Blastocystis: Investigations on host-pathogen interactions using in vitro model systems

Ph.DDOCTOR OF PHILOSOPH

Bacterial control of host gene expression through RNA polymerase II

The normal flora furnishes the host with ecological barriers that prevent pathogen attack while maintaining tissue homeostasis. Urinary tract infections (UTIs) constitute a highly relevant model of microbial adaptation in which some patients infected with Escherichia coil develop acute pyelonephritis, while other patients with bacteriuria exhibit an asymptomatic carrier state similar to bacterial commensalism. It remains unclear if the lack of destructive inflammation merely reflects low virulence or if carrier strains actively inhibit disease-associated responses in the host. Here, we identify a new mechanism of bacterial adaptation through broad suppression of RNA polymerase II-dependent (Pol II-dependent) host gene expression. Over 60% of all genes were suppressed 24 hours after human inoculation with the prototype asymptomatic bacteriuria (ABU) strain E. coil 83972, and inhibition was verified by infection of human cells. Specific repressors and activators of Pol II-dependent transcription were modified, Pol II phosphorylation was inhibited, and pathogen-specific signaling was suppressed in cell lines and inoculated patients. An increased frequency of strains inhibiting Pol II was epidemiologically verified in ABU and fecal strains compared with acute pyelonephritis, and a Pol II antagonist suppressed the disease-associated host response. These results suggest that by manipulating host gene expression, ABU strains promote tissue integrity while inhibiting pathology. Such bacterial modulation of host gene expression may be essential to sustain asymptomatic bacterial carriage by ensuring that potentially destructive immune activation will not occur

Slit2 prevents neutrophil recruitment and renal ischemia-reperfusion injury

Neutrophils recruited to the postischemic kidney contribute to the pathogenesis of ischemia-reperfusion injury (IRI), which is the most common cause of renal failure among hospitalized patients. The Slit family of secreted proteins inhibits chemotaxis of leukocytes by preventing activation of Rho-family GTPases, suggesting that members of this family might modulate the recruitment of neutrophils and the resulting IRI. Here, in static and microfluidic shear assays, Slit2 inhibited multiple steps required for the infiltration of neutrophils into tissue. Specifically, Slit2 blocked the capture and firm adhesion of human neutrophils to inflamed vascular endothelial barriers as well as their subsequent transmigration. To examine whether these observations were relevant to renal IRI, we administered Slit2 to mice before bilateral clamping of the renal pedicles. Assessed at 18 hours after reperfusion, Slit2 significantly inhibited renal tubular necrosis, neutrophil and macrophage infiltration, and rise in plasma creatinine. In vitro, Slit2 did not impair the protective functions of neutrophils, including phagocytosis and superoxide production, and did not inhibit neutrophils from killing the extracellular pathogen Staphylococcus aureus. In vivo, administration of Slit2 did not attenuate neutrophil recruitment or bacterial clearance in mice with ascending Escherichia coli urinary tract infections and did not increase the bacterial load in the livers of mice infected with the intracellular pathogen Listeriamonocytogenes. Collectively, these results suggest that Slit2 may hold promise as a strategy to combat renal IRI without compromising the protective innate immune response. Copyright © 2013 by the American Society of Nephrology

Antibacterial and Anti-Inflammatory Effects of Apolipoprotein E

Apolipoprotein E (APOE) is a lipid-transport protein that functions as a key mediator of lipid transport and cholesterol metabolism. Recent studies have shown that peptides derived from human APOE display anti-inflammatory and antimicrobial effects. Here, we applied in vitro assays and fluorescent microscopy to investigate the anti-bacterial effects of full-length APOE. The interaction of APOE with endotoxins from Escherichia coli was explored using surface plasmon resonance, binding assays, transmission electron microscopy and all-atom molecular dynamics (MD) simulations. We also studied the immunomodulatory activity of APOE using in vitro cell assays and an in vivo mouse model in combination with advanced imaging techniques. We observed that APOE exhibits anti-bacterial activity against several Gram-negative bacterial strains of Pseudomonas aeruginosa and Escherichia coli. In addition, we showed that APOE exhibits a significant binding affinity for lipopolysaccharide (LPS) and lipid A as well as heparin. MD simulations identified the low-density lipoprotein receptor (LDLR) binding region in helix 4 of APOE as a primary binding site for these molecules via electrostatic interactions. Together, our data suggest that APOE may have an important role in controlling inflammation during Gram-negative bacterial infection

PROBLEM OF MARINE INVASIVE SPECIES AND THEIR IMPACT ON SINGAPORE WATERS

Master'sMASTER OF SCIENC

Rapid in vitro and in vivo Evaluation of Antimicrobial Formulations Using Bioluminescent Pathogenic Bacteria

Basic and translational research needs rapid methods to test antimicrobial formulations. Bioluminescent bacteria and advanced imaging systems capable of acquiring bioluminescence enable us to quickly and longitudinally evaluate the efficacy of antimicrobials. Conventional approaches, such as radial diffusion and viable count assays, are time-consuming and do not allow for longitudinal analysis. Bioluminescence imaging is sensitive and gives vital spatial and temporal information on the infection status in the body. Here, using bioluminescent Pseudomonas aeruginosa, we describe an in vitro and an in vivo approach to rapidly evaluate the antimicrobial efficacy of the host-defense peptide TCP-25

Blastocystis ratti Contains Cysteine Proteases That Mediate Interleukin-8 Response from Human Intestinal Epithelial Cells in an NF-κB-Dependent Manner▿

Blastocystis is a ubiquitous enteric protozoan found in the intestinal tracts of humans and a wide range of animals. Evidence accumulated over the last decade suggests association of Blastocystis with gastrointestinal disorders involving diarrhea, abdominal pain, constipation, nausea, and fatigue. Clinical and experimental studies have associated Blastocystis with intestinal inflammation, and it has been shown that Blastocystis has potential to modulate the host immune response. Blastocystis is also reported to be an opportunistic pathogen in immunosuppressed patients, especially those suffering from AIDS. However, nothing is known about the parasitic virulence factors and early events following host-parasite interactions. In the present study, we investigated the molecular mechanism by which Blastocystis activates interleukin-8 (IL-8) gene expression in human colonic epithelial T84 cells. We demonstrate for the first time that cysteine proteases of Blastocystis ratti WR1, a zoonotic isolate, can activate IL-8 gene expression in human colonic epithelial cells. Furthermore, we show that NF-κB activation is involved in the production of IL-8. In addition, our findings show that treatment with the antiprotozoal drug metronidazole can avert IL-8 production induced by B. ratti WR1. We also show for the first time that the central vacuole of Blastocystis may function as a reservoir for cysteine proteases. Our findings will contribute to an understanding of the pathobiology of a poorly studied parasite whose public health importance is increasingly recognized