Ricardo Valladares

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.

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.

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

Structural and enzymatic characterization of NanS (YjhS), a 9-O-Acetyl N-acetylneuraminic acid esterase from Escherichia coli O157:H7.

There is a high prevalence of sialic acid in a number of different organisms, resulting in there being a myriad of different enzymes that can exploit it as a fermentable carbon source. One such enzyme is NanS, a carbohydrate esterase that we show here deacetylates the 9 position of 9‐O‐sialic acid so that it can be readily transported into the cell for catabolism. Through structural studies, we show that NanS adopts a SGNH hydrolase fold. Although the backbone of the structure is similar to previously characterized family members, sequence comparisons indicate that this family can be further subdivided into two subfamilies with somewhat different fingerprints. NanS is the founding member of group II. Its catalytic center contains Ser19 and His301 but no Asp/Glu is present to form the classical catalytic triad. The contribution of Ser19 and His301 to catalysis was confirmed by mutagenesis. In addition to structural characterization, we have mapped the specificity of NanS using a battery of substrates.

The structure of a putative S-formylglutathione hydrolase from Agrobacterium tumefaciens

The structure of the Atu1476 protein from Agrobacterium tumefaciens was determined at 2 Å resolution. The crystal structure and biochemical characterization of this enzyme support the conclusion that this protein is an S‐formylglutathione hydrolase (AtuSFGH). The three‐dimensional structure of AtuSFGH contains the α/β hydrolase fold topology and exists as a homo‐dimer. Contacts between the two monomers in the dimer are formed both by hydrogen bonds and salt bridges. Biochemical characterization reveals that AtuSFGH hydrolyzes CO bonds with high affinity toward short to medium chain esters, unlike the other known SFGHs which have greater affinity toward shorter chained esters. A potential role for Cys54 in regulation of enzyme activity through S‐glutathionylation is also proposed.

Complete Genome Sequences of Lactobacillus johnsonii Strain N6.2 and Lactobacillus reuteri Strain TD1

ABSTRACT We report here the complete genome sequences of Lactobacillus johnsonii strain N6.2, a homofermentative lactic acid intestinal bacterium, and Lactobacillus reuteri strain TD1, a heterofermentative lactic acid intestinal bacterium, both isolated from a type 1 diabetes-resistant rat model.

H2O2 production rate in Lactobacillus johnsonii is modulated via the interplay of a heterodimeric flavin oxidoreductase with a soluble 28 Kd PAS domain containing protein

Host and commensals crosstalk, mediated by reactive oxygen species (ROS), has triggered a growing scientific interest to understand the mechanisms governing such interaction. However, the majority of the scientific studies published do not evaluate the ROS production by commensals bacteria. In this context we recently showed that Lactobacillus johnsonii N6.2, a strain of probiotic value, modulates the activity of the critical enzymes 2,3-indoleamine dioxygenase via H2O2 production. L. johnsonii N6.2 by decreasing IDO activity, is able to modify the tryptophan/kynurenine ratio in the host blood with further systemic consequences. Understanding the mechanisms of H2O2 production is critical to predict the probiotic value of these strains and to optimize bacterial biomass production in industrial processes. We performed a transcriptome analysis to identify genes differentially expressed in L. johnsonii N6.2 cells collected from cultures grown under different aeration conditions. Herein we described the biochemical characteristics of a heterodimeric FMN reductase (FRedA/B) whose in vitro activity is controlled by LjPAS protein with a typical Per-Arnst-Sim (PAS) sensor domain. Interestingly, LjPAS is fused to the FMN reductase domains in other lactobacillaceae. In L. johnsonii, LjPAS is encoded by an independent gene which expression is repressed under anaerobic conditions (>3 fold). Purified LjPAS was able to slow down the FRedA/B initial activity rate when the holoenzyme precursors (FredA, FredB, and FMN) were mixed in vitro. Altogether the results obtained suggest that LjPAS module regulates the H2O2 production helping the cells to minimize oxidative stress in response to environmental conditions.

