Yong Q. Chen

Protective effects of Lactobacillus plantarum CCFM8610 against chronic cadmium toxicity in mice indicate routes of protection besides intestinal sequestration.

ABSTRACT Our previous study confirmed the ability of Lactobacillus plantarum CCFM8610 to protect against acute cadmium (Cd) toxicity in mice. This study was designed to evaluate the protective effects of CCFM8610 against chronic Cd toxicity in mice and to gain insights into the protection mode of this strain. Experimental mice were divided into two groups and exposed to Cd for 8 weeks via drinking water or intraperitoneal injection. Both groups were further divided into four subgroups, control, Cd only, CCFM8610 only, and Cd plus CCFM8610. Levels of Cd were measured in the feces, liver, and kidneys, and alterations of several biomarkers of Cd toxicity were noted. The results showed that when Cd was introduced orally, cotreatment with Cd and CCFM8610 effectively decreased intestinal Cd absorption, reduced Cd accumulation in tissue, alleviated tissue oxidative stress, reversed hepatic and renal damage, and ameliorated the corresponding histopathological changes. When Cd was introduced intraperitoneally, administration of CCFM8610 did not have an impact on tissue Cd accumulation or reverse the activities of antioxidant enzymes. However, CCFM8610 still offered protection against oxidative stress and reversed the alterations of Cd toxicity biomarkers and tissue histopathology. These results suggest that CCFM8610 is effective against chronic cadmium toxicity in mice. Besides intestinal Cd sequestration, CCFM8610 treatment offers direct protection against Cd-induced oxidative stress. We also provide evidence that the latter is unlikely to be mediated via protection against Cd-induced alteration of antioxidant enzyme activities.

Comparison of Biochemical Activities between High and Low Lipid-Producing Strains of Mucor circinelloides: An Explanation for the High Oleaginicity of Strain WJ11

The oleaginous fungus, Mucor circinelloides, is one of few fungi that produce high amounts of γ-linolenic acid (GLA); however, it usually only produces <25% lipid. Nevertheless, a new strain (WJ11) isolated in this laboratory can produce lipid up to 36% (w/w) cell dry weight (CDW). We have investigated the potential mechanism of high lipid accumulation in M. circinelloides WJ11 by comparative biochemical analysis with a low lipid-producing strain, M. circinelloides CBS 277.49, which accumulates less than 15% (w/w) lipid. M. circinelloides WJ11 produced more cell mass than that of strain CBS 277.49, although with slower glucose consumption. In the lipid accumulation phase, activities of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in strain WJ11 were greater than in CBS 277.49 by 46% and 17%, respectively, and therefore may provide more NADPH for fatty acid biosynthesis. The activities of NAD+:isocitrate dehydrogenase and NADP+:isocitrate dehydrogenase, however, were 43% and 54%, respectively, lower in WJ11 than in CBS 277.49 and may retard the tricarboxylic acid cycle and thereby provide more substrate for ATP:citrate lyase (ACL) to produce acetyl-CoA. Also, the activities of ACL and fatty acid synthase in the high lipid-producing strain, WJ11, were 25% and 56%, respectively, greater than in strain CBS 277.49. These enzymes may therefore cooperatively regulate the fatty acid biosynthesis in these two strains.

Proteomics analysis of high lipid-producing strain Mucor circinelloides WJ11: an explanation for the mechanism of lipid accumulation at the proteomic level

BackgroundThe oleaginous fungus, Mucor circinelloides, is attracting considerable interest as it produces oil rich in γ-linolenic acid. Nitrogen (N) deficiency is a common strategy to trigger the lipid accumulation in oleaginous microorganisms. Although a simple pathway from N depletion in the medium to lipid accumulation has been elucidated at the enzymatic level, global changes at protein levels upon N depletion have not been investigated. In this study, we have systematically analyzed the changes at the levels of protein expression in M. circinelloides WJ11, a high lipid-producing strain (36xa0%, lipid/cell dry weight), during lipid accumulation.ResultsProteomic analysis demonstrated that N depletion increased the expression of glutamine synthetase, involved in ammonia assimilation, for the supply of cellular nitrogen but decreased the metabolism of amino acids. Upon N deficiency, many proteins (e.g., fructose-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, enolase, pyruvate kinase) involved in glycolytic pathway were up-regulated while proteins involved in the tricarboxylic acid cycle (e.g., isocitrate dehydrogenase, succinyl-CoA ligase, succinate dehydrogenase, fumarate hydratase) were down-regulated, indicating this activity was retarded thereby leading to a greater flux of carbon into fatty acid biosynthesis. Moreover, glucose-6-phosphate dehydrogenase, transaldolase and transketolase, which participate in the pentose phosphate pathway, were up-regulated, leading to the increased production of NADPH, the reducing power for fatty acid biosynthesis. Furthermore, protein and nucleic acid metabolism were down-regulated and some proteins involved in energy metabolism, signal transduction, molecular chaperone and redox homeostasis were up-regulated upon N depletion, which may be the cellular response to the stress produced by the onset of N deficiency.ConclusionN limitation increased those expressions of the proteins involved in ammonia assimilation but decreased that involved in the biosynthesis of amino acids. Upon N deprivation, the glycolytic pathway was up-regulated, while the activity of the tricarboxylic acid cycle was retarded, thus, leading more carbon flux to fatty acid biosynthesis. Moreover, the pentose phosphate pathway was up-regulated, then this would increase the production of NADPH. Together, coordinated regulation of central carbon metabolism upon N limitation, provides more carbon flux to acetyl-CoA and NADPH for fatty acid biosynthesis.

