Graciela L. Lorca

Culture-independent identification of gut bacteria correlated with the onset of diabetes in a rat model

Bacteria associated with the onset of type 1 diabetes in a rat model system were identified. In two experiments, stool samples were collected at three time points after birth from bio-breeding diabetes-prone (BB-DP) and bio-breeding diabetes-resistant (BB-DR) rats. DNA was isolated from these samples and the 16S rRNA gene was amplified using universal primer sets. In the first experiment, bands specific to BB-DP and BB-DR genotypes were identified by automated ribosomal intergenic spacer analysis at the time of diabetes onset in BB-DP. Lactobacillus and Bacteroides strains were identified in the BB-DR- and BB-DP-specific bands, respectively. Sanger sequencing showed that the BB-DP and BB-DR bacterial communities differed significantly but too few reads were available to identify significant differences at the genus or species levels. A second experiment confirmed these results using higher throughput pyrosequencing and quantitative PCR of 16S rRNA with more rats per genotype. An average of 4541 and 3381 16S rRNA bacterial reads were obtained from each of the 10 BB-DR and 10 BB-DP samples collected at time of diabetes onset. Nine genera were more abundant in BB-DP whereas another nine genera were more abundant in BB-DR. Thirteen and eleven species were more abundant in BB-DP and BB-DR, respectively. An average of 23% and 10% of all reads could be classified at the genus and species levels, respectively. Quantitative PCR verified the higher abundance of Lactobacillus and Bifidobacterium in the BB-DR samples. Whether these changes are caused by diabetes or are involved in the development of the disease is unknown.

Lactobacillus johnsonii N6.2 mitigates the development of type 1 diabetes in BB-DP rats.

Background The intestinal epithelium is a barrier that composes one of the most immunologically active surfaces of the body due to constant exposure to microorganisms as well as an infinite diversity of food antigens. Disruption of intestinal barrier function and aberrant mucosal immune activation have been implicated in a variety of diseases within and outside of the gastrointestinal tract. With this model in mind, recent studies have shown a link between diet, composition of intestinal microbiota, and type 1 diabetes pathogenesis. In the BioBreeding rat model of type 1 diabetes, comparison of the intestinal microbial composition of diabetes prone and diabetes resistant animals found Lactobacillus species were negatively correlated with type 1 diabetes development. Two species, Lactobacillus johnsonii and L. reuteri, were isolated from diabetes resistant rats. In this study diabetes prone rats were administered pure cultures of L. johnsonii or L. reuteri isolated from diabetes resistant rats to determine the effect on type 1 diabetes development. Methodology/Principal Findings Results Rats administered L. johnsonii, but not L. reuteri, post-weaning developed type 1 diabetes at a protracted rate. Analysis of the intestinal ileum showed administration of L. johnsonii induced changes in the native microbiota, host mucosal proteins, and host oxidative stress response. A decreased oxidative intestinal environment was evidenced by decreased expression of several oxidative response proteins in the intestinal mucosa (Gpx1, GR, Cat). In L. johnsonii fed animals low levels of the pro-inflammatory cytokine IFNγ were correlated with low levels of iNOS and high levels of Cox2. The administration of L. johnsonii also resulted in higher levels of the tight junction protein claudin. Conclusions It was determined that the administration of L. johnsonii isolated from BioBreeding diabetes resistant rats delays or inhibits the onset of type 1 diabetes in BioBreeding diabetes prone rats. Taken collectively, these data suggest that the gut and the gut microbiota are potential agents of influence in type 1 diabetes development. These data also support therapeutic efforts that seek to modify gut microbiota as a means to modulate development of this disorder.

Inhibition of Type 1 Diabetes Correlated to a Lactobacillus johnsonii N6.2-Mediated Th17 Bias

Although it is known that resident gut flora contribute to immune system function and homeostasis, their role in the progression of the autoimmune disease type 1 diabetes (T1D) is poorly understood. Comparison of stool samples isolated from Bio-Breeding rats, a classic model of T1D, shows that distinct bacterial populations reside in spontaneous Bio-Breeding diabetes-prone (BBDP) and Bio-Breeding diabetes-resistant animals. We have previously shown that the oral transfer of Lactobacillus johnsonii strain N6.2 (LjN6.2) from Bio-Breeding diabetes-resistant to BBDP rodents conferred T1D resistance to BBDP rodents, whereas Lactobacillus reuteri strain TD1 did not. In this study, we show that diabetes resistance in LjN6.2-fed BBDP rodents was correlated to a Th17 cell bias within the mesenteric lymph nodes. The Th17 bias was not observed in the non-gut–draining axillary lymph nodes, suggesting that the Th17 bias was because of immune system interactions with LjN6.2 within the mesenteric lymph node. LjN6.2 interactions with the immune system were observed in the spleens of diabetes-resistant, LjN6.2-fed BBDP rats, as they also possessed a Th17 bias in comparison with control or Lactobacillus reuteri strain TD1–fed rats. Using C57BL/6 mouse in vitro assays, we show that LjN6.2 directly mediated enhanced Th17 differentiation of lymphocytes in the presence of TCR stimulation, which required APCs. Finally, we show that footpad vaccination of NOD mice with LjN6.2-pulsed dendritic cells was sufficient to mediate a Th17 bias in vivo. Together, these data suggest an interesting paradigm whereby T1D induction can be circumvented by gut flora-mediated Th17 differentiation.

