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Mannheimia haemolytica leukotoxin-host cell receptor interactions

Dissertation
Author: Sudarvili Shanthalingam
Abstract:
Mannheimia haemolytica is the primary bacterial pathogen of bovine pneumonic pasteurellosis, an economically important disease of cattle worldwide. Leukotoxin (Lkt) produced by M. haemolytica is the major virulence factor of this organism. The cytolytic activity of Lkt is specific for ruminant leukocytes. Lkt utilizes CD18, the β subunit of β2 -integrins, as its receptor on ruminant leukocytes. Previously, our laboratory mapped the Lkt-binding domain to lie between amino acids (aa) 1-291 of CD18. Therefore, the next logical step was to identify the precise Lkt binding site within this domain and to determine whether co-administration of CD18 peptide analogs would inhibit / mitigate M. haemolytica -caused lung injury. In this study, by using synthetic peptides spanning aa1-291 of bovine CD18 in Lkt-induced cytolysis assays, the precise binding site of Lkt was mapped to aa 5-17 of ruminant CD18. Surprisingly, all the aa of this peptide belong to the predicted signal peptide of CD18. This observation led to the finding that the signal peptide of ruminant CD18 is not cleaved, and that the intact signal peptide renders ruminants susceptible to M. haemolytica Lkt. Site-directed mutagenesis of a single aa in the signal peptide resulted in the cleavage of signal peptide and abrogation of Lkt-induced cytolysis of target cells. This finding indicates that engineering cattle and other ruminants to contain this mutation would provide a novel technology to render them less susceptible to pneumonic pasteurellosis and concomitant economic losses. The peptide spanning aa 5-17 (P17) was used in a calf challenge study which was designed as a 'proof of concept' experiment. Even though the difference in percent volume of lungs exhibiting gross pneumonic lesions between P17-inoculated calves and control peptide-inoculated calves was not statistically significant, M. haemolytica isolated from the lungs of P17-inoculated calves was 100- to 1000-fold less than those from the control peptide-inoculated calves, suggesting that P17 reduced leukotoxic activity in the lungs which enhanced bacterial clearance by phagocytes. It is likely that prolonging the presence and activity of CD18 peptide analog in the lungs, for example by means of a nanoparticle delivery system such as dextran nanospheres, would enhance its protective activity.

TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS……………………………………………………………iii

ABSTRACT………………………………………………………………………….....v

LIST OF TABLES………………………………………………………………..........ix

LIST OF FIGURES……………………………………………………………………..x

GENERAL INTRODUCTION…………………………………………………………1 1. REFERENCES……………………………………………………………….5

CHAPTER ONE

1. ABSTRACT……………………………………………………………………10

2. INTRODUCTION……………………………………………………………...11

3. MATERIALS AND METHODS………………………………………………12

4. RESULTS AND DISCUSSION……………………………………………….14

5. REFERENCES…………………………………………………………………17

6. TABLES………………………………………………………………………..21

7. FIGURES………………………………………………………………………22

CHAPTER TWO

1. ABSTRACT……………………………………………………………………35

2. INTRODUCTION…………………………………………………………….. 36

3. MATERIALS AND METHODS………………………………………………38

vi ii

4. RESULTS………………………………………………………………………42

5. DISCUSSION…………………………………………………………………..47

6. REFERENCES…………………………………………………………………51

7. FIGURES………………………………………………………………………57

CHAPTER THREE

1. ABSTRACT…………………………………………………………………....74

2. INTRODUCTION…………………………………………………………. ….76

3. MATERIALS AND METHODS………………………………………………78

4. RESULTS……………………………………………………………………....84

5. DISCUSSION…………………………………………………………………..87

6. REFERENCES…………………………………………………………………90

7. TABLES………………………………………………………………………..95

8. FIGURES………………………………………………………………………99

CONCLUSION……………………………………………………………………….102

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LIST OF TABLES

Page

CHAPTER ONE

1. Comparison of the amino acid sequence of the CD18 of bison, deer and elk with that of other ruminants and non-ruminants……………………….21

