Biological Characterization of Moraxella Bovis Lipopolysaccharide Kristine Ann Johansen Iowa State University

Iowa State University Capstones, Theses and Retrospective Theses and Dissertations Dissertations 1-1-1988 Biological characterization of Moraxella bovis lipopolysaccharide Kristine Ann Johansen Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/rtd Recommended Citation Johansen, Kristine Ann, "Biological characterization of Moraxella bovis lipopolysaccharide" (1988). Retrospective Theses and Dissertations. 18329. https://lib.dr.iastate.edu/rtd/18329 This Thesis is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Retrospective Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Biological charact e rization of Moraxella bovis lipopolysaccharide by Kris t ine Ann Johansen c.. 3 A Thesis Submitted t o the Graduate Faculty in Part ial Fulfillment of the Requirements f or the Degree of MASTER OF SCIENCE Interdepartmental Program: Immunobiology Major: Immunobiology Signatures have been redacted for privacy I owa St a t e Univers ity Ames, I owa 1988 ii TABLE OF CONTENTS Page INTRODUCTION 1 LITERATURE REVIEW 3 Perspect ive 3 LPS Structure and Re activity 3 LPS-Cellular Interactions 6 LPS and Biol ogi cal Reactions 14 LPS and Cy t okine Induction 22 Current Advances 26 MATERIALS AND METHODS 29 Organisms 29 Animals 29 Hot Phenol-wa ter Extra ction of LPS 30 Comp ositional Analysi s of LPS 3 1 SDS-PAGE Analysis 33 Silver Staining of LPS 33 We s tern Blo t Analysi s 34 Pyr ogenicity As say 34 Local Shwartzman React ion 35 Stimulation of Murine Pe ritoneal Exuda t e Cells 36 Peritonal Exudate Cell Toxi city 37 Interleukin 1 Assay 38 Tumor Necr osis Fact or Assay 38 Sp l enic Bl as t ogenesi s Assay 40 iii Page Lethality in Galactosamine-sensit ized mice 41 RESULTS 42 Moraxella LPS Compositional Analysis 42 SDS-PAGE Analysis 42 Western Blot Analysis 43 Pyrogenicity 47 Local Shwartzman Reaction 52 Splenic Blastogenesis 55 Tumor Necrosis Factor Assay 64 Interleukin 1 Assay 70 Lethality in Galac tosamine-sensitized Mice 70 Peritoneal Exudate Cell Toxicity 75 DISCUSSION 77 Compositional Analysis and SDS-PAGE Profiles 77 Western Blot Analysis 79 Pyrogenicity 80 Local Shwartzman Reaction 81 Splenic Blastogenesis 82 Tumor Necrosis Factor Assay 83 Interleukin 1 Assay 84 Lethal Toxicit y in Galactosamine-sensit ized Mice 85 Peritoneal Exudate Cell Toxicity 86 SUMMARY AND FUTURE DIRECTIONS 87 iv Page REFERENCES 90 ACKNOWLEDGEMENTS 102 1 INTRODUCTION Moraxella bovis, a small, gram-negative, nonfermenting cocco- bacillus , is recognized as the primary etiologic agent of infectious bovine keratoconjunctivitis (IBK), or pinkeye . IBK may result in temporary or permanent blindness of affected eyes, as well as reduced feed conversion, and so is a disease of humanitarian consideration and economic importance (Marshall et al . , 1985; Horsnell and Teale, 1987). Although o ther factors, such as ultraviolet irradiation (Kopecky et al., 1980) and infection with Mycoplasma bovoculi (Rosenbusch, 1983) and infectious bovine rhinotracheitis virus (Pugh et al., 1970), have been shown to contribute to l esion production, ~ · bovis has two recognized virulence factors . These are the beta hemolysin and pili, both of which are outer membrane constituents and are required for virulence (Sandhu et al. , 1977; Pedersen et al . , 1972) . Most attempts to develop a vaccine for IBK have met limited success in the field (Pugh et al., 1982) . Many of these trials have involved whole cell bacterins (from hemolytic and nonhemolytic strains) or pili preparations; however, although there is some protection against homologous challenge, heterologous protection is variable at best (Pugh et al., 1976). Additionally, treatment for IBK may be time consuming and expensive, especially when considering reports that all animals in an infected herd are most likely exposed to ~ · bovis within several days (Ostle and Rosenbusch, 1985) . The outer membrane has been the principal site for investigation of M. bovis virulence factors because of the importance of the gram-negative 2 outer membrane interface with t he host environment (Ostle and Rosenbusch , 1986) . The outer membrane is t he site f or many virulence and res i s tance factors for gr am- negative or ganisms ; it is a l so accessible for recognition by the host immune system and thus , outer membrane antigens may be valuable f or diagnosis. One ma jor component of the outer membrane of gr am- negative bacteri a is the lipopolysaccharide (LPS) , which is r esponsible for a l a r ge number of pathophysiologic effects. Li popol y- saccharide may a lso be protective to the organism, s ince it is import ant in serum resistance. Due t o its s truc t ure and outer membrane location, LPS i s highl y i mmunogenic . To date, t hough , research on t he LPS of Mo r axella bovis and its pot entia l r ole as a virulence or resis t ance factor in IBK has been limit ed. Few data on the LPS of Moraxella spp . are available (Adams e t a l., 1970; Veslemoy e t al. , 1980; Ho ris berger and De ntan, 1980) , although t he pi lin gene has already be c l oned (Ma rrs e t a l., 1985) . The purpose of this wo r k was t o l ay t he foundation for ascerta ining t he r ole of M. bovis LPS in the pathogenesis of I BK by char ac t erization of i t s LPS, which included analysis of i t s chemical composit ion, polyac r yl amide gel pr ofile , and a numbe r of biological assays . 