Lactobacillus strains reduce the toxic effects of a subchronic exposure to arsenite through drinking water.

The aim of the present study was to determine the efficacy of LAB strains in reducing the intestinal toxicity of arsenite [As(III)] and its tissue accumulation. For this purpose, Balb/c mice were randomly separated in four groups. One group received no treatment (control), one group received only As(III) (30 mg/L) via drinking water and the remaining two groups received As(III) via water and a daily dose of two LAB strains (Lactobacillus intestinalis LE1 and Lacticaseibacillus paracasei BL23) by gavage during 2 months. The results show that both strains reduce the pro-inflammatory and pro-oxidant response observed at the colonic level, partially restore the expression of the intercellular junction proteins (CLDN3 and OCLN) responsible for the maintenance of epithelial integrity, and increase the synthesis of the major mucin of the colonic mucus layer (MUC2), compared to animals treated with As(III) alone. Microbial metabolism of short-chain fatty acids also undergoes a recovery and the levels of fatty acids in the lumen reach values similar to those of untreated animals. All these positive effects imply the restoration of mucosal permeability, and a reduction of the marker of endotoxemia LPS binding protein (LBP). Treatment with the bacteria also has a direct impact on intestinal absorption, reducing the accumulation of As in the internal organs. The data suggest that the protective effect may be due to a reduced internalization of As(III) in intestinal tissues and to a possible antioxidant and anti-inflammatory activity of the bacteria through activation of pathways such as Nrf2 and IL-10. In vitro tests show that the protection may be the result of the combined action of structural and metabolic components of the LAB strains.

Intestinal homeostasis disruption in mice chronically exposed to arsenite-contaminated drinking water.

Chronic exposure to inorganic arsenic [As(III) and As(V)] affects about 200 million people, and is linked to a greater incidence of certain types of cancer. Drinking water is the main route of exposure, so, in endemic areas, the intestinal mucosa is constantly exposed to the metalloid. However, studies on the intestinal toxicity of inorganic As are scarce. The objective of this study was to evaluate the toxicity of a chronic exposure to As(III) on the intestinal mucosa and its associated microbiota. For this purpose, BALB/c mice were exposed during 6 months through drinking water to As(III) (15 and 30 mg/L). Treatment with As(III) increased reactive oxygen species (43-64%) and lipid peroxidation (8-51%). A pro-inflammatory response was also observed, evidenced by an increase in fecal lactoferrin (23-29%) and mucosal neutrophil infiltration. As(III) also induced an increase in the colonic levels of pro-inflammatory cytokines (24-201%) and the activation of some pro-inflammatory signaling pathways. Reductions in the number of goblet cells and mucus production were also observed. Moreover, As(III) exposure resulted in changes in gut microbial alpha diversity but no differences in beta diversity. This suggested that the abundance of some taxa was significantly affected by As(III), although the composition of the population did not show significant alterations. Analysis of differential taxa agreed with this, 21 ASVs were affected in abundance or variability, especially ASVs from the family Muribaculaceae. Intestinal microbiota metabolism was also affected, as reductions in fecal concentration of short-chain fatty acids were observed. The effects observed on different components of the intestinal barrier may be responsible of the increased permeability in As(III) treated mice, evidenced by an increase in fecal albumin (48-66%). Moreover, serum levels of Lipopolysaccharide binding proteins and TNF-α were increased in animals treated with 30 mg/L of As(III), suggesting a low-level systemic inflammation.

Lipoteichoic acid depletion in Lactobacillus impacts cell morphology and stress response but does not abolish mercury surface binding.

