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Relationship between heat-labile enterotoxin secretion capacity and virulence in wild type porcine-origin enterotoxigenic Escherichia coli strains.

Wijemanne P, Xing J, Berberov EM, Marx DB, Francis DH, Moxley RA - PLoS ONE (2015)

Bottom Line: Maximum Likelihood phylogenetic trees constructed using T2SS genes gspC, gspD, gspE and homologs showed that strains 2534-86 and 3030-2 clustered together in the same clade with other porcine-origin ETEC strains in the database, UMNK88 and UMN18.Protein modeling of the ATPase gene (gspE) further revealed a direct relationship between the predicted ATP-binding capacities and LT secretion levels as follows: H10407, -8.8 kcal/mol and 199 ng/ml; 3030-2, -8.6 kcal/mol and 133 ng/ml; and 2534-86, -8.5 kcal/mol and 80 ng/ml.This study demonstrated a direct relationship between predicted ATP-binding capacity of GspE and LT secretion, and between the latter and virulence.

View Article: PubMed Central - PubMed

Affiliation: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America.

ABSTRACT
Heat-labile enterotoxin (LT) is an important virulence factor secreted by some strains of enterotoxigenic Escherichia coli (ETEC). The prototypic human-origin strain H10407 secretes LT via a type II secretion system (T2SS). We sought to determine the relationship between the capacity to secrete LT and virulence in porcine-origin wild type (WT) ETEC strains. Sixteen WT ETEC strains isolated from cases of severe diarrheal disease were analyzed by GM1ganglioside enzyme-linked immunosorbent assay to measure LT concentrations in culture supernatants. All strains had detectable LT in supernatants by 2 h of culture and 1 strain, which was particularly virulent in gnotobiotic piglets (3030-2), had the highest LT secretion level all porcine-origin WT strains tested (P<0.05). The level of LT secretion (concentration in supernatants at 6-h culture) explained 92% of the variation in time-to-a-moribund-condition (R2 = 0.92, P<0.0001) in gnotobiotic piglets inoculated with either strain 3030-2, or an ETEC strain of lesser virulence (2534-86), or a non-enterotoxigenic WT strain (G58-1). All 16 porcine ETEC strains were positive by PCR analysis for the T2SS genes, gspD and gspK, and bioinformatic analysis of 4 porcine-origin strains for which complete genomic sequences were available revealed a T2SS with a high degree of homology to that of H10407. Maximum Likelihood phylogenetic trees constructed using T2SS genes gspC, gspD, gspE and homologs showed that strains 2534-86 and 3030-2 clustered together in the same clade with other porcine-origin ETEC strains in the database, UMNK88 and UMN18. Protein modeling of the ATPase gene (gspE) further revealed a direct relationship between the predicted ATP-binding capacities and LT secretion levels as follows: H10407, -8.8 kcal/mol and 199 ng/ml; 3030-2, -8.6 kcal/mol and 133 ng/ml; and 2534-86, -8.5 kcal/mol and 80 ng/ml. This study demonstrated a direct relationship between predicted ATP-binding capacity of GspE and LT secretion, and between the latter and virulence.

No MeSH data available.


Related in: MedlinePlus

Heat-labile enterotoxin (LT) secretion into the periplasm over time by human- and porcine-origin enterotoxigenic E. coli strains grown in CAYE-M medium.Strains were cultured at 37°C and 225 rpm in CAYE-M using a flask-to-medium ratio of 8.3:1. Periplasmic extracts were prepared from samples of cell pellets obtained at 2, 4 and 6 h of culture, and LT concentrations in these extracts at each time interval were determined by GM1-ELISA. *LT concentrations in H10407 periplasmic extracts are significantly different (P<0.05) from that of all other strains at the corresponding time interval. †LT concentrations in 3030–2 periplasmic extracts are significantly different (P<0.05) from that of all other porcine or 2534–86 derivative strains at the corresponding time interval. eltAB: strain is positive for the LT genes by PCR. estB: strain is positive for STb gene by PCR. H = human-origin strain, P = porcine-origin strain, D = 2534–86 derivative strain.
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pone.0117663.g002: Heat-labile enterotoxin (LT) secretion into the periplasm over time by human- and porcine-origin enterotoxigenic E. coli strains grown in CAYE-M medium.Strains were cultured at 37°C and 225 rpm in CAYE-M using a flask-to-medium ratio of 8.3:1. Periplasmic extracts were prepared from samples of cell pellets obtained at 2, 4 and 6 h of culture, and LT concentrations in these extracts at each time interval were determined by GM1-ELISA. *LT concentrations in H10407 periplasmic extracts are significantly different (P<0.05) from that of all other strains at the corresponding time interval. †LT concentrations in 3030–2 periplasmic extracts are significantly different (P<0.05) from that of all other porcine or 2534–86 derivative strains at the corresponding time interval. eltAB: strain is positive for the LT genes by PCR. estB: strain is positive for STb gene by PCR. H = human-origin strain, P = porcine-origin strain, D = 2534–86 derivative strain.