Genetically Engineered Lactococcus lactis Protect against House Dust Mite Allergy in a BALB/c Mouse Model

Background Mucosal vaccine based on lactic acid bacteria is an attractive concept for the prevention and treatment of allergic diseases, but their mechanisms of action in vivo are poorly understood. Therefore, we sought to investigate how recombinant major dust mite allergen Der p2-expressing Lactococcus lactis as a mucosal vaccine induced the immune tolerance against house dust mite allergy in a mouse model. Methods Three strains of recombinant L. lactis producing Der p2 in different cell components (extracellular, intracellular and cell wall) were firstly constructed. Their prophylactic potential was evaluated in a Der p2-sensitised mouse model, and immunomodulation properties at the cellular level were determined by measuring cytokine production in vitro. Results Der p2 expressed in the different recombinant L. lactis strains was recognized by a polyclonal anti-Der p2 antibody. Oral treatment with the recombinant L. lactis prior sensitization significantly prevented the development of airway inflammation in the Der p2-sensitized mice, as determined by the attenuation of inflammatory cells infiltration in the lung tissues and decrease of Th2 cytokines IL-4 and IL-5 levels in bronchoalveolar lavage. In addition, the serum allergen-specific IgE levels were significantly reduced, and the levels of IL-4 in the spleen and mesenteric lymph nodes cell cultures were also markedly decreased upon allergen stimulation in the mice fed with the recombinant L. lactis strains. These protective effects correlated with a significant up-regulation of regulatory T cells in the mesenteric lymph nodes. Conclusion Oral pretreatment with live recombinant L. lactis prevented the development of allergen-induced airway inflammation primarily by the induction of specific mucosal immune tolerance.

(13)C-metabolic flux analysis of lipid accumulation in the oleaginous fungus Mucor circinelloides.

The oleaginous fungus Mucor circinelloides is of industrial interest because it can produce high levels of polyunsaturated fatty acid γ-linolenic acid. M. circinelloides CBS 277.49 is able to accumulate less than 15% of cell dry weight as lipids, while M. circinelloides WJ11 can accumulate lipid up to 36%. In order to better understand the mechanisms behind the differential lipid accumulation in these two strains, tracer experiments with (13)C-glucose were performed with the growth of M. circinelloides and subsequent gas chromatography-mass spectrometric detection of (13)C-patterns in proteinogenic amino acids was carried out to identify the metabolic network topology and estimate intracellular fluxes. Our results showed that the high oleaginous strain WJ11 had higher flux of pentose phosphate pathway and malic enzyme, lower flux in tricarboxylic acid cycle, higher flux in glyoxylate cycle and ATP: citrate lyase, together, it might provide more NADPH and substrate acetyl-CoA for fatty acid synthesis.

Complete Genome Sequence of a High Lipid-Producing Strain of Mucor circinelloides WJ11 and Comparative Genome Analysis with a Low Lipid-Producing Strain CBS 277.49

The genome of a high lipid-producing fungus Mucor circinelloides WJ11 (36% w/w lipid, cell dry weight, CDW) was sequenced and compared with that of the low lipid-producing strain, CBS 277.49 (15% w/w lipid, CDW), which had been sequenced by Joint Genome Institute. The WJ11 genome assembly size was 35.4 Mb with a G+C content of 39.7%. The general features of WJ11 and CBS 277.49 indicated that they have close similarity at the level of gene order and gene identity. Whole genome alignments with MAUVE revealed the presence of numerous blocks of homologous regions and MUMmer analysis showed that the genomes of these two strains were mostly co-linear. The central carbon and lipid metabolism pathways of these two strains were reconstructed and the numbers of genes encoding the enzymes related to lipid accumulation were compared. Many unique genes coding for proteins involved in cell growth, carbohydrate metabolism and lipid metabolism were identified for each strain. In conclusion, our study on the genome sequence of WJ11 and the comparative genomic analysis between WJ11 and CBS 277.49 elucidated the general features of the genome and the potential mechanism of high lipid accumulation in strain WJ11 at the genomic level. The different numbers of genes and unique genes involved in lipid accumulation may play a role in the high oleaginicity of strain WJ11.

Role of pentose phosphate pathway in lipid accumulation of oleaginous fungus Mucor circinelloides

Lipid biosynthesis in oleaginous fungi requires acetyl-CoA, as the essential precursor of fatty acids, and a supply of reducing power, NADPH. Malic enzyme provides the majority of NADPH needed for lipid biosynthesis in most oleaginous fungi, while the reactions of the pentose phosphate pathway (PPP) provide additional NADPH. We have therefore overexpressed the genes coding for glucose 6-phosphate dehydrogenase (g6pd) and 6-phosphogluconate dehydrogenase (6pgd) from the PPP in the oleaginous fungus Mucor circinelloides to analyze its effect on lipid accumulation. The results showed that overexpressing g6pd or 6pgd increased the lipid content of cell dry weight by 20–30% compared to the control strain, and with higher G6PD and 6PGD activities and higher mRNA levels in the overexpressing strains. The results suggest that G6PD and 6PGD are important NADPH providers and play a key role on lipid accumulation in oleaginous fungus M. circinelloides.

