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2003 Identification of an immunogenic Mannheimia haemolytica immunoglobulin-, fibronectin- and fibrinogen-binding protein differentially expressed in vitro Ruth Smith Osmundson Iowa State University
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Identification of an immunogenic Mannheimia haemolytica immunoglobulin-, fibronectin- and fibrinogen-binding protein differentially expressed in vitro
by
Ruth Smith Osmundson
A dissertation submitted to the graduate faculty
in partial fulfillment of the requirements for the degree of
DOCTOR OF PHILOSOPHY
Major: Immunobiology
Program of Study Committee: Louisa B. Tabatabai, Co-major Professor James A. Roth, Co-major Professor Joan E. Cunnick James S. Dickson Merlin L. Kaeberle
Iowa State University
Ames, Iowa
2003 UMI Number: 3085935
UMI
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DEDICATION
To my children,
Anna Smith Osmundson
and
Caleb Smith Osmundson:
Reach for your stars! iv
TABLE OF CONTENTS
ABSTRACT vii
CHAPTER 1. GENERAL INTRODUCTION 1 Dissertation Organization 5 Literature Review 6 Mannheimia haemolytica 6 Taxonomy 6 Physical characteristics 7 Bovine Respiratory Disease 7 Etiology and epidemiology 7 Pathogenesis 8 Virulence factors 11 Leukotoxin 11 Lipopolysaccharides 12 Capsular polysaccharide 12 O-sialoglycoprotease 13 Neuraminidase 13 Fimbrae 14 Iron-regulated Proteins 14 Vaccines 15 History 16 Bacterins 16 Live vaccines 16 Subunit vaccines 17 Growth condition dependent protein expression 18 Immunoglobulin-binding proteins 20 History 20 Protein A 21 Immunoglobulin-binding protein classification 21 Immunoglobulin-binding protein binding specificity 22 Immunoglobulin-binding proteins and virulence 23 Superantigens 23 Mitogens 24 Complement activation and inhibition 25 Antiphagocytes 25 Antibody-dependent cellular cytotoxicity inhibition 25 Virulence in vivo 25 Therapeutic use of immunoglobulin-binding proteins 26 Extracellular matrix-binding proteins (ECMBPs) 27 Extracellular matrix proteins (ECMPs) 27 Fibronectin 27 Fibrinogen 28 V
Extracellular matrix-binding proteins 28 Identification 28 Virulence association 29
Vaccine potential 31 References 32
CHAPTER 2. IDENTIFICATION AND CHARACTERIZATION OF AN 50 IMMUNOGLOBULIN-BINDING PROTEIN EXPRESSED BY MANNHEIMIA
Abstract 50 Introduction 51 Materials and Methods 54 Bacterial isolates 54 Whole cell sonicate and supernatant preparations 54 Gel electrophoresis and immunoblotting 55 Bovine antisera 55 Affinity isolation 56 Flow cytometry 56 Results 57 Identification of M. haemolytica IgBP(s) 57 Isolation of M. haemolytica IgBP(s) 57 Surface expression of M. haemolytica IgBP(s) 58 Humoral immunogenicity of M. haemolytica IgBP(s) 58 Involvement of IgBP(s) from different serotypes with species-specific 58 infection Discussion 59 References 64 Figures and figure legends 69
CHAPTER 3. FIBRINOGEN- AND FIBRONECTIN-BINDING AND IN VITRO 75 GROWTH CONDITION-DEPENDENT EXPRESSION OF A MANNHEIMIA HAEMOLYTICA IMMUNOGLOBULIN-BINDING PROTEIN Abstract 75 Introduction 76 Materials and Methods 80 Bacterial isolate and growth conditions 80 Fetal bovine sera 80 IgBP isolation 80 Gel electrophoresis and immunoblotting 80 WCS and membrane protein preparation 81 2-D gel electrophoresis 82 2-D gel analysis 83 vi
Results 83 M. haemolytica growth in different in vitro growth conditions 83 M. haemolytica IgBP also binds fibrinogen and fibronectin 84 Comparative analysis of total IgBP pixel density 85 Comparative analysis of individual spot pixel density 86 Discussion 87 References 90 Figures and figure legends 93 Tables 1 and 2 97 CHAPTER 4. GENERAL DISCUSSION AND CONCLUSIONS 98 Table 1 105 Table 2 106 References 108 ACKNOWLEDGMENTS 110 Vil
ABSTRACT
Bovine respiratory disease (BRD), or "shipping fever", is the cause of large losses to
the cattle industry annually and is primarily caused by Mannheimia haemolytica serotype
(ST) 1. M. haemolytica, a normal flora respiratory tract bacterium, rapidly multiplies upon a
stressful occurrence and colonizes the upper respiratory tract. This can lead to
fibrinohemorrhagic pneumonia. Bacterial immunoglobulin-binding protein (IgBP)
expression is known to play a role in the pathogenesis of a variety of organisms. Far-
Western blot was used to demonstrate the presence of an IgBP(s) in whole cell sonicates
(WCSs) and a culture supernatant (CS) preparation from M. haemolytica ST 1. The IgBP(s)
was isolated by affinity chromatography and used in a far-Western blot to show that the
IgBP(s) also binds the extra-cellular matrix proteins (ECMPs) fibrinogen and fibronectin.
ECMPs are known to play a role in colonization of some bacteria by acting as a bridge enabling binding between bacteria expressing extracellular matrix-binding proteins and epithelial cells. Flow cytometry showed the surface expression of an IgBP(s) on whole cell
M. haemolytica. Immune sera from convalescent cattle showed an increased antibody response to the 75.0 kDa IgBP when compared to sera from naive or acutely infected cattle as seen by Western blot. It was demonstrated that all 12 serotypes (1,2, 5-9, 12-14, and 16) bound both bovine Fc IgG and sheep Fc IgG. Significant differences were not found between ST1, most frequently isolated in infections in cattle and ST2, isolated predominantly in sheep mannheimiosis. The effect of various M. haemolytica growth conditions on IgBP(s) expression was assessed by 2-D gel electrophoresis analysis. Membrane expression of M. haemolytica IgBP(s) was increased three-fold when M. haemolytica was grown in RPMI- 1640 with 10%FBS and in RPMI-1640 at 40°C and increased two-fold when grown in
RPMI-1640 with 50 jaM 2,2'-dipyridyl when compared to growth in RPMI-1640 at 37°C.
The IgBP(s) constituted up to 19.9% of M. haemolytica membrane proteins when M. haemolytica was grown in RPMI at 40°C. It is a highly expressed protein when M. haemolytica is grown under conditions mimicking factors of the in vivo environment. This study shows a possible role in virulence for the M. haemolytica IgBP. It is immunogenic, surface expressed and as an IgBP it may enable M. haemolytica to evade the host immune response and may play a role in colonization through binding fibronectin or fibrinogen. 1
CHAPTER L GENERAL INTRODUCTION
Bovine respiratory disease (BRD) or "shipping fever" is believed to result in the single largest loss annually to the cattle industry. BRD has a complex etiology but pulmonary fibrinous pneumonia, the ultimate cause of severe clinical disease and death, is primarily caused by Mannheimia haemolytica (Houghton and Gourlay, 1984). M. haemolytica serotype ST2 is a normal flora bacteria of the upper respiratory tract (URT) in cattle but on occurrence of a stressful incident such as viral infection or shipping from farm to feedlot, isolates of M. haemolytica shift from ST2 to ST1 and can grow explosively colonizing the URT (Frank 1979; Frank and Smith, 1983). The bacteria are believed to be inhaled into the lung resulting in macrophage activation and pulmonary serum flooding. An influx of neutrophils along with an M. haemolytica secreted leukotoxin exacerbates the response. This leads to lung lesions characteristic of M. haemolytica-induced fibrinohemorrhagic pneumonia. Some animals are able to recover while others succumb to pneumonic mannheimiosis (Whitely et al., 1992).
A number of vaccines are available for M. haemolytica induced BRD. Bacterins provide protection of variable efficacy. A number of experimental trials have even shown an exacerbation of BRD in bacterin vaccinates (Friend et al., 1977; Wilkie et al., 1980). While live vaccines generally perform better in field-tests than bacterins (Confer et al., 1985b), live vaccines still have variable efficacy with some trials showing no increased protection due to immunization (Purdy et al., 1986). Live vaccines have inherent problems with production and stability. While not providing full protection, bacterial component vaccines have demonstrated the importance of leukotoxin (Conlon et al., 1991), iron binding proteins 2
(Potter et al., 1999) and surface antigens (Shewen et al., 1988) in protection. There is a need for a more efficacious vaccine.
M. haemolytica expresses a number of known virulence factors. The best characterized is a leukotoxin (Kaehler et al., 1980), a member of the RTX toxin family.
Other virulence factors include the capsular polysaccharide (Carter, 1956), LPS (Rimsay et al., 1981), fimbrae (Morck et al., 1987; Potter et al., 1988), iron-regulated proteins,
(Donachie and Gilmour, 1988), neuraminidase (Frank and Tabatabai, 1981) and O- sialoglycoprotease (Abdullah et al., 1992). This study demonstrated the presence of an additional M. haemolytica virulence factor, an immunoglobulin-binding protein (IgBP).
IgBPs have been identified in a number of other Gram-positive and Gram-negative bacteria. Surface expressed protein A from Staphylococcus aureus Cowan 1 is the first IgBP identified (Forsgren and Sjôquist, 1966). Protein A is known inhibits phagocytosis and IgBP surface expression is responsible for this inhibition (Dosset et al., 1969). Protein A in solution fixes complement by forming IgG:protein A complexes (Langone et al., 1978) and to inhibit antibody-dependent cellular cytotoxicity (Rosenblatt et al., 1977). Acting as a B cell superantigen, protein A activates complement (Kozlowski et al., 1996), induces expression of Ig from the VH3 gene family (Kristiansen et al., 1994), increases antigen presentation through soluble protein A:Ig complexes associated with VH3 family surfaced expressed immunoglobulins (Leonetti et al., 1999) and is involved in inflammation as demonstrated by the elicitation of an Arthus reaction through administration of soluble protein A:Ig complexes (Kozlowski et al., 1998). In vivo experiments indicate a role for
IgBPs in virulence. Mice injected with an S. aureus protein A deletion mutant require a marginally higher dose of the mutant for inactivity and take significantly longer to succumb 3 to infection and die at a slightly decreased rate than mice infected with S. aureus expressing protein A (Patel et al., 1987). These results may have been more dramatic if a second more recently identified S. aureus IgBP, Sbi, had also been deleted (Zhang et al., 1998). These experiments demonstrate a role for IgBPs in virulence although their role has not been clearly elucidated.
Most bacteria initiate infection by binding to host cells. Bacterial cell surface components adhere to host cell components or to host extracellular matrix (ECM) proteins that bind host cells and act as a bridge between bacteria and host cells. Extracellular matrix- binding proteins (ECMBPs) from Staphylococcus aureus are best characterized and include proteins that bind fibronectin (Fn), collagen (Cn) and a secreted protein that binds fibrinogen
(Fg) (Flock, 1999). A number of Gram-negative organisms express ECMBPs including
Eschericia coli (Westerland et al., 1989), Haemophilus ducreyi (Abeck et al., 1992), H. influenza (Fink et al., 2002), Neisseria gonorrhoeae (van Putten et al., 1998) and Borellia burgdorferi (Probert and Johnson, 1998). In vitro models demonstrate several additional roles for ECMBPs in virulence. In the presence of Fn and an anti-CD3 monoclonal antibody.
S. aureus fibronectin-binding protein A (FnbpA) mediates adhesion to T cells and T cell costimulation through the T cell asPi integrin, which binds fibronectin and functions as a costimulatory molecule, leading to T cell activation (Miyamoto et al., 2001). A T helper type
2 inflammatory environment promotes S. aureus binding to skin and this binding is mediated by Fn and Fg (Cho et al., 2001a). ECMBPs appear to have a variety of roles in virulence.
The surface components used by M. haemolytica to adhere to airway epithelial cells are not known. Two types of fimbrae, a large, rigid fimbrae (Morck et al., 1987) as well a smaller, flexible fimbrae (Potter et al., 1988) have been reported. However, several 4 investigators have been unable to demonstrate the presence of fimbrae on newly isolated
M. haemolytica ST1 using the same procedures (Gonzalez and Maheswaran, 1993) and fimbrial adhesion has not been demonstrated for M. haemolytica. Identification of an M. haemolytica adhesive surface component would open the door to development of a treatment that could decrease colonization and result in reduced development of pneumonic pasteurellosis.
The mechanism for the abrupt change from the predominance of the normal flora M. haemolytica ST2 to explosive growth of stress-associated ST1 is not known. However, change in host environmental factors due to stress is likely to play a role. In an effort to recreate in vivo protein expression while using in vitro systems and also to define culture conditions that affect expression of particular proteins, a number of studies have been done to define protein expression under different culture conditions. Pathogens are known to up- regulate or down-regulate expression of adhesive components under varying conditions of the host environment (Finlay and Falkow, 1989; Francis et al., 1989; and Mekalanos, 1992).
Shewen and Wilkie (1982) reported that the addition- of 7% fetal bovine serum (FBS) to
RPMI-1640 tissue culture media achieves enhanced M. haemolytica leukotoxin production compared to tissue culture media alone. Iron-regulated outer membrane proteins of 71, 77 and 100 kDa are expressed at higher levels in iron-restricted media (Deneer et al., 1989;
Morck et al., 1991). No capsular polysaccharide production is detected when M. haemolytica is grown above 40°C (Puente-Polledo et al., 1998). Establishing in vitro growth conditions that closely mimic in vivo conditions may increase the efficacy of vaccines prepared from in vitro M. haemolytica cultures. 5
Preliminary results in our lab indicated that M. haemolytica might express an IgBP(s).
When normal bovine serum (NBS) is used as a negative control to label whole cell M.
haemolytica in an ELISA, similar absorbance values are obtained with the NBS and with an anti-sera specific for an M. haemolytica iron-regulated protein (L.B. Tabatabai, unpublished observations). The objectives of the present study were: 1) To identify and isolate an M. haemolytica IgBP(s); 2) To determine the presence or absence of IgBP surface expression; 3)
To determine whether there is an increased antibody response to the IgP in cattle convalescing from BRD; 4) To determine whether the IgBP may play a role in the dominance of ST1 association with BRD (Wessman and Hilker, 1968 and Frank, 1979) or the dominance of ST2 in sheep mannheimiosis (Fraser, 1982); 5) To establish whether the
IgBP binds one or more ECMPs; and 6) To assess whether this protein would be up- regulated when grown under in vitro growth conditions that more closely mimic factors of the in vivo environment.
Dissertation Organization
This dissertation consists of four chapters. Chapter 1 is a general introduction and review of the literature on Mannheimia haemolytica, immunoglobulin-binding proteins and extracellular matrix-binding proteins. Chapter 2 and 3 consist of two papers. The first paper,
"Identification, isolation and characterization of an immunoglobulin-binding protein expressed by Mannheimia haemolytica" is to be submitted for publication in the journal
Infection and Immunity. The second paper, "Fibrinogen- and fibronectin-binding of a
Mannheimia haemolytica immunoglobulin-binding protein differentially expressed in vitro," will also be submitted to Infection and Immunity. Chapter 4 consists of a general discussion 6
and conclusions. References for the general introduction and the literature review are in
alphabetical order and follow the literature review. References for each paper follow the
discussion section of each paper while the references for the general discussion and
conclusion references follow that section.
Literature Review
Mannheimia haemolytica
Taxonomy
Whether Perroncito in 1878, Pasteur in 1880, or Kitt in 1885, was the first to isolate and describe the bacillus associated with fowl cholera is not clear. Bacilli with similar characteristics were associated with diseases of rabbits, swine and cattle and with fowl cholera. These diseases were characterized as being associated with hemorrhagic septicemia.
Italian Count Trevisan first proposed the genus name Pasteurella (Holmes et al., 1999) for these organisms in honor of Pasteur's work on fowl cholera. After a series of name changes.
Newsom and Cross (1932) proposed that organisms associated with bovine hemorrhagic septicemia and pneumonia be given the name Pasteurella haemolytica. Shirlaw (1938) termed the disease bovine pasteurellosis and Smith (1961) described two biotypes of P. haemolytica that he termed A and T. These letters stood for arabinose and trehalose fermentation, respectively. Seventeen serotypes (ST) have been defined (Biberstein et. al,
1960; Fodor et al., 1988; Pegram et al., 1979; Younan, 1995), twelve associated with biotype
A and four associated with biotype T. The T biotypes were reclassified into a new species
Pasteurella trehalosi in 1990 and include serotypes 3, 4, 10 and 15 (Bingham et al., 1990). 7
Through DNA-DNA hybridizations and 16S rRNA sequencing, Angen proposed reclassifying Pasteurella haemolytica organisms into the new genus Mannheimia (Angen,
1999). This now includes serotypes 1, 2, 5, 6, 7, 8, 9, 12, 13, 14, 16 and 17. Mannheimia belongs to the Pasteurellaceae family that consists of the genera Haemophilus, Actinobacillus and Pasteurella, the HAP organisms.