CHAPTER THREE 1. Evaluation and scoring of clinical signs………………………………………..95 2. Bacteria isolated from calves pre-inoculation and at necropsy………………...96 3. Gross pneumonic lesions expressed as a % of total lung volume………………97 4. Number of M. haemolytica (CFU per gram of lung tissue) isolated from the lungs of calves at necropsy…………………………………..98

x

LIST OF FIGURES

Page

CHAPTER ONE 1. PMNs and PBMCs from bison, deer and lysed by leukotoxin………………….22 2. The nucleotide and deduced amino acid sequence of the CD18 of bison, deer and elk………………………………………………………………24 3. Comparison of the deduced amino acid sequences of CDS of bison, deer and elk CD18 with that of ruminants and non-ruminants………………….30

CHAPTER TWO 1. The CD18 signal peptide analog P1 (aa 1-20) and P5 (aa 5-24) inhibit Lkt-induced cytolysis of BL3 cells………………………………………57 2. N- and C-terminal truncations of peptide P5 identify aa 5-17 as the Lkt-binding domain on bovine CD18………………………………….....59 3. Inhibition of Lkt-induced cytolysis of ruminant PMNs by peptide P17 confirms aa 5-17 as the Lkt-binding domain on CD18 of ruminants………61 4. Anti-signal peptide serum binds to membrane CD18 of PMNs of all ruminants tested………………………………………………………………....63 5. The signal peptide of bovine CD18 is not cleaved……………………………...65 6. The signal peptide of CD18 of ruminants contains cleavage-inhibiting glutamine (Q) at aa position -5 relative to the cleavage site, whereas

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that of non-ruminants contain cleavage- conducive glycine (G)…………………67 7. Mutation of glutamine (Q) to glycine (G) at positions -5 of the signal peptide of bovine CD18 abrogates Lkt-induced cytolysis of transfectants expressing CD18 with Q(-5)G mutation…………………………………………69 8. Peptides spanning aa 500-600 of bovine CD18 fail to inhibit Lkt-induced cytolysis of bovine PMNs……………………………………..…...71

CHAPTER THREE 1. Co-incubation of peptide P17 with in vitro cultures of M. haemolytica abrogates leukotoxic activity…………………………………...........................99 2. Mean clinical scores of calves inoculated with M. haemolytica only (Group I), M. haemolytica along with PSC (Group II) and M. haemolytica along with peptide P17 (Group III)……………………………100 3. Representative gross- and histo-pathology of the lungs of calves infected with M. haemolytica with or without peptides………………………..101

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Mannheimia haemolytica leukotoxin – host cell receptor interactions

GENERAL INTRODUCTION

Bovine pneumonic pasteurellosis, more commonly known as shipping fever, is an economically significant respiratory disease of both beef and dairy cattle industry in North America and Western Europe (Mosier, 1997; Ames, 1997). The annual economic losses to the US cattle industry have been estimated to be as high as $1 billion (Bowland and Shewen, 2000). Mannheimia haemolytica is the primary bacterial agent responsible for the pathophysiological events leading to this acute fibrinonecrotizing pneumonia (Whiteley et al., 1992). M. haemolytica is commonly found as a commensal bacterium in the upper respiratory tract of healthy cattle. Exposure of cattle to stress factors or viral or other bacterial infections leads to proliferation of M. haemolytica in the upper respiratory tract. Once present in high levels, it enters the alveolar spaces through repeated aspiration of infected droplets and sloughed cells/tissues. Here, it initiates an inflammatory cascade, causing pneumonia with the massive neutrophil influx along with accumulation of fibrin and subsequent necrosis in the alveolar spaces. M. haemolytica has been isolated worldwide, but the prevalence of disease strongly correlates with Western animal management practices that include overcrowding and transport. The organism can also cause pneumonic and septic disease in other ruminants, including domestic sheep (Filion et al., 1984), goats (Msra et al., 1970), bighorn sheep (Foreyt et al., 1994, Dassanayake et al., 2009), and bison (Dyer and Ward, 1998). M. haemolytica produces several virulence factors which include the capsule, outer membrane proteins, adhesins, neuraminidase, lipopolysaccharide and leukotoxin (Lkt; Confer et al., 1990). Of these virulence factors, Lkt has been accepted as the most important one based on the observations that Lkt-deletion mutants induce much milder lung lesions and reduced mortality

