Clonal relatedness of enterotoxigenic Escherichia coli (ETEC) strains expressing LT and CS17 isolated from children with diarrhoea in La Paz, Bolivia

Background

Enterotoxigenic Escherichia coli (ETEC) is a major cause of traveller''s and infantile diarrhoea in the developing world. ETEC produces two toxins, a heat-stable toxin (known as ST) and a heat-labile toxin (LT) and colonization factors that help the bacteria to attach to epithelial cells.

Methodology/Principal Findings

In this study, we characterized a subset of ETEC clinical isolates recovered from Bolivian children under 5 years of age using a combination of multilocus sequence typing (MLST) analysis, virulence typing, serotyping and antimicrobial resistance test patterns in order to determine the genetic background of ETEC strains circulating in Bolivia. We found that strains expressing the heat-labile (LT) enterotoxin and colonization factor CS17 were common and belonged to several MLST sequence types but mainly to sequence type-423 and sequence type-443 (Achtman scheme). To further study the LT/CS17 strains we analysed the nucleotide sequence of the CS17 operon and compared the structure to LT/CS17 ETEC isolates from Bangladesh. Sequence analysis confirmed that all sequence type-423 strains from Bolivia had a single nucleotide polymorphism; SNP_bol in the CS17 operon that was also found in some other MLST sequence types from Bolivia but not in strains recovered from Bangladeshi children. The dominant ETEC clone in Bolivia (sequence type-423/SNP_bol) was found to persist over multiple years and was associated with severe diarrhoea but these strains were variable with respect to antimicrobial resistance patterns.

Conclusion/Significance

The results showed that although the LT/CS17 phenotype is common among ETEC strains in Bolivia, multiple clones, as determined by unique MLST sequence types, populate this phenotype. Our data also appear to suggest that acquisition and loss of antimicrobial resistance in LT-expressing CS17 ETEC clones is more dynamic than acquisition or loss of virulence factors.     

Establishment of a transgenic mouse model specifically expressing human serum amyloid A in adipose tissue

Obesity and obesity co-morbidities are associated with a low grade inflammation and elevated serum levels of acute phase proteins, including serum amyloid A (SAA). In the non-acute phase in humans, adipocytes are major producers of SAA but the function of adipocyte-derived SAA is unknown. To clarify the role of adipocyte-derived SAA, a transgenic mouse model expressing human SAA1 (hSAA) in adipocytes was established. hSAA expression was analysed using real-time PCR analysis. Male animals were challenged with a high fat (HF) diet. Plasma samples were subjected to fast protein liquid chromatography (FPLC) separation. hSAA, cholesterol and triglyceride content were measured in plasma and in FPLC fractions. Real-time PCR analysis confirmed an adipose tissue-specific hSAA gene expression. Moreover, the hSAA gene expression was not influenced by HF diet. However, hSAA plasma levels in HF fed animals (37.7±4.0 µg/mL, n = 7) were increased compared to those in normal chow fed animals (4.8±0.5 µg/mL, n = 10; p<0.001), and plasma levels in the two groups were in the same ranges as in obese and lean human subjects, respectively. In FPLC separated plasma samples, the concentration of hSAA peaked in high-density lipoprotein (HDL) containing fractions. In addition, cholesterol distribution over the different lipoprotein subfractions as assessed by FPLC analysis was similar within the two experimental groups. The established transgenic mouse model demonstrates that adipose tissue produced hSAA enters the circulation, resulting in elevated plasma levels of hSAA. This new model will enable further studies of metabolic effects of adipose tissue-derived SAA.     

Concentrations of cholestenoic acids in plasma from patients with liver disease

The concentrations of 3 beta-hydroxy-5-cholestenoic acid, 3 beta,7 alpha-dihydroxy-5-cholestenoic acid, and 7 alpha-hydroxy-3-oxo-4-cholestenoic acid were determined in plasma from patients with different liver diseases and compared with those of unconjugated and conjugated C24 bile acids. The levels of the cholestenoic acids were similar in patients with extrahepatic cholestasis and in controls (median concentration 153 and 162 ng/ml, respectively), whereas significantly elevated levels were found in plasma from patients with primary biliary cirrhosis (median concentration 298 ng/ml) and alcoholic liver cirrhosis (median concentration 262 ng/ml). As expected, conjugated C24 bile acids were elevated in most patients whereas the corresponding unconjugated compounds were low in cholestasis and elevated in alcoholic liver cirrhosis. The levels of the individual C27 acids were usually positively correlated to each other and also to the levels of conjugated C24 bile acids in plasma from patients with liver cirrhosis. In contrast, there was no correlation between the levels of C27 acids and conjugated bile acids in patients with extrahepatic cholestasis. The levels of unconjugated C24 bile acids were not correlated to C27 acids or conjugated bile acids in any of the groups. The results indicate that there is a close metabolic relationship between the individual C27 acids, that they do not participate in an enterohepatic circulation, and that the liver is important for their elimination/metabolism.     