Physical characteristics
M. haemolytica are small, non-motile, pleomorphic, gram-negative, cocco-bacilli.
The bacteria have a serotype specific capsular polysaccharide. Log phase cultures tend to be encapsulated while older cultures are generally not encapsulated (Corstvet et al., 1982).
After multiple passages in vitro, M. haemolytica ST1 maintains capsular polysaccharide production (Gentry et al., 1987). There are no flagella on M. haemolytica but two types of fimbrae have been reported (Morck et al., 1987; Potter et al., 1988). When grown on ovine or bovine blood agar a narrow zone of ^-hemolysis is seen and smooth gray colonies are seen.
Bovine respiratory disease
Etiology and epidemiology. Because BRD is brought on by multiple factors, the etiology of BRD is not always clear. However, it is generally agreed that M. haemolytica
ST1 is the primary causative agent of BRD or shipping fever (Frank, 1979 and Confer,
1988). Serotype 1 is the predominant serotype isolated in BRD (Frank, 1979 and Frank et al., 1983) and is almost solely isolated from cattle that have died from BRD (Fox et al., 1971;
Allan et al., 1985). 8
The incidence of BRD is associated with stress. Stress can be due to management practices such as shipping, weaning, crowding, comingling of herds, dehorning and castration. Viral infections are also considered to be predisposing factors in BRD. A study reveals that infectious bovine rhinotracheitis virus (IBR), bovine viral diarrhea virus (BVDV) parainfluenza type three virus (PI-3) and bovine bovine respiratory syncytial virus (BRSV) are associated with respiratory disease (Martin and Bohac, 1986). The greatest incidence of disease occurs in shipping yards and feedlots following shipment of animals; thus BRD is also called shipping fever. Dairy cattle are also known to have the disease. Serotype 2 is most frequently isolated from M. haemolytica-infected sheep (Fraser et al., 1982).
Pathogenesis. M. haemolytica serotype (ST2) is a normal flora bacteria of the upper respiratory tract (URT) in cattle but on occurrence of a stressful incident such as viral infection or shipping from farm to feedlot, isolates of M. haemolytica shift from ST2 to ST1 and can grow explosively colonizing the URT (Frank 1979; Frank and Smith, 1983; Jones,
1987; Frank, 1988). The mechanism for the abrupt change from the predominance of the normal flora M. haemolytica ST2 to explosive growth of stress-associated ST1 is not known.
However, change in host environmental factors due to stress is likely to play a role.
The mechanism used by M. haemolytica to colonize the URT is also not clear. The bacteria may adhere to mucosal epithelial cells or proliferate within the mucous layer.
Fimbrae have been reported in vitro on M. haemolytica (Morck et al., 1987, Potter et al.,
1988), but fimbrae are not found in M. haemolytica isolated from the lung or URT of cattle
(Gonzalez et al., 1993). In addition, their role in attachment has not been demonstrated. A number of Gram-negative bacteria express adhesins that are distinct from fimbrae (Minion et 9 al., 1986; Mooi et al., 1992). Healthy or non-stressed cattle make use of the mucociliary ladder to clear bacteria from the URT. However, in humans, the mucociliary ladder is impaired in Gram-negative bacterial pneumonia (Pavia, 1987). Viral infections may kill ciliated epithelial cells or alter cell ciliary function. Neuraminidase, expressed by M. haemolytica ST1 (Frank and Tabatabai, 1981) decreases the viscosity of bovine respiratory mucus (Milligan et al., 1978). Decreased viscosity could inhibit the clearance of M. haemolytica. Beyond the aforementioned possibilities, M. haemolytica may multiply so rapidly that clearance mechanisms are overwhelmed. It is hypothesized that the large number of M. haemolytica in the URT is then inhaled into the lung in aerosolized droplets.
In healthy cattle, macrophage activation initiates an inflammatory response and along with a humoral immune response, the bacteria are cleared. However, in immunologically compromised cattle, the immune response is ineffective at clearing the bacteria and the inflammatory response itself becomes part of the pathology. Activation of alveolar macrophages by bacterial lipopolysaccharide (LPS) and leukotoxin (Lkt) results in secretion of Interleukin-1 (IL-1), tumor necrosis factor alpha (TNFa) and Interleukin-8 (IL-8), proinflammatory cytokines (Yoo et al., 1995; Lafleur et al., 2001). Caswell (2001) demonstrated that IL-8 is an important neutrophil chemoattractant found in bronchoalveolar lavage (BAL) from calves with pneumonic pasteurellosis. After aerosol exposure of calves to M. haemolytica, lung lavage fluid contains an influx of neutrophils 30 minutes after exposure and by 60 minutes there is an equal percentage of alveolar macrophages and neutrophils. Four hours post-infection, more than 90% of cells are neutrophils (Walker et al.,
1985). In separate experiments macrophages from BAL fluid express IL-1, TNFa and IL-8 within the first four hours after infection with IL-8 being expressed to the greatest extent. By 10 eight hours post-infection the neutrophils are the predominant source of IL-8 in BAL fluid
(Malazdrewich et al., 2001). Neutrophils are stimulated by low concentrations of an M. haemolytica secreted Lkt to undergo a respiratory burst that results in the release of superoxide anion, hydrogen peroxide (Czuprynski et al., 1991) and lysosomal proteolytic enzymes (Styrt et al., 1990). The result of the release of oxygen products and proteases is tissue injury and necrosis. An indication of the central role neutrophil activation plays in lesion development is shown in experiments in which neutrophil depletion reduced lesion severity (Breider et al., 1988). The lipid A portion of LPS is known to be able to activate the classical complement pathway (Morrison and Rudback, 1981) while the polysaccharide component activates the alternate complement pathway (Morrison and Ulevitch, 1978).
Products of complement activation contribute to the inflammatory response.
Characteristic lesions of pneumonic mannheimiosis and associated lung damage are believed to occur as a result of host-pathogen interactions and an inflated inflammatory response. Typical lesions of shipping fever involve alveolar edema, hemorrhage, neutrophilic and fibrinous exudates and pulmonary vascular thrombosis. Inflammatory cells and fibrous tissue surround focal areas of coagulation necrosis. Within these lesions are characteristic "streaming macrophages" (Jubb et al., 1985). Experiments by Reeve-Johnson et al. (2001) noted higher clinical scores in calves with temperatures of 39.9 to 41,0°C compared to normal temperatures of 39.2°C. Death is thought to result from hypoxia or toxemia. 11
Virulence factors
A number of virulence factors have been identified in M. haemolytica induced BRD.
The role of some virulence factors is well studied while the mechanism of action of others remains to be determined. The role of several virulence factors are deduced from the role of similar virulence factors in other organisms. The most well characterized virulence factors are the leukotoxin and lipopolysaccharide.
Leukotoxin (Lkt). In 1978, it was determined that M. haemolytica is cytotoxic for bovine leukocytes (Benson et al., 1978). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) determined its molecular weight to be 101-105 kDa (Chang et al., 1987). Shewen and Wilkie (1982) showed that Lkt is specific for ruminant neutrophils, monocytes, macrophages and lymphocytes. It is produced during logarithmic phase but not in the stationery phase of growth (Shewen and Wilkie, 1985). In 1987 the gene was cloned and sequenced (Lo et al., 1987). Leukotoxin is a member of the RTX family of cytolysins that are pore-forming toxins (Whitely et al., 1992). Reports also demonstrate that the Lkt can apoptotically induce cell death (Wang et al., 1998).
The activity of the Lkt is responsible for a great deal of the pathological effects of
BRD. One group of researchers showed that subcytolytic concentrations of Lkt stimulate activation of neutrophils which results in a respiratory burst that releases superoxide anion and hydrogen peroxide. This is followed by degranulation and cytolysis (Maheswaran et al.,
1992). Release of these oxygen products and proteolytic enzymes would be expected to cause tissue necrosis. High concentrations of Lkt are lytic for ruminant leukocytes. More study is necessary to determine whether low or high concentrations of Lkt are more 12 detrimental to the host. Other activities of Lkt include stimulating the release of IL-1 and
TNFa from macrophages (Yoo et al., 1995) and histamine from mast cells (Adusu et al.,
1994). These cytokines are known to contribute to increased capillary permeability, neutrophil influx and the inflammatory response associated with BRD.
Lipopolysaccharide (LPS). M. haemolytica LPS induces a complex set of responses from a variety of cell types. Endotoxin from M. haemolytica LPS is released into the inflammatory exudate and is localized in neutrophils, macrophages, endothelial cells and epithelial cells (Whitely et al., 1990). The interaction of endotoxin and cells leads to cell activation or cell death. Some of the actions mediated by endotoxin include but are not limited to coagulation, inflammation and release of oxygen products. Lipopolysaccharide activated endothelial cells and macrophages produce a wide variety of proinflammatory and procoagulant mediators (Gartner et al., 1988; Cotran, 1987; Russo, 1980; Tipping et al.,
1988). Alveolar macrophages produce toxic oxygen radicals and proteases as well as nitric oxide in response to M. haemolytica LPS. (Werb, 1983; Dyer et al., 1985; Yoo et al., 1996).
Interestingly, Lafleur et al. (2001) reported a synergistic response between LPS and Lkt in inducing cytolysis and inflammatory cytokine (TNFa and IL-8) production in bovine alveolar macrophages. LPS primes neutrophils for release of toxic oxygen products and lysosomal enzymes. Even though LPS effects are multifaceted, antibodies to LPS are not associated with protection (Confer et al., 1988).
Capsular polysaccharide (CPS). The M. haemolytica CPS has been shown to augment the ability of the bacteria to resist phagocytosis and can hinder complement- mediated lysis (Confer et al., 1990b; Czuprynski et al., 1991). However, recent construction 13 of an acapsular mutant demonstrates that the mutant is as resistant to complement-mediated killing in colostrum-deprived calf serum as the encapsulated parent strain. Nonetheless, it is more sensitve to killing by immune bovine serum (McKerral and Lo, 2002). Barallo et al.
(1999) observed that production of the CPS in ST2 is strictly temperature regulated and that no production is detected above 40°C.
O-sialoglycoprotease. In 1992, Abdullah recognized that M. haemolytica produces a
35-kDa O-sialoglycoprotease found in the culture supernatant that cleaves O-glycosylated proteins but not N-glycosylated proteins. Antibodies to this enzyme are found in bovine sera
(Lee et al., 1994). Tabatabai (1981) observed that fetuin is a substrate for the M. haemolytica
O-sialoglycoprotease and can be used as an alternative substrate to identify the protease activity. M. haemolytica O-sialoglycoprotease enhances bovine platelet adhesion which would indicate a role in thrombosis during mannheimiosis (Nyarko et al., 1998).
Neuraminidase. Neuraminidase is implicated as a virulence factor in bacteria.
When sialic acid residues are removed from salivary glycoproteins, salivary secretions are less able to protect against pathogenic bacteria (Gottschalk, 1960). This would enhance the ability of microorganisms to survive in vivo. Neuraminidase production was first demonstrated in M. haemolytica by Scharmann et al. (1970). Frank and Tabatabai (1981) characterized the production of neuraminidase by different M. haemolytica serotypes and isolates. They also reported that antiserum to M. haemolytica is capable of neutralizing neuraminidase activity. Straus and Purdy (1995) established that neuraminidase is produced in vivo in market stressed cattle. Fimbrae. In order for rapidly multiplying M. haemolytica to colonize the nasopharynx the bacteria must either adhere to mucosal epithelial cells or proliferate within the mucous layer. Fimbrae are associated with epithelial cell adherence in a number of bacteria and Potter et al. (1988) and Morck et al. (1987) reported M. haemolytica associated fimbrae. Two types of fimbrae, a large, rigid fimbrae (Morck et al., 1987) as well a smaller, flexible fimbrae (Potter et al., 1988) have been reported but fimbrae have are not found in M. haemolytica isolated from the lung or URT of cattle (Gonzalez et al., 1993). In addition, their role in attachment has not been demonstrated. A number of Gram-negative bacteria express adhesins that are distinct from fimbrae (Minion et al., 1986; Mooi et al., 1992). It is possible that an unknown virulence factor or other known virulence factors such as neuraminidase play a role in adherence so that the involvement of fimbrae may not be essential for colonization.
Iron-regulated proteins. The host sequesters iron during infection whereas organisms up-regulate the expression of iron-regulated proteins for iron-uptake when the organism is in an iron-restricted environment such as the lung (Donachie and Gilmour, 1988;
Confer et al., 1995). Iron is essential in a variety of cellular functions for both the host and an invading organism. M. haemolytica expresses iron-regulated proteins, both outer membrane proteins (Donachie and Gilmour, 1988; Lainson et al., 1991; Confer et al., 1995) termed transferrin binding protein A and B (TbpA and TbpB) and periplasmic proteins
(Lainson et al.,1991; Tabatabai and Frank, 1997) termed ferric binding protein A and B
(FbpA and FbpB). TbpA and TbpB are receptors for transferring iron (Ogunnariwo and
Schryvers, 1990; Gray-Owen, 1996) while FbpA and FbpB function as iron-binding and 15 iron-transport proteins (Gray-Owen and Schryvers, 1996; Kirby et al., 1998). Davies et al.
(1992) showed that expression of outer membrane iron-regulated proteins increases when M. haemolytica are grown under iron-restricted conditions and that this increase occurrs only in disease-associated strains. Belzer et al. (2000) reported that convalescent calves have high antibody titers to the 35 kDa FbpA. The native FbpA does not cross-react with other
Pasteurella trehalosi serovars (Tabatabai and Frank 1997).
Vaccines
An ideal vaccine would provide long-term protection against disease, be easily administered and have no adverse reactions. A significant degree of protection from pneumonic mannheimiosis is achieved through passive transfer of humoral immunity
(Mosier et al., 1995) and calves challenged with M. haemolytica after an experimental infection shows no clinical signs of disease (Cho and Jericho, 1986). These results indicate that protection from an M. haemolytica challenge is possible. However, the history of vaccine development for M. haemolytica-induced BRD has shown that this is not an easy task and full protection for all animals may never be achieved. Although a number of important virulence factors are identified, none of them alone has been sufficient to provide protection. A variety of vaccine formulations have been tested including bacterins, live vaccines and subunit vaccines. Protection is variable. Often there is a significant difference in the efficacy of a particular vaccine between experimental trials and field trials and repetition of results from individual trials has been inconsistent. Experimental trials are hampered by a lack of a suitable laboratory animal model of BRD. Trials are limited by animal size and availability of accommodations keeping many trials to a small number of 16
animals. More knowledge about the pathogenesis of pneumonic mannheimiosis may be
necessary for development of a consistently effective vaccine.
History. Early vaccine effectiveness was limited due to confusion over which
organisms to incorporate into a vaccine. Vaccines included M. haemolytica and P. multocida
as well as the viruses infectious bovine rhinotracheitis (IBR) and parainfluenza-3 (Pl-3)
individually and in various combinations. M. haemolytica alone was shown to produce
fibrinous pneumonia which reduced the focus on bacterial vaccines to M. haemolytica
(Confer et al., 1989). Because virally induced stress is associated with BRD, viral vaccine
formulations remain an area of experimentation. Initial vaccines consisted of bacterins, viral
vaccines or bacterin-viral combinations. None of these provided consistent protection. In
the 1980s live vaccines at times were successful although at times no effect due to
vaccination was found (Purdy, et al., 1986). More recently, efforts have been focused on
subunit vaccines incorporating known virulence factors and bacterial cell extracts.
Bacterins. Bacterin vaccines in different trials are shown to be somewhat protective
(Mosier, 1989), have no effect (Confer et al., 1985b) or at other times result in more severe
lesions (Friend et al., 1977; Wilkie et al., 1980). Confer et al. (1985) showed that while no
effect is found with a bacterin vaccine, a live vaccine in that same trial results in significant
reduction in pulmonary lesions. The focus for vaccine production shifted to live vaccines.
Live vaccines. During the 1980s, the protective effect of live and modified-live vaccines was explored. In an experimental transthoracic challenge of calves following vaccination with either a bacterin or a live M. haemolytica vaccine, little reduction in mean lesion scores resulted from a formalin-killed bacterin vaccination (Confer et al., 1985).
However, a live M. haemolytica vaccine grown on brain-heart infusion agar results in at least
a four-fold reduction in lesion scores when compared to control animals. Although nearly
60% of the bacterin-treated calves had antibody titers that were as high or higher than live-
vaccinates, no correlation is found between these antibody titers and resistance to infection.
A similar effect was seen in experiments by Panciera et al. (1984). However, Purdy et al.
(1986) found no significant effect on performance, morbidity or mortality from a live, freeze-
dried vaccine in field trial conditions. A modified-live lyophilized M. haemolytica vaccine
trial resulted in greater than a two-fold reduction in lesion scores (Blanchard et al., 1987).
The reduction in protection afforded by modified-live vaccines compared to live vaccines
may be due to the loss of antigens important for protection or the presence of replicating M
haemolytica may be necessary. The effectiveness of live vaccines is limited by the concurrent administration of antibiotics and is sensitive to improper storage and administration of the vaccine preparations.
Subunit vaccines. More recent vaccination efforts have focused on subunit vaccines.
High antibody titers to various antigens of M. haemolytica correlate with protection. High antibody responses to both the leukotoxin and the CPS correlate with protection in cattle
(Confer et al., 1985a). As more is learned about the pathogenesis of pneumonic mannheimiosis and the role of different virulence factors, it is anticipated that vaccines incorporating or enriched in known virulence factors will provide better protection.
