IU Indianapolis ScholarWorks :: Browsing by Author "Dziarski, Roman"

Browsing by Author "Dziarski, Roman"

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Author Correction: Nod2 and Nod2-regulated microbiota protect BALB/c mice from diet-induced obesity and metabolic dysfunction
(SpringerNature, 2018-04-16) Rodriguez-Nunez, Ivan; Caluag, Tiffany; Kirby, Kori; Rudick, Charles N.; Dziarski, Roman; Gupta, Dipika; Medicine, School of Medicine
A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.
Bactericidal peptidoglycan recognition protein induces oxidative stress in Escherichia coli through a block in respiratory chain and increase in central carbon catabolism
(Wiley, 2017-09) Kashyap, Des R.; Kuzma, Marcin; Kowalczyk, Dominik A.; Gupta, Dipika; Dziarski, Roman; Medicine, School of Medicine
Mammalian Peptidoglycan Recognition Proteins (PGRPs) kill both Gram-positive and Gram-negative bacteria through simultaneous induction of oxidative, thiol and metal stress responses in bacteria. However, metabolic pathways through which PGRPs induce these bactericidal stress responses are unknown. We screened Keio collection of Escherichia coli deletion mutants and revealed that deleting genes for respiratory chain flavoproteins or for tricarboxylic acid (TCA) cycle resulted in increased resistance of E. coli to PGRP killing. PGRP-induced killing depended on the production of hydrogen peroxide, which required increased supply of NADH for respiratory chain oxidoreductases from central carbon catabolism (glycolysis and TCA cycle), and was controlled by cAMP-Crp. Bactericidal PGRP induced a rapid decrease in respiration, which suggested that the main source of increased production of hydrogen peroxide was a block in respiratory chain and diversion of electrons from NADH oxidoreductases to oxygen. CpxRA two-component system was a negative regulator of PGRP-induced oxidative stress. By contrast, PGRP-induced thiol stress (depletion of thiols) and metal stress (increase in intracellular free Zn2+ through influx of extracellular Zn2+ ) were mostly independent of oxidative stress. Thus, manipulating pathways that induce oxidative, thiol and metal stress in bacteria could be a useful strategy to design new approaches to antibacterial therapy.
Formate dehydrogenase, ubiquinone, and cytochrome bd-I are required for peptidoglycan recognition protein-induced oxidative stress and killing in Escherichia coli
(Nature, 2020-02-06) Kashyap, Des R.; Kowalczyk, Dominik A.; Shan, Yue; Yang, Chun-Kai; Gupta, Dipika; Dziarski, Roman; IU School of Medicine-Northwest
Mammalian Peptidoglycan Recognition Proteins (PGRPs) kill bacteria through induction of synergistic oxidative, thiol, and metal stress. PGRPs induce oxidative stress in bacteria through a block in the respiratory chain, which results in decreased respiration and incomplete reduction of oxygen (O2) to hydrogen peroxide (H2O2). In this study we identify the site of PGRP-induced generation of H2O2 in Escherichia coli. Tn-seq screening of E. coli Tn10 insertion library revealed that mutants in formate dehydrogenase (FDH) genes had the highest survival following PGRP treatment. Mutants lacking functional FDH-O had abolished PGRP-induced H2O2 production and the highest resistance to PGRP-induced killing, and formate enhanced PGRP-induced killing and H2O2 production in an FDH-dependent manner. Mutants in ubiquinone synthesis (but not menaquinone and demethylmenaquinone) and cytochrome bd-I (but not cytochromes bo3 and bd-II) also had completely abolished PGRP-induced H2O2 production and high resistance to PGRP-induced killing. Because electrons in the respiratory chain flow from dehydrogenases’ substrates through quinones and then cytochromes to O2, these results imply that the site of PGRP-induced incomplete reduction of O2 to H2O2 is downstream from dehydrogenases and ubiquinone at the level of cytochrome bd-I, which results in oxidative stress. These results reveal several essential steps in PGRP-induced bacterial killing.
How innate immunity proteins kill bacteria and why they are not prone to resistance
(Springer, 2018-02-01) Dziarski, Roman; Gupta, Dipika; Medicine, School of Medicine
Recent advances on antibacterial activity of peptidoglycan recognition proteins (PGRPs) offer some insight into how innate immunity has retained its antimicrobial effectiveness for millions of years with no frequent emergence of resistant strains. First, PGRP can bind to multiple components of bacterial envelope (peptidoglycan, lipoteichoic acid, and lipopolysaccharide). Second, PGRP simultaneously induces oxidative, thiol, and metal stress responses in bacteria, which individually are bacteriostatic, but in combination are bactericidal. Third, PGRP induces oxidative, thiol, and metal stress responses in bacteria through three independent pathways. Fourth, antibacterial effects of PGRP are enhanced by other innate immune responses. Thus, emergence of PGRP resistance is prevented by bacteriostatic effect and independence of each PGRP-induced stress response, as PGRP resistance would require simultaneous acquisition of three separate mechanisms disabling the induction of all three stress responses. By contrast, each antibiotic has one primary target and one primary antibacterial mechanism, and for this reason resistance to antibiotics can be generated by inhibition of this primary mechanism. Manipulating bacterial metabolic responses can enhance bacterial killing by antibiotics and elimination of antibiotic-tolerant bacteria, but such manipulations do not overcome genetically encoded antibiotic resistance. Pathogens cause infections by evading, inhibiting, or subverting host immune responses.
