Elicitation of epithelial-derived immune effectors by outer membrane vesicles of 1 nontypeable Haemophilus influenzae 2 3

24 Outer membrane vesicles (OMVs) are produced by all Gram negative 25 microorganisms studied to date. The contribution of OMVs to biological processes is 26 diverse; mediation of bacterial stress response, selective packaging and secretion of 27 virulence determinants, modulation of host immune response, and contribution to biofilm 28 formation and stability. First characterized as transformasomes in Haemophilus, these 29 membranous blebs facilitate transfer of DNA amongst bacteria. Nontypeable 30 Haemophilus influenzae (NTHI), an opportunistic pathogen of the upper and lower 31 respiratory tracts, produces OMVs in vivo, however, there is a paucity of information 32 regarding both the composition and role of OMVs during NTHI colonization and 33 pathogenesis. We demonstrated that purified NTHI vesicles are 20-200nm in size and 34 contain DNA, adhesin P5, IgA endopeptidase, serine protease, and heme utilization 35 protein, suggesting a multifaceted role in virulence. NTHI OMVs can bind to human 36 pharyngeal epithelial cells resulting in a timeand temperature-dependent aggregation 37 on the host cell surface with subsequent internalization. OMVs co-localized with the 38 endocytosis protein caveolin, indicating that internalization is mediated by caveolae, 39 cholesterol-rich lipid raft domains. Upon interaction with epithelial cells, NTHI OMVs 40 stimulate significant release of the immunomodulatory cytokine IL-8 as well as the 41 antimicrobial peptide LL-37. Thus, we demonstrated that NTHI OMVs contain 42 virulence-associated proteins that dynamically interact with and invade host epithelial 43 cells. Beyond their ability to mediate DNA transfer in Haemophilus, OMV stimulation of 44 host immunomodulatory cytokine and antimicrobial peptide release supports a dynamic 45 role for vesiculation in NTHI pathogenesis and clinically relevant disease progression. 46 on N ovem er 2, 2017 by gest http/iai.asm . rg/ D ow nladed fom Introduction 47 Nontypeable Haemophilus influenzae (NTHI) is a Gram negative commensal 48 inhabitant of the human nasopharynx that causes diseases of the mammalian upper 49 and lower airways. NTHI predominates in otitis media (OM) and other localized 50 respiratory diseases such as acute sinusitis and community-acquired pneumonia and 51 has important consequences in patients with chronic obstructive pulmonary disease 52 (COPD) or cystic fibrosis, with direct and indirect costs of diagnosing and managing OM 53 exceeding $5 billion annually in the U.S. alone (49), (36), (41), (21), (22), (42), (33). To 54 gain a more comprehensive understanding of the dynamic interplay between microbe55 expressed virulence factors and the host immune response, particularly during the 56 transition from commensal to pathogen, the secretion of virulence-associated bacterial 57 components must be further investigated. The blebbing of outer membrane vesicles is 58 one common and recently-appreciated mechanism of release of virulence-associated 59 proteins by Gram negative bacteria, such as NTHI. 60 Outer membrane vesicles (OMVs) are ubiquitously shed among pathogenic and 61 non-pathogenic Gram negative bacteria (26), (7), (23). Vesicles are composed of 62 lumenal periplasmic material bound by a layer of bacterial outer membrane components 63 (e.g. lipopolysaccharide, phospholipids, outer membrane proteins) and play an active 64 role in pathogenesis, proposed to be vehicles for virulence factor delivery to host cells 65 (17), (3), (15), (52), (19), (20), (4). The functions of bacterial OMVs are versatile, such 66 as regulation of the bacterial stress response, reduction of toxic compounds in the 67 environment, quorum sensing, and co-aggregation of bacteria thus enabling 68 colonization and biofilm formation (30), (24), (11), (54), (44), (35), (7), (56). In addition 69 on N ovem er 2, 2017 by gest http/iai.asm . rg/ D ow nladed fom to these functions, OMVs mediate an immunomodulatory role in bacterial pathogenesis 70 (4). Moraxella OMVs contain the immunostimulatory superantigen molecule MID that 71 induces a non-specific immune response that fails to target Moraxella whole bacterial 72 cells, thus contributing to pathogenesis (51). Further, OMVs stimulate TLR-2, TLR-4 73 and TLR-9 host cell signaling molecules and the release of immunomodulatory 74 cytokines such as IL-6, IL-8, and IL-12 (50), (23), (7). This ability of OMVs to modulate 75 robust host immune responses presents OMVs as attractive vaccine candidates; both 76 natural and engineered OMVs have been shown to confer immunity to challenges with 77 pathogenic bacteria (50), (51). 78 NTHI, a versatile opportunistic pathogen, has been reported to shed outer 79 membrane vesicles both in vitro (6) and in vivo (14) thus, NTHI OMVs are likely to be 80 immunomodulatory, contributing to NTHI pathogenesis. Outer membrane vesicles 81 produced by Haemophilus were described to have a role in natural competence (6) and 82 were characterized as transformasomes due to their ability to transport DNA, both 83 internally and on their surface, among bacterial cells (18). Importantly, Haemophilus 84 vesicles could induce blood-brain barrier permeability during experimental meningitis 85 (55). The biological role of outer membrane vesicles produced by clinically relevant 86 NTHI strains remains unknown and there is a paucity of comprehensive information 87 regarding the components of vesicles and their role in clinically relevant Haemophilus 88 disease states. Thus, we investigated OMV production, identified vesicle protein cargo, 89 and monitored OMV interaction and stimulation of host respiratory and middle ear 90 epithelial cells. We further monitored this interaction with host epithelial cells by 91 fluorescence microscopy and elucidated the mechanism of OMV internalization. The 92 on N ovem er 2, 2017 by gest http/iai.asm . rg/ D ow nladed fom ability of OMVs to modulate host cell responses suggests that outer membrane vesicles 93 are a potent contributor to NTHI pathogenesis and play a role in the delicate balance 94 between the commensal and pathogenic lifestyles of NTHI. 95