Presponse is a commercial vaccine incorporating an enriched leukotoxin fraction with culture supernatant. The culture supernatant contains ST1 specific antigens and other soluble 18 capsular material. A number of experiments involving Presponse show an approximately two fold increase in protection following Presponse vaccination (Shewen and Wilkie, 1989;
Shewen et. al., 1988; Jim et. al., 1988). In one experimental trial, a vaccine incorporating
Presponse enriched with recombinant Lkt is more efficacious than Presponse alone (Conlon et al., 1991).
A vaccine composed of outer membrane proteins of M. haemolytica with no leukotoxin resulted in lesion scores similar to scores found with live vaccinates (Morton, et al., 1995). This argues against the importance of Lkt in vaccine formulations. Gilmour et al.
(1991) and Potter et al. (1999) demonstrated the importance of iron-regulated proteins in protection. Incorporation of iron-regulated proteins in a sodium salicylate extract vaccine in sheep results in almost complete protection (Gilmour, 1991). Vaccination with iron- regulated proteins TbpA and TbpB alone resulted in a fourfold decrease in clinical scores compared to unvaccinated calves (Potter et al., 1999). There is also experimental evidence for a role for the CPS in protection of goats (Purdy et al., 1993). Vaccines containing M. haemolytica chemical extracts show some promise for protection. A recent experiment involving an M. haemolytica sodium salicylate extract enriched with LPS, Lkt, iron-regulated outer membrane proteins and CPS resulted in mean clinical lesion scores similar to that found with live vaccines (Sreevatsan et al., 1996). Subunit vaccines avoid the inherent problems a live vaccine presents and appear to hold promise in deriving an effective vaccine.
Growth condition dependent protein expression. Pathogenic bacteria may express different components when grown in vivo rather than in vitro (Davies et al., 1994). In an effort to recreate in vivo protein expression while using in vitro systems and also to define 19 culture conditions that affect expression of particular proteins, a number of studies have been done to define protein expression under different culture conditions. Conventional media for in vitro growth is usually selected for its ability to support M. haemolytica growth. Shewen and Wilkie (1982) reported that the addition of 7% fetal bovine serum (FBS) to RPMI-1640 tissue culture media achieves enhanced M. haemolytica leukotoxin production compared to tissue culture media alone. This may be due to the presence of iron in FBS, because Gentry et al. (1986) demonstrated that the addition of iron compounds to culture media results in leukotoxin production levels comparable to FBS induced leukotoxin production levels. Iron- regulated outer membrane proteins of 71, 77 and 100 kDa are expressed at higher levels in iron-restricted media (Deneer et al., 1989; Morck, 1991). Increased expression levels of outer membrane proteins (OMPs) are found in M. haemolytica grown in decomplemented fetal calf serum (Davies et al., 1992).
In another study, M. haemolytica OMP protein expression was compared between bacteria that were isolated from lungs of experimentally infected calves to bacteria cultured under various in vitro growth conditions. OMP expression profiles on SDS-PAGE are similar for M. haemolytica isolated from lungs and M. haemolytica grown in newborn calf serum (Davies et al., 1994). No CPS production is detected when M. haemolytica is grown above 40°C. When M. haemolytica is grown at 43°C, enzymes involved in CPS biosynthesis are found at a level at least 25 times lower than M. haemolytica grown at 37°C (Barallo,
1999). Establishing in vitro growth conditions that closely mimic in vivo conditions may increase the efficacy of vaccines prepared from in vitro M. haemolytica cultures. 20
Immunoglobulin-binding proteins (IgBPs)
Immunoglobulin-binding proteins are expressed by a number of bacteria and bind immunoglobulins non-specifically, typically on the Fc or Fab portion of the molecule. They are believed to be virulence factors.
History. The phenomenon of bacterial surface proteins binding to immunoglobulins in a nonspecific manner was described well before the basis for this reactivity was understood. Jensen (1958) demonstrated reactivity between Staphylococcus aureus and 500 normal human serum samples. This reactivity does not correlate with previous known exposure to the bacteria. He termed this a kind of natural immunity or pseudoimmune reactivity and called the bacterial surface antigen involved antigen A. In another demonstration of antigen A reactivity, Cohen et al. (1961) found S. aureus binds rabbit sera with no known previous exposure to the bacteria. Antigen A was termed protein A after its protein nature was determined by digestion with trypsin (Grov et al., 1964). Sjôquist combined new found biochemical knowledge of immunoglobulins with work to define the antigens on the surface of S. aureus and along with Fôrsgren determined protein A binds to the Fc portion of IgG (Fôrsgren and Sjôquist, 1966). In their research, gel filtration purified protein A and papain digested IgG Fc fragments precipitated in an agar gel Ouchterlony plate. Until recently, bacterial proteins that bind immunoglobulins were termed Fc receptors.
More recently, they have been termed immunoglobulin-binding proteins (IgBPs) in recognition of the fact that they have no signaling capability as other cell surface receptors have. 21
Protein A. Further characterization of protein A showed that it is a cell surface
protein (Sjôquist et al., 1972) with a molecular weight of 42-kDa and has a distinct extended
shape (Bjorck et al., 1972). Protein A is antiphagocytic and chemotactic in vitro (Dosset et
al., 1969), and IgG complexed with protein A activates complement via the classical pathway
(Sjôquist and Stâlenheim, 1969). The discovery of extracellular protein A in methicillin-
resistant S. aureus (Lindmark et al., 1977) led to the development of a method for
purification and production of soluble protein A. Work focused towards the widespread
utilization of protein A as an immunochemical regent in detecting and isolating antibodies
and little attention was paid to its role in S. aureus infections.
Immunoglobulin-binding protein classification. An IgBP was discovered on the surface of ^-hemolytic streptococci. This protein is called protein G and it is a cell surface expressed and secreted IgBP associated with groups A, C, and G streptococci (Kronvall.
1973). Protein G, like Protein A, is an elongated molecule (Âkerstrom and Bjorck. 1986).
The discovery of protein G led to the concept that proteins on the surface of bacteria that nonspecifically bind immunoglobulins may be a general feature of bacteria-host interactions.
Following this finding a systematic study determined that many Gram-positive bacteria express IgBPs that bind Ig from a variety of Ig sources. Myhre and Kronvall (1977, 1980,
1981), and Myhre et al. (1979) described five different types of IgBPs based on functional binding properties of bacteria to Igs from different species and antigenic relatedness. In
1988, Reiss identified a sixth type of IgBP based on these same criteria (Reiss, 1990).
Immunoglobulin binding proteins have been identified on other Gram-positive as well as Gram-negative bacteria. Coprococcus comes (Van de Merwe and Stegeman, 1985), 22
Haemophilus somnus (Widder et al., 1988), Brucella abortus (Bricker et al., 1991),
Actinobacillus actinomycetemcomitans (Mintz et al., 1994), Aeromonas salmonicida (Phipps
and Kay, 1988) and Escherichia coli (Sandt et al., 1997) are all known to express IgBPs. No
published information is available that describes classification of IgBPs from gram-negative
organisms.
Immunoglobulin-binding protein specificity. IgBPs bind immunoglobulins other
than Fc IgG. Protein L from Peptostreptococcus magnus binds k light chains (Bjorck, 1988)
and protein A and protein G bind Fab and F(ab')a respectively (Boyle, 2000). Clostridium
perfringes expresses an IgBP termed protein P that also binds F(ab')2 fragments (Boyle,
2000). Neisseria catarrhalis, group A, C and G streptococci, Haemophilus influenzae
(Forsgren and Grubb, 1979), Haemophilus haemolyticus and Haemophilus aegypticus
(Akkoyunlu, 1991) all bind human IgD. M proteins sir (Stenberg, 1994) and arp (Heden and
Lindahl, 1993) and a specific IgA receptor on group B streptococci (Lindahl, 1990) bind
human IgA. Protein sir also binds IgM. Additional specificity for IgM has been reported for
B. abortus (Nielsen, et al., 1981), and Borrelia burgdorfori (Dorward, et al., 1992).
Immunoglobulins are not the only proteins IgBPs bind. While it has been known since 1987 that protein G binds albumin and IgG (Bjorck, 1987), more recent work has shown that other IgBPs bind other proteins. Specificity for binding plasminogen (Berge and
Sjôbring, 1993), C4-binding protein (Thern, 1995) fibrinogen and fibronectin (Reichardt, et al., 1995) and factor H (Kotarsky, 1998) has been demonstrated. Protien G has separate binding sites for IgG and albumin (Frick et al., 1994, 1995). The Fc binding portions of protein G bind two plasma proteinase inhibitors, alpha-2 macroglobulin and kininogen. 23
Binding for all three proteins apparently occurs within the IgG binding domains (Bjorck et al., 1987; Sjôbring et al., 1989)
Immunoglobulin-binding proteins and virulence. Fc-mediated antibody binding to the surface of IgBP expressing bacteria has provided the classical picture for a role for IgBPs in virulence. Antibody bound to the surface of bacteria would provide a means for bacteria to evade the host immune response by inhibiting phagocytosis and complement-mediated killing. Additionally, soluble IgBPs bound to antibodies would decrease the number of specific antibodies available for a humoral immune response. While these effects have been demonstrated in vitro, the role of IgBPs in virulence in vivo has been more difficult to demonstrate. But this does not preclude a role for IgBPs in virulence. Continuing research shows a greater IgBP role in virulence than had originally been conceived.
Superantigens. In 1979, it was established that protein A bound Fab IgG and that this binding took place outside the antigen-binding site (Endressen, 1979). Protein A binds the Fab portion of IgA, IgM and IgE (Inganas, 1981). Protein A binding is restricted to antibodies coded by the VH3 family of genes (Sasso, et al., 1991) and 32-54% of all circulating B cells are capable of this binding. Protein A is also capable of binding membrane bound immunoglobulins. This binding is similar to that found with T cell superantigens.
Protein A, acting as a B cell superantigen, activates complement (Kozlowski et al.,
1996), induces expression of Ig from the Vh3 gene family (Kristiansen et al., 1994), increases antigen presentation through soluble protein A:Ig complexes associated with Vh3 family surfaced expressed immunoglobulins (Leonetti et al., 1999) and soluble protein A:Ig
L 24 complexes are involved in inflammation as demonstrated by the elicitation of an Arthus reaction (Kozlowski et al., 1998). Protein A and protein L, acting as superantigens, activate human heart mast cells by binding IgE on the surface of cells (Genovese, 2000).
Although these responses may appear to have a strong role in virulence, recently a perhaps more far reaching role has been found for protein A acting as a superantigen via suppression of Vh3 expressing B cells. Following intraperitoneal immunization of both neonatal and adult VH3 "knock in" mice with protein A, researchers demonstrated a selective suppression in Vh3 expressing IgM secreting cells in both the spleen and liver and a loss of detectable Vh3 expressing B-l lymphocytes in the peritoneum. This suppression is T cell independent and selectively suppresses a set of Vh3 antigen-specific natural antibodies. Mice treated with protein A are tolerant to a whole cell S. pneumonia extract as measured by
ELISPOT assay for specific IgM secreting cells. After cessation of protein A treatment, splenic lymphocytes (B-2 B cells) return to normal whereas the suppression of peritoneal lymphocytes (B-l B cells) is still present 7-21 weeks later (Silverman et al., 2000). This study demonstrates a potentially very important role for IgBP involvement in virulence.
Mitogens. Protein A activates lymphocyte proliferation. This activation is limited to B cells with no T cell stimulation found. Optimal stimulation results when protein
A is immobilized either on Sepharose or on the surface of S. aureus (Fôrsgren, et al., 1976).
Stimulation occurrs with IgG, IgD and IgM bearing B cells (Romagnani, et al., 1980).
Mitogenic activity of Protein A persists when tyrosil residues necessary for Fc IgG binding were inactivated and appeared to be due to Fab binding (Romagnani, et al., 1982). 25
Complement activation and inhibition. Early studies demonstrated that soluble protein A:IgG complexes fixes complement (Sjôquist and Stâlenheim, 1969).
Langone et al (1978) showed that protein A and IgG complexes in the molecular formula
[(IgG): protein A]2 act like IgM molecules in fixing complement and lysing erythrocytes.
More recently, protein H was shown to form soluble complement-activating complexes that cleave C3 but to inhibit complement activation by Ig-coated target. Protein H inhibits Clq binding to IgG immobilized on polyacrylamide beads (Berge, 1997).
Antiphagocytes. In 1969, Dosset et al. showed that protein A on the surface of S. aureus inhibits phagocytosis and that this effect is dose dependent. This phenomenon has been demonstrated with a number of IgBPs and more recently was shown with protein H from Streptococcus pyogenes (Kihlberg et al.,1999).
Antibody-dependent cellular cytotoxicity inhibition. IgBPs have been shown to inhibit antibody-dependent cellular cytotoxicity. Rosenblatt (1977) showed that incubation of antibody-coated target cells with protein A inhibits lysis by lymphocytes.
Virulence role in vivo. The association of disease and IgBP expression has been difficult to show in vivo. This may be because of the complex nature of virulence and because even though an IgBP may have been identified in an organism, it may not be the only IgBP expressed by that organism. This was demonstrated in a protein A deficient mutant S. aureus infection. The mutants were constructed with site-directed allelic replacement. The protein A-deficient mutants are marginally less virulent than protein A expressing S. aureus as measured by death after intraperitoneal injections of bacteria in mice. 26
The mice injected with protein A expressing S. aureus die significantly more quickly than mice injected with the mutant (Patel et al., 1987). The mechanism for the reduction in virulence is not known and the results may have been more dramatic if a second IgBP S. aureus has recently been shown to express, Sbi, (Zhang, 1998) had also been deleted.
Further evidence for IgBPs role in virulence was established in H. somnus infection in cattle.
Pathogenic and carrier isolates were checked for immunoglobulin-Fc binding activity. All isolates express binding activity except for carrier isolates from the prepuce of bulls. None of these binds IgG-Fc (Widders, 1989). Finally, the immunoglobulin-binding domains of protein L form P. magnus, a pathogenic vaginal colonizer, were expressed in Streptococcus godonii, an oral commensal. The recombinant bacteria show increased murine vaginal colonization and persisted for a longer time in the murine vagina (Ricci et al., 2001). These experiments show a role for IgBPs in bacterial virulence.
Therapeutic uses of immunoglobulin-binding proteins (IgBPs). IgBPs fused to antigens increase immunogenicity. This was seen as an increased humoral immune response in vivo (Lowenadler et al., 1990) and in vitro as increased T cell presentation by antigen presenting cells expressing immunoglobulins on their surface (Leonetti, et al., 1998). Protein
A has five Fc IgG binding sites (Hjelm et al., 1975 and Lôfdahl et al., 1983) and increased immunogenicity correlates with an increased number of immunoglobulin-binding sites on protein A fused to antigen (Leonetti, et al., 1998). These phenomena may prove important because it increases immunogenicity without an adjuvant.
Protein A also has a number of biomodulatory effects. Protein A acts as a mitogen or induces apoptosis in the same cell depending on concentration. At a particular dose level, it 28
each with a molecular weight between 235-270 kDa. Each subunit is made up largely of
three types of repeating modules, types I, II and III. A variable number of type III repeats
contributes to the variability in subunit molecular weight. Fibronectin is a flexible, extended
molecule. Fibronectin binds to cell surfaces and dimers polymerize to form fibrils. Cell
adhesion is mediated by an arg-gly-glu (RGD) sequence of fibronectin that binds (3,
integrins. With continued polymerization, Fn fibrils extend into the ECM. Matrix formation
is a cooperative process involving interactions between Fn and other ECM components such
as collagens, laminin and proteoglycans. Addition of heparin and collagen to cultures has
been shown to increase Fn binding to cells (McKeown-Longo and Mosher, 1985).
Transforming growth factor-(3 enhances expression of Fn matrix assembly sites and this is
accompanied by an accumulation of Fn fibrils (Allen-Hoffman et al., 1998). Fibronectin also
contains binding domains for fibrin, heparin, and collagen.
Fibrinogen. Fibrinogen is a 340 kDa glycoprotein synthesized in
hepatocytes. It is composed of three pairs of nonidentical chains that are linked by interchain
disulfide bonds to form an Fg molecule. Fibrinogen is cleaved by thrombin to form fibrin
and thus is involved in clotting. Fibrinogen also binds to a platelet receptor and mediates
platelet adhesion and aggregation. Fibrinogen interacts with integrin receptors on endothelial
cells (Cheresh, et al., 1989) and leukocytes (Altieri et al., 1990).
Extracellular matrix-binding proteins
Identification. Kuusela (1978) first reported that fibronectin would bind to S. aureus. Following this, two S. aureus Fn-binding proteins (FnBPs), FnbpA and FnbpB, were 29
identified (Signas et al., 1989 and Jônsson et al., 1991). FnbpA is known to bind Fg as well
as Fn. (Wann et al., 2000). ECMBPs of S. aureus are best characterized and along with
FnbpA and FnbpB include surface expressed type II collagen-binding protein (CnBP),
clumping factor A and B (ClfA and ClfB), Fg-binding proteins, and a secreted Fg-binding
protein (Efb) (in Flock review, 1999). One S. aureus ECMBP, extracellular adherence
protein (Eap), binds seven plasma proteins including Fn and Fg (Palma et al., 1999).