3 LITERATURE REVIEW Perspective Bacterial products may often have a profound effect on their hosts. One such class of bacterial products are endotoxins, which are part of the cell wall of gram- negative bacteria. This designation serves t o distinguish endotoxins from excreted toxic bacterial products which are termed "exotoxins". Endotoxins are composed of lipopol ysaccharide (LPS) and associated protein. Endotoxin (and LPS) may cause a tremendous variet y of reactions in the whole animal and on many different cell t ypes, which include the induction of cytokines and causing massive changes in the blood coagulation system. LPS has also been shown t o have a role in the pathogenicity of some gram-negative microorganisms. LPS Structure and Reac tivit y Before delving into the biological effects of LPS and endotoxin, it is perhaps worthwhile to discuss the structure of the molecule and relate it to the biological activity of the molecule . To begin such a review, it is important t o distinguish clearl y the difference between endotoxin and LPS . Lipopolysaccharide is comprised of three distinct regions, t he " 0- antigen", the core polysaccharide and lipid A. Endotoxin is LPS plus associated protein; this protein contributes to differences in biological act ivity seen between LPS and endotoxin (Morrison and Ulevitch, 1978; Br adley, 1979) . The amount of protein extracted along with the LPS depends upon the method employed for extraction (Morrison and Ulevitch, 4 1978) . So-called Boivin LPS is extracted using trichlor oacetic acid a nd produces LPS contaminated with proteins (endotoxin) (Staub, 1965), while so-called Westphal LPS i s extracted with phenol and yields r elatively pure preparations of LPS (Westphal and Jann, 1965) ; LPS may also be extracted successfully using aqueous butanol (Morrison and Leive , 1975) . In addit ion, endot oxin-associ a t ed protein (EAP) has been f ound t o exhibit interleukin 1 ( Il-1 ) properties , such as the direct stimulat ion of serum amyloid A production by res ting human T cells; its activity is no t blocked by polymyxin B (Johns et al . , 1988), an antibiotic polypeptide which binds the lipid A moiety of LPS and abr ogates its activity. Ot her s tudies have demonstrated that EAP has a gr anulopoietic colony- s timula ting activity on human peripheral blood lymphocytes and bone marrow precursor cells (Bjornson et al. , 1988). The lipid A regi on of LPS is generally considered t o be the mos t conserved part of the molecule and usually consist s of a diglucosamine backbone with amide and ester-linked l ong cha in fatty acids, as well as pyrophosphate groups (Wilkinson, 1977) . The amide linked fatty acids appear to be uniformly beta-hydroxy substituted. There appeared to be no particular significance, in terms of r eactivit y , for chain length of ester-linked fatty acids and bo th odd and even numbered chains are found; however, it has been recently found that fatty acid substituents of synthetic lipid A are important in imparting immunoreactivity (Kumazawa e t al., 1988) . In these s tudies, different length acyl gr oups (Cl2-Cl6) and different s t ereoisomers for lipid A subunit analogs (4- 0- phosphono- D- glucosamine derivatives) were used t o s tudy the mitogenicity, 5 pyrogenicit y , let hal toxicity, local Shwartzman reaction, as well as cytokine induction (e . g . , TNF-inducing activity); it was found that fatty acid substituents wit h the (RR) configuration had greater endotoxic activit ies than the corresponding (SS) stereoisomers and that acyl chain l ength also affected reactivity. Some investigations have suggest ed that protein may be covalently bound to the lipid A region (Wilkinson, 1977). Lipid A is also the most internal moiet y of LPS and is probably int ercalated in the lipid bilayer of the out er leaflet of t he cell (Osborn, 1979) . Lipid A is linked through a unique sugar to the core region. This unusual sugar is 2- keto 3- deoxy- octulosonate (KDO).

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