Targeting Streptococcus pneumoniae UDP-glucose pyrophosphorylase (UGPase): in vitro validation of a putative inhibitor

Monica Sharma; Swati Sharma; Pallab Ray; Anuradha Chakraborti

doi:10.33393/dti.2020.2103

Authors

Monica Sharma Molecular Genetics Laboratory, Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education & Research (PGIMER), Chandigarh - India
Swati Sharma Molecular Genetics Laboratory, Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education & Research (PGIMER), Chandigarh - India
Pallab Ray Bacteriology Laboratory, Department of Medical Microbiology, Postgraduate Institute of Medical Education & Research (PGIMER), Chandigarh - India
Anuradha Chakraborti Molecular Genetics Laboratory, Department of Experimental Medicine and Biotechnology, Postgraduate Institute of Medical Education & Research (PGIMER), Chandigarh - India

DOI:

https://doi.org/10.33393/dti.2020.2103

Keywords:

Capsule, galU, UDP, Virulence, Inhibitor, Pneumococcus

Abstract

Background: Genome plasticity of Streptococcus pneumoniae is responsible for the reduced efficacy of various antibiotics and capsular polysaccharide based vaccines. Therefore targets independent of capsular types are sought to control the pneumococcal pathogenicity. UcrDP-glucose pyrophosphorylase (UGPase) is one such desired candidate being responsible for the synthesis of UDP-glucose, a sugar-precursor in capsular biosynthesis and metabolic Leloir pathway. Being crucial to pneumococcal pathobiology, the effect of UGPase inhibition on virulence was evaluated in vitro.

Methods: A putative inhibitor (UDP) was evaluated for effective inhibitory concentration in S. pneumoniae and A549 cells, its efficacy and toxicity. Effect of UDP on adherence and phagocytosis was measured in human respiratory epithelial (A549 and HEp-2) and macrophage (THP1 and J774.A.1) cell lines respectively.

Results: A differential effective inhibitory concentration of UDP for UGPase inhibition was observed in S. pneumoniae and A549 cells i.e. 5 µM and 100 µM respectively. UDP treatments lowered percent cytotoxicity in pneumococcal infected monolayers and didn't exert adverse effects on viabilities. S. pneumoniae adherence to host cells was decreased significantly with UDP treatments. UDP induced the secretion of IL-1β, TNF-α, IL-6, and IL-8 and increased pneumococcal phagocytosis.

Conclusion: Our study shows UDP mediated decrease in the virulence of S. pneumoniae and demonstrates UDP as an effective inhibitor of pneumococcal UGPase.

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References

Musher DM. Streptococcus pneumoniae. In: Mandell GL, Bennett JE, Dolin R, eds. Mandell, Douglas, and Bennett’s principles and practice of infectious diseases. 7th ed. Philadelphia, PA: Churchill Livingstone Elsevier 2010; 2623-2642.

Troeger C, Blacker B, Khalil IA, et al., SRM GBD 2016 Lower Respiratory Infections Collaborators. Estimates of the global, regional, and national morbidity, mortality, and aetiologies of lower respiratory infections in 195 countries, 1990-2016: a systematic analysis for the Global Burden of Disease Study 2016. Lancet Infect Dis. 2018;18:1191-1210.

Wahl B, O’Brien KL, Greenbaum A, et al. Burden of Streptococcus pneumoniae and Haemophilus influenzae type b disease in children in the era of conjugate vaccines: global, regional, and national estimates for 2000-15. Lancet Glob Health. 2018;6:e744-e757.

Wahl B, Sharan A, Deloria Knoll M, et al. National, regional, and state-level burden of Streptococcus pneumoniae and Haemophilus influenzae type b disease in children in India: modeled estimates for 2000-15. Lancet Glob Health. 2019;7:e735-e747.

Kim SH, Song JH, Chung DR, et al. Changing trends in antimicrobial resistance and serotypes of Streptococcus pneumoniae isolates in Asian countries: an Asian Network for Surveillance of Resistant Pathogens (ANSORP) study. Antimicrob Agents Chemother. 2012;56:1418-1426.

Azarian T, Grant LR, Arnold BJ, et al. The impact of serotype-specific vaccination on phylodynamic parameters of Streptococcus pneumoniae and the pneumococcal pan-genome. PLoS Pathog. 2018;14:e1006966.

McLaughlin JM, Jiang Q, Isturiz RE, et al. Effectiveness of 13-valent pneumococcal conjugate vaccine against hospitalization for community-acquired pneumonia in older US adults: a test-negative design. Clin Infect Dis. 2018;67:1498-1506.

Mitchell AM, Mitchell TJ. Streptococcus pneumoniae: virulence factors and variation. Clin Microbiol Infect. 2010;16:411-418.

Brooks LRK, Mias GI. Streptococcus pneumoniae’s virulence and host immunity: aging, diagnostics, and prevention. Front Immunol. 2018;9:1366.

Magee AD, Yother J. Requirement for capsule in colonization by Streptococcus pneumoniae. Infect Immun. 2001;69:3755-3761.

Shainheit MG, Mulé M, Camilli A. The core promoter of the capsule operon of Streptococcus pneumoniae is necessary for colonization and invasive disease. Infect Immun. 2014;82:694-705.

García E, Llull D, López R. Functional organization of the gene cluster involved in the synthesis of the pneumococcal capsule. Int Microbiol. 1999;2:169-176.

García E, Llull D, Muñoz R, Mollerach M, López R. Current trends in capsular polysaccharide biosynthesis of Streptococcus pneumoniae. Res Microbiol. 2000;151:429-435.

