Maria L. Marco

Identification of Lactobacillus plantarum genes modulating the cytokine response of human peripheral blood mononuclear cells

BackgroundModulation of the immune system is one of the most plausible mechanisms underlying the beneficial effects of probiotic bacteria on human health. Presently, the specific probiotic cell products responsible for immunomodulation are largely unknown. In this study, the genetic and phenotypic diversity of strains of the Lactobacillus plantarum species were investigated to identify genes of L. plantarum with the potential to influence the amounts of cytokines interleukin 10 (IL-10) and IL-12 and the ratio of IL-10/IL-12 produced by peripheral blood mononuclear cells (PBMCs).ResultsA total of 42 Lactobacillus plantarum strains isolated from diverse environmental and human sources were evaluated for their capacity to stimulate cytokine production in PBMCs. The L. plantarum strains induced the secretion of the anti-inflammatory cytokine IL-10 over an average 14-fold range and secretion of the pro-inflammatory cytokine IL-12 over an average 16-fold range. Comparisons of the strain-specific cytokine responses of PBMCs to comparative genome hybridization profiles obtained with L. plantarum WCFS1 DNA microarrays (also termed gene-trait matching) resulted in the identification of 6 candidate genetic loci with immunomodulatory capacities. These loci included genes encoding an N-acetyl-glucosamine/galactosamine phosphotransferase system, the LamBDCA quorum sensing system, and components of the plantaricin (bacteriocin) biosynthesis and transport pathway. Deletion of these genes in L. plantarum WCFS1 resulted in growth phase-dependent changes in the PBMC IL-10 and IL-12 cytokine profiles compared with wild-type cells.ConclusionsThe altered PBMC cytokine profiles obtained with the L. plantarum WCFS1 mutants were in good agreement with the predictions made by gene-trait matching for the 42 L. plantarum strains. This study therefore resulted in the identification of genes present in certain strains of L. plantarum which might be responsible for the stimulation of anti- or pro-inflammatory immune responses in the gut.

Genetic Characterization of the Bile Salt Response in Lactobacillus plantarum and Analysis of Responsive Promoters In Vitro and In Situ in the Gastrointestinal Tract

In this paper we describe the growth, morphological, and genetic responses of Lactobacillus plantarum WCFS1 to bile. Growth experiments revealed that a stepwise increase in the porcine bile concentration led to a gradual decrease in the maximal growth rate. Moreover, the final density reached by an L. plantarum culture growing in MRS containing 0.1% bile was approximately threefold lower than that in MRS lacking bile. The morphology of the cells grown in MRS containing 0.1% bile was investigated by scanning electron microscopy, which revealed that cells clumped together and had rough surfaces and that some of the cells had a shrunken and empty appearance, which clearly contrasted with the characteristic rod-shaped, smooth-surface morphology of L. plantarum cells grown in MRS without bile. An alr complementation-based genome-wide promoter screening analysis was performed with L. plantarum, which led to identification of 31 genes whose expression was potentially induced by 0.1% porcine bile. Remarkably, 11 membrane- and cell wall-associated functions appeared to be induced by bile, as were five functions involved in redox reactions and five regulatory factors. Moreover, the lp_0237 and lp_0775 genes, identified here as genes that are inducible by bile in vitro, were previously identified in our laboratory as important for L. plantarum in vivo during passage in the mouse gastrointestinal tract (P. A. Bron, C. Grangette, A. Mercenier, W. M. de Vos, and M. Kleerebezem, J. Bacteriol. 186:5721-5729, 2004). A quantitative reverse transcription-PCR approach focusing on these two genes confirmed that the expression level of lp_0237 and lp_0775 was significantly higher in cells grown in the presence of bile and cells isolated from the mouse duodenum than in cells grown on laboratory medium without bile.

Food Formats for Effective Delivery of Probiotics

Probiotic bacteria are increasingly incorporated into food products intended to confer health benefits in the human gut and beyond. Little is known about how the food matrix and product formulation impacts probiotic functionality, even though such information is essential to scientific understanding and regulatory substantiation of health benefits. The food format has the potential to affect probiotic survival, physiology, and potentially efficacy, but few comparative studies in humans have been conducted. Human studies should account for the effects of the food base on human health and the bioactive components present in the foods that may augment or diminish interactions of the probiotic with the human host. Some studies show that food ingredients such as prebiotics and milk components can improve probiotic survival during the shelf life of foods, which may enhance probiotic efficacy through increased dose effects. Furthermore, there are indications that synbiotic products are more effective than either probiotics or prebiotics alone. Identification of probiotic adaptations to the food and gut environments holds promise for determining the specific cell components and potential bacterial-food interactions necessary for health benefits and determining how these factors are affected by changes in food formulation and host diet. These studies, combined with controlled human studies, are important future research activities for advancing this field.

