichia coli O157 and O157:H7 in raw milk samples and Van herby cheese samples. For this purpose, 100 samples of raw milk were collected and 100 samples of herby cheese sold for consumption in Van province in Turkey were obtained from grocers and markets in order to detect the presence of Escherichia coli O157 and O157:H7. The method of E. coli O157 and O157:H7 isolation proposed by the Food and Drug Administration (FDA) was used. E. coli O157 in raw milk and herby cheese samples was found in 11% and 6% of samples respectively, and E. coli O157:H7 was found in 2% of herby cheese samples. No E. coli O157:H7 was detected in raw milk samples. This study showed that raw milk was contaminated with E. coli O157 and herby cheese was contaminated with both E. coli O157 and E. coli O157:H7; therefore, herby cheese poses a serious risk to public health.

Evidence of Non-O157 Shiga Toxin–Producing Escherichia coli in the Feces of Meat Goats at a U.S. Slaughter Plant

2013 ◽  
Vol 76 (9) ◽  
pp. 1626-1629 ◽  
Author(s):  
M. E. JACOB ◽  
D. M. FOSTER ◽  
A. T. ROGERS ◽  
C. C. BALCOMB ◽  
X. SHI ◽  
...  
Keyword(s):  
United States ◽  
Escherichia Coli ◽  
Shiga Toxin ◽  
Virulence Genes ◽  
Southeastern United States ◽  
Human Pathogens ◽  
Fecal Samples ◽  
E Coli ◽  
Meat Goats ◽  
Two Samples

Shiga toxin–producing Escherichia coli (STEC) are important human pathogens, and attention to non-O157 serogroups has increased in recent years. Although cattle are normally considered the primary reservoir for STEC, recent illnesses associated with goat contact have indicated that these animals are important potential reservoirs for the organisms. The prevalence of STEC, particularly non-O157 serogroups, in U.S. goats has not been well described. Our objective was to determine the prevalence of six major non-O157 STEC serogroups in the feces of meat goats. Rectal contents from 296 goats were collected postevisceration at a slaughter plant in the southeastern United States over 9 days during a 12-week period from August through October 2012. Samples were enriched in E. coli broth, and DNA was extracted and used as template in an 11-gene multiplex PCR that detected six non-O157 serogroups (O26, O45, O103, O121, O111, and O145) and virulence genes. Samples were considered positive when at least one non-O157 STEC serotype was present with either stx1 or stx2. All six non-O157 serogroups were detected by PCR in our samples, and 14.5% of samples were positive for at least one serogroup. Prevalence of O26 was highest, with 6.4% of goat fecal samples positive. The prevalence of O45 was 3.4%, O103 was 4.4%, O111 was 4.1%, O121 was 1.4%, and O145 was 3.0%. Twenty-two (7.4%) of 296 fecal samples had more than one non-O157 serogroup detected in the feces. Two samples had evidence of three non-O157 STEC serogroups. Goats appear to be an important reservoir for non-O157 STEC, and further work to understand the characteristics, epidemiology, and ecology of STEC in these animals is warranted.

Growth of Escherichia coli O157:H7, Non-O157 Shiga Toxin–Producing Escherichia coli, and Salmonella in Water and Hydroponic Fertilizer Solutions

2016 ◽  
Vol 79 (12) ◽  
pp. 2179-2183 ◽  
Author(s):  
ANGELA SHAW ◽  
KARA HELTERBRAN ◽  
MICHAEL R. EVANS ◽  
CHRISTOPHER CURREY
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Water System ◽  
The United States ◽  
Plain Water ◽  
Escherichia Coli O157 ◽  
Human Pathogens ◽  
E Coli ◽  
Fertilizer Solution ◽  
Fertilizer Solutions

