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Histophilus somni IbpA DR2/Fic in virulence and immunoprotection at the 1

natural host alveolar epithelial barrier 2

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Running Title: Histophilus somni DR2/Fic cytotoxicity and immunoprotection 5

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Bereket Zekarias1, Seema Mattoo

2, Carolyn Worby

2, Jason Lehmann

1, Ricardo F. Rosenbusch

3, 7

Lynette B. Corbeil1,4*

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Department of Pathology, University of California San Diego, San Diego, CA, 921031, 10

Department of Pharmacology, University of California San Diego, La Jolla, CA, 920932, 11

Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State 12

University, Ames, IA, 500113, Department of Population Health and Reproduction, School of 13

Veterinary Medicine, University of California Davis, Davis, CA, 956164 14

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* Corresponding author: Lynette Corbeil, 1Department of Pathology, University of California 17

San Diego, 200 W. Arbor Drive, San Diego, CA, 92103-8416. Tel 619-543-7314. Fax 619-543-18

6614. E-mail: lcorbeil@ucsd.edu. 19

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Copyright © 2010, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Infect. Immun. doi:10.1128/IAI.01277-09 IAI Accepts, published online ahead of print on 22 February 2010

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ABSTRACT 21

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Newly recognized Fic family virulence proteins may be important in many bacterial pathogens. 23

To relate cellular mechanisms to pathogenesis and immune protection, we studied cytotoxicity of 24

Histophilus somni immunoglobulin binding protein-A (IbpA) DR2/Fic for natural host target 25

cells. Live virulent IbpA producing H. somni strain 2336, its cell free culture supernatant (CCS) 26

or recombinant (r) DR2/Fic caused dramatic retraction and rounding of bovine alveolar type 2 27

(BAT2) epithelial cells. The IbpA deficient H. somni strain 129Pt and a Fic motif His298Ala 28

mutant rDR2/Fic protein were not cytotoxic. The cellular mechanism of DR2/Fic cytotoxicity 29

was demonstrated by incubation of BAT2 cell lysates with strain 2336 CCS or rDR2/Fic in the 30

presence of [α-32

P]ATP, resulting in adenylylation of Rho GTPases and cytoskeletal disruption. 31

Since IbpA is not secreted by type III or type IV secretion systems, we determined whether 32

DR2/Fic entered the host cytoplasm to access its Rho GTPase targets. Although H. somni did 33

not invade BAT2 cells, DR2/Fic was internalized by cells treated with H. somni, CCS or 34

rDR2/Fic protein, as shown by confocal immunomicroscopy. Transwell bacterial migration 35

assays showed that strain 2336 migrated between retracted BAT2 cells in large numbers but the 36

IbpA deficient strain 129Pt did not cross unless the monolayer was pretreated with strain 2336 37

CCS or rDR2/Fic protein. Antibody to rDR2/ Fic or passively protective convalescent phase 38

serum blocked IbpA mediated cytotoxicity and inhibited H. somni transmigration across BAT2 39

monolayers, confirming the role of DR2/Fic in pathogenesis and corresponding to in vivo 40

protection in previous animal studies. 41

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INTRODUCTION 42

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New mechanisms of virulence due to Fic family proteins may be of important significance since 44

many bacterial pathogens have Fic sequences in their genomes (27). Mechanisms of action were 45

reported for the first time in 2009 in a few of these pathogens using cell lines (14, 18, 27, 28) but 46

their importance in relevant models of pathogenesis and immune protection remains to be 47

demonstrated. We chose to study the IbpA DR2/Fic cytotoxin of Histophilus somni, formerly 48

called Haemophilus somnus (1), because we previously reproduced pneumonia and septicemia in 49

animals with this pathogen (7,9) and demonstrated protection by immunizing with IbpA DR2/Fic 50

(7). Histophilus somni is an economically important pathogen of cattle and other ruminants that 51

causes respiratory disease, septicemia, thrombotic meningoencephalitis, myocarditis, arthritis 52

and abortion (5, 11, 16, 21, 26). The organism also can be a member of the normal flora of the 53

lower reproductive tract and, to a lesser extent, the upper respiratory tract (5, 12). The 54

pathogenesis of H. somni pneumonia, the most commonly reported syndrome in H. somni 55

infection, and the mechanisms by which the bacteria spread into the systemic circulation from 56

the respiratory tract are not clearly defined. 57

One of the virulence factors of H. somni is immunoglobulin-binding protein A (IbpA), a 58

secreted and surface associated fibrillar protein of 4,095 amino acid residues. It is transported to 59

the bacterial surface by a two-partner secretion pathway (13, 23). All tested isolates of H. somni 60

produce IbpA, except for four carrier strains including strain 129Pt which lacks the entire ibpA 61

gene locus (4, 25). The IbpA producing strain 2336 has been shown to be virulent in a bovine 62

pneumonia model (9, 10). Convalescent phase bovine serum which recognizes IbpA (6, 19, 29) 63

passively protects calves against pneumonia (8). The N-terminal region of IbpA has several 64

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putative adhesin domains with homology to those of filamentous haemagglutinin (FHA) of 65

Bordetella pertussis (23). The C-terminus of IbpA contains several repeat sequences including 66

two large (400 residue) direct repeats (DR1 and DR2) (6). Each DR contains a conserved Fic 67

(Filamentation induced by cAMP) motif (27). This motif was originally described in E. coli as a 68

stress-response protein associated with bacterial filamentous growth in the presence of excess 69

cAMP (15). The Fic family proteins all contain a conserved Fic motif, 70

H×F×(D/E)(A/G)N(K/G)R which is involved in virulence of several pathogens (14, 18, 27, 28). 71

We recently showed that expression of DR2/Fic in HeLa cells resulted in disruption of 72

the cellular cytoskeleton due to adenylylation and subsequent inactivation of the Rho GTPases 73

(27). The Fic motif in DR2 was critical since a substitution of the conserved His residue in the 74

