Desulfovibrio in the Gut: The Enemy within?

Desulfovibrio (DSV) are sulfate-reducing bacteria (SRB) that are ubiquitously present in the environment and as resident commensal bacteria within the human gastrointestinal tract. Though they are minor residents of the healthy gut, DSV are opportunistic pathobionts that may overgrow in the setting of various intestinal and extra-intestinal diseases. An increasing number of studies have demonstrated a positive correlation between DSV overgrowth (bloom) and various human diseases. While the relationship between DSV bloom and disease pathology has not been clearly established, mounting evidence suggests a causal role for these bacteria in disease development. As DSV are the most predominant genera of SRB in the gut, this review summarizes current knowledge regarding the relationship between DSV and a variety of diseases. In this study, we also discuss the mechanisms by which these bacteria may contribute to disease pathology.

Mucin and Agitation Shape Predation of Escherichia coli by Lytic Coliphage.

The ability of bacteriophage (phage), abundant within the gastrointestinal microbiome, to regulate bacterial populations within the same micro-environment offers prophylactic and therapeutic opportunities. Bacteria and phage have both been shown to interact intimately with mucin, and these interactions invariably effect the outcomes of phage predation within the intestine. To better understand the influence of the gastrointestinal micro-environment on phage predation, we employed enclosed, in vitro systems to investigate the roles of mucin concentration and agitation as a function of phage type and number on bacterial killing. Using two lytic coliphage, T4 and PhiX174, bacterial viability was quantified following exposure to phages at different multiplicities of infection (MOI) within increasing, physiological levels of mucin (0-4%) with and without agitation. Comparison of bacterial viability outcomes demonstrated that at low MOI, agitation in combination with higher mucin concentration (>2%) inhibited phage predation by both phages. However, when MOI was increased, PhiX predation was recovered regardless of mucin concentration or agitation. In contrast, only constant agitation of samples containing a high MOI of T4 demonstrated phage predation; briefly agitated samples remained hindered. Our results demonstrate that each phage-bacteria pairing is uniquely influenced by environmental factors, and these should be considered when determining the potential efficacy of phage predation under homeostatic or therapeutic circumstances.

Intestinal Alkaline Phosphatase Prevents Sulfate Reducing Bacteria-Induced Increased Tight Junction Permeability by Inhibiting Snail Pathway.

Tight junctions (TJs) are essential components of intestinal barrier integrity and protect the epithelium against passive paracellular flux and microbial translocation. Dysfunctional TJ leads to leaky gut, a condition associated with diseases including inflammatory bowel disease (IBD). Sulfate-Reducing Bacteria (SRB) are minor residents of the gut. An increased number of Desulfovibrio, the most predominant SRB, is observed in IBD and other diseases associated with leaky gut. However, it is not known whether Desulfovibrio contributes to leaky gut. We tested the hypothesis that Desulfovibrio vulgaris (DSV) may induce intestinal permeability in vitro. Snail, a transcription factor, disrupts barrier function by affecting TJ proteins such as occludin. Intestinal alkaline phosphatase (IAP), a host defense protein, protects epithelial barrier integrity. We tested whether DSV induced permeability in polarized Caco-2 cells via snail and if this effect was inhibited by IAP. Barrier integrity was assessed by measuring transepithelial electric resistance (TEER) and by 4kDa FITC-Dextran flux to determine paracellular permeability. We found that DSV reduced TEER, increased FITC-flux, upregulated snail protein expression, caused nuclear translocation of snail, and disrupted occludin staining at the junctions. DSV-induced permeability effects were inhibited in cells knocked down for snail. Pre-treatment of cells with IAP inhibited DSV-induced FITC flux and snail expression and DSV-mediated disruption of occludin staining. These data show that DSV, a resident commensal bacterium, can contribute to leaky gut and that snail may serve as a novel therapeutic target to mitigate DSV-induced effects. Taken together, our study suggests a novel underlying mechanism of association of Desulfovibrio bloom with diseases with increased intestinal permeability. Our study also underscores IAP as a novel therapeutic intervention for correcting SRB-induced leaky gut via inhibition of snail.

Norepinephrine induces growth of Desulfovibrio vulgaris in an iron dependent manner.