Role of malate transporter in lipid accumulation of oleaginous fungus Mucor circinelloides.

Fatty acid biosynthesis in oleaginous fungi requires the supply of reducing power, NADPH, and the precursor of fatty acids, acetyl-CoA, which is generated in the cytosol being produced by ATP: citrate lyase which requires citrate to be, transported from the mitochondrion by the citrate/malate/pyruvate transporter. This transporter, which is within the mitochondrial membrane, transports cytosolic malate into the mitochondrion in exchange for mitochondrial citrate moving into the cytosol (Fig.xa01). The role of malate transporter in lipid accumulation in oleaginous fungi is not fully understood, however. Therefore, the expression level of the mt gene, coding for a malate transporter, was manipulated in the oleaginous fungus Mucor circinelloides to analyze its effect on lipid accumulation. The results showed that mt overexpression increased the lipid content for about 70xa0% (from 13 to 22xa0% dry cell weight, CDW), whereas the lipid content in mt knockout mutant decreased about 27xa0% (from 13 to 9.5xa0% CDW) compared with the control strain. Furthermore, the extracellular malate concentration was decreased in the mt overexpressing strain and increased in the mt knockout strain compared with the wild-type strain. This work suggests that the malate transporter plays an important role in regulating lipid accumulation in oleaginous fungus M. circinelloides.

Bioinformatical analysis and preliminary study of the role of lipase in lipid metabolism in Mucor circinelloides

The filamentous fungus Mucor circinelloides has been widely used as a model organism to investigate the mechanisms of lipid accumulation. Although a lot of work has been done to analyze and explore many of the essential enzymes/genes related to lipid accumulation in M. circinelloides, the function of lipase in this fungus has not been studied at all. In this study, we report some important characteristics of all 30 lipases, and especially 4 lipases Lip6, Lip10, Lip19 and Lip24 with functional analyses in Mucor circinelloides CBS 277.49 based on the genome sequences. Transcriptional analyses revealed that the Lip6 or Lip10 gene expression increased significantly during 24 h to 72 h of lipid accumulation while Lip19 and Lip24 genes were down-regulated along with the increase in lipid accumulation. Over-expression of either lipase Lip6 or Lip10 led to a slight increase in cell dry weight, but no significant effect on lipid accumulation. More interestingly over-expression of Lip6 resulted in a 17% increase in C18:3 content of the lipids. However, over-expression of lipase Lip19 and Lip24 decreased the cell dry weight by 9.08–18.8% and lipid content by 37.86–41.42%, respectively. The fatty acid profiles of strains with Lip19 and Lip24 over-expression were also significantly changed as compared to control. Analysis of lipase genes revealed that the sequences of Lip6 and Lip10 not only contained the typical lipase motif (G/A) XSXG, but also the consensus sequence motif HXXXXD (a typical of acyltransferase motif). These results suggest that Lip6 or Lip10 may control acyltransferase activity in Mucor circienlloides and play a role in fatty acid reconstruction of TAG, while Lip19 or Lip24 may work as a TAG lipase involved in TAG metabolism/degradation.

Protective effects of lactic acid bacteria-fermented soymilk against chronic cadmium toxicity in mice

Our previous study confirmed that Lactobacillus plantarum CCFM8610 has protective effects against chronic cadmium (Cd) toxicity in mice, whereas L. bulgaricus CCFM8004 fails to provide similar protection. This study was designed to evaluate the protective effects of soymilk fermented with these lactic acid bacteria, against chronic Cd toxicity in mice, and to give an insight into the mechanism of the conjunct effect of soymilk and these strains. Experimental mice were divided into five groups as control, Cd only, non-fermented soymilk plus Cd, CCFM8610-fermented soymilk plus Cd, and CCFM8004-fermented soymilk plus Cd. The treatment of all groups was carried out for 8 weeks. Levels of Cd were measured in feces and tissues, and alterations in several biomarkers of Cd toxicity were noted. The results showed that non-fermented soymilk gave limited protection against chronic Cd toxicity in mice. However, oral administration of L. plantarum CCFM8610-fermented soymilk was able to increase fecal Cd excretion, reduce tissue Cd burden, alleviate tissue oxidative stress, reverse changes in hepatic and renal damage biomarkers, and ameliorate tissue histopathological changes in mice, indicating that L. plantarum CCFM8610-fermented soymilk could be considered as a dietary therapeutic strategy against chronic Cd toxicity. The treatment of L. bulgaricus CCFM8004-fermented soymilk provided similar protection, although the effects were less significant than for CCFM8610 treatment. The conjunct effects of the strains and the soymilk may be attributed to the increased Cd excretion ability and antioxidative capacity after fermentation.