Fic motif by Ala abrogated cytotoxicity (27). In that study, we transfected human HeLa cells 75

with DR2/Fic. This did not reflect a physiologically relevant interaction of H. somni with its 76

natural host cells. Therefore we developed systems for assessing the IbpA DR2/Fic function 77

relevant to natural disease and protective immunity. Based on the previously reported 78

attachment of H. somni to bovine turbinate (BT) cells (24) and the location of the organism in the 79

alveolus during pneumonia (3, 9), we compared the cytotoxic effects of recombinant DR2/Fic in 80

BT cells, primary bovine alveolar type 2 (BAT2) cells and the human HeLa cell line used 81

previously (27). We now report that treatment of these cells with virulent H. somni strain 2336, 82

IbpA enriched cell free culture supernatant (CCS) or recombinant DR2/Fic (rDR2/Fic) caused 83

cell rounding and retraction. BAT2 cells were most susceptible and HeLa cells least susceptible. 84

Retraction was shown to be due to adenylylation of Rho GTPases resulting in cytoskeletal 85

disruption in BAT2 cells. Paracellular migration of virulent H. somni across a BAT2 monolayer 86

was demonstrated, suggesting a route for invasion of the blood stream. A carrier strain of H. 87

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somni which lacks IbpA, 129Pt, did not cause cytotoxicity or transmigrate across the alveolar 88

epithelial monolayer. Convalescent phase serum or antibody to DR2/Fic neutralized toxicity and 89

prevented migration across BAT2 monolayers, indicating that the IbpA DR2/Fic domain is 90

relevant to in vivo pathogenesis and immune protection against disease in the natural host. 91

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MATERIALS AND METHODS 93

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Bacterial strains, growth and culture supernatant preparation. Histophilus somni strain 95

2336, a virulent pneumonic isolate previously used to induce experimental pneumonia in calves 96

(8, 9), and strain 129Pt, an asymptomatic carrier strain isolated from the prepuce of a normal bull 97

(4, 25), were grown on Brain-Heart Infusion (BHI) (BD Diagnostics, Sparks, MD) agar 98

containing 5% bovine blood in Alsevers solution (Colorado Serum Co., Denver, CO) at 37oC in a 99

candle jar. Culture supernatant was prepared from H. somni scraped from an 18 h BHI blood 100

agar plate, inoculated into in BHI-broth supplemented with 0.1% Tris-base and 0.01% thiamine 101

monophosphate and grown for 6 hours at 37 oC with shaking at 200 RPM. The inoculum for 102

each culture was standardized spectrophotometrically and confirmed by plate counting to be 103

approximately 5 x 107 CFU. Six-hour cultures of H. somni shed minimal detectable 104

lipooligosaccharide into the culture supernatant in preliminary studies (unpublished data). The 105

culture was centrifuged at 5,000 x g for 15 min, the supernatant filtered through a 0.22-µm-106

diameter filter and then concentrated 40-fold in a centrifugal filter concentrating device (Amicon 107

Ultra) with a 10-kDa molecular mass cutoff (Millipore, Billerica, MA) by about 2 h 108

centrifugation at 3,000 x g. The retentate was further washed twice in phosphate-buffered saline 109

(PBS) to produce cell free culture supernatant (CCS). Each preparation was monitored by 110

Western blotting against rabbit antibody to rDR2/Fic and bovine convalescent phase serum for 111

presence of IbpA. 112

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Recombinant DR2/Fic protein production. Expression and purification of recombinant 114

DR2/Fic protein and the mutant DR2/Fic has been described previously (27). Briefly, the ibpA 115

DR2/Fic encoding region was PCR amplified using primer sequences 116

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AAATCATCTCCGCAAGAAGGA and TTTTGCCAACTCTTTTAAAAAC (ibpA GenBank 117

accession no. CP000947, locus HSM_1489) and cloned into a GST-tag plasmid vector pET41a 118

(Novagen, Madison, WI). Recombinant DR2/Fic expressed in E. coli BL21 cells was purified by 119

glutathione-affinity chromatography (Sigma, St. Louis MO). A site-directed point mutation of 120

Histidine at residue 298 in DR2/Fic (residue 3717 in the IbpA sequence) replaced with Alanine 121

was constructed (rDR2/Fic H/A), as previously described (27), and purified as above. 122

Production of polyclonal antibodies. Polyclonal antibodies against rDR2/Fic protein were 123

produced by immunizing two rabbits with purified recombinant GST-DR2/Fic protein emulsified 124

with Freund’s adjuvant (Cocalico Biologicals, Reamstown, PA). Blood was collected before and 125

after immunization for preparation of pre- and post- immunization sera. Convalescent phase 126

bovine serum was obtained from two calves (E5 and E7) with experimental H. somni pneumonia 127

induced by intrabronchial inoculation of strain 2336 (9). Serum samples were collected before 128

infection (preimmune serum) and 6 weeks after the induction of pneumonia, during 129

convalescence. Specificities of these sera were evaluated by Western blotting. 130

Cell culture. Primary BAT2 cells were isolated from newborn calf lung collected in cold Hank's 131

balanced salt solution (HBSS) containing gentamycin (40 µg/ml), fungizone (5 µg/ml) and 132

cefoperazone (35 µg/ml). Finely minced tissue was stirred to release macrophages, and then 133

digested in 0.3% dispase II (Boehringer, Mannheim, IN) in HBSS, centrifuged at 500 × g for 10 134

min and the supernatant decanted. The pellet containing BAT2 cells was suspended in the last 135

drop of dispase solution and gently mixed with PBS containing10% horse serum. After 136

centrifuging at 500 × g for 5 min, the pellet was resuspended in 10 ml DMEM/Keratinocyte 137

medium at 1:1 (Invitrogen, Carlsbad, CA), tissue clumps were sedimented and the top layer 138

transferred to a tissue culture flask pre-coated with 0.1% gelatin containing 20% fetal bovine 139

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serum (FBS) v:v (gelatin/FBS). Cells were incubated at 37oC in 5% CO2 and fed with fresh 140

DMEM/Keratinocyte medium supplemented with 2% FBS, 5 mM L-glutamine, 0.02% 141

lactalbumin hydrolyzate, containing penicillin (100 U/ml) /streptomycin (100 µg/ml) 142