Spatial and temporal expression of Lactobacillus plantarum genes in the gastrointestinal tracts of mice.

ABSTRACT Lactobacillus plantarum is a common inhabitant of mammalian gastrointestinal tracts, and L. plantarum strain WCFS1 is a human isolate with a known genome sequence. L. plantarum WCFS1 survives intestinal passage in an active form, and its transit time and transcriptional activities were monitored in 15 BALB/c mice at 2, 4, 6, 8, and 24 h after being fed a single intragastric dose of this organism. Enumeration of viable cells isolated from fecal material revealed that the majority of the L. plantarum inoculum transited the mouse intestine within 4 h after ingestion. Three mice were sacrificed at each time point, and total RNA was isolated from the mouse intestinal compartments (stomach through colon). Quantification of L. plantarum 16S rRNA by quantitative real-time reverse-transcription-PCR revealed that L. plantarum was present at elevated levels in the stomach and small intestine for at least 4 h following ingestion and for over 8 h in the cecum and colon. We also examined the expression of 9 L. plantarum housekeeping genes and 15 L. plantarum in vivo-inducible (ivi) genes previously identified by recombination-based in vivo expression technology to be induced in the mouse gastrointestinal tract. The relative expression levels of the ivi genes increased up to 350-fold in the mouse intestine compared to levels observed for L. plantarum WCFS1 cells grown in a rich laboratory medium. Moreover, several genes displayed intestinal compartment-specific (small intestine versus colon) activities. These results confirm that L. plantarum displays specific and differential responses at various sites along the mammalian intestine.

The intestinal microbiota in aged mice is modulated by dietary resistant starch and correlated with improvements in host responses.

Dietary interventions might prevent or reverse age-related declines in health through modification of the activity and composition of the intestinal microbiota. As a first step toward more comprehensive evaluations of single dietary components on healthy aging, 16S rRNA gene amplicon sequencing was applied to determine the structure of the bacterial communities in the ceca of 20-month-old healthy mice fed energy-controlled diets containing 0, 18, or 36% type 2 resistant starch (RS) from high-amylose maize (HAM-RS2). The cecal microbiota of mice fed a diet depleted in RS and containing the readily digestible carbohydrate amylopectin were dominated by bacteria in the Firmicutes phylum and contained low levels of Bacteroidetes and Actinobacteria. In contrast, mice fed diets containing HAM-RS2 were colonized by higher levels of Bacteroidetes and Bifidobacterium, Akkermansia, and Allobaculum species in proportions that were dependent on the concentration of the dietary fiber. The proportions of Bifidobacterium and Akkermansia were positively correlated with mouse feeding responses, gut weight, and expression levels of proglucagon, the precursor of the gut anti-obesity/diabetic hormone GLP-1. This study showed that aging mice harbor a distinct microbiota, which can be modulated by RS and enriched for bacteria that are associated with improved health.

Lifestyle of Lactobacillus plantarum in the mouse caecum.

Lactobacillus plantarum is a common inhabitant of mammalian gastrointestinal tracts. Strains of L. plantarum are also marketed as probiotics intended to confer beneficial health effects upon delivery to the human gut. To understand how L. plantarum adapts to its gut habitat, we used whole genome transcriptional profiling to characterize the transcriptome of strain WCFS1 during colonization of the caeca of adult germ-free C57Bl/6 J mice fed a standard low-fat rodent chow diet rich in complex plant polysaccharides or a prototypic Western diet high in simple sugars and fat. Lactobacillus plantarum colonized the digestive tracts of these animals to high levels, although L. plantarum was found in 10-fold higher amounts in the caeca of mice fed the standard chow. Metabolic reconstructions based on the transcriptional data sets revealed that genes involved in carbohydrate transport and metabolism form the principal functional group that is upregulated in vivo compared with exponential phase cells grown in three different culture media, and that a Western diet provides a more nutritionally restricted, growth limiting milieu for the microbe in the distal gut. A set of bacterial genes encoding cell surface-related functions were differentially regulated in both groups of mice. This set included downregulated genes required for the d-alanylation of lipoteichoic acids, extracellular structures of L. plantarum that mediate interactions with the host immune system. These results, obtained in a reductionist gnotobiotic mouse model of the gut ecosystem, provide insights about the niches (professions) of this lactic acid bacterium, and a context for systematically testing features that affect epithelial and immune cell responses to this organism in the digestive tract.