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than the wild-type organisms (Petras et al., 1995; Tatum et al., 1998; Highlander et al., 2000; Dassanayake et al., 2009). Lkt produced by M. haemolytica is a member of the RTX (r epeats in t ox in) family of toxins produced by a number of Gram-negative bacteria. Toxins of the RTX family lyse their target cells primarily through formation of pores (Coote, 1992) which leads to the efflux of K + , influx of Ca 2+ , colloidal osmotic swelling and eventual cell lysis (Clinkenbeard et al., 1989). The cytolytic activity of M. haemolytica Lkt is specific for ruminant leukocytes (Kaehler et al., 1980; Shewen and Wilkie, 1982; Chang et al., 1986). While all subsets of leukocytes are susceptible to Lkt-induced cytolysis, polymorphonuclear leukocytes (PMNs) are the most susceptible subset. Moreover, PMN depletion in calves has been shown to drastically reduce the lung lesions. Lysis of alveolar macrophages and PMNs impairs the phagocytic ability of the host which facilitates proliferation and survival of bacteria within the lung. Cytolysis of these cells also results in the release of their proteolytic enzymes and pro-inflammatory substances, which cause structural degradation of the alveolar epitheilial linings. Therefore, Lkt-induced lysis and de- granulation of the alveolar macrophages and PMNs are responsible for the acute inflammation and lung injury characteristic of this disease. Previously, our laboratory and that of others have independently shown that the cytotoxic effect of Lkt on bovine and ovine leukocytes is mediated by Lkt-β 2 -integrin interactions (Wang et al., 1998; Ambagala et al., 1999; Li et al., 1999). β 2 -integrins are leukocyte-specific integrins that are critical for homing of leukocytes to the sites of inflammation, phagocytosis, antigen presentation, and cytotoxicity (Gamberg e al., 1998; Luscinskas et al., 1989). They are heterodimeric glycoproteins composed of α subunit (CD11), and β subunit (CD18). CD18 associates with four distinct α chains to give rise to four different β 2 -integrins: CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), CD11c/CD18 (CR4), and CD11d/CD18 (Gahmberg et al., 1998;

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and Noti et al., 2000). In previous studies in our laboratory, the recombinant expression of bovine or ovine CD18 in Lkt non-susceptible cell lines rendered them susceptible to Lkt-induced cytolysis, suggesting that CD18, the β subunit of β 2 -integrins, serves as the receptor for Lkt on leukocytes (Deshpande et al., 2002; Liu et al., 2007; Dassanayake et al., 2007a). Monomeric expression of CD18 on a transfectant cell-line confirmed that CD18 is the functional receptor for Lkt (2007b). Subsequently, by constructing bovine-murine CD18 chimeras, the Lkt-binding site was mapped to a domain on CD18 encompassing amino acids 1 to 291 (Gopinath et al., 2005). The next logical step, therefore, was to identify the precise binding site of Lkt within the CD18 domain encompassing amino acids 1-291, which formed the first objective of this study. Binding of a ligand to its receptor could potentially be inhibited by synthetic peptides representing the amino acids comprising the binding site of the ligand on the receptor (Tibetts et al., 1999, 2000). Therefore the first hypothesis of this study was that peptide analogs of bovine CD18 will inhibit the Lkt-induced cytolysis of bovine leukocytes. Cytotoxicity assays with a nested set of peptides spanning amino acids 1 to 291 of CD18 identified the Lkt-binding domain to lie between the amino acids 5 to 17. Surprisingly, these amino acids comprise most of the amino acids from signal peptide of CD18, which suggested that the signal peptide of CD18 of cattle, and possibly other ruminants may not be cleaved. However, the paradigm dictates that the signal peptides of membrane proteins are cleaved once the nascent proteins reach the endoplasmic reticulum for the post-translational modifications. Further experiments were designed to answer the question as to whether the signal peptide of CD18 of ruminants remains intact on the mature CD18 molecule, and if so, whether the intact signal peptide renders ruminants susceptible to M. haemolytica Lkt.

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The nucleotide and predicted amino acid sequence of CD18 of five ruminants were available in the GenBank (cattle, buffalo, domestic sheep, bighorn sheep and goat). In order to enhance the validity of theoretical observations that could be made, the cDNA encoding CD18 of bison, deer and elk were cloned. The molecular cloning of CD18 of bison, deer and elk, and their comparison with that of other ruminants and non-ruminants are described in the manuscript in Chapter 1 which is under review for publication in Veterinary Immunology and Immunopathology. The results of the other experiments which determined that the signal peptide of CD18 of ruminants is indeed not cleaved, and that the intact signal peptide of CD18 renders the ruminants susceptible to M. haemolytica Lkt, are described in the manuscript in Chapter 2 which has been published in the Proceedings of the National Academy of Science, USA (Volume 106, pages 15448-15453). The second hypothesis of this study was that the peptide analogs of CD18 will inhibit lung lesions caused by M. haemolytica in calves. The results of this ‗proof of concept‘ study involving the endobronchial co-inoculation of a CD18 peptide analog with M. haemolytica are described in the manuscript in Chapter 3.