(Invitrogen) every two days. BAT2 cells were identified by their star-shaped appearance before 143

flattening and by their Nile Red-stainable cytoplasmic vacuoles (lamellar bodies) (20) and were 144

used at the maximum of 13 passages. BT cells (kindly provided by L J Gershwin, UC Davis) 145

were grown in the DMEM/Keratinocyte medium and the HeLa cells were grown in DMEM 146

supplemented with 10% FBS, plus penicillin (100 U/ml) /streptomycin (100 µg/ml) at 37 oC in a 147

humidified atmosphere of 5% CO2. Cells were grown in 75 cm2

culture flask pre-coated with 148

gelatin/FBS and harvested by brief digestion with 0. 05% trypsin, 10 mM EDTA solution 149

(Invitrogen). For assays, cells were seeded either in 24-well tissue culture plates (5 × 104 150

cells/well), in 12-well culture plates containing coverslips (5 × 105 cells/ well) or in 8-well 151

chambered glass slides (Nunc, Naperville, IL) (5 × 103

cells/well). Glass slides and glass 152

coverslips were pre-coated with10 µg/ml bovine plasma fibronectin (Invitrogen) overnight at 4 153

oC. 154

Cytotoxicity assay. Cells grown to about 90 % confluence in chambered slides or on glass 155

coverslips in 12-well culture plates were treated with live bacteria, CCS, purified rDR2/Fic 156

proteins or the rHis/Ala mutant DR2/Fic protein. Bacteria were harvested from an 18 hr culture 157

on BHI blood agar, resuspended in tissue culture medium without antibiotics and incubated for 2 158

h at 37oC before adding to the cells. Bacterial counts was estimated by spectrophotometer and 159

confirmed by CFU counting. Cells were treated with bacteria at a multiplicity of infection (MOI 160

- bacteria: cell) of 10:1 or 100:1, CCS at a final concentration of 20× in culture medium or 161

recombinant proteins at 20 µg/ml of culture medium without FBS After this treatment for 4 h, 162

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cells were washed twice with PBS and fixed with 4% fresh paraformaldehyde for 20 min at 4 oC 163

before permeabilizing with 0.1% Triton X-100 for 5 min and staining with Rhodamine-164

phalloidin (Invitrogen) for 30 min at room temperature. Nuclei were counter-stained with DAPI 165

and slides were mounted in Prolong Anti-fade reagent (Invitrogen). The number of rounded or 166

retracted cells and the total number of cells in the field (as determined by the DAPI nuclear stain) 167

were counted in ten separate microscope fields by fluorescence microscopy and means 168

calculated. Mitotic figures distinguished normal retracted/rounded cells due to mitosis versus the 169

cytotoxic rounded or retracted phenotype. Toxicity refers to cell rounding or retraction, not 170

necessarily cell death. Experiments were repeated at least twice. 171

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In vitro Rho GTPase adenylylation assay with BAT2 and HEK293T cell lysates. 173

Mammalian cell extracts were prepared by lysing cells in lysis buffer (50 mM Tris, pH 7.5, 500 174

mM NaCl, 1% Triton-X 100, 0.1% SDS, 10 mM MgCl2, 1mM pefabloc, 1mM benzimidine 175

hydrochloride, 1 µM leupeptide, 1µM E64) and collecting the supernatant from a 13,000 rpm 176

centrifugation in a microfuge for 10 min. Approximately 1 µg of GST-DR2 was incubated with 177

30 µg of HEK293T or BAT2 cell extract in 40 µl adenylylation reactions containing 25mM Tris-178

HCl, pH 7.5, 3.0mM MgCl2, 1mM DTT, 0.5mM EDTA and 1 µCi [α-32

P]-ATP for 1 h at 30oC. 179

Reactions were stopped by adding Nupage loading buffer (Invitrogen). Extract samples 180

containing 30µg protein were used for the Western analyses. Reaction products were separated 181

by SDS-polyacrylamide gel electrophoresis and visualized by Coomassie staining (loading 182

controls), autoradiography or Western blot analyses with antibody to RhoA (Cell Signaling 183

Technology, Danvers, MA), Cdc42 (BD Transduction Laboratories, Lexington, KY) or Rac1 184

(Abcam Inc., Cambridge MA). 185

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Inhibition of cytotoxicity by serum antibody. To determine whether antibody neutralizes 186

cytotoxicity of live viable bacteria or CCS for BAT2 cells, heat decomplemented filtered 187

sterilized rabbit anti-DR2/Fic serum, rabbit pre-immune serum, bovine convalescent phase serum 188

or bovine pre-immune serum was added to a suspension of H. somni (106 CFU/ml) or 20× CCS 189

at a final serum concentration of 1:100. The bacteria or CCS and serum mixtures were incubated 190

with shaking for 45 min at room temperature for the bacterial preparation or at 4oC for the CCS 191

preparation. The mixtures were then added to the BAT2 cells. After 4 h of incubation cells were 192

washed, fixed and cytotoxicity was quantified by microscopy as described above. 193

Bacterial attachment and invasion assay. Confluent BAT2 and BT cell monolayers in 24-well 194

culture plates were washed three times in DMEM and infected with H. somni in tissue culture 195

medium without antibiotics at an MOI of 50. Plates were centrifuged at 500 × g for 5 min and 196

incubated for 1.5 h at 37°C in humidified 5% CO2 incubator. Cells were then washed gently five 197

times with PBS to remove non-adhering bacteria. Cell associated viable bacteria were 198

determined by lysing cells with 0.5% Triton X-100 for 5 min at room temperature, diluting in 199

PBS with vigorous pipetting and plating for counting CFU. Preliminary experiments showed that 200

treatment of H. somni with 0.5% Triton X-100 for 5 min did not decrease viable cell counts (data 201

not shown). Invasion was assessed by quantifying intracellular bacteria in a gentamicin 202

protection assay. After infection for 1.5 h and washing five times to remove non-adherent 203

bacteria as above, fresh medium containing gentamicin (100 µg/ml) was added. Cells were 204

incubated for a further 1 h, washed three times with PBS, lysed and dilutions plated to count 205

internalized bacterial CFU. 206

Confocal microscopy. BAT2 cells were grown on glass coverslips in a 12-well culture plate to 207

