Miguel A. Ferrero

Biosynthesis and production of polysialic acids in bacteria

Polysialic acids (PA) are protective capsular sialohomopolymers present in some bacteria which can invade the mammalian host and cause lethal bacteremia and meningitis. Biosynthesis and translocation of PA to the cell surface are equivalent in different species and bacterial strains which are produced. The diversity in PA structure is derived from the PA linkages and is a consequence of the specific sialyltransferase activities. The monomer acetylation and the polymer length could be important factors in the potential virulence. In vivo PA production is affected by different physical and chemical factors. The temperature of cellular growth strictly regulates PA genesis through a molecular complex and multifactorial mechanism that operate to transcription level.

Non-toxic plant metabolites regulate Staphylococcus viability and biofilm formation: a natural therapeutic strategy useful in the treatment and prevention of skin infections

In the present study, the efficacy of generally recognised as safe (GRAS) antimicrobial plant metabolites in regulating the growth of Staphylococcus aureus and S. epidermidis was investigated. Thymol, carvacrol and eugenol showed the strongest antibacterial action against these microorganisms, at a subinhibitory concentration (SIC) of ≤ 50 μg ml−1. Genistein, hydroquinone and resveratrol showed antimicrobial effects but with a wide concentration range (SIC = 50–1,000 μg ml−1), while catechin, gallic acid, protocatechuic acid, p-hydroxybenzoic acid and cranberry extract were the most biologically compatible molecules (SIC ≥ 1000 μg ml−1). Genistein, protocatechuic acid, cranberry extract, p-hydroxybenzoic acid and resveratrol also showed anti-biofilm activity against S. aureus, but not against S. epidermidis in which, surprisingly, these metabolites stimulated biofilm formation (between 35% and 1,200%). Binary combinations of cranberry extract and resveratrol with genistein, protocatechuic or p-hydroxibenzoic acid enhanced the stimulatory effect on S. epidermidis biofilm formation and maintained or even increased S. aureus anti-biofilm activity.

Regulation of capsular polysialic acid biosynthesis by temperature in Pasteurella haemolytica A2

The capsular polysaccharide of Pasteurella haemolytica A2 consists of a linear polymer of N‐acetylneuraminic acid (Neu5Ac) with α(2–8) linkages. The production of this polymer is strictly regulated by the growth temperature and above 40°C no production is detected. Analysis of the enzymatic activities directly involved in its biosynthesis reveals that Neu5Ac lyase, CMP‐Neu5Ac synthetase and polysialyltransferase are involved in this regulation. Very low activities were found in P. haemolytica grown at 43°C (at least 25 times lower than those observed when the growth temperature was 37°C). The synthesis of these enzymes increased rapidly when bacteria grown at 43°C were transferred to 37°C and decreased dramatically when cells grown at 37°C were transferred to 43°C. These findings indicate that the cellular growth temperature regulates the synthesis of these enzymes and hence the concentration of the intermediates necessary for capsular polysaccharide genesis in P. haemolytica A2.

Biochemical conditions for the production of polysialic acid by Pasteurella haemolytica A2.

The capsular polysaccharide of Pasteurella haemolytica A2 consists of a linear polymer of N-acetylneuraminic acid (Neu5Ac) with α(2–8) linkages. When the bacterium was grown at 37°C for 90 h in 250 ml shake flasks at 200 rpm in Brain heart infusion broth (BHIB), it accumulated, attaining a level of 60 μg/ml. Release of this polymer was strictly regulated by the growth temperature, and above 40° no production was detected. The pathway for the biosynthesis of this sialic acid capsular polymer was also examined in P. haemolytica A2 and was seen to involve the sequential presence of three enzymatic activities: Neu5Ac lyase activity, which synthesizes Neu5Ac by condensation of N-acetyl-D-mannosamine and pyruvate with apparent Km values of 91 mM and 73 mM, respectively; a CMP-Neu5Ac synthetase, which catalyzes the production of CMP-Neu5Ac from Neu5Ac and CTP with apparent Km values of 2 mM and 0.5 mM, respectively, and finally a membrane-associated polysialyltransferase, which catalyzes the incorporation of sialic acid from CMP-Neu5Ac into polymeric products with an apparent CMP-Neu5Ac Km of 250 μM.

Regulation of capsular polysialic acid biosynthesis by N-acetyl-d-mannosamine, an intermediate of sialic acid metabolism

N‐Acetyl‐d‐mannosamine (ManNAc) is a specific substrate for the synthesis of N‐acetylneuraminic acid, the essential precursor of bacterial capsular polysialic acid (PA). When Escherichia coli K92 used ManNAc as a carbon source, we observed a dramatic reduction (up to 90%) in in vivo PA production. Experiments in which the carbon source was changed revealed that the maximal inhibitory effect occurred when this sugar was present in the medium before the logarithmic phase of bacterial growth had started. Enzymatic analysis revealed that high concentrations of ManNAc‐6‐phosphate inhibit NeuAc lyase, the enzyme that synthesizes NeuAc for PA biosynthesis in E. coli. These results indicate that ManNAc‐6‐phosphate is able to regulate NeuAc lyase activity and modulate the PA synthesis.

Differences in gluten metabolism among healthy volunteers, coeliac disease patients and first-degree relatives.