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REFERENCES 1. Ambagala TC, Ambagala AP, Srikumaran S. 1999. The leukotoxin of Pasteurella haemolytica binds to beta(2) integrins on bovine leukocytes. FEMS Microbiol Lett 179:161-167. 2. Ames TR. 1997. Dairy calf pneumonia: the disease and its impact. Vet Clin N Am Food Anim Pract 13:379-391. 3. Bowland SL, Shewn PE. 2000. Bovine respiratory disease: commercial vaccines currently available in Canada. Can Vet J 41:33-48. 4. Chang YF, Renshaw HW, Martens RJ, Livingston Jr CW. 1986. Pasteurella haemolytica leukotoxin: chemiluminescent responses of peripheral blood leukocytes from several different mammalian species to leukotoxin- and opsonin-treated living and killed Pasteurella haemolytica and Staphylococcus aureus. Am J Vet Res 47: 67-74. 5. Clinkenbeard KD, Mosier DA, Confer AW. 1989. Transmembrane pore size and role of cell swelling in cytotoxicity caused by Pasteurella haemolytica leukotoxin. Infect Immun 57:420-425. 6. Clinkenbeard KD, Upton ML. 1991. Lysis of bovine platelets by Pasteurella haemolytica leukotoxin. Am J Vet Res 52:453-57. 7. Confer AW, Panciera RJ, Clinkenbeard KD, Mosier DA. 1990. Molecular aspects of virulence of Pasteurella haemolytica. Can J Vet Res 54:S48-52. 8. Coote JG. 1992. Structural and functional relationships among the RTX toxin determinants of Gram-negative bacteria. FEMS Microbiol Rev 88:137-62.

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9. Dassanayake RP, Maheswaran SK, Srikumaran S. 2007a. Monomeric expression of bovine ß 2 -integrin subunits reveals their role in Mannheimia haemolytica leukotoxin- induced biological effects. Infect Immun 75:5004-5010. 10. Dassanayake RP, Shanthalingam S, Davis WC, Srikumaran S. 2007b. Mannheimia haemolytica leukotoxin-induced cytolysis of ovine (Ovis aries ) leukocytes is mediated by CD18, the ß subunit of ß 2 -integrins. Microb Pathog 42:167-173. 11. Dassanayake RP, Shanthalingam S, Herndon CN, Lawrence PK, Frances CE, Potter KA, Foreyt WJ, Clinkenbeard KD, Srikumaran S. 2009. Mannheimia haemolytica serotype A1 exhibits differential pathogenicity in two related species Ovis Canadensis and Ovis aries. Vet Microbiol 133:366-371. 12. Deshpande MS, Ambagala TC, Ambagala APN, Kehrli Jr ME, Srikumaran S. 2002. Bovine CD18 is necessary and sufficient to mediate Mannheimia (Pasteurella ) haemolytica leukotoxin-induced cytolysis. Infect Immun 70:5058-5068. 13. Dyer NW, Ward AC. 1998. Pneumonic pasteurellosis associated with Pasteurella haemolytica serotype A6 in American bison (Bison bison). J Vet Diag Invest 10:360-362. 14. Filion LG, Willson PJ, bielefeldt-Ohmann H, Babiuk LA and Thomson RG. 1984. The possible role of stress in the induction of pneumonic pasteurellosis. Can J Comp Med 48:268-274. 15. Foreyt WJ, Snipes KP, Kasten RW. 1994. Fatal pneumonia following inoculation of healthy bighorn sheep with Pasteurella haemolytica from healthy domestic sheep. J Wildl Dis 30:137-145. 16. Gahmberg CG, Valmu L, Fagerholm S, Kotovuori P, Ihanus E, Tian L, Pessa-Morokawa