80% - 90% confluence. Cells were treated with virulent strain 2336 or IbpA negative strain 208

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129Pt at 10 MOI , with CCS at 20× concentration or rDR2/Fic protein (20 µg/ml) for 4 h as 209

described in cytotoxicity assay. This step was done to determine attachment and uptake of IbpA 210

protein. After treating with the above IbpA containing preparations, cells were washed three 211

times with PBS, fixed with 4% paraformaldehyde and permeabilized or not-permeabilized

with 212

0.1% Triton X-100 for 5 min, before reacting with rabbit anti-DR2/Fic at 1:100 in 0.5% BSA in 213

PBS for 2 h at room temperature. The Triton X 100 treatment was done to permeablize the cells 214

to the antibody in order to detect IbpA DR2/Fic which would have been taken up during the 215

incubation step before fixation and Triton X 100 treatment. Cells were then washed, incubated 216

for 2 h at room temperature with Alexa Fluor 488 conjugated goat anti-rabbit antibody at 1:500, 217

washed again, and stained with Rhodamine-phalloidin for F-actin labeling and TO-PRO-3 for 218

labeling of nuclei (Invitrogen). Slides were dried, mounted in Prolong Anti-fade reagent 219

(Invitrogen) and examined under a Leica TCS SP5 confocal microscope (Leica Microsystems, 220

Bannockburn, IL). Alexa Fluor 488 was excited with the 488 nm laser and emission was 221

measured at wavelengths of 515 ± 30 nm; Rhodamine was excited with

568 nm and emission was 222

measured at 600 ± 40 nm; TO-PRO-3 was excited at 635 nm, and emission

was measured at 223

wavelengths > 650 nm. A series of z-section images (0.25 µm) were collected and analyzed with 224

ImageJ software (http://www.rsb.info.nih.gov). 225

Transmigration assay. BAT2 cells were grown on polycarbonate Transwell inserts of 6.5 mm 226

diameter, with 3 µM pore size filters fitted into a 24-well plate (Corning, Cambridge, MA). The 227

Transwell filters were first coated with gelatin/FBS, dried and covered with DMEM for 1 h at 37 228

oC before seeding BAT2 cells at 2 × 10

3 cells/well. Cells were grown for 5 days to form a 229

complete monolayer. Then cells were washed and 105 CFU of H. somni strain 2336 or 129Pt in 230

80µl cell culture medium (without antibiotics) was added to the Transwell insert (approximately 231

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10 MOI). The lower chamber was filled with 250µl of cell culture medium. After 3 h of 232

incubation, the Tranwell-insert was removed and the contents of the lower chamber were diluted 233

and plated on BHI blood agar plates for viable bacterial counts. In order to assess the effect of 234

anti-serum on H. somni 2336 transmigration across a BAT2 monolayer bacteria were first 235

incubated with anti-DR2/Fic serum, convalescent phase bovine serum or the pre-immune serum 236

controls, at a 1:100 dilution, for 45 min at room temperature before transferring onto the cell 237

monolayer. In a separate experiment to determine whether IbpA DR2/Fic would result in 238

migration of strain 129Pt, the BAT2 monolayer were pre-treated for 4 h with either purified 239

rDR2/Fic protein or the mutant protein (both at 20µg/ml), 20× CCS of H. somni 2336 or cell 240

culture medium alone before adding the bacteria. 241

Statistical analysis. Data were analyzed with one-way ANOVA using GraphPad Prism software 242

(GraphPad Software, La Jolla, CA). Treatment groups were compared using Bonferroni’s 243

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RESULTS 245

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Live H. somni, CCS and purified rDR2/Fic protein causes retraction and rounding of 247

bovine respiratory epithelial cells and human HeLa cells 248

We previously showed that H. somni and its CCS are toxic for human HeLa cells (27) but it was 249

clear that a model relevant to the host specific disease was needed. Therefore, we developed an 250

in vitro model using cells from the bovine upper respiratory tract (BT cells) and the pulmonary 251

alveolar epithelial cells (BAT2 cells). In stark contrast to HeLa cells, treatment with live virulent 252

strain 2336 H. somni, its CCS or rDR2/Fic protein resulted in robust cell rounding and retraction 253

in BAT2 cells (Fig. 1A). Carrier strain 129Pt did not cause rounding or retraction (Fig 1A), nor 254

did its CCS (data not shown). Strain 2336 CCS contained abundant HMW proteins reacting with 255

anti-DR2/Fic or convalescent serum in Western blots (Fig. 1B), indicating the presence of IbpA. 256

CCS of strain 129Pt was negative for IbpA in the same blots. In cytotoxicty tests, the BAT2 cells 257

were most susceptible, followed by BT cells, and the human HeLa cell line was least susceptible 258

(Fig. 1C, D and E). A mutant rDR2/Fic protein (rDR2/Fic H/A) with the critical His residue at 259

position 298 in the Fic motif replaced by Ala did not cause cytotoxicity when used at equivalent 260

concentrations to rDR2/Fic (Fig. 1E). These results showed that an active Fic motif is required 261

for IbpA mediated cytotoxicity and that IbpA’s effects were strongest against bovine targets 262

relevant to H. somni disease. Since BAT2 cells were most susceptible and were most disease 263

relevant, these cells were used for the rest of the study. 264

Rho GTPases are the molecular targets of IbpA DR2/Fic in BAT2 cells 265

Previously we showed that the IbpA DR1 and DR2/Fic domains adenylylate Rho GTPases and 266

disrupt their downstream signaling cascades in human HEK293T epithelial cell extracts (27). 267

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Therefore, we now determined whether Rho GTPases were targets for IbpA DR2/Fic mediated 268

adenylylation in BAT2 cells. Like control HEK293T cells, rDR2/Fic2 adenylylated endogenous 269

proteins that migrated at the size of Rho GTPases (Table 1) when incubated with cell lysates of 270