Desulfovibrio spp. is a commensal sulfate reducing bacterium that is present in small numbers in the gastrointestinal tract. Increased concentrations of Desulfovibrio spp. (blooms) have been reported in patients with inflammatory bowel disease and irritable bowel syndrome. Since stress has been reported to exacerbate symptoms of these chronic diseases, this study examined whether the stress catecholamine norepinephrine (NE) promotes Desulfovibrio growth. Norepinephrine-stimulated growth has been reported in other bacterial taxa, and this effect may depend on the availability of the micronutrient iron. OBJECTIVES: This study tested whether norepinephrine exposure affects the in vitro growth of Desulfovibrio vulgaris in an iron dependent manner. METHODS: DSV was incubated in a growth medium with and without 1 µm of norepinephrine. An additional growth assay added the iron chelator deferoxamine in NE exposed DSV. Iron regulatory genes were assessed with and without the treatment of NE and Deferoxamine. RESULTS: We found that norepinephrine significantly increased growth of D. vulgaris. Norepinephrine also increased bacterial production of hydrogen sulfide. Additionally, norepinephrine significantly increased bacterial expression in three of the four tested iron regulatory genes. The iron chelator deferoxamine inhibited growth of D. vulgaris in a dose-dependent manner and reversed the effect of norepinephrine on proliferation of D. vulgaris and on bacterial expression of iron regulatory genes. CONCLUSION: The data presented in this work suggests that promotion of D. vulgaris growth by norepinephrine is iron dependent.

Role of Intestinal Alkaline Phosphatase in Innate Immunity.

Intestinal alkaline phosphatase (IAP) is a multi-functional protein that has been demonstrated to primarily protect the gut. The role of IAP in maintaining intestinal homeostasis is underscored by the observation that IAP expression is defective in many gastrointestinal-related disorders such as inflammatory bowel disease IBD, necrotizing enterocolitis, and metabolic syndrome and that exogenous IAP supplementation improves the outcomes associated with these disorders. Additionally, studies using transgenic IAP-knock out (IAP-KO) mouse models further support the importance of the defensive role of IAP in the intestine. Supplementation of exogenous IAP and cellular overexpression of IAP have also been used in vitro to dissect out the downstream mechanisms of this protein in mammalian cell lines. Some of the innate immune functions of IAP include lipopolysaccharide (LPS) detoxification, protection of gut barrier integrity, regulation of gut microbial communities and its anti-inflammatory roles. A novel function of IAP recently identified is the induction of autophagy. Due to its critical role in the gut physiology and its excellent safety profile, IAP has been used in phase 2a clinical trials for treating conditions such as sepsis-associated acute kidney injury. Many excellent reviews discuss the role of IAP in physiology and pathophysiology and here we extend these to include recent updates on this important host defense protein and discuss its role in innate immunity via its effects on bacteria as well as on host cells. We will also discuss the relationship between IAP and autophagy and how these two pathways may act in concert to protect the gut.

Notch Signaling Pathway Is Activated by Sulfate Reducing Bacteria.

Sulfate Reducing Bacteria (SRB), usually rare residents of the gut, are often found in increased numbers (called a SRB bloom) in inflammatory conditions such as Inflammatory Bowel Disease (IBD), pouchitis, and periodontitis. However, the underlying mechanisms of this association remain largely unknown. Notch signaling, a conserved cell-cell communication pathway, is usually involved in tissue development and differentiation. Dysregulated Notch signaling is observed in inflammatory conditions such as IBD. Lipolysaccharide and pathogens also activate Notch pathway in macrophages. In this study, we tested whether Desulfovibrio, the most dominant SRB genus in the gut, may activate Notch signaling. RAW 264.7 macrophages were infected with Desulfovibrio vulgaris (DSV) and analyzed for the expression of Notch signaling pathway-related proteins. We found that DSV induced protein expression of Notch1 receptor, Notch intracellular domain (NICD) and p21, a downstream Notch target, in a dose-and time-dependent manner. DSV also induced the expression of pro-IL1ß, a precursor of IL-1ß, and SOCS3, a regulator of cytokine signaling. The gamma secretase inhibitor DAPT or Notch siRNA dampened DSV-induced Notch-related protein expression as well the expression of pro-IL1ß and SOCS3. Induction of Notch-related proteins by DSV was not affected by TLR4 -IN -C34(C34), a TLR4 receptor antagonist. Additionally, cell-free supernatant of DSV-infected macrophages induced NICD expression in uninfected macrophages. DSV also activated Notch pathway in the human epithelial cell line HCT116 and in mouse small intestine. Thus, our study uncovers a novel mechanism by which SRB interact with host cells by activating Notch signaling pathway. Our study lays a framework for examining whether the Notch pathway induced by SRB contributes to inflammation in conditions associated with SRB bloom and whether it can be targeted as a therapeutic approach to treat these conditions.