Although S. aureus ECMBPs are the best characterized, a sizeable number of other organisms express ECMBPs. Streptococcus spp. express ECMBPs. In addition, a number of
Gram-negative organisms including Escherichia coli (Westerland et al., 1989), Haemophilus ducreyi (Abeck et al., 1992), H. influenza (Fink et al., 2002), Neisseria gonorrhoaea (van
Putten et al., 1998), and Borellia burgdorferi (Probert and Johnson, 1998) express ECMBPs.
Recently, ECMBP research has increased as their role in virulence is becoming more apparent.
A number of IgBPs are known to also bind ECMPs. For example, Protein H, present in some strains of S. pyogenes and first known to bind Fc IgG, also binds fibronectin. A fibronectin binding protein also from S. pyogenes, Sfbl, binds Fc IgG (Frick et al, 1995;
Medina et al, 1999). A Group C streptococci expresses protein FAI which binds fibrinogen, albumin and IgG (Talay et al 1996). In addition, FgBP, a fibrinogen-binding protein of
Streptococcus equi, has recently been shown to bind equine Fc IgG (Meehan et al, 2001).
Virulence association. Unlike IgBPs which were originally valued as reagents to isolate immunoglobulins, studies on extracellular matrix-binding proteins were focused on their role in virulence soon after these protein were identified. Although the mechanisms are 30 generally unclear, a number of roles for ECMBPs in virulence have been determined.
Fibronectin-binding proteins in particular have been associated with virulence and once again, most work has been done with S. aureus.
Exogenous Fn is required for internalization of S. aureus by HEp-2 cells. HEp-2 cells are unable to produce Fn. In this same study, monoclonal antibody to (3, integrins significantly reduced invasion which suggests a bridge is formed by Fn between an FnBP and
Pi integrins (Dziewanowska et al., 2000). Recent work by Dziewanowska (2002) demonstrated that invasion into human corneal epithelial cells isreduced by 99% with an
FnBP-deficient isogenic strain of S. aureus. Clearly, FnBPs play a role in S.aureus internalization.
In relation to respiratory diseases, two sets of experiments illustrate the importance of
S. aureus FnBPs in bacterial adherence to human endothelial and airway epithelial cells.
Mongodin et al. (2002) established that an FnBP-deficient strain of S. aureus have a five-fold decrease in the level of adherence to human airway epithelial cells suggesting that FnBPs are involved in adherence. In 1999, Peacock et al., using a number of strains deficient in individual ECMBPss or protein A showed that only the mutant deficient in FnbpA and B production lowered adherence to endothelial cells. They conclude that their results indicate that Fnbp binding to endothelial cell Fn is the dominant pathway for S. aureus adherence to human endothelial cells in vitro. Bacterial cell adherence to host cells is a required initial step in most bacterial infections and ECMBPs, particularly FnBPs, may play a large role in this process or may be required for infection.
In vivo and in vitro models demonstrate several additional roles for ECMBPs in virulence. Both FnBP and ClfA, a fibrinogen-binding protein, enhance S. aureus binding to 31
atopic skin as was found with S. aureus strains that did not produce one or the other of these
proteins (Cho et al., 2001a). On human T cells, integrins ot4pi and otspi bind fibronectin and
function as a costimulatory molecule for T cell activation. Miyamoto et al. (2001)
established that S. aureus FnbpA through the T cell a$Pi integrin mediates adhesion to T
cells and T cell activation in vitro in the presence of Fn and an anti-CD3 monoclonal
antibody. In relation to this, S. aureus preferentially binds to skin sites of T helpers-
mediated sites of inflammation when compared to T helper-1 -mediated sites of inflammation
in a murine model. Fibrinogen and Fn mediate this binding (Cho et al., 2001b). An isogenic
mutant unable to produce the Fn- and Fg-binding protein of Streptococcus suis ST2
decreases colonization of the joints and CNS of piglets while colonization of the tonsils is
unaffected (de Greeff et al., 2002). The role of ECMBPs is not limited to adherence.
Vaccine potential. In 1999, Brennan et al. demonstrated that antibodies raised to one
of three fibronectin-binding sites on S. aureus Fnbp are able to block adherence of S. aureus
to Fn in vitro. Work previous to this showed that antibodies to Fnbp opsonize S. aureus as seen in an in vitro phagocytosis assay. Also, mice clear opsonized bacteria from the
peritoneal cavity and liver more rapidly than controls (Rozalska and Wadstrom, 1993).
Mamo et al. (1995) described a 1000 fold reduction in the number of opsonized S. aureus
recovered from mammary glands of mice injected with opsonized bacteria as well as a
significant reduction in pathology. Mamo et al. (1994) showed that mice vaccinated with an
Fn-binding domain from S. aureus Fnbp have a decreased number of bacteria recovered from mammary glands as well as a significantly reduced incidence of severe mastitis. In an organism other than S. aureus, immunization with Streptococcus pyogenes fibronectin- 32
binding protein, FB54, results in mice that survive significantly longer than control mice. In
the same study, an increased IgG titer to FB54 is increased in actively infected human
patients compared to healthy volunteers (Kawabata et al., 2001). Hap is a broad-spectrum H.
influenza expressed ECMBP that binds fibronectin, laminin and collagen IV. Cutter, et al.
(2002) demonstrated that mice immunized intranasally with purified Hap significantly
reducted the density of nasopharyngeal colonization compared to controls challenged with a
heterologous strain of H. influenza. The overall results from the above-mentioned studies
suggest that a vaccine-induced immune response to ECMBPs may reduce the incidence of
disease.
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CHAPTER 2. IDENTIFICATION, ISOLATION, AND CHARACTERIZATION OF
AN IMMUNOGLOBULIN-BINDING PROTEIN EXPRESSED BY MANNHEIMIA
HAEMOLYTICA
Ruth Smith Osmundson and Louisa B. Tabatabai
A paper to be submitted to Infection and Immunity
Abstract
Mannheimia haemolytica is the primary causative agent of bovine respiratory disease
(BRD) or "shipping fever". Bacterial immunoglobulin-binding protein (IgBP) expression is known to play a role in the pathogenesis of a variety of organisms. This study demonstrated the presence of an IgBP in whole cell sonicates (WCSs) and a culture supernatant (CS) preparation from M. haemolytica serotype (ST) 1. Far-Western blot demonstrated binding of bovine Fc IgG to M. haemolytica expressed IgBP. An M. haemolytica IgBP(s) protein was isolated by affinity chromatography using bovine gamma globulin linked to Sepharose and flow cytometry showed the surface expression of an IgBP on whole cell M. haemolytica.
Immune sera from convalescent cattle showed an increased antibody response to the 75.0 kDa IgBP when compared to sera from naïve or acutely infected cattle as seen by Western blot. These results demonstrated the presence of an IgBP on the surface of M. haemolytica and that an immune response to the 75.0 kDa IgBP may play a role in protection from M. haemolytica infection. It was demonstrated that all 12 serotypes (1,2, 5-9, 12-14, and 16) bound both bovine Fc IgG and sheep Fc IgG without appreciable differences between ST1, most frequently isolated in infections in cattle and ST2, isolated predominantly in sheep 51 mannheimiosis. Serotype-specific Fc IgG binding did not appear to play a role in species- specific infection by IgBPs from different serotypes of M. haemolytica.
Introduction
Bovine respiratory disease (BRD) or "shipping fever" is believed to result in the single largest loss annually to the cattle industry. BRD has a complex etiology but pulmonary fibrinous pneumonia, the ultimate cause of severe clinical disease and death, is primarily caused by Mannheimia haemolytica (20). Calves are chiefly affected and are predisposed to M.haemolytica infection following stressful incidents such as shipping, weaning or viral infection. M. haemolytica, a normal flora bacterium of the nasopharynx, increases in number and colonizes the upper respiratory tract following a stressful incident.
The increased number of M. haemolytica are believed to be inhaled into the lung resulting in macrophage activation and pulmonary serum flooding. An influx of neutrophils along with an M. haemolytica secreted leukotoxin exacerbates the response. This leads to lung lesions characteristic of M. haemolytica-'mduced fibrinohemorrhagic pneumonia. Some animals are able to recover while others succumb to pneumonic mannheimiosis (45).
A number of vaccines are available for M. haemolytica induced BRD. Bacterins provide protection of variable efficacy. A number of experimental trials even show an exacerbation of BRD in bacterin vaccinates (17,47). While live vaccines generally perform better in field-tests than bacterins (7), live vaccines still have variable efficacy with some trials showing no increase in protection due to immunization (35). Also, live vaccines have inherent problems with production and stability. While not providing full protection, bacterial component vaccines demonstrate the importance of leukotoxin (8), iron binding 52
proteins (34) and surface antigens (39) in protection. There is a need for a more efficacious
vaccine.
M. haemolytica expresses a number of known virulence factors. The best
characterized is a leukotoxin (21), a member of the RTX toxin family. While the leukotoxin
is a major virulence factor, immunization with the leukotoxin alone is not sufficient to
provide protection (43). Other virulence factors include the capsular polysaccharide (5), LPS
(36), fimbrae (31), iron-regulated proteins, (9), neuraminidase (13) and O-sialoglycoprotease
(1). This study demonstrated the presence of an additional M. haemolytica virulence factor,
an immunoglobulin-binding protein (IgBP).
IgBPs have been identified in a number of other gram-positive and gram-negative
bacteria. Protein A from Staphylococcus aureus Cowan 1 was the first IgBP identified (12).
Protein A inhibits phagocytosis and protein A surface expression is responsible for this
inhibition (10). Protein A in solution fixes complement by forming IgG:protein A complexes
(27) and inhibits antibody-dependent cellular cytotoxicity (38). Streptococcal protein H, an
Fc IgG binding protein, forms soluble complement-activating complexes with IgG but decrease complement activation by inhibiting Clq binding to IgG of IgG-coated cells (2). A
Moraxella catarrhalis IgD-binding protein, MID, activates human B cells and in the presence of Th2 cytokines induces immunoglobulin (Ig) secretion (18). Acting as a B cell superantigen, protein A activates complement (23), induces expression of Ig from the Vh3 gene family (25) and increases antigen presentation through soluble protein A:Ig complexes associated with Vh3 family surface expressed immunoglobulins (28). It is involved in inflammation as demonstrated by the elicitation of an Arthus reaction through administration of soluble protein A:Ig complexes (24). Following intraperitoneal immunization of both 53
neonatal and adult Vh3 "knock in"mice with protein A, researchers demonstrated a selective suppression in Vh3 expressing IgM secreting cells in both the spleen and liver and a loss of detectable Vh3 expressing B-l lymphocytes in the peritoneum (40). In vivo and in vitro evidence shows that protein A acts as a B cell superantigen.
In vivo experiments also indicate a role for IgBPs in virulence. Mice injected with an
S. aureus protein A deletion mutant require a marginally higher dose of the mutant for infectivity and infected mice take significantly longer to succumb to infection and die at a slightly decreased rate than mice infected with S. aureus expressing protein A (32). These results may have been more dramatic if a recently discovered second S. aureus IgBP, Sbi, had also been deleted (49). When immunoglobulin-binding domains of peptostreptococcal protein L are expressed on Streptococcus gordonii, vaginal colonization is enhanced and colonization persists for a longer time (36). These experiments demonstrate a role for IgBPs in virulence although their role has not been clearly elucidated.
Early work with Gram-positive bacteria focused on utilizing IgBPs as immunochemical reagents. For example, protein G binds Igs from a wider range of species while protein A binds Igs from fewer species and with lower affinity (5). These features have been used to isolate Igs from different species. No work has been published concerning the possible relationship between species-specific IgBP binding and species-specific infection or concerning a possible relationship between serotype-specific IgBP binding and serotype-specific infection.
Preliminary results in our lab indicated that M. haemolytica might express an IgBP.
When normal bovine serum (NBS) is used as a negative control to label whole cell M. haemolytica in an ELISA, similar absorbance values are obtained with the NBS and with an anti-sera specific for an M. haemolytica iron-regulated protein (Tabatabai, unpublished results). The objectives of the present study were: 1) To identify and isolate an M. haemolytica IgBP, 2) To determine the presence or absence of IgBP surface expression, 3)
To determine whether there is an increased antibody response to the IgBP in cattle convalescing from BRD, 4) To determine whether the IgBP may play a role in the dominance of ST1 association with BRD or the dominance of ST2 in sheep mannheimiosis.
Materials and Methods
Bacterial isolates
M. haemolytica ST 1 (isolate LI01) was isolated from an infected bovine lung (14).
Serotypes 1,2, 5-9, 12-14, and 16 (3, 33, 11) were gifts from G. Frank (USDA/ARS National
Animal Disease Center, Ames, Iowa). Serotype 17 (48) was unavailable for this study.
Haemophilus somnus strain 8025 was a gift from Ron Griffith (Iowa State University).
Whole cell sonicate and supernatant preparations
Cells stored at -70°C were cultured overnight on blood agar plates, colonies transferred and cultured with shaking in 3 ml RPMI (Sigma; St. Louis, MO) with 20mM
HEPES (pH 7.2) for 6 hours at 37°C. Ten ml of these cultures were used to culture 1 L
RPMI for 20 hours with shaking at 37°C. The culture was centrifuged (6,000 x g for 20 minutes) and the CS was retained and stored at -70°C. The cell pellet was washed with 0.1M phosphate buffered saline (PBS), pH 7.2, and centrifuged (6,000 x g for 20 minutes) three times. The cells were lysed in water. The lysate was sonicated for four minutes (4 cycles of
1" on, 1 "off, power at 7, duty cycle at 8) using a Branson Sonifier 250. The lysate was 55 ultracentrifuged (50,000 x g for 2 hours at 4°C). The supernatant was retained as the whole cell sonicate (WCS) preparation, dialyzed in 5 mM NH4HCO3 (pH 7.2), aliquoted and stored at -70°C. Prior to IgBP affinity isolation, M. haemolytica WCS was dialyzed in 50 mM Tris
(pH 8.0). This procedure was used for all M. haemolytica serotypes and H. somnus 8025.
Gel electrophoresis and immunoblotting
SDS-PAGE was conducted on denatured M. haemolytica WCSs, CS and isolated
IgBP in 12.5% acrylamide gels with a 4% acrylamide stacking gel (26). Protein concentrations for all preparation were determined by Folin-Lowry (29). Proteins were transferred onto 0.45p,m Protran nitrocellulose (Schleicher & Schuell, Keene, NJ) using a
Bio-Rad Trans-Blot Cell apparatus. Nitrocellulose blots were blocked with 0.25% fish gelatin (Norland Products, Inc., New Brunswick, NJ ) in 0.1M PBS, pH 7.2 for 15 minutes.
In far-Western blots, which detect non-antibody mediated protein:protein interactions, transferred IgBP was bound to bovine Fc IgG by incubating nitrocellulose blots overnight in bovine Fc IgG (44 jj.g/10 ml 0.1M PBS, pH7.2, Jackson Laboratories, West Grove, PA) or sheep Fc IgG (180 p.g/10 ml 0.1 M PBS, pH7.2, Jackson Laboratories, West Grove, PA). In
Western blots, which detect antigen:antibody interactions, transferred WCS was incubated with cattle sera (1:100) in 0.25% fish gelatin, 0.1M PBS, pH 7.2. After washing (0.1 M PBS, pH 7.2 with 0.05% Tween 80) the blots were developed with horseradish-peroxidase (HRP)- conjugated rabbit anti-bovine Fc IgG, sheep Fc IgG or bovine IgG.
Bovine antisera
Sera from acutely infected animals were obtained from 10 feed yard calves three days after experimental infection with M. haemolytica. Sera from convalescent animals were 56
collected two weeks post-infection. The log2 indirect hemagglutination titers (14) ranged from 4 to 7 for the infected calves and from 7 to 10 for the convalescent calves.
Affinity isolation
Cyanogen bromide (CNBr)-activated Sepharose 4B (Sigma, St. Louis, MO) was coupled to bovine gamma globulin (Jackson Immunoresearch Labs, West Grove, PA) according to manufacturer's procedure. M. haemolytica WCS in 50 mM Tris (pH 8.0) was adsorbed with bovine gamma globulin-Sepharose 4B by rotating the sample for 48 hours at
4°C. IgBP was eluted from the Sepharose in a column with 0.9% NaCl-lM propionic acid.
IgBP was concentrated by Speedvac vacuum concentration (Savant, Holbrook, NY).