Nod2 and Nod2-regulated microbiota protect BALB/c mice from diet-induced obesity and metabolic dysfunction
(SpringerNature, 2017-04-03) Rodriguez-Nunez, Ivan; Caluag, Tiffany; Kirby, Kori; Rudick, Charles N.; Dziarski, Roman; Gupta, Dipika; Department of Medicine, School of Medicine
Genetics plays a central role in susceptibility to obesity and metabolic diseases. BALB/c mice are known to be resistant to high fat diet (HFD)-induced obesity, however the genetic cause remains unknown. We report that deletion of the innate immunity antibacterial gene Nod2 abolishes this resistance, as Nod2 -/- BALB/c mice developed HFD-dependent obesity and hallmark features of metabolic syndrome. Nod2 -/- HFD mice developed hyperlipidemia, hyperglycemia, glucose intolerance, increased adiposity, and steatosis, with large lipid droplets in their hepatocytes. These changes were accompanied by increased expression of immune genes in adipose tissue and differential expression of genes for lipid metabolism, signaling, stress, transport, cell cycle, and development in both adipose tissue and liver. Nod2 -/- HFD mice exhibited changes in the composition of the gut microbiota and long-term treatment with antibiotics abolished diet-dependent weight gain in Nod2 -/- mice, but not in wild type mice. Furthermore, microbiota from Nod2 -/- HFD mice transferred sensitivity to weight gain, steatosis, and hyperglycemia to wild type germ free mice. In summary, we have identified a novel role for Nod2 in obesity and demonstrate that Nod2 and Nod2-regulated microbiota protect BALB/c mice from diet-induced obesity and metabolic dysfunction.
Nod2 protects mice from inflammation and obesity-dependent liver cancer
(Nature, 2020-11-25) Gurses, Serdar A.; Banskar, Sunil; Stewart, Cody; Trimoski, Bill; Dziarski, Roman; Gupta, Dipika; IU School of Medicine-Northwest
Nod2 is a pattern recognition receptor that modulates host innate immune responses and protects from inflammation, steatosis, and obesity. Obesity and inflammation are risk factors for hepatocellular carcinoma, however, the role of Nod2 in obesity-dependent hepatic tumorigenesis is not known. Here we tested the hypothesis that Nod2 protects from high fat diet (HFD)-dependent hepatic cancer. We used an obesity-dependent hepatic tumor model. WT and Nod2−/− mice were treated with the carcinogen dimethylbenz[a]anthracene (DMBA) and maintained on HFD. Nod2−/− mice treated with DMBA and maintained on HFD gain significantly more weight and develop more liver tumors than similarly treated WT mice. Livers of Nod2−/− tumorigenic mice had increased expression of genes involved in cell proliferation, immune responses, and cholesterol biosynthesis, increased infiltration of neutrophils, inflammatory monocytes, and T cells, and increased activation of STAT3 and ERK during the later stages of tumorigenesis. Bioinformatic analyses of genes with differential expression predicted an increase in cancer, immune, and cholesterol biosynthesis pathways. In summary, we have identified a novel role for Nod2 and demonstrate that Nod2 protects from HFD-dependent liver malignancy and this protection is accompanied by decreased cell proliferation, inflammation, steroid biosynthesis, neutrophils and macrophages infiltration, and STAT3 and MAPK signaling in the liver.
Peptidoglycan recognition protein 3 and Nod2 synergistically protect mice from dextran sodium sulfate-induced colitis
(The American Association of Immunologists, 2014-09-15) Jing, Xuefang; Park, Shin Yong; Núñez, Gabriel; Dziarski, Roman; Gupta, Dipika; Department of Medicine, IU School of Medicine
Aberrant immune response and changes in the gut microflora are the main causes of inflammatory bowel disease (IBD). Peptidoglycan recognition proteins (Pglyrp1, Pglyrp2, Pglyrp3, and Pglyrp4) are bactericidal innate immunity proteins that maintain normal gut microbiome, protect against experimental colitis, and are associated with IBD in humans. Nucleotide-binding oligomerization domain 2 (Nod2) is an intracellular bacterial sensor and may be required for maintaining normal gut microbiome. Mutations in Nod2 are strongly associated with Crohn's disease, but the causative mechanism is not understood, and the role of Nod2 in ulcerative colitis is not known. Because IBD is likely caused by variable multiple mutations in different individuals, in this study, we examined the combined role of Pglyrp3 and Nod2 in the development of experimental colitis in mice. We demonstrate that a combined deficiency of Pglyrp3 and Nod2 results in higher sensitivity to dextran sodium sulfate-induced colitis compared with a single deficiency. Pglyrp3(-/-)Nod2(-/-) mice had decreased survival and higher loss of body weight, increased intestinal bleeding, higher apoptosis of colonic mucosa, elevated expression of cytokines and chemokines, altered gut microbiome, and increased levels of ATP in the colon. Increased sensitivity to dextran sodium sulfate-induced colitis in Pglyrp3(-/-)Nod2(-/-) mice depended on increased apoptosis of intestinal epithelium, changed gut microflora, and elevated ATP. Pglyrp3 deficiency contributed colitis-predisposing intestinal microflora and increased intestinal ATP, whereas Nod2 deficiency contributed higher apoptosis and responsiveness to increased level of ATP. In summary, Pglyrp3 and Nod2 are both required for maintaining gut homeostasis and protection against colitis, but their protective mechanisms differ.