Lipoteichoic acid (LTA) is a key component of the cell wall of most Gram-positive bacteria and plays many structural and functional roles. In probiotic lactobacilli, the function of LTA in mediating bacteria/host cross-talk has been evidenced and it has been postulated that, owing to its anionic nature, LTA may play a role in toxic metal sequestration by these bacteria. However, studies on this last aspect employing strains unable to synthesise LTA are lacking. We have inactivated the LTA polymerase encoding gene ltaS in two different Lactobacillus plantarum strains. Analysis of LTA contents in wild-type and ltaS mutant strains corroborated the role of this gene as a major contributor to LTA synthesis in L. plantarum. The mutant strains displayed strain-dependent anomalous cell morphologies that resulted in elongated or irregular cells with aberrant septum formation. They also exhibited higher sensitivity to several stresses (osmotic and heat) and to antimicrobials that target the cell wall. The toxicity of inorganic [(Hg(II)] and organic mercury (methyl-Hg) was also increased upon ltaS mutation in a strain-dependent manner. However, the mutant strains showed 0 to 50% decrease in their capacity of Hg binding compared to their corresponding parental strains. This result suggests a partial contribution of LTA to Hg binding onto the cell surface that was dependent on the strain and the Hg form.

The malate sensing two-component system MaeKR is a non-canonical class of sensory complex for C4-dicarboxylates.

Microbial colonization of different environments is enabled to a great extent by the plasticity of their sensory mechanisms, among them, the two-component signal transduction systems (TCS). Here, an example of TCS plasticity is presented: the regulation of L-malate catabolism via malic enzyme by MaeRK in Lactobacillales. MaeKR belongs to the citrate family of TCS as the Escherichia coli DcuSR system. We show that the Lactobacillus casei histidine-kinase MaeK is defective in autophosphorylation activity as it lacks a functional catalytic and ATP binding domain. The cognate response regulator MaeR was poorly phosphorylated at its phosphoacceptor Asp in vitro. This phosphorylation, however, enhanced MaeR binding in vitro to its target sites and it was required for induction of regulated genes in vivo. Elucidation of the MaeR structure revealed that response regulator dimerization is accomplished by the swapping of α4-ß5-α5 elements between two monomers, generating a phosphoacceptor competent conformation. Sequence and phylogenetic analyses showed that the MaeKR peculiarities are not exclusive to L. casei as they are shared by the rest of orthologous systems of Lactobacillales. Our results reveal MaeKR as a non-canonical TCS displaying distinctive features: a swapped response regulator and a sensor histidine kinase lacking ATP-dependent kinase activity.

The use of lactic acid bacteria to reduce mercury bioaccessibility.

Mercury in food is present in either inorganic [Hg(II)] or methylmercury (CH3Hg) form. Intestinal absorption of mercury is influenced by interactions with other food components. The use of dietary components to reduce mercury bioavailability has been previously proposed. The aim of this work is to explore the use of lactic acid bacteria to reduce the amount of mercury solubilized after gastrointestinal digestion and available for absorption (bioaccessibility). Ten strains were tested by addition to aqueous solutions containing Hg(II) or CH3Hg, or to food samples, and submission of the mixtures to gastrointestinal digestion. All of the strains assayed reduce the soluble fraction from standards of mercury species under gastrointestinal digestion conditions (72-98%). However their effectiveness is lower in food, and reductions in bioaccessibility are only observed with mushrooms (⩽68%). It is hypothesized that bioaccessible mercury in seafood forms part of complexes that do not interact with lactic acid bacteria.

Characterization of a fibronectin-binding protein from Lactobacillus casei BL23.

AIMS: To characterize the functionality of the Lactobacillus casei BL23 fbpA gene encoding a putative fibronectin-binding protein. METHODS AND RESULTS: Adhesion tests showed that L. casei BL23 binds immobilized and soluble fibronectin in a protease-sensitive manner. A mutant with inactivated fbpA showed a decrease in binding to immobilized fibronectin and a strong reduction in the surface hydrophobicity as reflected by microbial adhesion to solvents test. However, minor effects were seen on adhesion to the human Caco-2 or HT-29 cell lines. Purified 6X(His)FbpA bound to immobilized fibronectin in a dose-dependent manner. Western blot experiments with FbpA-specific antibodies showed that FbpA could be extracted from the cell surface by LiCl treatment and that protease digestion of the cells reduced the amount of extracted FbpA. Furthermore, surface exposition of FbpA was detected in other L. casei strains by LiCl extraction and whole-cell ELISA. CONCLUSIONS: FbpA can be found at the L. casei BL23 surface and participates in cell attachment to immobilized fibronectin. We showed that FbpA is an important, but not the only, factor contributing to fibronectin binding in BL23 strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report showing the involvement of FbpA in fibronectin binding in L. casei BL23 and represents a new contribution to the study of attachment factors in probiotic bacteria.