Mentions: To confirm that other porcine-origin ETEC strains could secrete LT and determine whether LT secretion levels were detectably related to virulence, 16 ETEC strains isolated from pigs with severe disease (S1 Table) were compared with that of prototypic human-origin strain H10407. All strains were cultured in CAYE-M and sampled at 2, 4, and 6 h of culture to avoid significant contribution of LT to the supernatant by bacterial lysis. In addition, the supernatants at 18 h of culture were tested to determine whether any differences among strains remained so throughout the growth curve. LT was detected in the culture supernatants of all porcine WT strains at 2, 4 and 6 h, confirming the capacity to secrete the toxin (Fig. 1). At 2 h, the concentration of LT in culture supernatant for each of the strains did not differ significantly from that of H10407; however, at 4 and 6 h the concentrations were significantly lower than that of H10407 (P<0.05; Fig. 1). A notable finding was that by 6 h, strain 3030–2 had secreted a significantly higher concentration of LT into the supernatant compared to that of all other porcine WT strains tested (P<0.05) and this difference remained so through 18 h of culture (Fig. 1). Over all time points, all strains tested had a higher concentration of LT in the supernatant than in the periplasm (Fig. 2). As in the previous experiment, the highest concentration of LT in the periplasm was at 4 h (Fig. 2), and at this time the 3030–2 periplasmic LT concentration was significantly higher than that of the other WT porcine strains (P<0.05). To confirm that bacterial lysis was not a significant contributor to LT in the culture supernatant nor resulted in loss of LT from the periplasm during the exponential growth phase, each fraction was tested for alkaline phosphatase activity in the samples at 4 and 6 h of culture. No alkaline phosphatase activity was detected in the culture supernatants of any of the 16 WT porcine strains at either time interval, and all periplasmic extracts contained alkaline phosphatase activity, as expected. In contrast, at 12, 18 and 24 h of culture, alkaline phosphatase activity was present in both the culture supernatant and periplasmic fractions. These results confirmed that LT was present in the supernatants during the exponential phase by secretion and not by bacterial lysis, but during the stationary and death phases, a portion of the LT activity was present in the supernatants as a result of cell lysis.


Relationship between heat-labile enterotoxin secretion capacity and virulence in wild type porcine-origin enterotoxigenic Escherichia coli strains.

Wijemanne P, Xing J, Berberov EM, Marx DB, Francis DH, Moxley RA - PLoS ONE (2015)

Heat-labile enterotoxin (LT) secretion into the periplasm over time by human- and porcine-origin enterotoxigenic E. coli strains grown in CAYE-M medium.Strains were cultured at 37°C and 225 rpm in CAYE-M using a flask-to-medium ratio of 8.3:1. Periplasmic extracts were prepared from samples of cell pellets obtained at 2, 4 and 6 h of culture, and LT concentrations in these extracts at each time interval were determined by GM1-ELISA. *LT concentrations in H10407 periplasmic extracts are significantly different (P<0.05) from that of all other strains at the corresponding time interval. †LT concentrations in 3030–2 periplasmic extracts are significantly different (P<0.05) from that of all other porcine or 2534–86 derivative strains at the corresponding time interval. eltAB: strain is positive for the LT genes by PCR. estB: strain is positive for STb gene by PCR. H = human-origin strain, P = porcine-origin strain, D = 2534–86 derivative strain.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4358887&req=5