Flow cytometry
M. haemolytica whole cells were blocked with 0.25% fish gelatin in 0.1 M PBS (pH
7.2) for 15 minutes at room temperature before labeling. Cells (0.5x 106) were labeled with either 0.1 M PBS (pH 7.2) or 1.9 mg bovine Fc IgG (Jackson Immunoresearch Laboratories,
West Grove, PA) for two hours at room temperature. Fluorescein isothiocyanate (FITC) labeled goat F(ab')a anti-bovine IgG, Fc fragment specific (Jackson Immunoresearch
Laboratories) was conjugated to all cells for two hours at room temperature. Cells were washed across a 0.22 p.m filter five times (0.1M PBS, pH 7.2 with 0.05% Tween-80) before and after adding conjugate. The final wash after incubation of cells in conjugate was done
with 0.01 M PBS, pH 7.2. Fluorescence was detected on a Beckman Coulter EPICS XL-
MCL (Miami, FL). Absorbance was at 488 nm and emission was read at 550 DL/525 BP. 57
Results
Identification of M. haemolytica-^pressed IgBP(s)
Both WCS and CS preparations from M. haemolytica ST1 expressed at least one
IgBP as seen on an immunoblot (Fig. 1). A dominant band at 75.0 kDa, a band at 40.7 kDa and several less distinct bands ranging from 74.9 kDa to 62.0 kDa were present in both WCS and CS preparations. A band at 107.5 kDa was present only in the supernatant. Although the
M, haemolytica supernatant was at a higher concentration than the WCS preparation in this immunoblot, the 107.5 kDa band appeared to be unique to the supernatant as this band did not appear when concentrated isolated IgBP(s) was run on SDS-PAGE at higher concentrations (Fig. 2). The H. somnus WCS preparation was a positive control known to express IgBPs (46). No binding was evident between the conjugate and the WCS or supernatant preparations (results not shown).
Isolation of M. haemolytica-expressed IgBP
M. haemolytica IgBP(s) was isolated by affinity chromatography (Fig. 2). An M. haemolytica WCS preparation was adsorbed with CnBr activated-Sepharose 4-B to which bovine gamma globulin had been bound. IgBP(s) was eluted in a column with 1 M propionic acid in 0.9% NaCl. At the IgBP concentration collected in the fractions, bands are seen at
77.2, 63.2 and 40.2 kDa. After IgBP fractions were concentrated by vacuum centrifugation, additional bands were evident at 168.7, 132.5, 56.5, and 20.0 kDa. All bands were able to bind bovine Fc IgG as seen on far-Western blot (results not shown). Efforts to determine the
N-terminal sequence of the 77.2 and 20.0 kDa bands were unsuccessful. The N- terminus might be blocked or there may have been insufficient protein to be sequenced. Surface expression of M. haemolytica IgBP
Whole cell M. haemolytica (0.5x10* cells) were labeled with bovine Fc IgG or PBS.
Bovine Fc IgG bound to whole cells was detected with FITC-conjugated goat F(ab'): anti- bovine IgG, Fc fragment specific. Propidium iodide incorporated M. haemolytica cells were used to the set the gate identifying M. haemolytica whole cells. Flow cytometry measurements showed that the number of FITC positive cells was increased by 64.9 % on M. haemolytica whole cells labeled with bovine Fc IgG compared to PBS labeled cells (Fig. 3).
Humoral immunogenicity of an M. haemolytica IgBP
Sera from naïve cattle, acutely infected cattle and convalescent cattle were used to probe an M. haemolytica WCS preparation in a Western blot. Binding was detected with an
HRP-conjugated rabbit anti-bovine Fc IgG. The humoral immune response to the dominant
75.0 kDa IgBP was increased in convalescent cattle when compared to naïve or acutely infected cattle (Fig. 4). The location of the IgBP in the WCS preparation was identified in lane c. This lane shows the WCS preparation probed with bovine Fc IgG. H. somnus in lane b was a positive control.
Involvement of IgBP from different serotypes with species-specific infection
M. haemolytica WCS preparations from all serotypes (1,2, 5-9, 12-14, and 16) were probed with either bovine Fc IgG or sheep Fc IgG in a far-Western blot (Fig. 5 and 6).
Detection was with HRP-conjugated rabbit anti-bovine or anti-sheep Fc IgG. The same
WCS preparation was used for both blots and all lanes have the same concentration of M. haemolytica WCS. At the conjugate dilution used, rabbit immunoglobulin (Ig) binding to M. haemolytica IgBP(s) was not apparent (Results not shown). 59
All serotypes bound both bovine Fc IgG and sheep Fc IgG (Fig. 5 and 6). The banding patterns found by probing with either bovine Fc IgG or sheep Fc IgG were quite dissimilar. More bands were evident with sheep Fc IgG binding than with bovine Fc IgG binding. The dominant bands detected from bovine Fc IgG binding were 74.1 63.5 and 33.2 kDa whereas the dominant sheep Fc IgG IgBP(s) bands were 58.4 and 50.1 kDa.
Serotypes 1 and 2 both bound bovine Fc IgG with dominant bands at 74.1 and 63.5 kDa (Fig. 5). There was no significant difference between the serotypes in the intensity of the staining of either band. The 74.1 kDa band was more intense in both serotypes. Serotype
2 had an additional band at 33.2 kDa that was not evident in ST 1. The remaining serotypes had various similar IgBP binding patterns and no serotype has a band that is unique to that serotype alone.
Serotype 1 bound sheep Fc IgG less intensely than ST2 binds sheep Fc IgG (Fig. 6).
Although both serotypes had similar binding patterns, ST2 IgBPs bound sheep Fc IgG more intensely at 79.4, 58.4, 50.1, 30.1 and 24.7 kDa than did ST1. Serotypes 5, 8, 13 and 14 had intense bands 58.4 kDa although all serotypes expressed an IgBP that binds at 58.4 kDa.
Generally, all serotypes had a similar sheep Fc binding pattern.
Discussion
This study showed that an IgBP(s) present in WCS and CS preparations binds bovine
Fc IgG as shown on far-Western blot (Fig. 1). Both preparations have a dominant band at
75.0 kDa and a less intensely staining band at 40.2 kDa. This suggests that the same protein or proteins is expressed in the WCS and supernatant. Both bands are of similar intensity in the WCS and CS preparations although the CS has almost twice the amount of total protein 60 in lane b as compared to the WCS in lane a. This could indicate that less IgBP(s) is secreted than is cell bound. The supernatant preparation has an additional band at 107.5 kDa. This band appears unique to the supernatant because isolated, concentrated IgBP(s) from a WCS preparation in figure two shows a number of additional bands on SDS-PAGE but no band comparable to the 107.5 kDa band found in the supernatant.
Numerous molecular weight (MW) determinations by SDS-PAGE have shown the dominant M. haemolytica IgBP to have MW ranging from 58.0 kDA to 77.2 kDa. Molecular weight variability is indicative of elongated molecules. Protein A displayed a variable MW
(40.2 kDa to 56.0 kDa) and is an elongated molecule (4). This suggests that the dominant M. haemolytica IgBP may be an elongated molecule.
There are a number of possible explanations for the appearance of more than one
IgBP band on far-Western blot. IgBPs from other organisms have repeat binding sites for Fc
Ig. Protein A has five (30) while protein G has three repeat Fc Ig binding regions (19). It is
possible that the different M. haemolytica IgBP(s) bands evident on far-Western blot are due to the IgBP(s) expressing a different number of Fc IgG binding sites which would result in
number of different molecular weight bands. Multiple bands could also be due to a unique
protein in each Western blot band or the bands might be degradation products of one or more
proteins. There is evidence for the latter possibility as seen by SDS-PAGE of the isolated,
concentrated IgBP (Fig. 2).
Mannheimia haemolytica IgBP(s) was isolated by affinity chromatography by
binding bovine gamma globulin to CNBr-activated Sepharose (Fig. 2). When affinity-
isolated IgBP(s) is concentrated (Fig. 2, lane c), five additional bands are evident. The 63.2
and 22.4 kDa bands appear to be breakdown products of larger proteins because these bands 61 are much more intense when the affinity-isolated IgBP(s) was concentrated while the 77.2
KDA band is of equal intensity in both preparations. Further work with the isolated protein
is planned in an effort to compare the M. haemolytica IgBP(s) to other better-characterized
IgBPs from gram-positive bacteria.
Several M. haemolytica IgBP(s) characteristics determined in this study indicate a
role for the IgBP in virulence. First, flow cytometry shows the IgBP(s) to be surface
expressed on M. haemolytica (Fig. 3). Surface expression of protein H, an Fc IgG-binding
protein from Streptococcus pyogenes, reduces phagocytosis of the bacteria (22) and inhibits
complement activation by blocking C3 deposition on the bacterial surface (2). Increased
protein A surface expression on S. aureus leads to increased inhibition of phagocytosis (10).
These virulence mechanisms may be active in M. haemolytica. Secondly, this study
identifies a secreted form of the M. haemolytica IgBP. Soluble protein A reduces antibody-
dependent cellular (38) and fixes complement in solution thus consuming complement
components (42). Soluble protein H interacts with IgG to form complement-activating
complexes (2). Thus, the CS IgBP(s) may play a role in inhibiting phagocytosis and limiting
complement activation as well as consuming soluble complement components. Lastly,
convalescent cattle had an increased antibody response to the 70.5 kDa M. haemolytica IgBP
when compared to naïve or acutely infected cattle (Fig. 4). The increased antibody response
in convalescent cattle suggests that an immune response to the IgBP(s) may be important in
protection.. These results indicate a role in virulence for the M. haemolytica IgBP(s).
M. haemolytica ST1 is generally associated with BRD in cattle (15, 44) and ST2 is
most frequently associated with pneumonic mannheimiosis in sheep (16). In an effort to
determine whether the M. haemolytica IgBP(s) is a factor in serotype-specificity of species- 62 specific infection, far-Western blots were run to determine whether differences are evident between binding of bovine Fc IgG or sheep Fc IgG to STl and ST2 and whether bovine Fc
IgG and sheep Fc IgG binds to all 12 serotype WCS preparations regardless of whether they are known to be involved in infection. Differences among infectious serotypes or between infectious and noninfectious serotypes might indicate a role for serotype-specific infection in cattle and sheep. Serotypes 1 and 2 both bind bovine Fc IgG and have protein bands at 74.1 and 63.5 kDa that stain with similar intensity in the two serotypes (Fig. 5). The 74.1 kDa band appears equally dominant in these two serotypes. A 33.2 kDa band is evident in ST2 that is not found in STl. It is possible that this difference is responsible for serotype- specificity of infection in cattle and sheep. However, the remaining serotypes express the
33.2 kDa protein band and these are not typically isolated from infections in sheep. This suggests that M. haemolytica expressed IgBPs do not play a role in serotype-specificity in infection of cattle. All 12 serotypes express an IgBP that binds bovine Fc IgG and all serotypes have similar banding patterns. No serotype has a protein band that is unique to that serotype. This is further evidence that M. haemolytica IgBP(s) does not play a role in the dominance of STl found in infections in cattle. Differences among the serotypes in binding bovine Fc IgG may be due to; 1) serotypes expressing dissimilar amounts of IgBP(s), 2) different serotypes expressing IgBP(s) with diverse numbers of Fc IgG binding sites, 3) M. haemolytica expressing more than one bovine Fc IgG IgBP in different serotypes.
Serotype 2 binds sheep Fc IgG more intensely than does STl (Fig. 6). However, as in bovine Fc IgG binding, other serotypes not known to be infective have protein-banding patterns as intense as found with ST 2. This would suggest that the difference found between
ST land ST2 is not responsible for serotype-specific infection in sheep. All serotypes bind 63 sheep Fc IgG in a similar protein-banding pattern. Four serotypes (5, 8, 13 and 14) have a particularly dominant band at 62.1 kDa. What role, if any, this may play in bacterial virulence or lack of virulence in these serotypes is not known.
Bovine Fc IgG bound to M. haemolytica IgBP(s) in a different protein-band pattern than sheep Fc IgG in all STs. M. haemolytica may express only one IgBP that has separate
binding sites for sheep Fc IgG and bovine Fc IgG or M. haemolytica may express different
proteins for binding bovine Fc IgG and sheep Fc IgG. Whether or not this plays any role in virulence is not known and is again doubtful because these same differences occur among infective serotypes and non-infective STs.
The fact that all serotypes express an IgBP(s) but not all serotypes are infective could indicate that the IgBP(s) are not involved in virulence. However, several lines of evidence
demonstrate that this may not be true: 1) Protein A from S. aureus (41) and protein H from
Streptococcus pyogenes are surface expressed. The M. haemolytica IgBP(s) was also shown
to be surface expressed. Protein A and protein H surface expression inhibits phagocytosis
(10, 22). 2) Convalescent cattle show an increased antibody response to M. haemolytica ST
1 IgBP. 3) M haemolytica IgBP(s) may be a virulence factor in infection of cattle and sheep
but may not be a factor sufficient by itself to cause infection.
In summary, this study identifies an M. haemolytica surface expressed IgBP(s) as
well as a secreted IgBP(s). The IgBP(s) does not appear to play a role in serotype-specificity
of species-specific infection as all serotypes bind both bovine Fc IgG and sheep Fc IgG.
However, sheep Fc IgG binds M. haemolytica IgBP(s) with a different protein band pattern
on far-Western blot than does bovine Fc IgG. This difference may indicate a different
virulence role for the IgBP(s) in cattle and sheep but more likely it may be that more than one 64
IgBP may be expressed by M. haemolytica or there are different binding sites for sheep Fc
IgG and bovine Fc IgG. The M. haemolytica IgBP(s) from the WCS preparation was isolated by affinity chromatography. Western blot results in this study showed an increased antibody response to M. haemolytica IgBP in cattle recovering from mannheimiosis indicating a
Gpossible role for the IgBP in protection from M. haemolytica induced BRD. The in vivo and in vitro virulence association of protein A and other previously identified IgBPs, the similarity in surface expression of protein A, protein H and M. haemolytica IgBP(s), the presence of a secreted M. haemolytica IgBP(s) and the increased immune response to an M. haemolytica IgBP in convalescent cattle suggest a role for M. haemolytica IgBP in virulence.
The M. haemolytica IgBP may be an important factor in future subunit vaccines.