Standard Bacteriophage Purification Procedures Cause Loss in Numbers and Activity.

For decades, bacteriophage purification has followed structured protocols focused on generating high concentrations of phage in manageable volumes. As research moves toward understanding complex phage populations, purification needs have shifted to maximize the amount of phage while maintaining diversity and activity. The effects of standard phage purification procedures such as polyethylene glycol (PEG) precipitation and cesium chloride (CsCl) density gradients on both diversity and activity of a phage population are not known. We have examined the effects of PEG precipitation and CsCl density gradients on a number of known phage (M13, T4, and ΦX 174) of varying structure and size, individually and as mixed sample. Measurement of phage numbers and activity throughout the purification process was performed. We demonstrate that these methods, used routinely to generate "pure" phage samples, are in fact detrimental to retention of phage number and activity; even more so in mixed phage samples. As such, minimal amounts of processing are recommended to introduce less bias and maintain more of a phage population.

Intestinal Alkaline Phosphatase Exerts Anti-Inflammatory Effects Against Lipopolysaccharide by Inducing Autophagy.

Intestinal alkaline phosphatase (IAP) regulates bicarbonate secretion, detoxifies lipopolysaccharide (LPS), regulates gut microbes, and dephosphorylates proinflammatory nucleotides. IAP also exhibits anti-inflammatory effects in a Toll-like Receptor-4 (TLR-4) dependent manner. However, it is not known whether IAP induces autophagy. We tested the hypothesis that IAP may induce autophagy which may mediate the anti-inflammatory effects of IAP. We found that exogenous IAP induced autophagy in intestinal epithelial cells and in macrophages. TLR4INC34 (C34), a TLR4 signaling inhibitor, suppressed IAP-induced autophagy. IAP also inhibited LPS-induced IL-1ß mRNA expression and activation of NF-κB. When autophagy was blocked by 3-methyladenine (3MA) or by Atg5 siRNA, IAP failed to block LPS-mediated effects. IAP also upregulated autophagy-related gene expression in small intestine in mice. We administered either vehicle or IAP (100 U/ml) in drinking water for 14 days in C57BL/6 mice. Mice were sacrificed and ileal tissues collected. Increased expression of Atg5, Atg16, Irgm1, Tlr4, and Lyz genes was observed in the IAP treated group compared to the vehicle treated group. Increase in Atg16 protein expression and fluorescence intensity of LC3 was also observed in IAP-treated tissues compared to the vehicle-treated tissues. Thus, our study lays the framework for investigating how IAP and autophagy may act together to control inflammatory conditions.

Autophagy counters LPS-mediated suppression of lysozyme.

Impaired Paneth cell expression of antimicrobial protein (AMP) lysozyme is found in patients with Crohn's disease with the autophagy gene ATG16L1 risk allele, in mice with mutations in autophagy genes Atg16L1, Atg5 and Atg7, and in Irgm1 knockout mice. Defective autophagy is also associated with expansion of resident Gram-negative bacteria in the intestinal lumen. These findings suggest that autophagy may control extracellular resident microbes by governing expression of lysozyme. To test the hypothesis that autophagy may have a defensive role in host response to resident extracellular microbes, we investigated the relationship between gut microbes, autophagy, and lysozyme. RAW 264.7 macrophages were treated with fecal slurry (FS), representing the resident microbial community; lipopolysaccharide (LPS); or butyrate, representing microbial products; or a representative resident Gram-negative bacterium Desulfovibrio vulgaris (DSV). FS, LPS, and DSV inhibited lysozyme expression, whereas butyrate had no effect. Induction of autophagy by rapamycin countered this inhibition, whereas silencing of the autophagy gene Irgm1 exacerbated the inhibitory effects of LPS on lysozyme expression. LPS also inhibited lysozyme activity against DSV and autophagy reversed this effect. Our results provide a novel insight into an interaction between gut bacteria, autophagy and AMP whereby autophagy may defend the host by countering the suppression of antimicrobial protein by Gram-negative bacteria.