BAT2 cells as determined by autoradiography and Western blot analysis for RhoA (Fig 2A) or 271

for Rac1 and Cdc42 (Fig 2B). Similar results were obtained by treating both cell lysates with 272

CCS from H. somni 2336 but not with CCS from IbpA deficient strain 129Pt (Fig. 2C). Since 273

bovine and human Rho GTPases are 100% identical at the amino acid level (Table 1) and, as 274

previously shown (27), both rDR2/Fic and H. somni strain 2336 CCS adenylylate purified human 275

GST-RhoA, -Rac and -Cdc42 in vitro, results described in Fig 2 confirm Fic mediated 276

adenylylation of bovine Rho GTPases. Interestingly, H. somni 2336 CCS and rDR2 displayed 277

much higher intensity of radiolabelled Rho GTPases in BAT2 cell extracts than in HEK293T cell 278

extracts, which correlates with the much higher concentrations of RhoA, Rac1 and Cdc42 in 279

BAT2 cells than in HEK239T cells as determined by Western blotting (Fig 2A, B and C). 280

Antibody neutralizes cytotoxicity 281

Convalescent phase serum is passively protective against bovine pneumonia and immunization 282

with rDR2 protein protects mice against septicemia, so we tested the ability of bovine 283

convalescent phase serum and rabbit antibody to rDR2/Fic to neutralize cytotoxicity in vitro. 284

Live H. somni strain 2336 and its CCS were treated with the anti-sera for 45 min prior to 285

treatment of BAT2 cells. Both anti-sera reacted with high molecular weight IbpA from strain 286

2336 in Western blots (Fig 1B). The rabbit anti-DR2/Fic and convalescent bovine sera 287

significantly decreased the cytotoxicity of live bacteria or CCS for BAT2 cells as compared with 288

the corresponding pre-immune sera (p < 0.05) (Figs. 3A and 3B). 289

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H. somni adheres to but does not invade BAT2 cells 291

Since H. somni is found in alveoli during experimental and spontaneous pneumonia (3, 9), 292

attaches to BT cells in vitro (24) and causes septicemia (5, 11), we investigated the possibility 293

that H. somni attaches to BT and/or BAT2 cells and then invades the cells in order to cross the 294

epithelial barrier. After 1.5 h incubation with cells and washing, H. somni was found to attach in 295

large numbers to both BT and BAT2 cells (Fig. 4). However, H. somni did not invade the cells in 296

the gentamycin protection assay (Fig. 4). This raises two questions. Firstly, does IbpA get into 297

cells to access Rho GTPases? Secondly, how does H. somni cross the alveolar epithelial barrier 298

to cause septicemia? 299

IbpA DR2/Fic attaches to BAT2 cells and is internalized 300

Confocal immunomicroscopy was used to determine whether IbpA DR2/Fic protein binds to cell 301

surfaces and whether it is internalized into BAT2 cells. Confocal images of BAT2 control cells, 302

not treated with H. somni or CCS but permeablized and labeled with antibody to rDR2/Fic, 303

showed no green immunofluorescence or retraction (Fig. 5A). Cells infected with IbpA negative 304

strain 129Pt also showed no immunofluorescence or retraction (Fig. 5B). However, in cells 305

infected with H. somni 2336 and immunolabelled for DR2/Fic antigen, DR2/Fic was detected on 306

retracting cell surfaces in non-permeablized cells (Fig. 5C). Much more DR2/Fic antigen was 307

located in the cytoplasm of the z-sections of retracting cells treated with either H. somni, CCS or 308

rDR2/Fic protein and permeabilized with Triton X-100 before staining with the primary rabbit 309

anti-rDR2/Fic antibody (Fig. 5D, E and F, respectively). Control cells treated with rGST protein 310

alone did not stain, whether permeabilized or not permeabilized (data not shown). These studies 311

show that IbpA DR2/Fic attaches to the cell surface and is internalized into BAT2 cells. The 312

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model provides a means for investigating the mechanism of IbpA internalization in relevant host 313

cells. 314

IbpA DR2/Fic induces paracellular migration of H. somni across BAT2 cell monolayers 315

Our data thus far indicate that H. somni does not gain access to the bloodstream by invading 316

through BAT2 cells, so we determined whether H. somni crosses the alveolar epithelial barrier 317

between the retracted cells by paracellular migration to cause septicemia. Virulent H. somni 318

strain 2336 was recovered in large numbers from the lower chamber of Transwell plates after 3 h 319

incubation (about 50% of the inoculum), whether cells were pretreated with medium or pre-320

immune serum (Fig. 6A). However, the transmigration was significantly inhibited by the 321

presence of antibody to rDR2/Fic or passively protective convalescent phase serum (p < 0.05) 322

(Fig. 6A). Almost none of the IbpA deficient strain 129Pt was recovered from the lower 323

compartment, unless the BAT2 monolayers were pretreated with rDR2/Fic protein or the CCS of 324

strain 2336 for 4 h to induce cell retraction before adding the bacteria (Fig. 6B). Pretreatment 325

with the mutant rDR2/Fic H/A (with inactive Fic) did not significantly increase transmigration 326

when compared with the untreated control (p > 0.05). Both the antibody neutralization of strain 327

2336 transmigration and the facilitation of strain 129Pt transmigration by pretreatment with 328

rDR2/Fic or strain 2336 CCS implicate DR2/Fic induced BAT2 cell retraction in allowing 329

paracellular invasion through the alveolar epithelial barrier, as presented in a simplified model of 330

H. somni migration between both alveolar epithelial cells and endothelial cells to cause 331

septicemia (Fig. 7). 332

333

334

335

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DISCUSSION 336

337

This study shows that IbpA producing H. somni strain 2336, its IbpA enriched culture 338

supernatant and the rDR2/Fic protein are cytotoxic for relevant host BT cells and BAT2 cells but 339

only have a minimal toxic effect on the human cervical carcinoma HeLa cell line, in keeping 340

with the strict ruminant host specificity of H. somni (5, 11). The greater sensitivity of BAT2 341

cells than BT cells corresponds to the severe pulmonary disease but mild upper respiratory 342

disease in bovine H. somni infection (5, 11). The previously reported detection of H. somni or 343

the IbpA antigen primarily in the pulmonary alveoli of animals with pneumonia also is consistent 344

with the high susceptibility of BAT2 cells (3, 9). Recently, we demonstrated that the mechanism 345

of IbpA DR2/Fic cytotoxicity in human cell lines is due to inactivation of the Rho GTPases by 346

adenylylation, resulting in disruption of the cytoskeletal network (27). Now we show that IbpA 347