Identification and Characterization of Feruloyl Esterases Produced by Probiotic Bacteria

A variety of phenolic compounds are naturally available, and contain one or more phenolic rings with or without substituents such as hydroxyl or methoxy groups. The term phytophenol, or phytochemical, is also used due to the widespread distribution of these chemicals throughout the plant kingdom (Huang et al., 2007). Phytophenols are secondary metabolites of plants, which are primarily used in defense against ultraviolet radiation and pathogens (Beckman, 2000). These chemicals also participate in the formation of macromolecular structures in plant cell walls, and are naturally present in the form of monophenols or polyphenols with ester linkages. The presence of phenolic ester linkages limits the hydrolytic activity of enzymes such as xylanases, cellulases, and pectinases, by shielding the site of hydrolysis on plant cell walls from these enzymes. Hydrolyzing the ester linkages within the phytophenols releases the phenolic acids and relaxes the structure of the plant cell wall, aiding in the degradation and maximizing the nutritional value of dietary fiber. Phenolic acids such as ferulic acid, caffeic acid, chlorogenic acid, and rosmarinic acid are studied extensively due to their anti-oxidative, anti-inflammatory, and other health related properties which have been demonstrated both in vitro and in vivo (Srinivasan et al., 2007). Even though phenolic acids can be easily found in dietary fiber, the ester linkages prevent their absorption in the human intestine. It has been demonstrated that only small monophenolic acids, but not esterified phenolic acids, can be absorbed efficiently by the monocarboxylic acid transporter (Konishi et al., 2005). Thus, an enzymatic step is required to convert the esterified phenolic acids into monophenolic acids prior to absorption. In the presence of water, a specific type of enzyme, feruloyl esterases (FAEs), is able to hydrolyze the phenolic compounds into respective alcohols and phenolic acids. Thus, FAEs become one of the target fields of study to improve the bioavailiability and assimilation of phenolic acids in the human diet.

Determination of Francisella tularensis AcpB acid phosphatase substrate preferences.

The Francisella speciesencode 4 main acid phosphatases (Acp) that are potentially involved in pathogenesis through currently unknown mechanisms. Only 2 of these enzymes, AcpA and AcpC, have been biochemically characterized to date. In this work we describe the catalytic properties of Francisella tularensis AcpB utilizing an array of 120 phosphorylated substrates. In contrast to most acid phosphatases, the purified enzyme showed a narrow range of substrate preferences, with the highest affinity towards thiamine phosphate (Km = 150 µM). Francisella species do not possess a thiamine biosynthetic pathway even though vitamin B1 is indispensable in numerous cellular functions. Consequently, thiamine should be incorporated from the environment, in this case, from the host cell. Our results suggested that AcpB could provide the hydrolytic activity necessary to transform the nontransportable phosphorylated vitamin B1 present in tissues to a form that can be absorbed by the intracellular pathogen.

The Escherichia coli yjfP Gene Encodes a Carboxylesterase Involved in Sugar Utilization during Diauxie.

Background: Acetylation and efflux of carbohydrates during cellular metabolism is a well-described phenomenon associated with a detoxification process to prevent metabolic congestion. It is still unclear why cells discard important metabolizable energy sources in the form of acetylated compounds. Methods: We describe the purification and characterization of an approximately 28-kDa intracellular carboxylesterase (YjfP) and the analysis of gene and protein expression by qRT-PCR and Western blot. Results: qRT-PCR and Western blot, respectively, showed that yjfP is upregulated during the diauxic lag in cells growing with a mixture of glucose and lactose. The β-galactosidase activity in the ΔyjfP strain was both delayed and half the magnitude of that of the wild-type strain. YjfP-hyperproducing strains displayed a long lag phase when cultured with glucose and then challenged to grow with lactose or galactose as the sole carbon source. Conclusion: Our results suggest that YjfP controls the intracellular concentration of acetyl sugars by redirecting them to the main metabolic circuits. Instead of detoxification, we propose that sugar acetylation is utilized by the cell for protection and to prevent the metabolism of a necessary minimal intracellular sugar pool. Those sugars can eventually be exported as a side effect of these mechanisms.