T. 1998. Leukocyte integrins and inflammation. Cell Mol Life Sci 54:549-555.

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17. Gopinath RS, Ambagala TC, Deshpande MS, Donis RO, Srikumaran S. 2005. Mannheimia (Pasteurella) heamolytica leukotoxin binding domain lies within amino acids 1 to 291 of bovine CD18. Infect Immun 73:6179-6182. 18. Highlander SK, Fedorova MD, Dusek DM, Panciera R, Alvarez LE, Renehart C. 2000. Inactivation of Pasteurella (Mannheimia) haemolytica leukotoxin causes partial attenuation of virulence in a calf challenge model. Infect Immun 68:3916-3922. 19. Kaehler KL, Markham RJF, Muscoplat CC, Johnson DW. 1980. Evidence of species specificity in the cytocidal effectes of Pasteurella haemolytica. Infect Immun 30:615-18. 20. Li J, Clinkenbeard KD, Ritchey JW. 1999. Bovine CD18 identified as a species specific receptor for Pasteurella haemolytica leukotoxin. Vet Microbiol 67:91-97. 21. Liu W, Brayton KA, Davis WC, Mansfield K, Lagerquist J, Foreyt WJ, Srikumaran S. 2007. Mannheimia (Pasteurella) haemolytica leukotoxin utilizes CD18 as its receptor on bighorn sheep leukocytes. J Wildl Dis 43:75-81. 22. Luscinskas FW, Brock AF, Arnaout MA, Gimbrone Jr MA. 1989. Endothelial- leukocyte adhesion molecule-1-dependent and leukocyte (CD11/CD18)- dependent mechanisms contribute to polymorphonuclear leukocyte adhesion to cytokine- activated human vascular endothelium. J Immunol 142:2257-2263. 23. Misra HN, Pandurangarao CC, and Khera SS. 1970. An outbreak of highly fatal pneumonia in kids due to Pasteurella haemolytica. Indian Vet J 47:808-809. 24. Mosier DA. 1997. Bacterial pneumonia. Vet Clin N Am Food Anim Pract 13:483-493. 25. Noti JD, Johnson AK, Dillon JD. 2000. Structural and functional characterization of the leukocyte integrin gene CD11d. Essential role of Sp1 and Sp3. J boil Chem 275:8959- 8969.

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26. Petras SF, Chidambaram M, Illyes EF, Forshauer S, Weinstock GM, Reese CP. 1995. Antigenic and virulence properties of Pasteurella haemolytica leukotoxin mutants. Infect Immun 63:1033-1039. 27. Shewen PE, Wilkie BN. 1982. Cytotoxin of Pasteurella haemolytica acting on bovine leukocytes. Infect Immun 35:91-94. 28. Tatum FM, Briggs RE, Sreevatsan SS, Zehr ES, Whiteley LO, Ames TR, Maheswaran SK. 1998. Construction of an isogenic leukotoxin deletion mutant of Pasteurella haemolytica serotype 1: characterization and virulence. Microb Pathog 24:37-46. 29. Tibbetts SA, et al 1999. Peptides derived from ICAM-1 and LFA-1 modulates T cell adhesion and immune function in a mixed lymphocyte culture. Transplantation 68: 685- 692. 30. Tibbetts SA, Seetharama JD, Siahaan TJ, Benedict SH, Chan MA. 2000. Linear and cyclic LFA-1 and ICAM-1 peptides inhibit T cell adhesion and function. Peptides 21:1161-1167. 31. Wang JF, Kieba IR, Korostoff J, Guo TL, Yamaguchi N, Rozmiarek H, Billings PC, Shenker BJ, Lally ET. 1998. Molecular and biochemical mechanisms of Pasteurella haemolytica leukotoxin-induced cell death. Microb Pathog 25:317-331. 32. Whiteley LO, Maheswaran Sk, Weiss DJ, Ames RP, Kannan MS. 1992. Pasteurella haemolytica A1 and bovine respiratory disease: pathogenesis. J Vet Int Med 6:11-22.

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CHAPTER ONE

Molecular cloning of CD18 of bison, deer and elk, and comparison with that of other ruminants and non-ruminants

Sudarvili Shanthalingam 1 , Junzo Norimine 1 , Wendy C. Brown 1,2 , and Subramaniam Srikumaran 1*

1 Department of Veterinary Microbiology and Pathology, 2 School for Global Animal Health. College of Veterinary Medicine, Washington State University, Pullman, WA 99164-7040, USA.

* Corresponding author

Note: This manuscript has been accepted for publication and „in press‟ in Veterinary Immunology and Immunopathology.