Autophagy Genes of Host Responds to Disruption of Gut Microbial Community by Antibiotics.

BACKGROUND: Defective autophagic machinery, such as that in Crohn's disease patients homozygous for ATG16L1 risk allele, is associated with alteration of resident gut bacterial communities. However, whether or not host autophagy responds to changes in the resident gut microbial community is not known. Here, we investigated the effect of antibiotic-induced disruption of the gut microbiome (dysbiosis) on autophagy gene expression and the expression of antimicrobial peptides/protein (AMP) over time. AIM: To test the hypothesis that antibiotic treatment may cause time-dependent changes in gut bacterial density, autophagy genes, and antimicrobial protein/peptide gene expression. METHODS: Mice (n = 8 per group) were treated with antibiotic cocktail and sacrificed at different intervals of recovery (days 3, 7, 10, 14, 21, 28, 35, and 42) post-antibiotics. DNA and RNA were extracted from small intestinal tissues. Bacterial density, expression of host autophagy genes, and AMP genes were analyzed by relative quantitative PCR. Fold change difference in comparison with untreated control group was calculated using 2-ΔΔCt method. Statistical analysis was performed using nonparametric Mann-Whitney test. RESULTS: Gut bacterial density changed in a time-dependent fashion in response to antibiotic treatment. These changes were concurrent with upregulation of autophagy genes and antimicrobial peptide/protein gene expression. We further showed that an oral gavage of a resident microbe Desulfovibrio, which bloomed in antibiotic-treated animals, induced Atg5 and lysozyme (Lyz) gene expression. CONCLUSION: Autophagy genes respond to dysbiosis induced by antibiotics. This response may be a host mechanism to detect and possibly correct dysbiosis by activating antimicrobial peptides/proteins that control the microbial load in the gut.

Sulfate-reducing bacteria impairs working memory in mice.

The ability of gut microbes to bi-directionally communicate with the brain and vice versa form the basis of the gut microbiome-central nervous system axis. It has been shown that inoculation with pathogenic gut bacteria alters the behavior of mice; however, it is not known whether or not non-pathogenic resident microbes have similar effects. In this study, we tested the hypothesis that the administration of sulfate-reducing bacteria (SRB), a specific group of resident gut bacteria that generate hydrogen sulfide (H2S), impair learning and memory performance in mice tested in an 8-arm radial maze and Morris water maze. We found that mice spent more time in the center of the maze when they were gavaged with live SRB as compared to mice given saline (control), lactulose+mannitol (L/M), or killed SRB. SRB-gavaged mice were also tested using the Morris water maze and were found to take longer to complete the test, spend more time further from the platform, and have a longer path length to reach the platform. This effect of SRB on maze performance was associated with a higher concentration of H2S in the small intestine and cecum. We conclude that SRB, a specific resident gut bacterial species, could impair cognitive function in mice.

Hydrogen Sulfide in Physiology and Diseases of the Digestive Tract.

Hydrogen sulfide (H2S) is a Janus-faced molecule. On one hand, several toxic functions have been attributed to H2S and exposure to high levels of this gas is extremely hazardous to health. On the other hand, H2S delivery based clinical therapies are being developed to combat inflammation, visceral pain, oxidative stress related tissue injury, thrombosis and cancer. Since its discovery, H2S has been found to have pleiotropic effects on physiology and health. H2S is a gasotransmitter that exerts its effect on different systems, such as gastrointestinal, neuronal, cardiovascular, respiratory, renal, and hepatic systems. In the gastrointestinal tract, in addition to H2S production by mammalian cystathionine-ß-synthase (CBS), cystathionine-γ-lyase (CSE), H2S is also generated by the metabolic activity of resident gut microbes, mainly by colonic Sulfate-Reducing Bacteria (SRB) via a dissimilatory sulfate reduction (DSR) pathway. In the gut, H2S regulates functions such as inflammation, ischemia/ reperfusion injury and motility. H2S derived from gut microbes has been found to be associated with gastrointestinal disorders such as ulcerative colitis, Crohn's disease and irritable bowel syndrome. This underscores the importance of gut microbes and their production of H2S on host physiology and pathophysiology.