Catabolite Repression and Activation in Bacillus subtilis: Dependency on CcpA, HPr, and HprK

Previous studies have suggested that the transcription factor CcpA, as well as the coeffectors HPr and Crh, both phosphorylated by the HprK kinase/phosphorylase, are primary mediators of catabolite repression and catabolite activation in Bacillus subtilis. We here report whole transcriptome analyses that characterize glucose-dependent gene expression in wild-type cells and in isogenic mutants lacking CcpA, HprK, or the HprK phosphorylatable serine in HPr. Binding site identification revealed which genes are likely to be primarily or secondarily regulated by CcpA. Most genes subject to CcpA-dependent regulation are regulated fully by HprK and partially by serine-phosphorylated HPr [HPr(Ser-P)]. A positive linear correlation was noted between the dependencies of catabolite-repressible gene expression on CcpA and HprK, but no such relationship was observed for catabolite-activated genes, suggesting that large numbers of the latter genes are not regulated by the CcpA-HPr(Ser-P) complex. Many genes that mediate nitrogen or phosphorus metabolism as well as those that function in stress responses proved to be subject to CcpA-dependent glucose control. While nitrogen-metabolic genes may be subject to either glucose repression or activation, depending on the gene, almost all glucose-responsive phosphorus-metabolic genes exhibit activation while almost all glucose-responsive stress genes show repression. These responses are discussed from physiological standpoints. These studies expand our appreciation of CcpA-mediated catabolite control and provide insight into potential interregulon control mechanisms in gram-positive bacteria.

Biochemical Properties of Two Cinnamoyl Esterases Purified from a Lactobacillus johnsonii Strain Isolated from Stool Samples of Diabetes-Resistant Rats

ABSTRACT Cinnamic acids (i.e., ferulic and caffeic acids) that are esterified to the vegetable cell walls should be enzymatically released to be absorbed in a mammals intestines. A low dosage of ferulic acid in rodent diets stimulates insulin production and alleviates symptoms caused by diabetes (M. Sri Balasubashini, R. Rukkumani, and V. P. Menon, Acta Diabetol. 40:118-122, 2003). Several lactic acid bacteria are able to display ferulic acid esterase (FAE) activity, suggesting that their probiotic activity could be, in part, mediated by the slow release of ferulic acid. In the present work, we describe the isolation of one strain identified as being Lactobacillus johnsonii that displayed strong FAE activity in stool samples from diabetes-resistant biobreeding rats. These animals are genetically susceptible to becoming diabetic but do not develop the disease. By using genomic analysis coupled to protein purification and catalytic screening, we were able to purify two proteins with FAE activity. The enzymes displayed 42% sequence identity and a broad range of substrate preferences. High affinities and catalytic efficiencies toward aromatic compounds such as ethyl ferulate (Km = 20 to 60 μM) and chlorogenic acid (Km = 10 to 50 μM) were observed. The strain isolated herein as well as the enzymes studied could be potentially useful for the formulation of probiotics to ameliorate diabetes symptoms.

Glyoxylate and Pyruvate Are Antagonistic Effectors of the Escherichia coli IclR Transcriptional Regulator

The Escherichia coli isocitrate lyase regulator (IclR) regulates the expression of the glyoxylate bypass operon (aceBAK). Founding member of a large family of common fold transcriptional regulators, IclR comprises a DNA binding domain that interacts with the operator sequence and a C-terminal domain (C-IclR) that binds a hitherto unknown small molecule. We screened a chemical library of more than 150 metabolic scaffolds using a high-throughput protein stability assay to identify molecules that bind IclR and then tested the active compounds in in vitro assays of operator binding. Glyoxylate and pyruvate, identified by this method, bound the C-IclR domain with KD values of 0.9 ± 0.2 and 156.2 ± 7.9 μm, as defined by isothermal titration calorimetry. Both compounds altered IclR interactions with operator DNA in electrophoretic mobility shift assays but showed an antagonistic effect. Glyoxylate disrupted the formation of the IclR/operator complex in vitro by favoring the inactive dimeric state of the protein, whereas pyruvate increased the binding of IclR to the aceBAK promoter by stabilizing the active tetrameric form of the protein. Structures of the C-IclR domain alone and in complex with each effector were determined at 2.3 Å, confirming the binding of both molecules in the effector recognition site previously characterized for the other representative of the family, the E. coli AllR regulator. Site-directed mutagenesis demonstrated the importance of hydrophobic patch formed by Met-146, Leu-154, Leu-220, and Leu-143 in interactions with effector molecules. In general, our strategy of combining chemical screens with functional assays and structural studies has uncovered two small molecules with antagonistic effects that regulate the IclR-dependent transcription of the aceBAK operon.