Mollerach M, López R, García E. Characterization of the galU gene of Streptococcus pneumoniae encoding a uridine diphosphoglucose pyrophosphorylase: a gene essential for capsular polysaccharide biosynthesis. J Exp Med. 1998;188:2047-2056.

Chang HY, Lee JH, Deng WL, Fu TF, Peng HL. Virulence and outer membrane properties of a galU mutant of Klebsiella pneumoniae CG43. Microb Pathog. 1996;20:255-261.

Priebe GP, Dean CR, Zaidi T, et al. The galU gene of Pseudomonas aeruginosa is required for corneal infection and efficient systemic spread following pneumonia but not for infection confined to the lung. Infect Immun. 2004;72:4224-4232.

Bonofiglio L, García E, Mollerach M. Biochemical characterization of the pneumococcal glucose 1-phosphate uridylyltransferase (GalU) essential for capsule biosynthesis. Curr Microbiol. 2005;51:217-221.

Bonofiglio L, García E, Mollerach M. The galU gene expression in Streptococcus pneumoniae. FEMS Microbiol Lett. 2012;332:47-53.

Cools F, Torfs E, Vanhoutte B, et al. Streptococcus pneumoniae galU gene mutation has a direct effect on biofilm growth, adherence and phagocytosis in vitro and pathogenicity in vivo. Pathog Dis. 2018;76.

Frey PA. The Leloir pathway: a mechanistic imperative for three enzymes to change the stereochemical configuration of a single carbon in galactose. FASEB J. 1996;10:461-470.

Alvaro Berbis M, Maria Sanchez-Puelles J, Javier Canada F, Jimenez-Barbero J. Structure and function of prokaryotic UDP-glucose pyrophosphorylase, a drug target candidate. Curr Med Chem. 2015;22:1687-1697.

Mollerach M, García E. The galU gene of Streptococcus pneumoniae that codes for a UDP-glucose pyrophosphorylase is highly polymorphic and suitable for molecular typing and phylogenetic studies. Gene. 2000;260:77-86.

Zavala A, Kovacec V, Levín G, et al. Screening assay for inhibitors of a recombinant Streptococcus pneumoniae UDP-glucose pyrophosphorylase. J Enzyme Inhib Med Chem. 2017;32:203-207.

Twentyman PR, Luscombe M. A study of some variables in a tetrazolium dye (MTT) based assay for cell growth and chemosensitivity. Br J Cancer. 1987;56:279-285.

Hara-Kaonga B, Pistole TG. A dual fluorescence flow cytometric analysis of bacterial adherence to mammalian host cells. J Microbiol Methods. 2007;69:37-43.

Hu BT, Yu X, Jones TR, et al. Approach to validating an opsonophagocytic assay for Streptococcus pneumoniae. Clin Diagn Lab Immunol. 2005;12:287-295.

Kim B, Jeong HK, Kim JH, Lee SY, Jou I, Joe EH. Uridine 5′-diphosphate induces chemokine expression in microglia and astrocytes through activation of the P2Y6 receptor. J Immunol. 2011;186:3701-3709.

Cox MA, Gomes B, Palmer K, et al. The pyrimidinergic P2Y6 receptor mediates a novel release of proinflammatory cytokines and chemokines in monocytic cells stimulated with UDP. Biochem Biophys Res Commun. 2005;330:467-473.

Grbic DM, Degagné É, Larrivée JF, et al. P2Y6 receptor contributes to neutrophil recruitment to inflamed intestinal mucosa by increasing CXC chemokine ligand 8 expression in an AP-1-dependent manner in epithelial cells. Inflamm Bowel Dis. 2012;18:1456-1469.

Ginsburg-Shmuel T, Haas M, Grbic D, et al. UDP made a highly promising stable, potent, and selective P2Y6-receptor agonist upon introduction of a boranophosphate moiety. Bioorg Med Chem. 2012;20:5483-5495.

Hao Y, Liang JF, Chow W, Cheung W, Ko W. P2Y6 receptor-mediated proinflammatory signaling in human bronchial epithelia. PLoS ONE. 2014;9:e106235.

Subramanian K, Henriques-Normark B, Normark S. Emerging concepts in the pathogenesis of the Streptococcus pneumoniae: from nasopharyngeal colonizer to intracellular pathogen. Cell Microbiol. 2019;2:e13077.

Zhang Z, Wang Z, Ren H, et al. P2Y6 agonist uridine 5′-diphosphate promotes host defense against bacterial infection via monocyte chemoattractant protein-1–mediated monocytes/macrophages recruitment. J Immunol. 2011;186:5376-5387.

Idzko M, Ferrari D, Eltzschig HK. Nucleotide signalling during inflammation. Nature. 2014;509:310-317.

Koizumi S, Shigemoto-Mogami Y, Nasu-Tada K, et al. UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis. Nature. 2007;446:1091-1095.

Inoue K, Koizumi S, Kataoka A, Tozaki-Saitoh H, Tsuda M. P2Y(6)-evoked microglial phagocytosis. Int Rev Neurobiol. 2009;85:159-163.

Neher JJ, Neniskyte U, Hornik T, Brown GC. Inhibition of UDP/P2Y6 purinergic signaling prevents phagocytosis of viable neurons by activated microglia in vitro and in vivo. Glia. 2014;62:1463-1475.

Zierhut M, Dyckhoff S, Masouris I, et al. Role of purinergic signaling in experimental pneumococcal meningitis. Sci Rep. 2017;7:44625.