Influence of the carbohydrate source on beta-glucan production and enzyme activities involved in sugar metabolism in Pediococcus parvulus 2.6.

The influence of carbohydrate source on growth, exopolysaccharide (EPS) production and on the activity of the enzymes implicated in energy generation and UDP-glucose synthesis in Pediococcus parvulus 2.6 was evaluated. The highest EPS production was obtained on glucose, while fructose was a poor substrate for EPS synthesis. HPLC and NMR analysis on monomer composition and structure of the EPS showed that this strain produced the same beta-glucan, regardless of the carbohydrate source. The alpha-phosphoglucomutase specific activities were dependent on the carbohydrate source and a high correlation between the activity of this enzyme and the amount of EPS was found in glucose- and maltose-grown cultures. alpha-UDP-glucose pyrophosphorylase activity, necessary for the activation of glucose, was very low, but significantly higher on glucose as sugar source. In vitro phosphorylation assays and transport activities showed that glucose is taken up by a proton motive force-dependent permease, while fructose is internalized by an inducible phosphotransferase system, which renders fructose-6-phosphate. The levels of 6-phosphofructokinase activity and alpha-phosphoglucomutase activities determined on fructose were higher and lower, than those found on glucose or maltose, respectively. This suggests that fructose-6-phosphate is mainly diverted to glycolysis and explains the low EPS synthesis on fructose. Results indicate that alpha-phosphoglucomutase and/or alpha-UDP-glucose pyrophosphorylase might be the bottlenecks for EPS biosynthesis, opening the field for metabolic-engineering strategies aimed to improve EPS production.

Induction of systemic and mucosal immune response and decrease in Streptococcus pneumoniae colonization by nasal inoculation of mice with recombinant lactic acid bacteria expressing pneumococcal surface antigen A

Mucosal epithelia constitute the first barriers to be overcome by pathogens during infection. The induction of protective IgA in this location is important for the prevention of infection and can be achieved through different mucosal immunization strategies. Lactic acid bacteria have been tested in the last few years as live vectors for the delivery of antigens at mucosal sites, with promising results. In this work, Streptococcus pneumoniae PsaA antigen was expressed in different species of lactic acid bacteria, such as Lactococcus lactis, Lactobacillus casei, Lactobacillus plantarum, and Lactobacillus helveticus. After nasal inoculation of C57Bl/6 mice, their ability to induce both systemic (IgG in serum) and mucosal (IgA in saliva, nasal and bronchial washes) anti-PsaA antibodies was determined. Immunization with L. lactis MG1363 induced very low levels of IgA and IgG, possibly by the low amount of PsaA expressed in this strain and its short persistence in the nasal mucosa. All three lactobacilli persisted in the nasal mucosa for 3 days and produced a similar amount of PsaA protein (150-250 ng per 109 CFU). However, L. plantarum NCDO1193 and L. helveticus ATCC15009 elicited the highest antibody response (IgA and IgG). Vaccination with recombinant lactobacilli but not with recombinant L. lactis led to a decrease in S. pneumoniae recovery from nasal mucosa upon a colonization challenge. Our results confirm that certain Lactobacillus strains have intrinsic properties that make them suitable candidates for mucosal vaccination experiments.

Expression of Streptococcus pneumoniae antigens, PsaA (pneumococcal surface antigen A) and PspA (pneumococcal surface protein A) by Lactobacillus casei

number of recent research works in lactic acid bacteria aim towards the design of new strains that could be used as live vectors for the delivery of antigens for oral vaccination, or other therapeutic molecules. In this work, an inducible expression system based on the Lactobacillus casei lactose operon promoter was used to express three important surface antigens of Streptococcus pneumoniae in this lactic acid bacterium: a virulence-related pneumococcal surface antigen (PsaA) and two variants of the virulence factor PspA (pneumococcal surface protein A). Expression of the three proteins was induced upon growth on lactose and strongly repressed by glucose. These proteins were produced intracellularly. Also, secretion to the growth medium was achieved by means of a fusion to the secreting and processing signals from the L. casei surface proteinase. Interestingly, while secreted PspA proteins were found in the culture supernatants, PsaA remained trapped in the cell wall. Expression of pneumococcal antigens in a food-grade organism opens an alternative for mucosal vaccination against this important pathogen. © 2003 Federation of European Microbiological Societies.