Lactobacillus johnsonii N6.2 Stimulates the Innate Immune Response through Toll-Like Receptor 9 in Caco-2 Cells and Increases Intestinal Crypt Paneth Cell Number in BioBreeding Diabetes-Prone Rats

Lactobacillus johnsonii (Ljo) N6.2 has been shown to mitigate the development of type 1 diabetes when administered to diabetes-prone rats. The specific mechanisms underlying this observed response remain under investigation. The objective of this study was to assess the effect of Ljo N6.2 on mucosal inflammatory response using differentiated Caco-2 monolayers. The mRNA expression levels of CCL20, CXCL8, and CXCL10 chemokines were determined by qRT-PCR. Ljo at 10(11) CFU/L induced a strong response in all chemokines examined. To assess the specific host-signaling pathways involved, we performed RT-PCR amplification of Toll-like receptors (TLR) and nucleotide-binding oligomerization domain-like receptors. TLR7 and TLR9 expression levels were induced 4.2- and 9-fold, respectively, whereas other TLR and nucleotide-binding oligomerization domain receptors were not modified. A similar effect was observed in Caco-2 monolayers treated with Ljo cell-free extract or purified nucleic acids (NA). Increased levels of IFN type 1 and IFN regulators Stat1 and IRF7 followed the upregulation of TLR9. Activation of TLR9 was also evidenced by increased Frizzled 5 expression in Ljo-treated Caco-2 cells and an increase in the number of Paneth cells in Ljo-fed, diabetes-prone rats. These results are in agreement with the polarizing-tolerizing mechanism recently described in which the apical stimulation of TLR9 in intestinal epithelial cells leads to a higher state of immunologic alertness. Furthermore, these results suggest that live probiotics could be, in the future, replaced with select cellular components.

The Intestinal Microbiome: Relationship to Type 1 Diabetes

This article discusses recent evidence that associates the developing intestinal microbiome to the pathogenesis of autoimmune T1D. It attempts to identify avenues that should be pursued that relate this new evidence to interventions that eventually could result in prevention.

Lactobacillus johnsonii inhibits indoleamine 2,3-dioxygenase and alters tryptophan metabolite levels in BioBreeding rats.

In our previous work, we found that feeding Lactobacillus johnsonii to BioBreeding diabetes‐prone (BBDP) rats decreased the incidence of diabetes development. The aim of this study was to investigate host pathways affected by L. johnsonii, with specific focus on the rate‐limiting enzyme of tryptophan catabolism, indoleamine 2,3‐dioxygenase (IDO). Suspensions of L. johnsonii or an equal volume of vehicle were orally administered to BBDP rats. Tissue IDO was investigated using quantitative RT‐PCR and Western blot, whereas tryptophan, kynurenine, and 5‐hydroxytryptamine (5‐HT) concentrations were quantified by HPLC and ELISA. IDO activity was also investigated using L. johnsonii culture cell‐free supernatant (CFS) with affinity‐purified IDO and HT‐29 intestinal epithelial cells. L. johnsonii feeding resulted in a 17% reduction in serum kynurenine compared with that in vehicle‐fed controls, correlating with a 1.4‐fold elevation in 5‐HT levels. H2O2 produced by L. johnsonii abolished IDO activity in vitro, and L. johnsonii feeding resulted in a 3.9‐fold increase in ileum lumen H2O2. L. johnsonii CFS significantly reduced IDO activity in HT‐29 intestinal epithelial cells (47% reduction) compared with that in vehicle‐treated controls, an effect abolished by catalase treatment. These data support the role of H2O2 in commensal bacteria‐host interactions and highlight the influence of commensal bacteria‐derived H2O2 on host physiology.—Valladares, R., Bojilova, L., Potts, A. H., Cameron, E., Gardner, C., Lorca, G., Gonzalez, C. F. Lactobacillus johnsonii inhibits indoleamine 2,3‐dioxygenase and alters tryptophan metabolite levels in BioBreeding rats. FASEB J. 27, 1711–1720 (2013). www.fasebj.org

Biological approaches for controlling shellfish-associated pathogens

As the consumption of seafood and shellfish increases around the world, so is the incidence of associated outbreaks of illness. Various postharvest treatments are effective at killing seafood-associated bacteria, but most of these treatments also kill the mollusks. Because consumer preferences for raw live shellfish persist, biological approaches for promoting microbiological safety of live product are being considered. Applications of probiotic bacteria to reduce human pathogens in live shellfish could augment current practices for preharvest monitoring of water quality. Postharvest, biological controls will be important to remove shellfish-associated commensal Vibrio spp. that are pathogenic to humans. Further investigations will reveal whether combining depuration with chemical disruption of bacterial attachment or cell-to-cell signaling may accomplish this goal.