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ABSTRACT Pneumonia caused by Mannheimia haemolytica is an important disease of cattle, domestic sheep, bighorn sheep and goats. Leukotoxin (Lkt) produced by M. haemolytica is cytolytic to all leukocyte subsets of these species. Lkt utilizes CD18, the subunit of 2- integrins, as its functional receptor on leukocytes of these species. Cytotoxicity assays revealed that leukocytes from bison, deer, and elk are also susceptible to Lkt-induced cytolysis. The availability of cDNA encoding CD18 of bison, deer and elk would facilitate the comparison of a greater number of ruminant CD18 cDNA with that of non-ruminants as a means of elucidation of the molecular basis for the specificity of M. haemolytica Lkt for ruminant leukocytes. Herein, we report the cloning and characterization of bison, deer, and elk CD18. The full length cDNA of bison and deer consists of 2310 bp with an ORF encoding 769 amino acids while elk CD18 consists of 2313 bp with an ORF encoding 770 amino acids. This gene is highly conserved among ruminants compared with non-ruminants. Phylogenetic analysis based on amino acid sequences showed that CD18 of bison is most closely related to that of cattle while CD18 of deer and elk are more closely related to each other.

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INTRODUCTION Mannheimia (Pasteurella) haemolytica is the most important bacterial pathogen of respiratory disease in cattle, domestic sheep (DS), bighorn sheep (BHS), and other domestic and wild ruminants. M. haemolytica causes an acute fibrino-necrotic pleuropneumonia resulting in extensive economic losses world-wide (Ackermann and Brogden, 2000; Miller, 2001; Odugbo et al., 2004). This Gram-negative bacterium produces several virulence factors which include an exotoxin that is cytolytic to all subsets of leukocytes, and hence referred to as leukotoxin (Lkt). Based on the observation that Lkt-deletion mutants of M. haemolytica cause reduced mortality and much milder lung lesions than the wild-type organisms, Lkt has been accepted as the most important virulence factor (Petras et al., 1995; Tatum et al., 1998; Highlander et al., 2000; Dassanayake et al., 2009). Cytolytic activity of M. haemolytica Lkt is specific for ruminant leukocytes (Kaehler et al., 1980; Chang et al., 1986). Earlier studies by us and others (Wang et al., 1998; Ambagala et al., 1999, Li et al., 1999; Jayaseelan et al., 2000) identified 2 -integrins as the receptor for Lkt of M. haemolytica on bovine leukocytes. β 2 -integrins, expressed exclusively on leukocytes, are composed of two non- covalently associated subunits, α (CD11) and β (CD18). CD18 associates with four distinct α chains to give rise to four different β 2 -integrins: CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), CD11c/CD18 (CR4), and CD11d/CD18. Studies in our laboratory have demonstrated that CD18, the subunit of 2 -integrins, is necessary and sufficient to mediate Lkt-induced cytolysis of bovine and ovine leukocytes (Deshpande et al., 2002; Liu et al., 2007; Dassanayake et al., 2007a, 2007b). Furthermore, we have mapped the Lkt binding site on bovine CD18 to lie between amino acids 1-291 (Gopinath et al., 2005). The nucleotide and deduced amino acid sequence of CD18 of cattle, DS, BHS, goats, and buffalo have been determined (Shuster et al., 1992; Zechinon et al.,

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2004a; Liu et al., 2006; Zechinon et al., 2004b). Availability of sequence information on CD18 of additional ruminant species would facilitate the comparison of a greater number of ruminant CD18 cDNAs with that of non-rumninants as a means of elucidation of the molecular basis underlying the specificity of M. haemolytica Lkt for ruminant leukocytes, which in turn should pave the way for the development of strategies to control this economically important disease in ruminants. Therefore the objective of this study was to clone and sequence the cDNA encoding the CD18 of bison (Bison bison), deer (Odocoileus hemionus), and elk (Cervus canadensis), and compare that with CD18 of other ruminants and non-ruminants.

MATERIALS AND METHODS PMNs and PBMCs of bison, deer and elk were isolated from peripheral blood by Ficoll - Paque (Amersham, NJ) density gradient centrifugation as described previously (Deshpande et al., 2002). The susceptibility of these cells to M. haemolytica Lkt-mediated cytolysis was confirmed by a previously described cytotoxicity assay {MTT [3-(4,5-dimethylthiazoyl-2-Yl)-2,5-diphenyl tetrazolium bromide] dye reduction assay}(Ambagala et al., 1999). The total RNA from PBMCs was extracted using TRIzol reagent and cDNA was synthesized using Superscript TM III first-strand synthesis system for RT-PCR following the manufacturer‘s instructions (Invitrogen Inc., Carlsbad, CA).