pone.0117663.g002: Heat-labile enterotoxin (LT) secretion into the periplasm over time by human- and porcine-origin enterotoxigenic E. coli strains grown in CAYE-M medium.Strains were cultured at 37°C and 225 rpm in CAYE-M using a flask-to-medium ratio of 8.3:1. Periplasmic extracts were prepared from samples of cell pellets obtained at 2, 4 and 6 h of culture, and LT concentrations in these extracts at each time interval were determined by GM1-ELISA. *LT concentrations in H10407 periplasmic extracts are significantly different (P<0.05) from that of all other strains at the corresponding time interval. †LT concentrations in 3030–2 periplasmic extracts are significantly different (P<0.05) from that of all other porcine or 2534–86 derivative strains at the corresponding time interval. eltAB: strain is positive for the LT genes by PCR. estB: strain is positive for STb gene by PCR. H = human-origin strain, P = porcine-origin strain, D = 2534–86 derivative strain.
Mentions: To confirm that other porcine-origin ETEC strains could secrete LT and determine whether LT secretion levels were detectably related to virulence, 16 ETEC strains isolated from pigs with severe disease (S1 Table) were compared with that of prototypic human-origin strain H10407. All strains were cultured in CAYE-M and sampled at 2, 4, and 6 h of culture to avoid significant contribution of LT to the supernatant by bacterial lysis. In addition, the supernatants at 18 h of culture were tested to determine whether any differences among strains remained so throughout the growth curve. LT was detected in the culture supernatants of all porcine WT strains at 2, 4 and 6 h, confirming the capacity to secrete the toxin (Fig. 1). At 2 h, the concentration of LT in culture supernatant for each of the strains did not differ significantly from that of H10407; however, at 4 and 6 h the concentrations were significantly lower than that of H10407 (P<0.05; Fig. 1). A notable finding was that by 6 h, strain 3030–2 had secreted a significantly higher concentration of LT into the supernatant compared to that of all other porcine WT strains tested (P<0.05) and this difference remained so through 18 h of culture (Fig. 1). Over all time points, all strains tested had a higher concentration of LT in the supernatant than in the periplasm (Fig. 2). As in the previous experiment, the highest concentration of LT in the periplasm was at 4 h (Fig. 2), and at this time the 3030–2 periplasmic LT concentration was significantly higher than that of the other WT porcine strains (P<0.05). To confirm that bacterial lysis was not a significant contributor to LT in the culture supernatant nor resulted in loss of LT from the periplasm during the exponential growth phase, each fraction was tested for alkaline phosphatase activity in the samples at 4 and 6 h of culture. No alkaline phosphatase activity was detected in the culture supernatants of any of the 16 WT porcine strains at either time interval, and all periplasmic extracts contained alkaline phosphatase activity, as expected. In contrast, at 12, 18 and 24 h of culture, alkaline phosphatase activity was present in both the culture supernatant and periplasmic fractions. These results confirmed that LT was present in the supernatants during the exponential phase by secretion and not by bacterial lysis, but during the stationary and death phases, a portion of the LT activity was present in the supernatants as a result of cell lysis.

Bottom Line: Maximum Likelihood phylogenetic trees constructed using T2SS genes gspC, gspD, gspE and homologs showed that strains 2534-86 and 3030-2 clustered together in the same clade with other porcine-origin ETEC strains in the database, UMNK88 and UMN18.Protein modeling of the ATPase gene (gspE) further revealed a direct relationship between the predicted ATP-binding capacities and LT secretion levels as follows: H10407, -8.8 kcal/mol and 199 ng/ml; 3030-2, -8.6 kcal/mol and 133 ng/ml; and 2534-86, -8.5 kcal/mol and 80 ng/ml.This study demonstrated a direct relationship between predicted ATP-binding capacity of GspE and LT secretion, and between the latter and virulence.

View Article: PubMed Central - PubMed

Affiliation: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America.

ABSTRACT
Heat-labile enterotoxin (LT) is an important virulence factor secreted by some strains of enterotoxigenic Escherichia coli (ETEC). The prototypic human-origin strain H10407 secretes LT via a type II secretion system (T2SS). We sought to determine the relationship between the capacity to secrete LT and virulence in porcine-origin wild type (WT) ETEC strains. Sixteen WT ETEC strains isolated from cases of severe diarrheal disease were analyzed by GM1ganglioside enzyme-linked immunosorbent assay to measure LT concentrations in culture supernatants. All strains had detectable LT in supernatants by 2 h of culture and 1 strain, which was particularly virulent in gnotobiotic piglets (3030-2), had the highest LT secretion level all porcine-origin WT strains tested (P<0.05). The level of LT secretion (concentration in supernatants at 6-h culture) explained 92% of the variation in time-to-a-moribund-condition (R2 = 0.92, P<0.0001) in gnotobiotic piglets inoculated with either strain 3030-2, or an ETEC strain of lesser virulence (2534-86), or a non-enterotoxigenic WT strain (G58-1). All 16 porcine ETEC strains were positive by PCR analysis for the T2SS genes, gspD and gspK, and bioinformatic analysis of 4 porcine-origin strains for which complete genomic sequences were available revealed a T2SS with a high degree of homology to that of H10407. Maximum Likelihood phylogenetic trees constructed using T2SS genes gspC, gspD, gspE and homologs showed that strains 2534-86 and 3030-2 clustered together in the same clade with other porcine-origin ETEC strains in the database, UMNK88 and UMN18. Protein modeling of the ATPase gene (gspE) further revealed a direct relationship between the predicted ATP-binding capacities and LT secretion levels as follows: H10407, -8.8 kcal/mol and 199 ng/ml; 3030-2, -8.6 kcal/mol and 133 ng/ml; and 2534-86, -8.5 kcal/mol and 80 ng/ml. This study demonstrated a direct relationship between predicted ATP-binding capacity of GspE and LT secretion, and between the latter and virulence.

No MeSH data available.


Related in: MedlinePlus