PARP1 is required for chromosomal translocations.

Chromosomal translocations are common contributors to malignancy, yet little is known about the precise molecular mechanisms by which they are generated. Sequencing translocation junctions in acute leukemias revealed that the translocations were likely mediated by a DNA double-strand break repair pathway termed nonhomologous end-joining (NHEJ). There are major 2 types of NHEJ: (1) the classical pathway initiated by the Ku complex, and (2) the alternative pathway initiated by poly ADP-ribose polymerase 1 (PARP1). Recent reports suggest that classical NHEJ repair components repress translocations, whereas alternative NHEJ components were required for translocations. The rate-limiting step for initiation of alternative NHEJ is the displacement of the Ku complex by PARP1. Therefore, we asked whether PARP1 inhibition could prevent chromosomal translocations in 3 translocation reporter systems. We found that 2 PARP1 inhibitors or repression of PARP1 protein expression strongly repressed chromosomal translocations, implying that PARP1 is essential for this process. Finally, PARP1 inhibition also reduced both ionizing radiation-generated and VP16-generated translocations in 2 cell lines. These data define PARP1 as a critical mediator of chromosomal translocations and raise the possibility that oncogenic translocations occurring after high-dose chemotherapy or radiation could be prevented by treatment with a clinically available PARP1 inhibitor.

Human IRGM regulates autophagy and cell-autonomous immunity functions through mitochondria.

IRGM, a human immunity-related GTPase, confers autophagic defence against intracellular pathogens by an unknown mechanism. Here, we report an unexpected mode of IRGM action. IRGM demonstrated differential affinity for the mitochondrial lipid cardiolipin, translocated to mitochondria, affected mitochondrial fission and induced autophagy. Mitochondrial fission was necessary for autophagic control of intracellular mycobacteria by IRGM. IRGM influenced mitochondrial membrane polarization and cell death. Overexpression of IRGMd, but not IRGMb splice isoforms, caused mitochondrial depolarization and autophagy-independent, but Bax/Bak-dependent, cell death. By acting on mitochondria, IRGM confers autophagic protection or cell death, explaining IRGM action both in defence against tuberculosis and in the damaging inflammation caused by Crohn's disease.

Autophagy pathway intersects with HIV-1 biosynthesis and regulates viral yields in macrophages.

Autophagy is a cytoplasmic degradative pathway that can participate in biosynthetic processes, as in the yeast Cvt pathway, but is more commonly known for its functions in removing damaged or surplus organelles and macromolecular complexes. Here, we find that autophagy intersects with human immunodeficiency virus (HIV) biogenesis, mirroring the above dichotomy. Early, nondegradative stages of autophagy promoted HIV yields. HIV Gag-derived proteins colocalized and interacted with the autophagy factor LC3, and autophagy promoted productive Gag processing. Nevertheless, when autophagy progressed through maturation stages, HIV was degraded. This, however, does not occur, as the HIV protein Nef acts as an antiautophagic maturation factor through interactions with the autophagy regulatory factor Beclin 1, thus protecting HIV from degradation. The dual interaction of HIV with the autophagy pathway enhances viral yields by using the early stages while inhibiting the late stages of autophagy. The role of Nef in the latter process enhances yields of infectious HIV and may be of significance for progression to clinical AIDS.

Leishmania requires Rab7-mediated degradation of endocytosed hemoglobin for their growth.

Leishmania is unable to synthesize heme and must acquire it from exogenous source, the mechanism of which is not known. We have shown that Leishmania endocytoses hemoglobin (Hb) and subsequently degrade it probably to generate heme. To understand how internalized Hb is degraded, we have cloned and expressed Rab7 homolog from Leishmania donovani. Interestingly, Rab7 in Leishmania is found to be localized both on early and late endocytic compartment and regulates both uptake and degradation of endocytosed Hb demonstrating that Rab7 in Leishmania play a very unique role connecting both early and late events of Hb endocytosis. Our data also indicate that overexpression of Rab7:WT in Leishmania induces transport of Hb to lysosomes and rapidly degrade internalized Hb. Whereas Hb transport to lysosomes and its degradation is significantly inhibited in cells overexpressing Rab7:T21N, a GDP locked mutant of Rab7. Moreover, cells overexpressing Rab7:T21N grow at a slower rate (<50%) compared with control Leishmania. Addition of exogenous hemin recovers the growth of Rab7:T21N mutant cells almost to the control level, suggesting that intracellular heme generated by Rab7-mediated Hb degradation is required for optimal growth of the parasites. Thus, our results identify a potential target which might be exploited to suppress the growth of Leishmania.