Forward and reverse primers to amplify the CD18 gene of bison and deer were designed based on multiple alignment of human ( NM_000211 ), mouse ( X14951 ), pig ( U13941 ), chimpanzee ( NM_001034122 ), cattle ( M81233 ), DS (AY484425), BHS ( DQ104444 ), goat ( AY452481 ) and Buffalo ( AY842449 ) cDNA sequences available in the GenBank. The primers designed to amplify CD18 cDNAs of bison and deer were: CD18 For; 5'-GGCATCCAGGGGACATGC-3' and CD18 Rev; 5'-

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CCCCTAACTCTCGGCAAAC-3'. Gene was amplified using a high fidelity polymerase, PfuUltra TM II fusion HS (Strategene, La Jolla, CA). Single band PCR amplicons were gel-purified and cloned into pCR R 4 Blunt-TOPO vector (Invitrogen). Following transformation of TOP10 chemically competent cells, positive clones were selected on LB-ampicillin plates and the insert was identified by colony PCR. Plasmid DNA from selected positive colonies was isolated and purified with QIAprep Spin Miniprep kit (Qiagen, Valencia, CA) by following the manufacturer‘s protocol. A total of 5 clones were sequenced using BigDye Terminator Chemistries and an ABI Prism 377 DNA sequencer (Applied Biosystems, CA). The bison and deer CD18 cDNA sequences have been deposited at GenBank (accession no. EU553919 and EU623794, respectively). The sequences of 5‘- and 3‘- untranslated regions (UTR) of elk CD18 were obtained by using a 2 nd generation 5‘/3‘ RACE kit (Roche applied Science, Germany). Gene-specific primers were designed based on the sequence alignment of cloned cattle, DS, BHS and mouse, human, and pig CD18. A gene-specific sense primer (5‘-GACAACAGCTCCATCATCTGCTC-3‘) and an anti-sense primer (5‘-GTCCTGGTCGCAAGTAAAGTGTC-3‘) were used to amplify the 3‘ and 5‘ ends of the elk CD18 cDNA, respectively, according to the manufacturer‘s instructions. The RACE amplicons were cloned into the pCR R 4 Blunt-TOPO vector. The positive clones containing inserts were identified by colony PCR and sequenced completely. cDNA from total RNA was synthesized using Superscript TM III first-strand synthesis system for RT-PCR following the manufacturer‘s instructions to obtain the full length CD18 coding sequences (CDS) using primers designed to amplify deer and bison CD18 cDNAs. Full length elk CD18 was cloned and sequenced by a protocol similar to that used for the CD18 of bison and deer, and the sequence was deposited at GenBank (accession no. EU553918).

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DNA sequence analysis, fragment assembly, and amino acid sequence prediction were performed with the ContigExpress module of Vector NTI AdvanceTM 9.1 (Invitrogen). SignalP V.2.0b2 ( http://www.cbs.dtu.dk/services/SignalP/ ) (Nielsen et al., 1997) and NetNGlyc V.1.0 ( http://www.cbs.dtu.dk/services/NetNGlyc/ ) (Jensen et al., 2002) provided signal peptide and N- glycosylation sites prediction, respectively. BLAST ( http://www.ncbi.nlm.nih-gov/ ) was used for homology and % identity, and open reading frame (ORF) were confirmed by ExPASY ( http://us.expasy.org ). Alignment of nucleotide and amino acid sequences and similarity analyses were performed with CLUSTAL-W1.8 ( http://www.ebi.ac.uk ; Thompson et al., 1994). Protein statistics were analyzed by EMBOSS server ( http://www.bioinformatics.wsu.edu/emboss/ ).

RESULTS AND DISCUSSION Lkt lysed PMNs and PBMCs of bison, deer, and elk in a concentration-dependent manner, as observed with the leukocytes of other ruminants (Fig. 1). The Fig 2 shows the complete nucleotide and derived amino acid sequences of bison, deer, and elk CD18. The bison and deer CD18 cDNA contain an ORF of 2307 bp which codes for 769 amino acids. The deduced polypeptides were 95% identical to each other. Elk CD18 cDNA contains 2310 bp coding for 770 amino acids. The deduced amino acid sequence from the coding region of elk CD18 shows 95% and 97% identity with that of bison and deer, respectively. The predicted molecular masses of the CD18 of bison, deer, and elk are 84.3 kD, 84.5 kD, and 84.7 kD, respectively. The predicted isoelectric points of bison, deer, and elk CD18 proteins are 6.0, 7.4, and 7.2, respectively. The comparison of deduced amino acid sequence of bison, deer, and elk CD18 with that of cattle, goats, DS, BHS, buffalo, pigs, humans, chimpanzees, mice and rats is shown in Table 1. The