DR2/Fic adenylylates the Rho GTPases of BAT2 cells as well. The higher intensity of 348

adenylylation in BAT2 cells, as compared with HEK239T cells, correlates with higher 349

concentration of Rho GTPases in BAT2 cells. Perhaps the severe retraction and rounding of 350

BAT2 cells is related to the high concentration of Rho GTPases. Adenylylation of host Rho 351

GTPases by bacterially secreted Fic family virulence proteins is a new frontier in microbial 352

pathogenesis, as reported in the last year (14, 18, 27, 28). Two such Fic containing virulence 353

proteins, VopS, of V. parahemolyticus and AnkX, of L. pneumophilia have been shown to access 354

the cellular cytoplasm by type III and type IV secretion systems, respectively (17, 28). However, 355

IbpA is secreted to the bacterial surface by a two-partner secretion system (23), so it is not clear 356

how IbpA gains access to the host cell cytoplasm to inhibit Rho GTPase signaling. Our 357

experiments show that IbpA does not reach the cytoplasm by invasion of these bovine epithelial 358

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cells by H. somni. Rather, IbpA released from the bacterial surface is internalized by cells as 359

determined by detection of DR2/Fic antigen intracellularly by confocal immunomicroscopy after 360

treatment of cells with live bacteria, CCS or rDR2/Fic protein. 361

The lack of H. somni invasion of bovine alveolar epithelial cells also raised the question 362

of how the organism reaches the blood stream to cause septicemia. Experiments with BAT2 363

monolayers in Transwells indicate that H. somni passes between retracted BAT2 cells to cross 364

the epithelial barrier by paracellular migration. A similar paracellular route may be used to cross 365

the endothelial cells beneath the alveolar epithelial cells. Indeed, Behling-Kelly et al. (2) showed 366

that H. somni increased permeability of a bovine brain microvascular endothelial monolayer. It is 367

likely that H. somni crosses the pulmonary microvascular endothelium in a similar fashion to 368

cause septicemia followed by myocarditis where the organism is found in large numbers on the 369

endothelial cells (16). Antibody to rDR2/Fic and convalescent phase serum from calves with 370

experimental H. somni pneumonia neutralized IbpA DR2/Fic cytotoxicity. This confirms the role 371

of the Fic domain in cell rounding and retraction. Antibody neutralization of in vitro cytotoxicity 372

due to rDR2/Fic also corresponds to the previously demonstrated immunoprotection of animals 373

against H. somni in active immunization studies with rDR2/Fic (7) and in passive immunization 374

studies with the same convalescent phase serum (8), which recognizes IbpA (29). The DR2/Fic 375

anti-serum and the bovine convalescent phase serum also blocked bacterial transmigration across 376

the BAT2 monolayer suggesting that the protection against pneumonia and subsequent 377

septicemia may be due to prevention of bacterial entry into the sub-epithelium at early stages of 378

infection in the lungs. Neutralization of both cytotoxicty and paracellular migration by antibody 379

in this relevant bovine respiratory alveolar barrier model in parallel with in vivo protection is 380

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convincing evidence that IbpA DR2/Fic is involved in both virulence of H. somni and 381

immunoprotection in the natural host. 382

In conclusion, IbpA DR2/Fic is cytotoxic for natural host epithelial target cells with the 383

relevant BAT2 cells being most susceptible. IbpA adenylylates and inactivates BAT2 Rho 384

GTPases causing cell rounding and retraction due to collapse of cellular cytoskeleton. The 385

retraction of alveolar epithelial cells allows H. somni to cross the respiratory alveolar epithelial 386

barrier by paracellular migration to reach the microvasculature. Neutralization by anti-sera 387

recognizing IbpA DR2/Fic likely accounts for immunoprotection in vivo and confirms the role of 388

IbpA DR2/Fic in pathogenesis. 389

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ACKNOWLEDGMENTS 390

This work was supported by USDA NRI grant 2005-35204-6257. 391

We thank J. E. Dixon for his critical reading of the manuscript, L. J. Gershwin for kind provision 392

of BT cells and J. C. Mendez for technical assistance. 393

394

COMPETING INTERESTS 395

The authors have no competing interests to disclose. 396

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REFERENCES 397

398

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16. O'Toole, D., T. Allen, R. Hunter, and L. B. Corbeil. 2009. Diagnostic exercise: 443

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18. Roy, C. R., and S. Mukherjee. 2009. Bacterial FIC Proteins AMP Up Infection. Sci. 449

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22. Sylte, M. J., L. B. Corbeil, T. J. Inzana, and C. J. Czuprynski. 2001. Haemophilus 459

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24. Ward, G. E., J. R. Nivard, and S. K. Maheswaran. 1984. Morphologic features, 465

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26. Widders, P. R., L. G. Paisley, R. P. Gogolewski, J. F. Evermann, J. W. Smith, and L. 471

B. Corbeil. 1986. Experimental abortion and the systemic immune response to 472

"Haemophilus somnus" in cattle. Infect. Immun. 54:555-560. 473

27. Worby, C. A., S. Mattoo, R. P. Kruger, L. B. Corbeil, A. Koller, J. C. Mendez, B. 474

Zekarias, C. Lazar, and J. E. Dixon. 2009. The fic domain: regulation of cell signaling 475

by adenylylation. Mol. Cell. 34:93-103. 476

28. Yarbrough, M. L., Y. Li, L. N. Kinch, N. V. Grishin, H. L. Ball, and K. Orth. 2009. 477

AMPylation of Rho GTPases by Vibrio VopS disrupts effector binding and downstream 478

signaling. Science 323:269-272. 479

29. Yarnall, M., and L. B. Corbeil. 1989. Antibody response to Haemophilus somnus Fc 480

receptor. J. Clin. Microbiol. 27:111-117. 481

482

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FIGURE LEGENDS 483

484

Figure 1. Cell rounding and retraction caused by H. somni, IbpA enriched culture 485

supernatant (CCS) and rDR2/Fic protein. (A) Micrographs of BAT2 cells treated with live H. 486

somni virulent strain 2336 or carrier strain 129Pt, 20× CCS or purified rDR2/Fic protein 487

