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Histophilus somni IbpA DR2/Fic in virulence and immunoprotection at the 1
natural host alveolar epithelial barrier 2
3
4
Running Title: Histophilus somni DR2/Fic cytotoxicity and immunoprotection 5
6
Bereket Zekarias1, Seema Mattoo
2, Carolyn Worby
2, Jason Lehmann
1, Ricardo F. Rosenbusch
3, 7
Lynette B. Corbeil1,4*
8
9
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: [email protected]. 19
20
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
22
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
43
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
92
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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
113
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
172
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
290
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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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398
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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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