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identity in amino acid sequence among the ruminants ranges from 94% to 99%. The identity in amino acid sequence between the ruminants and non-ruminants ranges from 81% to 88%. All three cloned CD18 proteins have a predicted N-terminal signal peptide sequence of 22 amino acids (maximum probability of 0.963). The predicted transmembrane domain is 23 amino acids long, which is followed by a cytoplasmic domain of 46 amino acids. Both regions are 100% identical among ruminants. The overall protein structure and domains are in agreement with previously identified CD18 of other species. The extracellular domains of CD18 of bison, deer, and elk contain 4, 7, and 6 N-linked glycosylation sites (Asn-X-Ser/Thr), respectively (Fig. 2). As with the CD18 of other species, the extracellular domain of bison, deer and elk CD18 also consists of an I-like domain of 240 amino acids (from amino acid 126 to 365 in elk and from amino acid 124 to 363 in bison and deer; Fig. 3A). It is identical to that found in other ruminant CD18, and quite similar to that of the CD18 of non-ruminants sequenced to-date. The I-like domain contains a metal ion-dependent adhesion site (MIDAS, DLSYS; Fig. 3A). CD18 of bison, deer and elk also contain 56 cysteine residues at identical positions, possibly involved in the formation of disulfide bridges. A phylogenetic tree was constructed with the Neighbor-joining algorithm (Saitou and Nei, 1987) included in the MEGA 4 program. Distance was estimated using the p-distance method. The robustness of the inferred trees was assessed by bootstrap (1000 replicates) analysis (Felsenstein, 1985). Fig 3B shows the phylogenetic relationship among the CD18 of different species. Deer and elk sequences are more closely related to each other compared to that of other species, and the bison sequence shows a closer relationship to that of cattle and then buffalo, than to CD18 from other species.

Full document contains 115 pages
Abstract: Mannheimia haemolytica is the primary bacterial pathogen of bovine pneumonic pasteurellosis, an economically important disease of cattle worldwide. Leukotoxin (Lkt) produced by M. haemolytica is the major virulence factor of this organism. The cytolytic activity of Lkt is specific for ruminant leukocytes. Lkt utilizes CD18, the β subunit of β2 -integrins, as its receptor on ruminant leukocytes. Previously, our laboratory mapped the Lkt-binding domain to lie between amino acids (aa) 1-291 of CD18. Therefore, the next logical step was to identify the precise Lkt binding site within this domain and to determine whether co-administration of CD18 peptide analogs would inhibit / mitigate M. haemolytica -caused lung injury. In this study, by using synthetic peptides spanning aa1-291 of bovine CD18 in Lkt-induced cytolysis assays, the precise binding site of Lkt was mapped to aa 5-17 of ruminant CD18. Surprisingly, all the aa of this peptide belong to the predicted signal peptide of CD18. This observation led to the finding that the signal peptide of ruminant CD18 is not cleaved, and that the intact signal peptide renders ruminants susceptible to M. haemolytica Lkt. Site-directed mutagenesis of a single aa in the signal peptide resulted in the cleavage of signal peptide and abrogation of Lkt-induced cytolysis of target cells. This finding indicates that engineering cattle and other ruminants to contain this mutation would provide a novel technology to render them less susceptible to pneumonic pasteurellosis and concomitant economic losses. The peptide spanning aa 5-17 (P17) was used in a calf challenge study which was designed as a 'proof of concept' experiment. Even though the difference in percent volume of lungs exhibiting gross pneumonic lesions between P17-inoculated calves and control peptide-inoculated calves was not statistically significant, M. haemolytica isolated from the lungs of P17-inoculated calves was 100- to 1000-fold less than those from the control peptide-inoculated calves, suggesting that P17 reduced leukotoxic activity in the lungs which enhanced bacterial clearance by phagocytes. It is likely that prolonging the presence and activity of CD18 peptide analog in the lungs, for example by means of a nanoparticle delivery system such as dextran nanospheres, would enhance its protective activity.