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* ê// * MW a b c MW
195.9- 125.6- -107.5 89.4- -75.0 64.9- 52.8- m -40.7 39.8-
FIG. 1. Far-Western blot identification of an IgBP expressed by M. haemolytica. Lanes: a, 7.0 (ig M. haemolytica WCS; b, 12.0|ig M. haemolytica supernatant; c, 36.0 jag H. somnus WCS. All lanes were probed with 0.58 mg bovine Fc IgG and developed with HRP-conjugated rabbit anti- bovine IgG, Fc fragment specific (1:1000). Location of molecular size markers is indicated on the left. All molecular weights are in kDa. 70
MW a b c Mw 195.9- -168.7
125.6- -132.5
89.4-
64.9- -77.2 -63.2 52.8- -56.5
-40.2 39.8-
27.7-
21.8- -20.0
FIG. 2. SDS-PAGE of IgBP isolation from M. haemolytica WCS. Lane: a, MW markers; b, 0.35 \ig IgBP isolated from M. haemolytica WCS; c, 3.50 |ig isolated, speed-vacuum concentrated IgBP from M. haemolytica. IgBP was isolated on a bovine gamma globulin-CNBr Sepharose column. All molecular weights are in kDa. 71
PBS Bovine Fc IgG
5.4% 70.3%
i 11 mil 11 urn 1 i mill 1888 FLl LOG FLl LOG Bovine Fc IgG
FIG. 3. Flow cytometry analysis of whole cell M. haemolytica IgBP surface expression. (A) Whole cell M. haemolytica (0.5x106) labeled with 0.1M PBS, pH 7.2. (B) Whole cell M. haemolytica (0.5x10*) labeled with 1.9 mg bovine Fc IgG in PBS. FITC-conjugated rabbit F(ab')2 anti-bovine IgG, Fc fragment specific revealed the presence of bound bovine Fc IgG. 72
\ 125.6- 89.4- 64.9- IgBP 52.8- 39.8- 27.7- 21.8-MNp ^ 16.2- FIG. 4. Antibody responses to M. haemolytica WCS IgBP in pooled sera from naïve, acutely infected and convalescent cattle. Lanes: a, MW markers; b, H. somnus WCS; c, 7.2 (ig isolated M. haemolytica IgBP; d, e, f, 7.0 j-ig M. haemolytica WCS. Lanes a, b and c were probed with 0.58 mg bovine Fc IgG. Lane d was probed with naïve cattle sera. Lane e was probed with sera from cattle with acute M. haemolytica infection. Lane f was probed with sera from cattle convalescing from M. haemolytica induced BRD. Cattle sera were diluted 1:100. All binding was detected with HRP- conjugated rabbit anti-bovine IgG, Fc fragment specific. 73 Serotype MW 1 2 5 6 7 8 9 11 12 13 14 16 MW 125.6- -121.3 89.4 - i , : , -99.4 64.9 -a# IV f"*" "r ' ' t#» -74.1 -63.5 52.8- " T- (' ITT -52.8 39.8 -•«* -36.8 - 33.2 27.7- -22.9 21.8- 16.2- Bovine Fc IgG FIG. 5. Bovine Fc IgG binds to all serotypes of M. haemolytica WCS preparations. In a far-Western blot, SDS-PAGE separated proteins from lO.Ojug M. haemolytica WCS preparations from all serotypes were transferred onto nitrocellulose and probed with bovine Fc IgG (0.5 mg). Blots were developed with HRP-conjugated rabbit F(ab')a anti-bovine Fc IgG. Molecular weight markers are in the first lane. The serotype of each M. haemolytica WCS preparation is identified above succeeding lanes. The molecular weight of all bands resulting from bovine Fc IgG binding is indicated on the right. 74 Serotype MW 12 5 6 7 8 9 11 12 13 14 16 125 6 - 89.4- -79:% 64.9 -1 52.8 WW -62'1 58.4 -50.1 #0* 39.8- ~41.6 44 3 - 36.3 -30.1 27.7- -26.3 00È000^ -24.7 21.8- Sheep Fc IgG FIG. 6. Sheep Fc IgG binds to all serotypes of M. haemolytica WCS preparations. In a far-Western blot, SDS-PAGE separated proteins from M. haemolytica WCS preparations from all serotypes (10.0 jug each) were transferred onto nitrocellulose and probed with sheep Fc IgG (0.5 mg). Blots were developed with HRP-conjugated rabbit F(ab')2 anti-sheep Fc Ig. Molecular weight markers are in the first lane. The serotype of each M. haemolytica WCS preparation is identified above each lane. The molecular weight of all bands resulting from bovine Fc IgG binding is indicated on the right. 75 CHAPTER 3. FIBRINOGEN- AND FIBRONECTIN-BINDING AND IN VITRO GROWTH CONDITION-DEPENDENT EXPRESSION OF A MANNHEIMIA HAEMOLYTICA IMMUNOGLOBULIN-BINDING PROTEIN Ruth Smith Osmundson and Louisa B. Tabatabai A paper to be submitted to Infection and Immunity Abstract Bovine respiratory disease (BRD) is the cause of large losses to the cattle industry annually and is primarily caused by Mannheimia haemolytica serotype (ST) 1. M. haemolytica, a normal flora respiratory tract bacteria, rapidly multiplies upon a stressful occurrence and colonizes the upper respiratory tract. Extra-cellular matrix proteins (ECMPs) are known to play a role in colonization by some bacteria by acting as a bridge enabling binding between bacteria expressing extracellular matrix-binding proteins (ECMBPs) and epithelial cells. A previously identified immunoglobulin-binding protein (IgBP) expressed by M. haemolytica was shown here to have bound the ECMPs fibrinogen (Fg) and fibronectin (Fn) Far-Western blot results demonstrated that the isolated IgBP bound bovine Fc IgG and either Fg or Fn simultaneously. The effect of various M. haemolytica growth conditions on IgBP expression was assessed by 2-D gel electrophoresis analysis. Membrane expression of M. haemolytica IgBP was increased in growth conditions more closely replicating in vivo conditions. IgBP expression was increased a minimum of twofold when M. haemolytica was grown in RPMI-1640 media with 2,2'-dipyridyl, and threefold when M. haemolytica was grown in RPMI-1640 supplemented with 10% fetal bovine sera (FBS) and 76 RPMI-1640 at 40°C when compared to IgBP expression when M. haemolytica was grown in RPMI-1640 at 37°C. Up to 19.9% of M. haemolytica membrane proteins were IgBPs when M. haemolytica was grown in RPMI at 40°C. This study shows a possible increased role in virulence for the previously identified M. haemolytica IgBP. It may play a role in colonization through binding Fg or Fn and is a highly expressed protein when M. haemolytica is grown under conditions mimicking aspects of in vivo conditions. Introduction Bovine respiratory disease (BRD) has a complex etiology but pneumonic pasteurellosis, more commonly called shipping fever, is believed to be caused by Mannheimia haemolytica. M. haemolytica serotype (ST2) is a normal flora bacteria of the upper respiratory tract (URT) in cattle but on occurrence of a stressful incident such as viral infection or shipping from farm to feedlot, isolates of M. haemolytica shift from ST2 to ST1 and can grow explosively colonizing the URT (Frank 1979; Frank and Smith, 1983). The bacteria are believed to be inhaled into the lungs. This is followed by an influx of neutrophils into the lungs, serum flooding, and an inflammatory response that leads to fibrinohemorrhagic pneumonia. Morbidity and mortality due to shipping fever are among the leading causes of economic loss in the cattle industry. Adherence to epithelial cells is a necessary component for M. haemolytica colonization of the URT in cattle. The surface components used by M. haemolytica to adhere to airway epithelial cells are not known. Two types of fimbrae, a large, rigid fimbrae (Morck et al., 1987) as well as smaller, flexible fimbrae (Potter, et al., 1988) have been reported but fimbrae have not yet been found in M. haemolytica isolated from the lung or URT of cattle 77 (Gonzalez and Maheswaran, 1993). In addition, their role in attachment has not been demonstrated. A number of Gram-negative bacteria express adhesins that are distinct from fimbrae (Minion et al., 1986 and Moo et al., 1992). Identification of an M. haemolytica adhesive surface component would open the door to development of a treatment that could decrease colonization and result in reduced development of pneumonic pasteurellosis. Bacteria bind to host cells when bacterial cell surface components adhere to host cell components or to host extracellular matrix proteins (ECMPs) that bind host cells and act as a bridge between bacteria and host cells. The latter group of adhesive bacterial cell surface components is termed extracellular matrix-binding proteins (ECMBPs), adhesins, microbial surface components recognizing adhesive matrix molecules (MSCRAMMs), or receptins. ECMBPs from Staphylococcus aureus are best characterized and include proteins that bind fibronectin (Fn), collagen and a secreted protein that binds fibrinogen (Fg) (Flock, 1999). One S. aureus ECMBP, extracellular adherence protein (Eap), binds seven plasma proteins including Fg and Fn (Palma et al., 1999). A number of Gram-negative organisms express ECMBPs including Eschericia coli (Westerland et al., 1989), Haemophilus ducreyi (Abeck et al., 1992), H. influenza (Fink et al., 2002), Neisseria gonorrhoeae (van Putten et al., 1998) and Borellia burgdorferi (Probert and Johnson, 1998). In addition, a number of immunoglobulin-binding proteins are known to bind ECMPs. For example, Protein H, present in some strains of S. pyogenes and first known to bind Fc IgG, also binds fibronectin. A fibronectin binding protein also from S. pyogenes, Sfbl, has been shown to bind Fc IgG (Frick et a., 1995; Medina et al., 1999). A Group C streptococci has been shown to express protein F AI which binds fibrinogen, albumin and IgG (Talay et al., 1996). In addition, 78 FgBP, a fibrinogen-binding protein of Streptococcus equi, has recently been shown to bind equine Fc IgG (Meehan et al., 2001). ECMBPs are believed to contribute to virulence in a number of bacteria. Mongodin et al. (2002) established that a fibronectin-binding protein-deficient strain of S. aureus has a fivefold-decrease in the level of adherence to human airway epithelial cells suggesting that Fnbps are involved in adherence. An isogenic mutant unable to produce the Fn- and Fg- binding protein of Streptococcus suis ST2 shows decreased colonization of piglets in the joints and central nervous system, organs specifically involved in S. suis infection (de Greeff, et al., 2002). In vitro models demonstrate several additional roles for ECMBPs in virulence. In the presence of Fn and an anti-CD3 monoclonal antibody, S. aureus FnbpA mediates adhesion to T cells and T cell costimulation through the T cell asPi integrin, which binds Fn and functions as a costimulatory molecule, leading to T cell activation (Miyamoto et al., 2001). A T helper type 2 inflammatory environment promotes S. aureus binding to skin and this binding is mediated by Fn and Fg (Cho et al., 2001). ECMBPs appear to have a variety of roles in virulence. The mechanism for the abrupt change from the predominance of the normal flora M. haemolytica ST2 to explosive growth of stress-associated ST1 is not known. However, change in host environmental factors due to stress is likely to play a role. In an effort to recreate in vivo protein expression while using in vitro systems and also to define culture conditions that affect expression of particular proteins, a number of studies have been done to define protein expression under different culture conditions. Shewen and Wilkie (1982) reported that the addition of 7% fetal bovine serum (FBS) to RPMI-1640 tissue culture media achieves enhanced M. haemolytica production of leukotoxin, a secreted RTX cytolysin, 79 compared to tissue culture media alone. Iron-regulated outer membrane proteins of 71, 77 and 100 kDa are expressed at higher levels in iron-restricted media (Deneer and Potter, 1989; Morck et al., 1991). M. haemolytica OMP protein expression was compared between bacteria that were isolated from lungs of experimentally infected calves to bacteria cultured under various in vitro growth conditions. OMP expression profiles on SDS-PAGE arre similar for M. haemolytica isolated from lungs and M. haemolytica grown in newborn calf serum (Davies et al., 1994). No capsular polysaccharide production is detected when M. haemolytica is grown above 40°C (Puente-Polledo et al., 1998). Experiments by Reeve- Johnson et al. (2001) noted higher clinical scores in calves with temperatures of 39.9-41.0°C compared to normal temperatures of 39.2°C. In addition, pathogens are known to upregulate or downregulate expression of adhesive components under varying conditions of the host environment (Finlay and Falkow, 1989; Francis et al., 1989; Mekalanos, 1992). Establishing in vitro growth conditions that closely mimic in vivo conditions may increase the efficacy of vaccines prepared from in vitro M. haemolytica cultures. The hypotheses for this study were that a previously identified M. haemolytica ST1 immunoglobulin-binding protein (IgBP) binds one or more ECMPs and that this protein would be upregulated when grown under m vitro growth conditions that more closely mimic in vivo conditions. 80 Material and Methods Bacterial isolate and growth conditions M. haemolytica ST1 (isolate LI01) was isolated from an infected bovine lung (Frank and Smith 1983). Fetal bovine sera FBS was obtained from National Animal Disease Center, Ames, Iowa. It was heated to 56°C for 25 min before use. IgBP isolation Cyanogen bromide (CNBr)-activated Sepharose 4B (Sigma, St. Louis. MO) was coupled to bovine gamma globulin (Jackson Immunoresearch Labs, West Grove. PA) according to manufacturer's procedure. M. haemolytica WCS in 50 mM Tris (pH 8.0) was adsorbed with bovine gamma globulin-Sepharose 4B by rotating the sample for 48 hours at 4°C. IgBP was eluted from the Sepharose in a column with 0.9% NaCl-lM propionic acid. IgBP was concentrated by Speeedvac vacuum concentration (Savant, Holbrook, NY). Gel electrophoresis and immunoblotting SDS-PAGE was conducted on denatured M. haemolytica WCS, isolated IgBP, Fn (from bovine plasma, Sigma, St. Louis MO) and Fg (from bovine plasma, Sigma, St. Louis, MO) in 12.5% acrylamide gels with a 4% acrylamide stacking gel (Laemmli, 1970) or 4-12% Bis-Tris polyacrylamide gels (Invitrogen, Carlsbad, CA). Protein concentrations for all preparations were determined by Folin-Lowry (Lowry et al., 1951). Proteins were 81 transferred onto 0.45(im Protran nitrocellulose (Schleicher & Schuell, Keene, NJ) using a Bio-Rad Trans-Blot Cell apparatus. Nitrocellulose blots were blocked with 0.25% fish gelatin (Norland Products, Inc., New Brunswick, NJ ) in 0.1M PBS, pH 7.2 for 15 minutes. Transferred IgBP was detected by incubating nitrocellulose blots overnight in bovine Fc IgG (44 jug/10 ml, Jackson Laboratories, West Grove, PA). After washing (0.1M PBS, pH 7.2 with 0.05% Tween 80) the blots were developed using horseradish-peroxidase (HRP)- conjugated rabbit antibodies to fibronectin (1:1000) or bovine Fc IgG (1:1000). WCS and membrane protein preparation Cells stored at -70°C were cultured overnight on blood agar plates (Becton Dickinson, Sparks, MD), colonies transferred and cultured in 3 ml RPMI-1640 (Sigma; St. Louis, MO) with 20mM HEPES (pH 7.2) for 6 hours at 37°C with shaking and 10 ml was used to culture 1 L RPMI-1640 at 37°C, RPMI-1640 with 50 |aM 2,2'-dipyridyl at 37°C. RPMI-1640 with 10% FBS at 37°C, or RPMI-1640 at 40°C for 20 hours with shaking. Optical density readings were taken at 600 nm on a Beckman DU 640 spectrophotometer (Beckman, Fullerton, CA). Streaking on blood agar plates checked purity. The culture was centrifuged (6,000 x g for 20 min). The cell pellet was washed with 0.01 M phosphate buffered saline (PBS), pH 7.2, and centrifuged (6,000 x g RPMI-1640 for 20 minutes) three times. After washing the pellet two more times, the pellet was resuspended in 0.01 M PBS (pH 7.2) and sonicated on ice for four minutes (8 cycles of 30 sec on, 30 sec off, power at 7, duty cycle at 5) using a Branson Sonifier 250. The sonicated cells were centrifuged (6000 x g for 20 min) and the supernatant was ultracentrifuged (50,000 x g for 2 hours at 4°C). The supernatant was retained as the WCS and the pellet, the membrane preparation, was 82 resuspended in 50 mM Tris HC1, pH 7.5 containing 50 jag /ml phenylmethylsolfonylfluoride (PMSF) (Sigma). The solution was set on ice for 1 h and protein concentrations were determined using Amersham Bioscience's PlusOne 2-D Quant kit (San Francisco, CA). 2-D gel electrophoresis Membrane samples (50 pig protein/50 (il) were added to 75 |il rehydration buffer (0.5% v/v carrier ampholytes, pH 3-10 (Amersham Bioscience, San Francisco, CA), 2M thiourea, 2mM tributylphosphine (TBP), 1 % w/v tetradecanoylamido-propyl-dimethyl ammonia-propane-sulfonate (ASB-14), 2% w/v CHAPS, 8M urea, 0.5% IPG buffer, pH3-10 (Amersham Bioscience), 10 ml E-pure water and 0.2% Pefabloc (Boehringer Ingelheim (Ingelheim, Germany), and 0.28 mg dithiothriotol (Sigma) were added. The samples were briefly centrifuged to remove particulate matter and 125 jo.1 of each sample was loaded onto Immobiline IPG DryStrip gels (7 cm, pH 3-10,) (Amersham Biosciences). Strips were rehydrated for lOh at 30°C and proteins were focused on an IPGphor electrophoresis unit (Amersham Bioscience) for approximately 33kVh as follows: Volts 500 1000 2000 4000 6000 8000 Hours 1:30 1:30 1:00 1:00 1:00 5:00 IPG strips were equilibrated in SDS equilibration buffer [50mM Tris-HCl (pH 8.8), 6 M urea, 30% (v/v) 87% glycerol, 2% (w/v) SDS, trace bromophenol blue in E-pure water] with 100 mg dithiothreitol/10 ml buffer for 15 min. Then IPG strips were equilibrated in SDS equilibration buffer with 250 mg iodoacetamide/10 ml solution for 15 min. Strips were sealed in place on top of an SDS-PAGE gel using 0.5% agarose in SDS electrophoresis 83 buffer containing bromophenol blue, SDS-PAGE was carried out on 4-12% Bis-Tris gel (Invitrogen) polyacrylamide gels using MOPS Running Buffer (Invitrogen). NuPAGE antioxidant (0.25%) (Invitrogen) was added to the cathode buffer. Second-dimension electrophoresis was carried out using 15mA/gel constant current for 15 min then at 200V (constant voltage)for 45 min in a Mighty Small II electrophoresis unit (Hoeffer Scientific Instruments, San Francisco, CA). Gels were stained overnight with 0.1% Coomassie Brilliant Blue R-250 in 50% methanol and 10% acetic acid. Gels were destained in 25% ethanol and 10% acetic acid and imaged on an Amersham Biosciences Image Scanner. 