X-ray structure of HPr kinase: a bacterial protein kinase with a P-loop nucleotide-binding domain.

HPr kinase/phosphatase (HprK/P) is a key regulatory enzyme controlling carbon metabolism in Gram- positive bacteria. It catalyses the ATP-dependent phosphorylation of Ser46 in HPr, a protein of the phosphotransferase system, and also its dephosphorylation. HprK/P is unrelated to eukaryotic protein kinases, but contains the Walker motif A characteristic of nucleotide-binding proteins. We report here the X-ray structure of an active fragment of Lactobacillus casei HprK/P at 2.8 A resolution, solved by the multiwavelength anomalous dispersion method on a seleniated protein (PDB code 1jb1). The protein is a hexamer, with each subunit containing an ATP-binding domain similar to nucleoside/nucleotide kinases, and a putative HPr-binding domain unrelated to the substrate-binding domains of other kinases. The Walker motif A forms a typical P-loop which binds inorganic phosphate in the crystal. We modelled ATP binding by comparison with adenylate kinase, and designed a tentative model of the complex with HPr based on a docking simulation. The results confirm that HprK/P represents a new family of protein kinases, first identified in bacteria, but which may also have members in eukaryotes.

Mutations lowering the phosphatase activity of HPr kinase/phosphatase switch off carbon metabolism.

The oligomeric bifunctional HPr kinase/P-Ser-HPr phosphatase (HprK/P) regulates many metabolic functions in Gram-positive bacteria by phosphorylating the phosphocarrier protein HPr at Ser46. We isolated Lactobacillus casei hprK alleles encoding mutant HprK/Ps exhibiting strongly reduced phosphatase, but almost normal kinase activity. Two mutations affected the Walker motif A of HprK/P and four a conserved C-terminal region in contact with the ATP-binding site of an adjacent subunit in the hexamer. Kinase and phosphatase activity appeared to be closely associated and linked to the Walker motif A, but dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr) is not simply a reversal of the kinase reaction. When the hprKV267F allele was expressed in Bacillus subtilis, the strongly reduced phosphatase activity of the mutant enzyme led to increased amounts of P-Ser-HPr. The hprKV267F mutant was unable to grow on carbohydrates transported by the phosphoenolpyruvate:glycose phosphotransferase system (PTS) and on most non-PTS carbohydrates. Disrupting ccpA relieved the growth defect only on non-PTS sugars, whereas replacing Ser46 in HPr with alanine also restored growth on PTS substrates.

Catabolite regulation of the cytochrome c550-encoding Bacillus subtilis cccA gene.

In Gram-positive bacteria, catabolite control protein A (CcpA)-mediated catabolite repression or activation regulates not only the expression of a great number of catabolic operons, but also the synthesis of enzymes of central metabolic pathways. We found that a constituent of the Bacillus subtilis respiratory chain, the small cytochrome c550 encoded by the cccA gene, was also submitted to catabolite repression. Similar to most catabolite-repressed genes and operons, the Bacillus subtilis cccA gene contains a potential catabolite response element cre, an operator site recognized by CcpA. The presumed cre overlaps the -35 region of the cccA promoter. Strains carrying a cccA'-IacZ fusion formed blue colonies when grown on rich solid medium, whereas white colonies were obtained when glucose was present. beta-Galactosidase assays with cells grown in rich medium confirmed the repressive effect of glucose on cccA'-lacZ expression. Introduction of a ccpA or hprK mutation or of a mutation affecting the presumed cccA cre relieved the repressive effect of glucose during late log phase. An additional glucose repression mechanism was activated during stationary phase, which was not relieved by the ccpA, hprK or cre mutations. An interaction of the repressor/corepressor complex (CcpA/seryl-phosphorylated HPr (P-Ser-HPr)) with the cccA cre could be demonstrated by gel shift experiments. By contrast, a DNA fragment carrying mutations in the presumed cccA cre was barely shifted by the CcpA/P-Ser-HPr complex. In footprinting experiments, the region corresponding to the presumed cccA cre was specifically protected in the presence of the CcpA/P-Ser-HPr complex.