Plasma bikunin: Half-life and tissue uptake

Bikunin is a chondroitin sulfate-containing plasma protein synthesized in the liver. In vitro, it has been shown to inhibit proteases and to have additional activities, but its biological function is still unclear. Here we have studied the dynamics of plasma bikunin in rats and mice. A half-life of 7 ± 2 min was obtained from the time course of the decrease of the plasma level of bikunin following hepatectomy. Clearance experiments with intravenously injected radiolabeled bikunin with or without the chondroitin sulfate chain showed that the polysaccharide had little influence on the elimination rate of the protein. The uptake of bikunin by different tissues was studied using bikunin labeled with the residualizing agent ¹²⁵I-tyramine cellobiose; 60 min after intravenous injection, 49% of the radioactivity was recovered in the kidneys and 6–11% in the liver, bones, skin, intestine and skeletal muscle. The uptake in the liver was analyzed by intravenous injection of radiolabeled bikunin followed by collagenase perfusion and dispersion of the liver cells. These experiments indicated that bikunin is first trapped extracellularly within the liver before being internalized by the cells. (Mol Cell Biochem 271: 61–67, 2005)     

An optimized CZE method for analysis of mono- and oligomeric aldose mixtures

An optimized capillary electrophoresis (CZE) method to analyze complex mixtures of aldoses was developed. The approach allows simultaneous quantitative analysis of all four isomeric aldopentoses, eight aldohexoses, as well as xylo- and cellooligosaccharides up to the tetraoses. UV tagging with 4-aminobenzoic acid ethyl ester (ABEE) in combination with reductive amination was used as pre-column derivatization. With optimum baseline separation and short run times, the method is very robust, and especially suited to follow reaction and isomerization kinetics of monosaccharides.     

Recognition of fold and sugar linkage for glycosyltransferases by multivariate sequence analysis

Glycosyltransferases (GTs) are among the largest groups of enzymes found and are usually classified on the basis of sequence comparisons into many families of varying similarity (CAZy systematics). Only two different Rossman-like folds have been detected (GT-A and GT-B) within the small number of established crystal structures. A third uncharacterized fold has been indicated with transmembrane organization (GT-C). We here use a method based on multivariate data analyses (MVDAs) of property patterns in amino acid sequences and can with high accuracy recognize the correct fold in a large data set of GTs. Likewise, a retaining or inverting enzymatic mechanism for attachment of the donor sugar could be properly revealed in the GT-A and GT-B fold group sequences by such analyses. Sequence alignments could be correlated to important variables in MVDA, and the separating amino acid positions could be mapped over the active sites. These seem to be localized to similar positions in space for the alpha/beta/alpha binding motifs in the GT-B fold group structures. Analogous, active-site sequence positions were found for the GT-A fold group. Multivariate property patterns could also easily group most GTs annotated in the genomes of Escherichia coli and Synechocystis to proper fold or organization group, according to benchmarking comparisons at the MetaServer. We conclude that the sequence property patterns revealed by the multivariate analyses seem more conserved than amino acid types for these GT groups, and these patterns are also conserved in the structures. Such patterns may also potentially define substrate preferences.     

Cerebrospinal Fluid Steroidomics: Are Bioactive Bile Acids Present in Brain?

In this study we have profiled the free sterol content of cerebrospinal fluid by a combination of charge tagging and liquid chromatography-tandem mass spectrometry. Surprisingly, the most abundant cholesterol metabolites were found to be C₂₇ and C₂₄ intermediates of the bile acid biosynthetic pathways with structures corresponding to 7α-hydroxy-3-oxocholest-4-en-26-oic acid (7.170 ± 2.826 ng/ml, mean ± S.D., six subjects), 3β-hydroxycholest-5-en-26-oic acid (0.416 ± 0.193 ng/ml), 7α,x-dihydroxy-3-oxocholest-4-en-26-oic acid (1.330 ± 0.543 ng/ml), and 7α-hydroxy-3-oxochol-4-en-24-oic acid (0.172 ± 0.085 ng/ml), and the C₂₆ sterol 7α-hydroxy-26-norcholest-4-ene-3,x-dione (0.204 ± 0.083 ng/ml), where x is an oxygen atom either on the CD rings or more likely on the C-17 side chain. The ability of intermediates of the bile acid biosynthetic pathways to activate the liver X receptors (LXRs) and the farnesoid X receptor was also evaluated. The acidic cholesterol metabolites 3β-hydroxycholest-5-en-26-oic acid and 3β,7α-dihydroxycholest-5-en-26-oic acid were found to activate LXR in a luciferase assay, but the major metabolite identified in this study, i.e. 7α-hydroxy-3-oxocholest-4-en-26-oic acid, was not an LXR ligand. 7α-Hydroxy-3-oxocholest-4-en-26-oic acid is formed from 3β,7α-dihydroxycholest-5-en-26-oic acid in a reaction catalyzed by 3β-hydroxy-Δ⁵-C₂₇-steroid dehydrogenase (HSD3B7), which may thus represent a deactivation pathway of LXR ligands in brain. Significantly, LXR activation has been found to reduce the symptoms of Alzheimer disease (Fan, J., Donkin, J., and Wellington C. (2009) Biofactors 35, 239–248); thus, cholesterol metabolites may play an important role in the etiology of Alzheimer disease.