Peptidoglycan Recognition Proteins Kill Bacteria by Inducing Oxidative, Thiol, and Metal Stress
(Public Library of Science, 2014-07-17) Kashyap, Des Raj; Rompca, Annemarie; Gaballa, Ahmed; Helmann, John D.; Chan, Jefferson; Chang, Christopher J.; Hozo, Iztok; Gupta, Dipika; Dziarski, Roman; Medicine, School of Medicine
Mammalian Peptidoglycan Recognition Proteins (PGRPs) are a family of evolutionary conserved bactericidal innate immunity proteins, but the mechanism through which they kill bacteria is unclear. We previously proposed that PGRPs are bactericidal due to induction of reactive oxygen species (ROS), a mechanism of killing that was also postulated, and later refuted, for several bactericidal antibiotics. Here, using whole genome expression arrays, qRT-PCR, and biochemical tests we show that in both Escherichia coli and Bacillus subtilis PGRPs induce a transcriptomic signature characteristic of oxidative stress, as well as correlated biochemical changes. However, induction of ROS was required, but not sufficient for PGRP killing. PGRPs also induced depletion of intracellular thiols and increased cytosolic concentrations of zinc and copper, as evidenced by transcriptome changes and supported by direct measurements. Depletion of thiols and elevated concentrations of metals were also required, but by themselves not sufficient, for bacterial killing. Chemical treatment studies demonstrated that efficient bacterial killing can be recapitulated only by the simultaneous addition of agents leading to production of ROS, depletion of thiols, and elevation of intracellular metal concentrations. These results identify a novel mechanism of bacterial killing by innate immunity proteins, which depends on synergistic effect of oxidative, thiol, and metal stress and differs from bacterial killing by antibiotics. These results offer potential targets for developing new antibacterial agents that would kill antibiotic-resistant bacteria.
Recognition of bacterial peptidoglycan by the innate immune system
(Springer, 2003) Dziarski, Roman; Microbiology and Immunology, School of Medicine
The innate immune system recognizes microorganisms through a series of pattern recognition receptors that are highly conserved in evolution. Peptidoglycan (PGN) is a unique and essential component of the cell wall of virtually all bacteria and is not present in eukaryotes, and thus is an excellent target for the innate immune system. Indeed, higher eukaryotes, including mammals, have several PGN recognition molecules, including CD14, Toll-like receptor 2, a family of peptidoglycan recognition proteins, Nod1 and Nod2, and PGN-lytic enzymes (lysozyme and amidases). These molecules induce host responses to microorganisms or have direct antimicrobial effects.
Respiratory chain components are required for peptidoglycan recognition protein-induced thiol depletion and killing in Bacillus subtilis and Escherichia coli
(Springer Nature, 2021-01-08) Yang, Chun‑Kai; Kashyap, Des R.; Kowalczyk, Dominik A.; Rudner, David Z.; Wang, Xindan; Gupta, Dipika; Dziarski, Roman; Medicine, School of Medicine
Mammalian peptidoglycan recognition proteins (PGRPs or PGLYRPs) kill bacteria through induction of synergistic oxidative, thiol, and metal stress. Tn-seq screening of Bacillus subtilis transposon insertion library revealed that mutants in the shikimate pathway of chorismate synthesis had high survival following PGLYRP4 treatment. Deletion mutants for these genes had decreased amounts of menaquinone (MK), increased resistance to killing, and attenuated depletion of thiols following PGLYRP4 treatment. These effects were reversed by MK or reproduced by inhibiting MK synthesis. Deletion of cytochrome aa3-600 or NADH dehydrogenase (NDH) genes also increased B. subtilis resistance to PGLYRP4-induced killing and attenuated thiol depletion. PGLYRP4 treatment also inhibited B. subtilis respiration. Similarly in Escherichia coli, deletion of ubiquinone (UQ) synthesis, formate dehydrogenases (FDH), NDH-1, or cytochrome bd-I genes attenuated PGLYRP4-induced thiol depletion. PGLYRP4-induced low level of cytoplasmic membrane depolarization in B. subtilis and E. coli was likely not responsible for thiol depletion. Thus, our results show that the respiratory electron transport chain components, cytochrome aa3-600, MK, and NDH in B. subtilis, and cytochrome bd-I, UQ, FDH-O, and NDH-1 in E. coli, are required for both PGLYRP4-induced killing and thiol depletion and indicate conservation of the PGLYRP4-induced thiol depletion and killing mechanisms in Gram-positive and Gram-negative bacteria.

Browsing by Author "Dziarski, Roman"

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