2-D gel analysis Far-Western blot and 2-D gel analysis were done using PDQuest 6.1 gel analysis software (Bio-Rad Laboratories, Hercules, CA). A match set was created with all four gels from the four different growth conditions and the far-Western immunoblot identifying immunoglobulin-binding protein. Spots identified on the immunoblot were matched to spots on the four gels. Spot analysis was performed for each gel. Normalization was done for each spot by dividing the raw pixel density quantity of each spot in a member gel by the total pixel density quantity of all pixels in the gel image. Pixel density was determined from a Gaussian image of each gel. Results M. heamolytica growth in different in vitro growth conditions In vitro growth conditions are usually chosen to support growth. Typical laboratory culture conditions for M. haemolytica are growth in a chemically defined media, RPMI-1640, at 37°C. To mimic aspects of in vivo growth conditions, M. haemolytica was grown in three different culture conditions: RPMI-1640 with 50 pM 2,2'-dipyridyl, RPMI-1640 with 10% fetal bovine serum and RPMI-1640 cultured at 40°C. The iron-chelator 2,2'-dipyridyl was added to RPMI-1640, a medium without added iron, to chelate any exogenous iron. Most pathogenic organisms upregulate a number of proteins under iron-restricted conditions (Gray-Owen and Schryvers, 1996). Fetal bovine sera (10%) was added to RPMI-1640 given that expression of leukotoxin, a cytolytic toxin secreted by M. haemolytica, is increased in vitro when 7% FBS is added to the media (Shewen and Wilkie, 1982). In addition, pneumonic mannheimiosis is known to lead to serum flooding in the lungs. Finally, M. haemolytica was grown in RPMI-1640 at 40°C because infected cattle have temperatures of 40°C or above. Normal cattle temperature range is 38.3-38.9°C (discussions with G. Frank, NADC, Ames, IA). Growth in RPMI-1640 at 40°C resulted in a culture density almost half that of M. haemolytica grown in the same media at 37°C (Table 1). When grown at 40°C, pellets of M. haemolytica cells appeaed more viscous than when grown at 37°C. When 10% FBS was added to RPMI-1640, cell culture density was nearly twice that of the density of cells when grown in RPMI at 37°C. Culture density of M. haemoltyica grown in RPMI-1640 with the iron-chelator 2,2'-dipyridyl was slightly less than growth in RPMI-1640 at 37°C. M. haemolytica IgBP also binds Fg and Fn Earlier work indicated that the M. haemolytica IgBP(s) might bind fibronectin. A mass-peptide fingerprint of a tryptic digest of one band of the IgBP(s) from an SDS-PAGE gel did not establish identity of the IgBP(s) with other proteins in the database (MS-Fit, University of California, San Francisco, CA). However, a degree of homology was found to 85 other Fn-binding proteins. Far-Western blots validated that isolated M. haemolytica IgBP(s) does bind Fn (Fig. 1, lane 2). One band was evident at 29.8 kDa. To determine whether the IgBP(s) would concurrently bind Fn and bovine Fc IgG, Fn was electrophoresed by SDS- PAGE and electroblotted to nitrocellulose. The blot was incubated with the IgBP(s) in 0.1 M PBS and after washing was incubated with bovine Fc IgG in 0.1 M PBS. After washing and development with conjugate [rabbit F(ab')z anti-bovine Fc IgG-HRP], the blots revealed that the IgBP bound bovine Fc IgG and Fn at the same time (Fig. 1, lane 4). Bands were evident at 214.9 and 36.8 kDa (Fig. 1A). No binding was seen on far-Western blot between Fn and bovine Fc IgG or Fn and the conjugate (results not shown). The same procedure showed that isolated IgBP(s) binds bovine Fc IgG and Fg simultaneously (Fig. 2, lane 2). This lane had nine bands ranging from 35.1-159.0 kDa. The control (Fig. 2, lane 5) showed slight evidence of conjugate-Fg binding. This lane revealed three faintly evident bands on the blot (71.0, 84.5, and 92.2 kDa). Comparative analysis of total IgBP pixel density IgBP expression was determined by 2-D gel analysis from M. haemolytica grown under culture conditions that more closely mimic in vivo conditions. The identity and location of the IgBP(s) are shown on the far-Western blot (Fig. 3A). The MW markers are indicated (Fig. 3A). The cross hairs indicate the location of the IgBP(s) on the blot and gels (Fig. 3, B through F). The pixel density of IgBP spots and total gel pixel density was determined with PDQuest 2-D gel analysis software. Dividing spot pixel density by total gel pixel density normalized the pixel density value of each spot. Normalization was necessary to account for differences in gel loading and/or staining efficiency. 86 Expression of membrane IgBP(s) was increased a minimum of twofold under all test conditions while the addition of 10% FBS or growth at 40°C increased IgBP(s)expression at least threefold (Table 2). When membrane IgBP expression was analyzed as a percentage of total membrane expression, all three test conditions show an approximately twofold increase in the percentage of the IgBP(s) compared to the control. Growth conditions of RPMI-1640 at 40°C yielded the highest percentage membrane IgBP(s)at 19.9%. Comparative analysis of individual spot pixel density The quantitative relatedness of IgBP spots from each of the three gels generated from M. haemolytica grown in RPMI-1640 with 50 (iM 2,2'-dipyridyl, RPMI-1640 with 10% FBS and RPMI-1640 at 40°C to the IgBP spots from M. haemolytica grown in RPMI-1640 at 37°C was determined with PDQuest 2-D gel analysis software. The pixel density of each IgBP spot in the gel from M. haemolytica grown in RPMI-1640 at 37° C (x-axis) was plotted on a log scale against the corresponding IgBP spot density in each of the three remaining gels containing proteins expressed under different conditions. If the spot quantities from the gels being compared are equal, the indicating dot will appear on the regression line with a slope of one. Deviations indicate an increase or decrease in expression for a given spot between the two gels. The correlation coefficient indicating the relatedness of all the spots between two gels as well as the equation for a linear regression defining the indicating dots from the plot is shown (Fig. 4). The IgBP(s) spots from the gels generated by M. haemolytica grown in RPMI-1640 at 37°C and RPMI-1640 with 10% FBS showed the largest amount of deviation (correlation coefficient of 0.20). Neither of the remaining analysis sets had a correlation coefficient greater than 0.50. 87 Discussion Bacterial cell adherence to host cells is a required initial step in most bacterial infections and ECMBPs may play an important role in this process. ECMBPs may be required for infection. The S. aureus FnbpA is known to bind Fg as well as Fn (Wann et al., 1999), and FnbpA has been shown to be involved in adherence to epithelial cells (Mongodin et al., 2002). The mechanism used by M. haemolytica to adhere to the URT in colonization is not known. Whereas M. haemolytica ST2 is most frequently found as a commensal of the nasopharynx, ST1 is most frequently isolated in infection. This change is initiated by stress and possibly involves a change in the host environment. Results here indicatde that the M haemolytica IgBP binds Fg and Fn. Fibrinogen and Fn are involved in bacterial cell adhesion as extracellular matrix proteins and experiments indicate a role for ECMBPs in bacterial adhesion to host cells. Up to 19.9 % of M. haemolytica membrane proteins were lgBPs in conditions modeling in vivo conditions which could indicate an important IgBP role in virulence. The M. haemolytica IgBP(s) might play a role in adherence and colonization. Additional roles for ECMBPs in virulence are being found and as a result the M. haemolytica IgBP(s) may have other roles in virulence. Far-Western blot results demonstrated that the M. haemolytica IgBP can bind bovine Fc IgG and Fg as well as bovine Fc IgG and Fn simultaneously. The significance of this is not known. There has been no previously demonstrated virulence role that IgBPs and ECMBPs have in common. It is possible that the M. haemolytica IgBP(s) binds one protein with a higher affinity than the other proteins or that steric hindrance could result in preferential binding of one or more proteins, particularly when IgBP(s) expression is upregulated. 88 Bacterial growth in RPMI-1640 with 10% FBS resulted in the highest number of bacteria while the least growth occurred when bacteria were grown in RPMI-1640 at 40°C. However, both of these conditions resulted in the greatest and nearly equal relative IgBP expression. It is possible that one environmental factor trigger induces upregulation under both conditions or the upregulation may be due to independent factors. Regardless, bacterial growth or lack of growth can occur independently of IgBP expression and IgBP expression can be upregulated under conditions that are not optimal for growth. The membrane proteins were separated over a pH 3-10 range. The predominance of the IgBP(s) spots were identified at 24 kDa and at higher pi values. These spots were 60% of the IgBP(s) from M. haemolytica grown in RPMI-1640 at 37°C and 76% of the IgBP(s) from M. haemolytica grown in RPMI-1640 with 10% FBS. It is not known if the multiple spots of M. haemolytica IgBP(s) are breakdown products of one protein or consist of a number of different proteins. Previous research suggested that the M. haemolytica IgBP(s) may proteolyze with products found at approximately 20 kDa (Osmundson, 2003, manuscript in preparation). A number of other IgBPs have multiple binding sites for immunoglobulins (Moks et al., 1986; Sjôbring et al., 1989). It is possible that breakdown products bind bovine Fc IgG as long as the binding sites are intact. When M. haemolytica was grown in control media, 8.2% of membrane proteins were IgBP(s). Up to 19.9% of membrane proteins consisted of the IgBP(s) when grown in media mimicking factors of in vivo growth. The smallest increase was seen when M. haemolytica were grown in RPMI-1640 with 50 (xM 2,2-dipyridyl where the percentage of IgBP(s) is 1.9 times that found in minimal media alone. These results suggest that the M. haemolytica 89 IgBP(s) may have a significant role in virulence under various growth conditions and any role it may play in virulence mightbe increased during M. haemolytica infection. Growth in both RPMI-1640 with 10% FBS and RPMI-1640 at 40°C resulted in a threefold increase in total IgBP pixel density and growth in RPMI-1640 with 2,2'-dipyridyl resulted in a twofold increase compared to IgBP expression in controlled conditions. This again argues for a role in the IgBP(s) in virulence. Even though there was a threefold increase in IgBP pixel density in the first two growth conditions, the scatter plot analysis indicated a differential distribution at the level of expression. The correlation coefficient of pixel density of individual spots from IgBP(s) grown from cells in RPMI-1640 with 10% FBS compared to IgBP(s) from cells grown in RPMI at 37°C was 0.2. This compares to a correlation coefficient of 0.4 in the same comparison in conditions involving RPMI-1640 at 40°C and RPMI-1640 at 37°C. The average pixel density deviation of upregulated protein(s) was 5494.4 for IgBPs from RPMI-1640 with 10% FBS grown M. haemolytica. This same value for IgBPs from RPMI-1640 at 40°C was 2986.9. Therefore, both growth conditions resulted in similar absolute total pixel density values, but there was a larger average deviation in individual spot density from M. haemolytica grown in RPMI-1640 with 10% FBS. 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Biochem. J. 315:577-582. Van Putten, J., T. Duencsing, and R. Cole. 1998. Entry of OpaA+ gonococci into HEp-2 cells requires concerted action of glycosaminoglycans, fibronectin integrin receptors. Mol. Microbiol. 29:369-379. Wann, E., B. Dassy, J. Fournier, and T. Foster. 1999. Genetic analysis of the cap5 locus of Staphylococcus aureus. FEMS Microbiol. Lett. 170:97-103. Westerland, B., P. Kusela, T. Vartix, I. van Die, and A. Korhonen. 1989. A novel lectin- dependent interaction of P fimbriae of Escherichia coli with immobilized fibronectin. FEBS Lett. 243:199-204. 93 MW 1 2 MW 3 4 5 -214.9 172.6 184.5- a* W 111.4-4#» 121.3- 79.6-* 85.9- 4# 61.3- 68.8- 49.0-*» 52.5- 36.4-* 40.0— -29.8 36.8 24.7 —- - 28.4- 4 19.2-m- FIG. 1. Far-Western blot identifying IgBP(s) binding to Fn. (A) Lanes: 1, MW markers; 2, Far-Western blot of SDS-PAGE of 7.0 jag isolated IgBP(s) blotted to nitrocellulose and bound with 300.0 ^g Fn followed by labeling with rabbit anti-Fn-HRP (1:1000); 3, MW markers; 4, Far-Western blot of SDS-PAGE of 10.0 ng Fn blotted to nitrocellulose and bound to 44.0 fig isolated IgBP(s) followed by binding to 44.0 (j.g bovine Fc IgG and rabbit F(ab')a anti-bovine Fc IgG-HRP; 5, SDS-PAGE of 10.0 (xg Fn 94 MW 1 2 3 MW 4 5 6 •1Q4 r —159.0 184.5 104iD -152.3 121 3- -118.8 ocn qpp 121.3- 85.9- M^-845 68.8- *-71.0 85.9- " a-84.5 52.5- •-71.0 40.0 — 68.8 52.5 -63.7 -46.9 28.4- 40.0 35.1 21.7 28.4— 16.8 - 4-12% gel 12% gel FIG. 2. Far-Western blot identifying IgBP(s) binding to Fg. Lanes: 1, MW markers; 2, Far-Western blot of SDS-PAGE of 10.0 (ig Fg blotted to nitrocellulose and bound to 7.0 gg isolated IgBP(s) followed by binding to 44.0 (ig bovine Fc IgG and rabbit F(ab')2 anti-bovine Fc IgG-HRP; 3, SDS-PAGE of 10.0 (xg Fg; 4, MW markers; 5, Far-Western blot of SDS-PAGE of 10.0 (j,g Fg blotted to nitrocellulose followed by binding to 44 p,g bovine Fc IgG and rabbit F(ab')z anti-bovine Fc IgG-HRP; 6 SDS- PAGE of 10.0 ng Fg. 95 ief igBP(s) 3 (184.5) •-(85.9) •-(68.8) •-(52.5) 42.5- - (40.0) 39.0- A p' -10 *++ * * . ^ «•» * Jk + + #r m*# D FIG. 3. 2-D SDS-PAGE gels and far-Western blot of M. haemolytica membrane proteins (50 p,g) grown in four in vitro growth conditions. (A,B) Far-Western blots of 2-D SDS-PAGE gel of membrane proteins (50 |ig) labeled with 44 jag bovine Fc IgG and labeled with rabbit F(ab')2 anti-bovine Fc IgG-HRP (1:1000). (A) MW of spots and MW markers. 2-D SDS- PAGE of membrane proteins (50 (xg) grown in (C) RPMI-1640 at 37°C (D) RPMI-1640 with 50 \xM 2,2'-dipyridyl (E) RPMI-1640 with 10% FBS (F) RPMI-1640 at 40°C. Crosshairs mark IgBP(s) location. 96 A B S A -- f s 1 5 CL Line A DC B/ Line A Line A 0.47X+ 236.0 y = 0.22x + 350.9 y = 0.45X + 239.2 pixel density units log10 11,000 0 pixel density units log,0 11,000 pixel density units log10 11,000 RPMI-1640 at 37°C RPMI-1640 at 37°C RPMI-1640 at 37°C FIG.4. Comparative analysis of individual spot pixel density. Individual spot pixel density values for IgBP(s) from M. haemolytica grown in RPMI-1640 (x-axis, panel A-C) is plotted versus individual spot pixel density for IgBP(s) from M. haemolytica grown under different conditions (y-axis): panel A, RPMI-1640 with 50pM 2,2'-dipyridyl; panel B, RPMI-1640 with 10% FBS; panel C, RPMI-1640 at 40°C. 97 TABLE 1. Bacterial growth in vitro RPMI-1640 RPMI-1640 Growth RPMI-1640 RPMI-1640 50\xM 2,2'- with 10% Condition 37° C 40°C dipyridyl FBS Afioo 0.60 0.54 1.12 0.31 TABLE 2. Comparison of total IgBP expression from four in vitro growth conditions Growth IgBP Fold-increase of % IgBP pixel density Condition pixel density IgBP pixel density of total pixel density RPMI-1640 at 37°C 30,393 1.00 8.2 RPMI-1640 with 50iiM 2,2-dipyridyl 64,265 2.11 15.8 RPMI-1640 with 94,880 3.12 16.1 10% FBS RPMI-1640 3.18 19.9 at 40°C 96,710 98 CHAPTER 4. GENERAL DISCUSSION AND CONCLUSIONS This study identified an IgBP expressed by M. haemolytica. Results based on virulence studies of IgBPs of other organisms suggest that the M. haemolytica IgBP(s) may have a significant role in virulence. IgBPs are present in a number of Gram-positive and Gram-negative bacteria. Experiments in vitro and in vivo point to an IgBP role in virulence both in terms of evasion of the host immune response and modulation of the immune response. The M. haemolytica IgBP was shown to be expressed in the WCS and also to be secreted. Both preparations had a dominant band on SDS-PAGE at 75.0 kDa and a less intensely staining band at 40.2 kDa. The supernatant preparation has an additional band at 107.5 kDa. This band appears unique to the supernatant because isolated, concentrated IgBP in Chapter 2. Fig. 2 shows a number of additional bands on SDS-PAGE but no band comparable to the 107.5 kDa band found in the supernatant. Additionally, 2-0 gel results from M haemolytica membrane preparations have no spots greater than 95 kDa. This additional supernatant band could be due to post-translational modification or perhaps an additional secreted IgBP that is not found in the WCS or membrane preparations. There are a number of possible explanations for the appearance of more than one IgBP band and multiple spots on SDS-PAGE and 2-D far-Western blots. IgBPs from other organisms have repeat binding sites for Ig. Protein A has five (Mocks et al., 1986) while protein G has three repeat Ig binding regions (Guss et al., 1986). It is possible that the different M. haemolytica IgBP bands evident on far-Western blot are due to a single protein differing only in the number of Fc IgG binding sites at a particular molecular weight band. 99 Multiple bands could also be due to a unique protein in each Western blot band or the bands might be degradation products of one or more proteins. There is evidence for the latter possibility as seen by SDS-PAGE of the isolated, concentrated IgBP (Chapter 2, Fig. 2). Bacterial cell adherence to host cells is a required initial step in most bacterial infections and ECMBPs may play a large role in this process or may be required for infection. The S. aureus FnbpA is known to bind Fg as well as Fn (Wann et al., 2000) and FnbpA is involved in adherence to epithelial cells (Mongodin et al., 2002). The mechanism used by M. haemolytica to adhere to the URT initiating colonization is not known. M haemolytica ST2 is most frequently found as a commensal of the nasopharynx while ST1 is most frequently isolated in infection. This change is initiated by stress and possibly involves a change in the host environment. Results here indicate that the M. haemolytica IgBP binds Fg and Fn. Fibrinogen and Fn are involved in bacterial cell adhesion as extracellular matrix proteins and experiments indicate a role for ECMBPs in bacterial adhesion to host cells. Up to 19.9 % of M. haemolytica membrane proteins were IgBPs in conditions modeling in vivo conditions which could indicate an important IgBP role in virulence. The M. haemolytica IgBP(s) might play a role in adherence and colonization. Additional roles for ECMBPs in virulence are being found and as a result the M. haemolytica IgBP(s) may have other roles in virulence. Far-Western blot results demonstrated that the M. haemolytica IgBP bound bovine Fc IgG and Fg as well as bovine Fc IgG and Fn simultaneously. The significance of this is not known. There is no previously demonstrated virulence role that IgBPs and ECMBPs have in common. It is possible that the M. haemolytica IgBP(s) binds one protein with a higher 