Convergence in probiotic Lactobacillus gut-adaptive responses in humans and mice

Probiotic bacteria provide unique opportunities to study the global responses and molecular mechanisms underlying the effects of gut-associated microorganisms in the human digestive tract. In this study, we show by comparative transcriptome analysis using DNA microarrays that the established probiotic Lactobacillus plantarum 299v specifically adapts its metabolic capacity in the human intestine for carbohydrate acquisition and expression of exopolysaccharide and proteinaceous cell surface compounds. This report constitutes the first application of global gene expression profiling of a commensal microorganism in the human gut. A core L. plantarum transcriptome expressed in the mammalian intestine was also determined through comparisons of L. plantarum 299v activities in humans to those found for L. plantarum WCFS1 in germ-free mice. These results identify the niche-specific adaptations of a dietary microorganism to the intestinal ecosystem and provide novel targets for molecular analysis of microbial–host interactions which affect human health.

Health benefits of fermented foods: microbiota and beyond

Fermented foods and beverages were among the first processed food products consumed by humans. The production of foods such as yogurt and cultured milk, wine and beer, sauerkraut and kimchi, and fermented sausage were initially valued because of their improved shelf life, safety, and organoleptic properties. It is increasingly understood that fermented foods can also have enhanced nutritional and functional properties due to transformation of substrates and formation of bioactive or bioavailable end-products. Many fermented foods also contain living microorganisms of which some are genetically similar to strains used as probiotics. Although only a limited number of clinical studies on fermented foods have been performed, there is evidence that these foods provide health benefits well-beyond the starting food materials.

Falcipain inhibitors: optimization studies of the 2-pyrimidinecarbonitrile lead series.

Falcipain-2 and falcipain-3 are papain-family cysteine proteases of the malaria parasite Plasmodium falciparum that are responsible for host hemoglobin hydrolysis to provide amino acids for parasite protein synthesis. Different heteroarylnitrile derivatives were studied as potential falcipain inhibitors and therefore potential antiparasitic lead compounds, with the 5-substituted-2-cyanopyrimidine chemical class emerging as the most potent and promising lead series. Through a sequential lead optimization process considering the different positions present in the initial scaffold, nanomolar and subnanomolar inhibitors at falcipains 2 and 3 were identified, with activity against cultured parasites in the micromolar range. Introduction of protonable amines within lead molecules led to marked improvements of up to 1000 times in activity against cultured parasites without noteworthy alterations in other SAR tendencies. Optimized compounds presented enzymatic activities in the picomolar to low nanomolar range and antiparasitic activities in the low nanomolar range.

Season, Irrigation, Leaf Age, and Escherichia coli Inoculation Influence the Bacterial Diversity in the Lettuce Phyllosphere

The developmental and temporal succession patterns and disturbance responses of phyllosphere bacterial communities are largely unknown. These factors might influence the capacity of human pathogens to persist in association with those communities on agriculturally-relevant plants. In this study, the phyllosphere microbiota was identified for Romaine lettuce plants grown in the Salinas Valley, CA, USA from four plantings performed over 2 years and including two irrigation methods and inoculations with an attenuated strain of Escherichia coli O157:H7. High-throughput DNA pyrosequencing of the V5 to V9 variable regions of bacterial 16S rRNA genes recovered in lettuce leaf washes revealed that the bacterial diversity in the phyllosphere was distinct for each field trial but was also strongly correlated with the season of planting. Firmicutes were generally most abundant in early season (June) plantings and Proteobacteria comprised the majority of bacteria recovered later in the year (August and October). Comparisons within individual field trials showed that bacterial diversity differed between sprinkler (overhead) and drip (surface) irrigated lettuce and increased over time as the plants grew. The microbiota were also distinct between control and E. coli O157:H7-inoculated plants and between E. coli O157:H7-inoculated plants with and without surviving pathogen cells. The bacterial inhabitants of the phyllosphere therefore appear to be affected by seasonal, irrigation, and biological factors in ways that are relevant for assessments of fresh produce food safety.