(20µg/ml) for 4 h. F-actin fibers stained with Rhodamine-phalloidin. The IbpA negative strain 488

129Pt does not induce retraction but live bacteria, CCS and rDR2/fic cause cells to retract and 489

round up. (B) Western blot analysis of strain 2336 or 129Pt CCS reacted with rabbit antibody to 490

rDR2/Fic or bovine convalescent phase serum. (C and D) Percent of BAT2, BT and HeLa cells 491

rounded/retracted after treatment with (C) live H. somni 2336 (100 MOI) or (D) CCS (20×) 492

expressed as number of affected cells out of total number of cells in ten separate microscope 493

field (each field contains on average 70 cells). Means and standard deviations are shown for one 494

representative of two independent replica experiments. Live virulent H. somni and CCS were 495

most toxic for BAT2 cells and least toxic for HeLa cells. The amount of DR2/Fic on the surface 496

or shed by live bacteria or in CCS was not measured; therefore the percent cytotoxicity can not 497

be compared among these treatment groups. The comparison is between cell types with the same 498

treatment. (E) Percent of cells (BAT2, BT and HeLa) rounded/retracted after treatment with 499

rDR2/Fic or rDR2/Fic H/A mutant, both at 20 µg/ml. Percent cytotoxicity calculated and data 500

presented as in (C). Replacement of the critical His with Ala in the Fic motif abrogates toxicity. 501

502

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Figure 2. In vitro adenylylation of Rho GTPases by rDR2/Fic protein. 503

(A) rDR2/Fic treatment of BAT2 or HEK293T cell lysate in the presence of α32

P-ATP. Transfer 504

of [32

P]AMP as visualized by autoradiography was much higher in the BAT2 cell extracts than in 505

the HEK293T cell extracts, though the Coomassie stain (loading control) shows similar protein 506

concentration for BAT2 and HEK293T cell lysates. Western analysis with anti-human RhoA was 507

carried out to determine the molecular mass and migration of endogenous RhoA in BAT2 and 508

HEK293T cell lysates. (B) Western analysis of the same extracts with anti-Rac1 and anti-Cdc42. 509

Note the much higher concentration of Rho GTPases in the BAT2 cell lysate compared with the 510

HEK293T lysate. (C) Virulent H. somni 2336 or IbpA deficient strain 129Pt CCS treatment of 511

BAT2 and HEK293T cell lysates. A radiolabeled band corresponding to adenylylated Rho 512

GTPases is observed only in strain 2336 treated samples. As with rDR2/Fic treatment, radiolabel 513

is much higher in BAT2 than in HEK293T cell extracts. 514

515

Figure 3. Antibody mediated neutralization of cytotoxicity due to live H. somni or CCS. 516

(A) H. somni infected BAT2 cells. (B) CCS treated BAT2 cells. Rabbit (Rab.) anti-DR2/Fic and 517

bovine convalescent (conv.) phase serum significantly reduce cytotoxicty as compared with the 518

corresponding pre-immune (pre.) serum treated cells. Bars marked with asterisks differ 519

significantly from pre-immune serum controls (p < 0.05). Results in A and B are shown for one 520

experiment and were reproduced in a second experiment. 521

522

Figure 4. H. somni attachment to and invasion of BAT2 and BT cells. Cell associated or 523

attached bacteria were determined after incubating with bacteria for 1.5 h, washing and lysing 524

cells with 0.5% Triton X-100. The number of intracellular bacteria was determined similarly 525

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after treatment with gentamicin at 100 µg/ml for 1 h before lysis. In both cases viable bacterial 526

counts were done to quantitate attached or internalized bacteria. Data are expressed as means of 527

six wells (+/- the standard deviations) for one of three representative experiments. Note that H. 528

somni attaches to but does not invade either cell type. 529

530

Figure 5. Confocal immunomicroscopy of BAT2 cells treated with H. somni, CCS or 531

rDR2/Fic protein, localizing DR2 antigen on the cell surface or in the cytoplasm. Cells were 532

treated with H. somni, CCS or rDR2/Fic protein and fixed before either permeablized with Triton 533

X-100 or not permeablized, then immunostained with anti-DR2/Fic serum, in order to detect 534

internalized or surface associated antigen, respectively. Blue = TO-ORO3 stained nuclei, Green 535

= DR2 antigen labeled with Alexa 448, Red = F actin stained with Rhodamine-phalloidin. (A) 536

Control BAT2 cells for non-specific antibody binding to normal non-retracted 537

uninfected/untreated cells. (B) H. somni 129Pt infected cells, permeablized. No DR2/Fic antigen 538

was detected in the BAT2 cell cytoplasm. (C) H. somni 2336 infected cells, non-permeabilized. 539

Green fluorescence shows DR2/Fic antigen on the retracted cell surface. (D) H. somni 2336 540

infected cells, permeabilized with Triton X-100. Green fluorescence shows internalized DR2/Fic 541

antigen in a retracted cell. (E) CCS treated cells, permeabilized with Triton X-100. Green 542

fluorescence shows internalized DR2/Fic antigen in retracted cells. (F) rDR2/Fic protein treated 543

cells, permeablized with Triton X-100. Green fluorescence shows internalized DR2/Fic antigen 544

in retracted cells. 545

546

Figure 6. Migration of H. somni across BAT2 cell monolayers in Transwells. (A) Virulent H. 547

somni strain 2336 crossed the BAT2 monolayer in large numbers and pretreatment of the 548