Regulatory functions of serine-46-phosphorylated HPr in Lactococcus lactis.

In most low-G+C gram-positive bacteria, the phosphoryl carrier protein HPr of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) becomes phosphorylated at Ser-46. This ATP-dependent reaction is catalyzed by the bifunctional HPr kinase/P-Ser-HPr phosphatase. We found that serine-phosphorylated HPr (P-Ser-HPr) of Lactococcus lactis participates not only in carbon catabolite repression of an operon encoding a beta-glucoside-specific EII and a 6-P-beta-glucosidase but also in inducer exclusion of the non-PTS carbohydrates maltose and ribose. In a wild-type strain, transport of these non-PTS carbohydrates is strongly inhibited by the presence of glucose, whereas in a ptsH1 mutant, in which Ser-46 of HPr is replaced with an alanine, glucose had lost its inhibitory effect. In vitro experiments carried out with L. lactis vesicles had suggested that P-Ser-HPr is also implicated in inducer expulsion of nonmetabolizable homologues of PTS sugars, such as methyl beta-D-thiogalactoside (TMG) and 2-deoxy-D-glucose (2-DG). In vivo experiments with the ptsH1 mutant established that P-Ser-HPr is not necessary for inducer expulsion. Glucose-activated 2-DG expulsion occurred at similar rates in wild-type and ptsH1 mutant strains, whereas TMG expulsion was slowed in the ptsH1 mutant. It therefore seems that P-Ser-HPr is not essential for inducer expulsion but that in certain cases it can play an indirect role in this regulatory process.

The ptsH gene from Bacillus thuringiensis israelensis. Characterization of a new phosphorylation site on the protein HPr.

The ptsH gene from Bacillus thuringiensis israelensis (Bti), coding for the phosphocarrier protein HPr of the phosphotransferase system has been cloned and overexpressed in Escherichia coli. Comparison of its primary sequence with other HPr sequences revealed that the conserved His15 and Ser46 residues were shifted by one amino acid and located at positions 14 and 45, respectively. The biological activity of the protein was not affected by this change. When expressed in a Bacillus subtilis ptsH deletion strain, Bti HPr was able to complement the functions of HPr in sugar uptake and glucose catabolite repression of the gnt and iol operons. A modified form of HPr was detected in Bti cells, and also when Bti ptsH was expressed in E. coli or B. subtilis. This modification was identified as phosphorylation, because alkaline phosphatase treatment converted the modified form to unmodified HPr. The phosphoryl bond in the new form of in vivo phosphorylated HPr was resistant to alkali treatment but sensitive to acid treatment, suggesting phosphorylation at a histidine residue. Replacement of His14 with alanine in Bti HPr prevented formation of the new form of phosphorylated HPr. The phosphorylated HPr was stable at 60 degrees C, in contrast with HPr phosphorylated at the N delta 1 position of His14 with phosphoenolpyruvate and enzyme I. (31)P-NMR spectroscopy was used to show that the new form of P-HPr carried the phosphoryl group bound to the N epsilon 2 position of His14 of Bti HPr. Phosphorylation of HPr at the novel site did not occur when Bti HPr was expressed in an enzyme I-deficient B. subtilis strain. In addition, P-(N epsilon 2)His-HPr did not transfer its phosphoryl group to the purified glucose-specific enzyme IIA domain of B. subtilis.

Enzyme I and HPr from Lactobacillus casei: their role in sugar transport, carbon catabolite repression and inducer exclusion.