Coeliac disease (CD) is an immune-mediated enteropathy resulting from exposure to gluten in genetically predisposed individuals. Gluten proteins are partially digested by human proteases generating immunogenic peptides that cause inflammation in patients carrying HLA-DQ2 and DQ8 genes. Although intestinal dysbiosis has been associated with patients with CD, bacterial metabolism of gluten has not been studied in depth thus far. The aim of this study was to analyse the metabolic activity of intestinal bacteria associated with gluten intake in healthy individuals, CD patients and first-degree relatives of CD patients. Faecal samples belonging to twenty-two untreated CD patients, twenty treated CD patients, sixteen healthy volunteers on normal diet, eleven healthy volunteers on gluten-free diet (GFD), seventy-one relatives of CD patients on normal diet and sixty-nine relatives on GFD were tested for several proteolytic activities, cultivable bacteria involved in gluten metabolism, SCFA and the amount of gluten in faeces. We detected faecal peptidasic activity against the gluten-derived peptide 33-mer. CD patients showed differences in faecal glutenasic activity (FGA), faecal tryptic activity (FTA), SCFA and faecal gluten content with respect to healthy volunteers. Alterations in specific bacterial groups metabolising gluten such as Clostridium or Lactobacillus were reported in CD patients. Relatives showed similar parameters to CD patients (SCFA) and healthy volunteers (FTA and FGA). Our data support the fact that commensal microbial activity is an important factor in the metabolism of gluten proteins and that this activity is altered in CD patients.

Transport of N-acetyl-D-mannosamine and N-acetyl-D-glucosamine in Escherichia coli K1: effect on capsular polysialic acid production

N‐Acetyl‐D‐mannosamine (ManNAc) and N‐acetyl‐D‐glucosamine (GlcNAc) are the essential precursors of N‐acetylneuraminic acid (NeuAc), the specific monomer of polysialic acid (PA), a bacterial pathogenic determinant. Escherichia coli K1 uses both amino sugars as carbon sources and uptake takes place through the mannose phosphotransferase system transporter, a phosphoenolpyruvate‐dependent phosphotransferase system that shows a broad range of specificity. Glucose, mannose, fructose, and glucosamine strongly inhibited the transport of these amino‐acetylated sugars and GlcNAc and ManNAc strongly affected ManNAc and GlcNAc uptake, respectively. The ManNAc and the GlcNAc phosphorylation that occurs during uptake affected NeuAc synthesis in vitro. These findings account for the low in vivo PA production observed when E. coli K1 uses ManNAc or GlcNAc as a carbon source for growth.

N-Acetylneuraminic acid uptake in Pasteurella (Mannheimia) haemolytica A2 occurs by an inducible and specific transport system

The N‐acetylneuraminic acid (NeuAc) transport system of Pasteurella (Mannheimia) haemolytica A2 was studied when this bacterium was grown in both complex and chemically defined media. Kinetic measurements were carried out at 37°C in 50 mM Tris–HCl buffer, pH 8.0, containing 50 μg/ml bovine serum albumin. Under these conditions, the uptake rate was linear for at least 3 min and the calculated K m for NeuAc was 0.1 μM. The transport rate was increased by the addition of several cations and was inhibited by sodium arsenite (95%), N,N′‐dicyclohexyl‐carbodiimide (50%), and 2,4‐dinitrophenol (40%) at final concentration of 1 mM (each). These results support the notion that NeuAc uptake is an active sugar cation symporter. Study of specificities showed that glucosamine, mannose and mannosamine inhibited the transport of NeuAc in this bacterium. Analysis of expression revealed that the NeuAc transport system was induced by NeuAc and by the simultaneous presence of glucose and galactose in the growth medium.

Polysialic and colanic acids metabolism in Escherichia coli K92 is regulated by RcsA and RcsB

We have shown previously that Escherichia coli K92 produces two different capsular polymers known as CA (colanic acid) and PA (polysialic acid) in a thermoregulated manner. The complex Rcs phosphorelay is largely related to the regulation of CA synthesis. Through deletion of rscA and rscB genes, we show that the Rcs system is involved in the regulation of both CA and PA synthesis in E. coli K92. Deletion of either rcsA or rcsB genes resulted in decreased expression of cps (CA biosynthesis cluster) at 19°C and 37°C, but only CA production was reduced at 19°C. Concerning PA, both deletions enhanced its synthesis at 37°C, which does not correlate with the reduced kps (PA biosynthesis cluster) expression observed in the rcsB mutant. Under this condition, expression of the nan operon responsible for PA catabolism was greatly reduced. Although RcsA and RcsB acted as negative regulators of PA synthesis at 37°C, their absence did not reestablish PA expression at low temperatures, despite the deletion of rcsB resulting in enhanced kps expression. Finally, our results revealed that RcsB controlled the expression of several genes (dsrA, rfaH, h-ns and slyA) involved in the thermoregulation of CA and PA synthesis, indicating that RcsB is part of a complex regulatory mechanism governing the surface appearance in E. coli.

Growth temperature regulation of some genes that define the superficial capsular carbohydrate composition of Escherichia coli K92

We studied growth temperature as a factor controlling the expression of genes involved in capsular polymers of Escherichia coli K92. These genes are shown to be regulated by growth temperature. Expression levels of genes belonging to the kps cluster, responsible for polysialic acid (PA) biosynthesis, were significantly increased at 37 °C compared with at 19 °C, being up to 500-fold increased for neuE and neuS genes. Similarly, the genes for the nan operon, responsible for PA catabolism, also reached higher expression levels at 37 °C, although with slightly lower values (39-141-fold). In contrast, genes of the cps operon, which are implicated in colanic acid (CA) metabolism, were upregulated when the bacteria were grown at 19 °C, albeit to a much lesser extent (around twofold). This different regulation of genes involved in the biosynthesis of polysialic and CAs correlates with the reported maximal production temperatures for the two polymers. The results suggest that the metabolism of PA is predominantly regulated by changes in gene expression, while CA production may be regulated mainly by post-transcriptional processes such as phosphorylation-dephosphorylation reactions.