Azurophil Granule Proteins Constitute the Major Mycobactericidal Proteins in Human Neutrophils and Enhance the Killing of Mycobacteria in Macrophages.docVIP

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Azurophil Granule Proteins Constitute the Major Mycobactericidal Proteins in Human Neutrophils and Enhance the Killing of Mycobacteria in Macrophages.doc

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Azurophil Granule Proteins Constitute the Major Mycobactericidal Proteins in Human Neutrophils and Enhance the Killing of Mycobacteria in Macrophages

AzurophilGranuleProteinsConstitutetheMajor MycobactericidalProteinsinHumanNeutrophilsand EnhancetheKillingofMycobacteriainMacrophages PrajnaJena1,SoumitraMohanty1,TirthankarMohanty2,StephanieKallert3,MatthiasMorgelin2, ThomasLindstr?m4,NielsBorregaard5,SteffenStenger3,AvinashSonawane1*.,OleE.S?rensen2*. 1School of Biotechnology, Campus-11, KIIT University, Bhubaneswar, Orissa, India, 2Division of Infection Medicine, Department of Clinical Sciences, Lund University, Lund, Sweden, 3Institute for Medical Microbiology and Hygiene, University of Ulm, Ulm, Germany, 4Department of Infectious Disease Immunology, Statens Serum Institute,Copenhagen,Denmark,5DepartmentofHematology,UniversityofCopenhagen,Copenhagen,Denmark Abstract Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophilsalsoplayacriticalroleininnateimmunitytotuberculosis,presumablybytheirdifferentantibacterialgranule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophilgranuleproteins(AZP)weremoreactiveagainstmycobacteriacomparedtoothergranuleproteinsandcytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. ExogenousadditionofAZPtomurinemacrophageRAW264.7,THP-1andperipheralbloodmonocyte-derivedmacrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescencestudiesshowedthatmacrophagesactivelyendocytoseneutrophilgranularproteins.Treatmentwith AZPresultedinincreaseinco-localiz