Human IRGM induces autophagy to eliminate intracellular mycobacteria.

Immunity-related p47 guanosine triphosphatases (IRG) play a role in defense against intracellular pathogens. We found that the murine Irgm1 (LRG-47) guanosine triphosphatase induced autophagy and generated large autolysosomal organelles as a mechanism for the elimination of intracellular Mycobacterium tuberculosis. We also identified a function for a human IRG protein in the control of intracellular pathogens and report that the human Irgm1 ortholog, IRGM, plays a role in autophagy and in the reduction of intracellular bacillary load.

Hemoglobin receptor in Leishmania is a hexokinase located in the flagellar pocket.

Hb endocytosis in Leishmania is mediated through a 46-kDa protein located in the flagellar pocket. To understand the nature of the Hb receptor (HbR), we have purified the 46-kDa protein to homogeneity from Leishmania promastigote membrane. Purified HbR specifically binds Hb. The gene for HbR was cloned, and sequence analysis of the full-length HbR gene indicates the presence of hexokinase (HK) signature sequences, ATP-binding domain, and PTS-II motif. Four lines of evidence indicate that HbR in Leishmania is a hexokinase: 1) the recombinant HbR binds Hb, and the Hb-binding domain resides in the N terminus of the protein; 2) recombinant proteins and cell lysate prepared from HbR-overexpressing Leishmania promastigotes show enhanced HK activity in comparison with untransfected cells; 3) immunolocalization studies using antibodies against the N-terminal fragment (Ld-HbR-DeltaC) of Ld-HbR indicate that this protein is located in the flagellar pocket of Leishmania; and 4) binding and uptake of (125)I-Hb by Leishmania is significantly inhibited by anti-Ld-HbR-DeltaC antibody and Ld-HbR-DeltaC, respectively. Taken together, these results indicate that HK present in the flagellar pocket of Leishmania is involved in Hb endocytosis.

Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages.

Mycobacterium tuberculosis is an intracellular pathogen persisting within phagosomes through interference with phagolysosome biogenesis. Here we show that stimulation of autophagic pathways in macrophages causes mycobacterial phagosomes to mature into phagolysosomes. Physiological induction of autophagy or its pharmacological stimulation by rapamycin resulted in mycobacterial phagosome colocalization with the autophagy effector LC3, an elongation factor in autophagosome formation. Autophagy stimulation increased phagosomal colocalization with Beclin-1, a subunit of the phosphatidylinositol 3-kinase hVPS34, necessary for autophagy and a target for mycobacterial phagosome maturation arrest. Induction of autophagy suppressed intracellular survival of mycobacteria. IFN-gamma induced autophagy in macrophages, and so did transfection with LRG-47, an effector of IFN-gamma required for antimycobacterial action. These findings demonstrate that autophagic pathways can overcome the trafficking block imposed by M. tuberculosis. Autophagy, which is a hormonally, developmentally, and, as shown here, immunologically regulated process, represents an underappreciated innate defense mechanism for control of intracellular pathogens.

Endosomal membrane traffic: convergence point targeted by Mycobacterium tuberculosis and HIV.

Inhibition of phagolysosome biogenesis in infected macrophages is a classical pathogenesis determinant of Mycobacterium tuberculosis. In this review we primarily cover the cellular mechanisms of M. tuberculosis phagosome maturation arrest. A detailed picture is beginning to emerge, involving regulators of membrane trafficking in mammalian cells and phagosomal interactions with endosomal organelles and the trans-Golgi network. We also present a hypothesis that overlaps may exist between the mycobacterial interference with the host cell membrane trafficking processes and the targeting of the late endosomal sorting machinery by HIV during viral budding in macrophages. We propose that interference with the endosomal sorting machinery contributes to the synergism between the two significant human diseases--AIDS and tuberculosis.