100 affinity than the other proteins or that steric hindrance could result in preferential binding of one or more proteins, particularly when IgBP(s) expression is upregulated. Several physical characteristics of the IgBP(s) determined in these experiments are consistent with the proposal that the IgBP(s) plays a role in virulence. First, flow cytometry showed the IgBP to be surface expressed on M. haemolytica (Chapter 2, Fig. 3). Surface expression of protein H, an Fc IgG-binding protein from Streptococcus pyogenes, reduces phagocytosis of the bacteria (Kihlberg, et al., 1999) and inhibites complement activation by blocking C3 deposition on the bacterial surface (Berge, et al., 1997). Increased protein A surface expression on S. aureus leads to increased inhibition of phagocytosis (Dosset, et al., 1969). These virulence mechanisms may be active in M haemolytica. Second, this study identified a secreted form of the M. haemolytica IgBP. Soluble protein A reduces antibody- dependent cellular (Rosenblatt, et al., 1977) and fixes complement in solution thus consuming complement components (Sjôquist and Stâlenheim, 1969). Soluble protein H interacts with IgG to form complement-activating complexes (Berge, et al., 1997). Thus, the CS IgBP may play a role in inhibiting phagocytosis and limiting complement activation as well as consuming soluble complement components. Additionally, convalescent cattle were shown to have an increased antibody response to the 70.5 kDa M. haemolytica IgBP when compared to naïve or acutely infected cattle (Chapter 2, Fig. 4). The increased antibody response in acutely infected cattle suggests that inhibiting the effectiveness of the IgBP may play a role in recovery. Lastly, growth in both RPMI-1640 with 10% FBS and RPMI-1640 at 40°C resulted in a three-fold increase in total IgBP pixel density on 2-D gel analysis and growth in RPMI-1640 with 2,2'-dipyridyl resulted in a two-fold increase compared to IgBP expression in controlled conditions. When M. haemolytica was grown in control media, 101 8.2% of membrane proteins were IgBP(s). Up to 19.9% of membrane proteins consisted of the IgBP(s) when grown in media simulating factors of in vivo growth. The smallest increase was seen when M. haemolytica are grown in RPMI-1640 with 50 (iM 2,2-dipyridyl where the percentage of IgBP(s) was 1.9 times that found in minimal media alone (Chapter 3, Table 2). These results suggest that the M. haemolytica IgBP(s) may have a significant role in virulence under various growth conditions and any role it may play in virulence may be increased during M. haemolytica infection. The mechanism for the abrupt change from the predominance of the normal flora M. haemolytica ST2 to explosive growth of stress-associated ST1 is not known. However, change in host environmental factors due to stress is likely to play a role. There is no significant difference in the bovine Fc IgG SDS-PAGE banding patterns between ST1 and ST2 WCSs prepared with the same growth conditions. However, IgBP(s)membrane expression was up-regulated in growth conditions that mimic factors of in vivo growth during infection. If the change from the commensal ST2 to infective ST1 is due to a phase variation in M. haemolytica and is due to environmental factors, increased IgBP(s) expression may be part of that change. M. haemolytica ST1 is generally associated with BRD in cattle (Whitely, et al., 1992 and (Frank, 1979) and ST2 is most frequently associated with pneumonic mannheimiosis in sheep (Fraser, et al., 1982). In an effort to determine whether the M. haemolytica IgBP is a factor in serotype-specificity of species-specific infection, far-Western blots were run to determine whether differences were evident between binding of bovine Fc IgG or sheep Fc IgG to ST1 and ST2 and whether bovine Fc IgG and sheep Fc IgG binds to all 12 serotype WCS preparations regardless of whether they are known to be involved in infection. Differences among infectious serotypes or between infectious and noninfectious serotypes might indicate a role for serotype-specific infection in cattle and sheep. Serotypes 1 and 2 both bound bovine Fc IgG and they all had protein bands at 74.1 and 63.5 kDa that stain with similar intensity in the two serotypes (Chapter 2, Fig. 5). The 74.1 kDa band appeared equally dominant in these two serotypes. A 33.2 kDa band was evident in ST2 that is not found in ST1. It is possible that this difference is responsible for serotype-specificity of infection in cattle and sheep. However, the remaining serotypes expressed the 33.2 kDa protein band and these are not implicated in infection in cattle. This suggests that M. haemolytica expressed IgBPs do not play a role in serotype-specificity in infection of cattle. All 12 serotypes expressed an IgBP that bound bovine Fc IgG and all serotypes had similar banding patterns. No serotype had a protein band that is unique to that serotype. This is further evidence that M. haemolytica IgBP does not play a role in the dominance of ST1 found in infections in cattle. Differences among the serotypes in binding bovine Fc IgG may be due to: 1) Serotypes expressing dissimilar amounts of IgBP; 2) different serotypes expressing IgBPs with diverse numbers of Fc IgG binding sites; or 3) M. haemolytica expressing more than one bovine Fc IgG IgBP in different serotypes. Serotype 2 bound sheep Fc IgG more intensely than does ST1 (Chapter 2, Fig. 6). However, as in bovine Fc IgG binding, other serotypes not known to be infective had protein-banding patterns as intense as found with ST 2. This would suggest that the difference found between ST land ST 2 is not responsible for serotype-specific infection in sheep. All serotypes bound sheep Fc IgG in a similar protein-banding pattern. Four serotypes (5, 8, 13 and 14) have a particularly dominant band at 62.1 kDa. What role if any this may play in bacterial virulence or lack of virulence in these serotypes is not known. 103 Interestingly, bovine Fc IgG bound to M. haemolytica IgBP in a different protein- band pattern than sheep Fc IgG in all STs. M. haemolytica may express only one IgBP that has separate binding sites for sheep Fc IgG and bovine Fc IgG or M. haemolytica may express different proteins for binding bovine Fc IgG and sheep Fc IgG. Whether or not this plays any role in virulence is not known and is again doubtful because these same differences occur among infective serotypes and noninfective STs. The fact that all serotypes expressed an IgBP(s) but not all serotypes are infective could indicate that the IgBP(s) are not involved in virulence. However, several lines of evidence demonstrate that this may not be true: 1) Protein A from S. aureus (Sjoquist et al., 1972), protein H from Streptococcus pyogenes are surface expressed. These experiments showed M. haemolytica IgBP(s) to be surface expressed. Protein A surface expression on S.aureus is involved in inhibiting phagocytosis (Dosset et al., 1969) and Protein H surface expression inhibits phagocytosis (Kihlberg et al., 1999). 2) Convalescent cattle showed an increased antibody response to M. haemolytica ST1 IgBP. 3) M. haemolytica IgBP may be a virulence factor in infection of cattle and sheep but may not be a factor sufficient by itself to cause infection. Bacterial growth in RPMI-1640 with 10% FBS resulted in the highest number of bacteria while the least growth occurred when bacteria were grown in RPMI-1640 at 40°C. However, both of these conditions resulted in the greatest and nearly equal relative IgBP expression. It is possible that one environmental factor trigger induces upregulation under both conditions or the upregulation may be due to independent factors. Regardless, bacterial growth or lack of growth can occur independently of IgBP expression and IgBP expression can be upregulated under conditions that are not optimal for growth. 104 Even though there was a three-fold increase in IgBP pixel density in the first two growth conditions the scatter plot analysis indicated a differential distribution in the level of expression. The correlation coefficient of pixel density of individual spots from IgBP(s) grown in RPMI-1640 with 10% FBS compared to IgBP(s) grown in RPMI at 37°C was 0.2 (Chapter 3, Fig. 3) This compared to a correlation coefficient of 0.4 in the same comparison in condition involving RPMI-1640 at 40°C and RPMI-1640 at 37°C. The average pixel density deviation of upregulated protein(s) was 5494.4 for IgBPs from RPMI-1640-FBS grown M. haemolytica. This same value for IgBPs from RPMI-1640 at 40°C was 2986.9. Therefore, both growth conditions result in similar absolute total pixel density values, but there is a larger average deviation from the spot density of individual IgBP(s) spots grown in control by IgBP(s) grown in RPMI-1640 with 10% FBS. There is an apparent difference in the upregulation of IgBP(s) expressed in the two different growth conditions. Even though M. haemolytica ST2 is found as a commensal in cattle while ST1 is generally isolated in M. haemolytica induced BRD, there was no apparent difference in the bovine Fc IgG SDS-PAGE banding patterns between ST1 and ST2 WCSs prepared with the same growth conditions. However, IgBP(s)membrane expression was upregulated in growth conditions that mimic factors of in vivo growth during infection. If the change from the commensal ST2 to infective ST1 is due to a phase variation in M. haemolytica and is due to environmental factors, expression may be part of that change. The bovine bacterial pathogenesis in Table 1 is consistent with information demonstrated in bacterial infections and M. haemolytica induced BRD. Table 1. Bovine bacterial pathogenesis 105 Body Iron Supply Bovine Fibrinogen and Bovine Temperature Serum Fibronectin Fc IgG Nasopharynx Normal Adequate Absent Present Absent (Commensal) 38.3-38.9°C URT Increasing Decreasing Absent Present Absent (Colonization) LRT Iron Serum >40.0°C Present Present (Infection) Sequestered Flooding The proposed IgBP role in virulence model in Table 2 is consistent with what has been shown about bacterial infections, BRD pathogenesis and with the results of this study. In this scenario, whether attachment or evasion dominates when Fn, Fg and bovine Fc IgG are available might depend on several factors. First, the protein bound to IgBPs would be a determining factor in the outcome and Fn or Fg binding would facilitate attachment while bovine Fc IgG binding would provide protection from the host immune response. Secondly, the degree of upregulation may determine which protein is bound as one protein may have a steric advantage or disadvantage in binding at a given expression level. Finally, the affinity of the IgBP(s) for a particular protein would affect which protein is bound. Perhaps in the LRT when serum IgG, Fn, and Fg would be abundant during infection, the IgBP(s) would have a greater affinity for Fc IgG than the ECMPs and evasion of the host immune response would be the dominant role of the IgBPs. Because pathogenesis is a process and because it is not likely that only one moiety of protein will be bound to IgBPs at a given time, the proportion of any of these proteins bound at a particular time might affect the virulence role of the IgBP(s). 106 Table 2. Proposed IgBP role in virulence Nasopharynx URT LRT (Commensal) (Colonization) (Infection) IgBP(s) Homeostatic Increased Increased Expression ECMP Binding Fn/Fg binding Fn/Fg binding Fn/Fg binding No Fc IgG binding No Fc IgG binding Bovine Fc IgG binding Function Attachment Attachment Attachment Evasion Future research could take a number of interesting directions. Biochemical and biophysical characterization of the IgBP(s) would establish similarities or dissimilarities between the M. haemolytica IgBP(s) and other known IgBP(s). This information could help to determine whether roles in virulence established for other IgBPs might also be true for the M. haemolytica IgBP(s). Biologically, it can be determined whether or not Fn or Fg can act as a bridge between the M. haemolytica IgBP(s) and airway epithelial cells thus facilitating adhesion. Genetic analysis would clarify whether the IgBP(s) is one protein or more than one protein. This work would open the door to creation of a deletion mutant lacking expression of the IgBP(s). Infectivity trials with the deletion mutant would help determine the importance of the IgBP(s) in virulence. In conclusion, this study identified an M. haemolytica surface expressed IgBP as well as a secreted IgBP. The IgBP did not appear to play a role in serotype-specificity of species- specific infection as all serotypes bound both bovine Fc IgG and sheep Fc IgG. However, sheep Fc IgG binds M. haemolytica IgBP with a different protein band pattern on far- Western blot than did bovine Fc IgG. This difference may indicate a different virulence role for the IgBP in cattle and sheep but more likely it may be that more than one IgBP may be 107 expressed by M. haemolytica or there are different binding sites for sheep Fc IgG and bovine Fc IgG. The M. haemolytica IgBP from the WCS preparation was isolated by affinity chromatography. Western blot results in this study showed an increased antibody response to M. haemolytica IgBP in cattle recovering from mannheimiosis indicating a role for the IgBP in protection from M. haemolytica induced BRD. The in vivo and in vitro virulence association of protein A and other previously identified IgBPs, the similarity in surface expression of protein A, protein H and M. haemolytica IgBP, the presence of a secreted M haemolytica IgBP and the increased immune response to M. haemolytica IgBP in convalescent cattle suggest a role for M. haemolytica IgBP in virulence. The M. haemolytica IgBP(s) bound Fg and Fn. These results suggest that the IgBP(s) has a further role in virulence. Other bacterial ECMBPs are involved in cell adhesion to airway epithelial cells. Similarly, upregulation of IgBP(s) expression in conditions mimicking in vivo conditions further suggests an important role in virulence. The M. haemolytica IgBP(s) might be an important component of an M. haemolytica subunit vaccine. 108 REFERENCES Berge, A., B. Kihlberg, A. Sjoholm, and L Bjôrck. 1997. Streptococcal protein H forms soluble complement-activating complexes with IgG, but inhibits complement activation by IgG-coated targets. J. Biol. Chem. 272:20774-20781. Dosset, J.H., G. Kronvall, R.C. Williams Jr., andP.G. Quie. 1969. Antiphagocytic effects of Staphylococcal protein A. 103:1405-1410. Frank, G. 1979. Pasteurella haemolytica and respiratory disease in cattle. Proc. Annu. Meet. US Anim. Health Assoc. 83:153-160. Fraser, J., N. Gilmour, S. Laird, and W. Donachie. 1982. Prevalence of Pasteurella haemolytica serotypes isolated from ovine pasteurellosis in Britain. Vet. Rec. 110: 560-561. Guss, B., M. Eliasson, A. Olsson, M. Uhln, A. Frej, H. Jôrnvall, J. Flock, and M. Lindberg. 1986. Structure of the IgG-binding regions of streptococcal protein G. EMBO. 5:1567-1575. Kihlberg, B., M. Collin, A. Olsèn, and L. Bjôrck. 1999. Protein H, an antiphagocytic surface protein m Streptococcus pyogenes. 67:1708-1714. Mocks, T., L. Abrahmsen, B. Nilsson, U. Hellman, J. Sjoquist, and M. Uhlén. 1986. Staphylococcal protein A consists of five IgG-binding domains. Eur. J. Biochem. 156:637- 643. Mongodin, E., O. Bajolet, J. Cutrona, N. Bonnet, F. Dupuit, E. Puchelle, and S. de Bentzmann. 2002. Fibronectin-binding proteins of Staphylococcus aureus are involved in adherence to human airway epithelium. Infect. Immun. 70:620-630. Rosenblatt, J., P. Zeltzer, J. Portaro, and R. Seeger. 1977. Inhibition of antibody- dependent cellular cytotoxicity by protein A from Staphylococcus aureus. J. Immunol. 118:981-985. Sjoquist, J., J. Movitz, I. Johansson, and H. Hjelm. 1972. Localization of protein A in the bacteria. Eur. J. Biochem. 30:190-194. Sjoquist. J., and G. Stâlenheim. 1969. Protein A from Staphylococcus aureus. IX. Complement-fixing activity of protein A-IgG complexes. J. Immunol. 103:467-473. Wann, E.R., G. Sivashankarappa, and M. Hôôk. 2000. The fibronectin-binding MSCRAMM FnbpA of Staphylococcus aureus is a Afunctional protein that also binds to fibrinogen. J. Biol. Chem. 275:13863-13871. 109 Whitely, L.O., S.K. Maheswaram, D.J. Weiss, T.R. Ames, and M.S. Kannan. 1992. Pasteurella haemolytica Al and bovine respiratory disease: pathogenesis. J. Vet. Intern. Med. 6:11-22. 110 ACKNOWLEDGMENTS I am ever grateful to my children. They have been with me in the good times and the bad while I've gone through graduate school. They shared their pride about my accomplishments and encouraged me when the going was rough. They have sacrificed at times they may not have wanted to sacrifice. And they've shared in the joy as I've accomplished my goals. May I be as good at supporting them toward their goals in life as they've been with mine. I am grateful to my major professor, Dr. Louisa B. Tabatabai. She had faith in me before I had faith in myself. At times when I wasn't sure whether I wanted to take the next step, she showed me where to put my foot. Her guidance in my research has often opened a door for exploration when the next stage looked dark to me. She has encouraged me to look for and try new, creative solutions. She taught me by her example to find different ways to generate more information so that more questions can be answered. She has freely shared her initiative and curiosity with me, and helped me to learn to use those same qualities. Thank you, Louisa. I would like to thank my committee members; my co-major professor Dr. James Roth who has been helpful with discussions, Dr. Joan Cunnick who has shared her expertise about flow cytometry, Dr. Merlin Kaeberle who has added his wisdom, and Dr. James Dickson who has made extra efforts to learn about my research after joining my committee after much of the research was done. I wasn't able to thank Dr. Donald Graves who retired before the completion of my research. He helped me keep perspective about my work. Ill Dr. Carol Belzer was a graduate student in our lab when I started. I'm grateful that she helped me learn many practical aspects about lab work and shared her roadmap through graduate school. Thank you, Carol. I also want to thank my parents, Isabelle Smith and Gordon Smith. I would not be the person I am today without the start they gave me in life.