ABSTRACT The desire for local, fresh produce year round is driving the growth of hydroponic growing systems in the United States. Many food crops, such as leafy greens and culinary herbs, grown within hydroponics systems have their root systems submerged in recirculating nutrient-dense fertilizer solutions from planting through harvest. If a foodborne pathogen were introduced into this water system, the risk of contamination to the entire crop would be high. Hence, this study was designed to determine whether Escherichia coli O157:H7, non-O157 Shiga toxin–producing E. coli, and Salmonella were able to survive and reproduce in two common hydroponic fertilizer solutions and in water or whether the bacteria would be killed or suppressed by the fertilizer solutions. All the pathogens grew by 1 to 6 log CFU/ml over a 24-h period, depending on the solution. E. coli O157:H7 reached higher levels in the fertilizer solution with plants (3.12 log CFU/ml), whereas non-O157 Shiga toxin–producing E. coli and Salmonella reached higher levels in the fertilizer solution without plants (1.36 to 3.77 log CFU/ml). The foodborne pathogens evaluated here survived for 24 h in the fertilizer solution, and populations grew more rapidly in these solutions than in plain water. Therefore, human pathogens entering the fertilizer solution tanks in hydroponic systems would be expected to rapidly propagate and spread throughout the system and potentially contaminate the entire crop.

scholarly journals Virulence Potential of Activatable Shiga Toxin 2d–Producing Escherichia coli Isolates from Fresh Produce

2015 ◽  
Vol 78 (11) ◽  
pp. 2085-2088 ◽  
Author(s):  
ANGELA R. MELTON-CELSA ◽  
ALISON D. O'BRIEN ◽  
PETER C. H. FENG
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Fresh Produce ◽  
Vero Cells ◽  
Human Pathogens ◽  
E Coli ◽  
Intestinal Mucus ◽  
Virulence Potential ◽  
Beef Products ◽  
Uremic Syndrome

Shiga toxin (Stx)–producing Escherichia coli (STEC) strains are food- and waterborne pathogens that are often transmitted via beef products or fresh produce. STEC strains cause both sporadic infections and outbreaks, which may result in hemorrhagic colitis and hemolytic uremic syndrome. STEC strains may elaborate Stx1, Stx2, and/or subtypes of those toxins. Epidemiological evidence indicates that STEC that produce subtypes Stx2a, Stx2c, and/or Stx2d are more often associated with serious illness. The Stx2d subtype becomes more toxic to Vero cells after incubation with intestinal mucus or elastase, a process named “activation.” Stx2d is not generally found in the E. coli serotypes most commonly connected to STEC outbreaks. However, STEC strains that are stx2d positive can be isolated from foods, an occurrence that gives rise to the question of whether those food isolates are potential human pathogens. In this study, we examined 14 STEC strains from fresh produce that were stx2d positive and found that they all produced the mucus-activatable Stx2d and that a subset of the strains tested were virulent in streptomycin-treated mice.

scholarly journals Presence and characteristics of sorbitol-negative Escherichia coli O157 in healthy sheep faeces

2008 ◽  
Vol 52 (No. 7) ◽  
pp. 301-307 ◽  
Author(s):  
H. Turutoglu ◽  
D. Ozturk ◽  
L. Guler ◽  
F. Pehlivanoglu
Keyword(s):  
Escherichia Coli ◽  
Multiplex Pcr ◽  
Shiga Toxin ◽  
Human Pathogens ◽  
Isolation Rate ◽  
E Coli ◽  
Awassi Sheep ◽  
Healthy Sheep

The presence of sorbitol-negative <i>Escherichia coli</i> O157 was investigated in healthy Awassi sheep faeces from 175 randomly selected animals in Burdur province of Turkey. Out of 175 animals, 16 (9.1%) were faecal shedding of sorbitol-negative <i>E. coli</i> O157. Out of the 15 flocks included in the study, 7 (47%) had at least one sheep positive for sorbitol-negative <i>E. coli</i> O157. The isolation rate of sorbitol-negative <i>E. coli</i> O157 ranged from 8.3 to 60% among the animals tested in the flocks. A total of 16 ovine sorbitol-negative <i>E. coli</i> O157 strains were characterized by a multiplex PCR. Results showed that 6 (37.7%) strains carried <i>stx1</i> gene, 3 (18.8%) <i>stx2</i> gene and 1 (6.3%) both <i>stx1</i> and <i>stx2</i> genes. Intimin (<i>eaeA</i>) gene was detected in 4 (25%) of the strains. None of the strains encoding for <i>stx</i> genes was positive for <i>eaeA</i> gene. The results demonstrate that the majority of sorbitol-negative <i>E. coli</i> O157 strains (62.5%) isolated from Awassi sheep in Burdur province of Turkey are Shiga toxin-producing <i>E. coli</i> that have a potential as human pathogens.

scholarly journals Functional Comparison of Serine Protease Autotransporters of Enterobacteriaceae