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bacteria with rabbit (Rab) antibody to rDR2/Fic or with bovine (Bov) convalescent (conv.) phase 549

bovine serum significantly (asterisk) decreases transmigration compared to wells treated with 550

medium or corresponding pre-immune (Pre) serum (p < 0.05). (B) IbpA negative H. somni strain 551

129Pt did not transmigrate across the BAT2 cell monolayer (control treated with medium). 552

Pretreatment of monolayers with either rDR2/Fic protein or CCS of strain 2336 caused a 553

significant (asterisk) increase in 129Pt transmigration compared to the medium control group (p 554

< 0.05). The mutant rDR2/Fic H/A protein, with the critical His replaced by Ala, did not 555

significantly increase transmigration of strain 129Pt. Data are presented as mean (+/-standard 556

deviation) from six wells in one experiment. Results were reproduced in a second experiment. 557

558

Figure 7. Model of H. somni crossing of the bovine alveolar barrier due to IbpA DR2/Fic 559

cytotoxicty. Live H. somni and shed IbpA (orange dots) cause retraction of alveolar epithelial 560

cells due to IbpA DR2/Fic mediated inactivation of the cellular Rho GTPases. Bacteria pass 561

between retracted alveolar cells. Others have shown that H. somni causes endothelial cell 562

retraction (2) and apoptosis (22). Crossing both alveolar epithelial and endothelial barriers would 563

result in entry to the circulation and septicemia. Our studies show that convalescent bovine 564

serum which protects against pneumonia in vivo (8) and antibody to IbpA DR2/Fic prevents 565

transmigration across alveolar epithelial cell monolayers, consistent with in vivo protection. 566

567

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FIGURES 1

2

3

4

5

6

7

8

Figure 1

(A)

2336DMEM/control 129Pt (100 MOI) (100 MOI)

2336 (10 MOI) rDR2 (20o g)CCS·20

(B)

CCS 1

29Pt

Rab. anti-DR2/Fic Bov. conv. serum

CCS 2

336

CCS 1

29Pt

CCS 2

336

116

200

97

kD

9

10

11

1

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Figure 1 (contin.) 12

0

20

40

60

80

100

% c

yto

tox

icit

y

(C)

BA

T2

HeL

a

BT

0

15

30

45

60

75

% c

yto

tox

icit

y

BA

T2

HeL

a

BT

(D)

13

0

10

20

30

40

50

% c

yto

tox

icit

y

DR2/Fic

DR2/Fic H/A mutant

BA

T2

HeL

a

BT

(E)

14

15

16

17

2

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Figure 2 18

19 (A)

HE

K2

93

T

BA

T2

250kD

75

50

37

25

20

15

BA

T2

BA

T2

HE

K2

93

T

HE

K2

93T

150

32P cATP anti-hRhoA Coomassie 20

anti-hRac1 anti-hCdc42

HE

K2

93

T

BA

T2

HE

K2

93

T

BA

T2

(B)

kD

20

25

15

21

22 (C)

KDKD

23

3

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Figure 3 24

25

20

40

60

80

100

20

40

60

80

100

Cyto

toxic

ity (

%)

Bov

. pre

.B

ov. c

onv.

Rab

. ant

i-

DR

2/Fi

c

*

*

Med

ium

(A)

Rab

. pre

.

26

20

40

60

80

100

20

40

60

80

100

Cyto

toxic

ity(%)

(B)

**

Bov

. con

v.

Rab

. ant

i-

DR

2/Fi

c

Rab

. pre

.

Bov

. pre

.

Med

ium

27

28

4

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Figure 4 29

30

31

0

2

4

6

8 Inoculum 1.3 · 107 CFU

CF

U (

log

10)

Atta

ched

Intra

-ce

llula

r

BAT2

1.5 · 105

0.7 · 105

(< 10)A

ttach

ed

Intra

-ce

llula

rBT

(< 10)

0

2

4

6

8

0

2

4

6

8 Inoculum 1.3 · 107 CFU

CF

U (

log

10)

Atta

ched

Intra

-ce

llula

r

BAT2

1.5 · 105

0.7 · 105

(< 10)A

ttach

ed

Intra

-ce

llula

rBT

(< 10)

32

33

5

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Figure 5 34

35

36

37

38

A

C D

B

E

F

A B

C D

FE

6

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Figure 6 39

40

(A)

**

10

20

30

40

50

60

10

20

30

40

50

60

% i

no

culu

m

Bov

. con

v.

Rab

. ant

i-

DR

2/Fi

c

Med

ium

Rab

. pre

.

Bov

. pre

.

Tra

nsm

igra

ted

CF

U

(x1

,00

0)

(A)

**

10

20

30

40

50

60

10

20

30

40

50

60

**

10

20

30

40

50

60

10

20

30

40

50

60

% i

no

culu

m

Bov

. con

v.

Rab

. ant

i-

DR

2/Fi

c

Med

ium

Rab

. pre

.

Bov

. pre

.

Tra

nsm

igra

ted

CF

U

(x1

,00

0)

41

0

2

4

6

8

10

12

0

2

4

6

8

10

12

(B)

Tra

nsm

igra

ted C

FU

(x1,0

00

)

% i

nocu

lum

DR

2/Fi

c

Med

ium

DR

2/Fi

c H

/A

Hs.

2336

C

CS

*

*

42

43

44

7

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8

45

46

47

Figure 7

Alveolar cell

Retracted alveolar cell

Retracted/apoptotic endothelium

Alveolus (air space)

H. somni with shed and surface IbpA

Septicemia

Alveolar cell

Retracted alveolar cell

Retracted/apoptotic endothelium

Alveolus (air space)

H. somni with shed and surface IbpA

Septicemia

48

49

50

51

52

53

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54

55

56

Table 1. Homology of predicted amino acid sequences of human and bovine Rho GTPase

family proteins.

Rho GTPase Sequence %

identity

(human vs. bovine)

Molecular mass

(kDa)

Database accession numbers

(human/bovine)

RhoA 100 21.8 NP_001655.1/NP_788818.1

Rac1 100 21.5 NP_008839/NP_776588

Cdc42 -

human isoform1

100 21.2 NP_001782/NP_001039797

57

9

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