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Glycogen in the female lower reproductive tract is a major carbon source for colonization and acidification by common vaginal Lactobacillus species, such as Lactobacillus crispatus . Previously, we identified the amylopullulanase encoding gene pulA of Lactobacillus crispatus to correlate with the ability to autonomously utilize glycogen for growth. Here, we further characterize genetic variation and differential regulation of pulA affecting the presence of its gene product on the outer surface layer. We show that alpha-glucan degrading activity dissipates when Lactobacillus crispatus is grown on glucose, maltose and maltotriose, in agreement with carbon catabolite repression elements flanking the pulA gene. Proteome analysis of the S-layer confirmed that the amylopullulanase protein is highly abundant in an S-layer enriched fraction, but not in a strain with a defective amylopullulanase variant or in an amylopullulanase-sufficient strain grown on glucose. In addition, we provide evidence that Lactobacillus crispatus pulA mutants are relevant in vivo, as they are commonly observed in metagenome datasets of human vaginal microbial communities. Analysis of the largest publicly available dataset of 1507 human vaginal metagenomes indicates that among the 270 samples that contain a Lactobacillus crispatus pulA gene, 62 samples (23%) had a defective variant of this gene. Taken together, these results demonstrate that both environmental, as well as genetic factors explain the variation of Lactobacillus crispatus alpha-glucosidases in the vaginal environment.

Intestinal infections are a global challenge, connected to malnutrition and inadequate hygiene in developing countries, and to expanding antibiotic resistance in developed countries. In general, a healthy host is capable of fighting off gut pathogens or at least to recover from infections quickly. The underlying protective mechanism, termed colonization resistance, is provided by indigenous commensal communities (microbiota) that are shaped and aided by the host's epithelial and innate immune system. Commensal-pathogen interactions are governed by competition for a suitable niche for replication and stable colonization, nutrient availability, species-specific alterations of the metabolic environment, changes in oxygen tension and release of chemicals and proteinaceous toxins (bacteriocins). This protective intestinal milieu is further reinforced by antimicrobial factors and chemicals secreted by the epithelial barrier, by dendritic cell sensing and by homeostasis between T-cell subsets (Treg/Th17) in the lamina propria. The 3 players (host-microbiota-pathogen) communicate via direct interactions or secreted factors. Our recent manuscript illustrates that reactive oxygen species (ROS) are an integral part of colonization resistance and should be considered an interkingdom antivirulence strategy.

NOX/DUOX family of NADPH oxidases are expressed in diverse tissues and are the primary enzymes for the generation of reactive oxygen species (ROS). The intestinal epithelium expresses NOX1, NOX4, and DUOX2, whose functions are not well understood. To address this, we generated mice with complete or epithelium-restricted deficiency in the obligatory NOX dimerization partner Cyba (p22(phox)). We discovered that NOX1 regulates DUOX2 expression in the intestinal epithelium, which magnified the epithelial ROS-deficiency. Unexpectedly, epithelial deficiency of Cyba resulted in protection from C. rodentium and L. monocytogenes infection. Microbiota analysis linked epithelial Cyba deficiency to an enrichment of H2O2-producing bacterial strains in the gut. In particular, elevated levels of lactobacilli physically displaced and attenuated C. rodentium virulence by H2O2-mediated suppression of the virulence-associated LEE pathogenicity island. This transmissible compensatory adaptation relied on environmental factors, an important consideration for prevention and therapy of enteric disease., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Hydrogen peroxide production is a well-known trait of many bacterial species associated with the human body. In the presence of oxygen, the probiotic lactic acid bacterium Lactobacillus johnsonii NCC 533 excretes up to 1 mM H(2)O(2), inducing growth stagnation and cell death. Disruption of genes commonly assumed to be involved in H(2)O(2) production (e.g., pyruvate oxidase, NADH oxidase, and lactate oxidase) did not affect this. Here we describe the purification of a novel NADH-dependent flavin reductase encoded by two highly similar genes (LJ_0548 and LJ_0549) that are conserved in lactobacilli belonging to the Lactobacillus acidophilus group. The genes are predicted to encode two 20-kDa proteins containing flavin mononucleotide (FMN) reductase conserved domains. Reductase activity requires FMN, flavin adenine dinucleotide (FAD), or riboflavin and is specific for NADH and not NADPH. The Km for FMN is 30 ± 8 μM, in accordance with its proposed in vivo role in H(2)O(2) production. Deletion of the encoding genes in L. johnsonii led to a 40-fold reduction of hydrogen peroxide formation. H(2)O(2) production in this mutant could only be restored by in trans complementation of both genes. Our work identifies a novel, conserved NADH-dependent flavin reductase that is prominently involved in H(2)O(2) production in L. johnsonii.

Southern blot analysis of DNA from paired samples of synovial compartment (membrane and/or fluid) and peripheral blood T cells from nine children with juvenile rheumatoid arthritis (JRA) was carried out. Using a T cell receptor C beta probe, dominant TCR rearrangements were discovered in specimens from three patients: synovial fluid T cells from one, synovial fluid and synovial membrane cells from a second, and synovial membrane and peripheral blood cells from a third. The patient showing dominant bands in peripheral blood as well as in synovium was the only child in the series with systemic disease. Since non-specific T cell recruitment is likely to dilute antigen specific clones to low levels, the finding of dominant rearrangements in three of nine patients may indicate that oligoclonality is indeed a feature of JRA.