We have cloned and sequenced the Lactobacillus casei ptsH and ptsI genes, which encode enzyme I and HPr, respectively, the general components of the phosphoenolpyruvate-carbohydrate phosphotransferase system (PTS). Northern blot analysis revealed that these two genes are organized in a single-transcriptional unit whose expression is partially induced. The PTS plays an important role in sugar transport in L. casei, as was confirmed by constructing enzyme I-deficient L. casei mutants, which were unable to ferment a large number of carbohydrates (fructose, mannose, mannitol, sorbose, sorbitol, amygdaline, arbutine, salicine, cellobiose, lactose, tagatose, trehalose and turanose). Phosphorylation of HPr at Ser-46 is assumed to be important for the regulation of sugar metabolism in Gram-positive bacteria. L. casei ptsH mutants were constructed in which phosphorylation of HPr at Ser-46 was either prevented or diminished (replacement of Ser-46 of HPr with Ala or Thr respectively). In a third mutant, Ile-47 of HPr was replaced with a threonine, which was assumed to reduce the affinity of P-Ser-HPr for its target protein CcpA. The ptsH mutants exhibited a less pronounced lag phase during diauxic growth in a mixture of glucose and lactose, two PTS sugars, and diauxie was abolished when cells were cultured in a mixture of glucose and the non-PTS sugars ribose or maltose. The ptsH mutants synthesizing Ser-46-Ala or Ile-47-Thr mutant HPr were partly or completely relieved from carbon catabolite repression (CCR), suggesting that the P-Ser-HPr/CcpA-mediated mechanism of CCR is common to most low G+C Gram-positive bacteria. In addition, in the three constructed ptsH mutants, glucose had lost its inhibitory effect on maltose transport, providing for the first time in vivo evidence that P-Ser-HPr participates also in inducer exclusion.

Phosphorylation of HPr by the bifunctional HPr Kinase/P-ser-HPr phosphatase from Lactobacillus casei controls catabolite repression and inducer exclusion but not inducer expulsion.

We have cloned and sequenced the Lactobacillus casei hprK gene encoding the bifunctional enzyme HPr kinase/P-Ser-HPr phosphatase (HprK/P). Purified recombinant L. casei HprK/P catalyzes the ATP-dependent phosphorylation of HPr, a phosphocarrier protein of the phosphoenolpyruvate:carbohydrate phosphotransferase system at the regulatory Ser-46 as well as the dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr). The two opposing activities of HprK/P were regulated by fructose-1,6-bisphosphate, which stimulated HPr phosphorylation, and by inorganic phosphate, which stimulated the P-Ser-HPr phosphatase activity. A mutant producing truncated HprK/P was found to be devoid of both HPr kinase and P-Ser-HPr phosphatase activities. When hprK was inactivated, carbon catabolite repression of N-acetylglucosaminidase disappeared, and the lag phase observed during diauxic growth of the wild-type strain on media containing glucose plus either lactose or maltose was strongly diminished. In addition, inducer exclusion exerted by the presence of glucose on maltose transport in the wild-type strain was abolished in the hprK mutant. However, inducer expulsion of methyl beta-D-thiogalactoside triggered by rapidly metabolizable carbon sources was still operative in ptsH mutants altered at Ser-46 of HPr and the hprK mutant, suggesting that, in contrast to the model proposed for inducer expulsion in gram-positive bacteria, P-Ser-HPr might not be involved in this regulatory process.

Elements involved in catabolite repression and substrate induction of the lactose operon in Lactobacillus casei.

In Lactobacillus casei ATCC 393, the chromosomally encoded lactose operon, lacTEGF, encodes an antiterminator protein (LacT), lactose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) elements (LacE and LacF), and a phospho-beta-galactosidase. lacT, lacE, and lacF mutant strains were constructed by double crossover. The lacT strain displayed constitutive termination at a ribonucleic antiterminator (RAT) site, whereas lacE and lacF mutants showed an inducer-independent antiterminator activity, as shown analysis of enzyme activity obtained from transcriptional fusions of lac promoter (lacp) and lacpDeltaRAT with the Escherichia coli gusA gene in the different lac mutants. These results strongly suggest that in vivo under noninducing conditions, the lactose-specific PTS elements negatively modulate LacT activity. Northern blot analysis detected a 100-nucleotide transcript starting at the transcription start site and ending a consensus RAT sequence and terminator region. In a ccpA mutant, transcription initiation was derepressed but no elongation through the terminator was observed in the presence of glucose and the inducing sugar, lactose. Full expression of lacTEGF was found only in a man ccpA double mutant, indicating that PTS elements are involved in the CcpA-independent catabolite repression mechanism probably via LacT.