2002 ◽  
Vol 70 (12) ◽  
pp. 7105-7113 ◽  
Author(s):  
Pinaki R. Dutta ◽  
Renato Cappello ◽  
Fernando Navarro-García ◽  
James P. Nataro
Keyword(s):  
Serine Protease ◽  
Morphological Changes ◽  
Shigella Flexneri ◽  
Coagulation Factor ◽  
Factor V ◽  
Substrate Specificities ◽  
Cell Monolayers ◽  
E Coli ◽  
Cytopathic Effects ◽  
Related Proteins

ABSTRACT The plasmid-encoded toxin (Pet) of enteroaggregative Escherichia coli (EAEC) belongs to a family of high-molecular-weight serine protease autotransporters of Enterobacteriaceae (SPATEs) which also includes Pic from EAEC and Shigella flexneri, EspC from enteropathogenic E. coli, EspP from enterohemorrhagic E. coli, Sat from uropathogenic E. coli, Tsh from avian pathogenic E. coli, and SepA from S. flexneri. Phylogenetic analysis shows the SPATE proteins to represent a distinct subfamily of autotransporters with amino acid identities ranging from 35 to 55%, providing a powerful resource to direct structure-function studies. In this study, we show that these related proteins are proteases with divergent substrate specificities, suggesting different functions. The cleavage profile of oligopeptides was found to be unique for each SPATE protein. The SPATEs showed proteolytic activity for several substrates, namely mucin, pepsin, human coagulation factor V, and erythroid spectrin. The cleavage of spectrin has been hypothesized as the mechanism through which Pet induces cytopathic effects. However, whereas Pet, Sat, and EspC cleaved spectrin, only Pet and Sat elicited cytopathic effects; the remaining SPATEs did not cause any morphological changes to HEp-2 cell monolayers. EspC and Pet exhibited acid-dissociable binding to HEp-2 cells. However, Pet was more efficient at entering HEp-2 cells, suggesting a basis for the different abilities of these two proteases to damage cells. Our data suggest that, despite the homologies observed among these proteins, the SPATEs have different pathogenetic functions only partly dependent on their substrate specificities.

scholarly journals L-Alanine Prototrophic Suppressors Emerge from L-Alanine Auxotroph through Stress-Induced Mutagenesis in Escherichia coli

2021 ◽  
Vol 9 (3) ◽  
pp. 472
Author(s):  
Harutaka Mishima ◽  
Hirokazu Watanabe ◽  
Kei Uchigasaki ◽  
So Shimoda ◽  
Shota Seki ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pyruvate Dehydrogenase ◽  
Pyruvate Dehydrogenase Complex ◽  
Spontaneous Mutation ◽  
Point Mutations ◽  
Whole Genome Analysis ◽  
E Coli ◽  
Clone Pair ◽  
Induced Mutagenesis ◽  
Isogenic Mutants

In Escherichia coli, L-alanine is synthesized by three isozymes: YfbQ, YfdZ, and AvtA. When an E. coli L-alanine auxotrophic isogenic mutant lacking the three isozymes was grown on L-alanine-deficient minimal agar medium, L-alanine prototrophic mutants emerged considerably more frequently than by spontaneous mutation; the emergence frequency increased over time, and, in an L-alanine-supplemented minimal medium, correlated inversely with L-alanine concentration, indicating that the mutants were derived through stress-induced mutagenesis. Whole-genome analysis of 40 independent L-alanine prototrophic mutants identified 16 and 18 clones harboring point mutation(s) in pyruvate dehydrogenase complex and phosphotransacetylase-acetate kinase pathway, which respectively produce acetyl coenzyme A and acetate from pyruvate. When two point mutations identified in L-alanine prototrophic mutants, in pta (D656A) and aceE (G147D), were individually introduced into the original L-alanine auxotroph, the isogenic mutants exhibited almost identical growth recovery as the respective cognate mutants. Each original- and isogenic-clone pair carrying the pta or aceE mutation showed extremely low phosphotransacetylase or pyruvate dehydrogenase activity, respectively. Lastly, extracellularly-added pyruvate, which dose-dependently supported L-alanine auxotroph growth, relieved the L-alanine starvation stress, preventing the emergence of L-alanine prototrophic mutants. Thus, L-alanine starvation-provoked stress-induced mutagenesis in the L-alanine auxotroph could lead to intracellular pyruvate increase, which eventually induces L-alanine prototrophy.

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