Lynette B. Corbeil

The Fic Domain: Regulation of Cell Signaling by Adenylylation

We show that the secreted antigen, IbpA, of the respiratory pathogen Histophilus somni induces cytotoxicity in mammalian cells via its Fic domains. Fic domains are defined by a core HPFxxGNGR motif and are conserved from bacteria to humans. We demonstrate that the Fic domains of IbpA catalyze a unique reversible adenylylation event that uses ATP to add an adenosine monophosphate (AMP) moiety to a conserved tyrosine residue in the switch I region of Rho GTPases. This modification requires the conserved histidine of the Fic core motif and renders Rho GTPases inactive. We further demonstrate that the only human protein containing a Fic domain, huntingtin yeast-interacting protein E (HYPE), also adenylylates Rho GTPases in vitro. Thus, we classify Fic domain-containing proteins as a class of enzymes that mediate bacterial pathogenesis as well as a previously unrecognized eukaryotic posttranslational modification that may regulate key signaling events.

Induction of immunity against lethal Haemophilus influenzae type b infection by Escherichia coli core lipopolysaccharide.

Efforts to prevent Haemophilus influenzae type b (HIB) infections in infancy have been hampered by the low immunogenicity of capsular polysaccharide vaccines in children younger than 18 mos. In searching for alternate immunogens, we have studied the protective potential of polysaccharide-poor, lipid-rich endotoxin (LPS) core in experimental HIB infections. Because all gram-negative bacteria have similar LPS core structures, we were able to use as vaccine the J5 mutant of Escherichia coli 0111, the LPS of which consists only of core components, and thus to avoid problems in interpretation arising from vaccine contamination with non-LPS HIB immunogens. Mice were given graded inocula of HIB and developed lethal infection analogous to human HIB disease when virulence was enhanced with mucin and hemoglobin. After active immunization with heat-killed E. coli J5, 40/50 (80%) of infected mice survived, compared with 14/50 (28%) of saline-immunized controls (P less than 0.005). Passive immunization with rabbit antiserum against E. coli J5 prevented lethal HIB infection when administered 24 or 72 h before or 3 h after infection. This protection was abolished by adsorption of antiserum with purified J5 LPS, with survival reduced from 14/24 to 0/24 (P less than 0.005). Furthermore, rabbit antiserum to purified J5 LPS gave just as potent protection against death as antiserum to whole J5 cells. These studies demonstrate that immunity to core LPS confers protection against experimental murine HIB infection and provide the framework for a new approach to prevention of human disease from HIB.

Immune and inflammatory responses to reproductive tract infection with Tritrichomonas foetus in immunized and control heifers.

Histopathologic changes and local antibody responses were studied in immunized and control heifers after intravaginal challenge with 10(6) Tritrichomonas foetus. Animals were given 3 intramuscular inoculations of immunoaffinity-purified superficial antigen, TF 1.17, in 2 different adjuvant combinations (incomplete Freunds adjuvant or dextran sulfate plus IFA-8 animals each) or adjuvant alone at 3-wk intervals and were challenged with T. foetus 2 wk later. Histologically, a nonsuppurative endometritis with nodular lymphoid aggregates in the stratum spongiosum was present in 9 of 24 heifers. Twice as many control heifers as immunized had moderate to severe endometritis at 10 wk and the rate of clearance of the organism was significantly faster in immunized than in control heifers. Furthermore, time of clearance was statistically correlated with severity of endometritis at 10 wk postinfection, when necropsies were done (P < 0.02). Because 9-10 wk postinfection is thought to be the critical period for determining fetal loss associated with endometritis, this correlation with early clearance is important to protection against disease. In heifers with moderate to severe infiltration of mononuclear cells in the endometrium, lymphoid nodules and some secondary follicles were detected. In the subgroup of 12 animals from which uterine secretions were collected. IgA antibody responses to antigen were detected by 6 wk in infected animals with increases in mean responses at 8 and 10 wk, but not in uninfected animals. A rationale is presented for consideration of the lymphoid nodules as a possible inductive site for this local antibody response to T. foetus.

Experimental Haemophilus somnus Pneumonia in Calves and Immunoperoxidase Localization of Bacteria

Pneumonia was produced in nine, conventionally reared calves by intrabronchial inoculation with Haemophilus somnus. Volumes of pneumonic lung were determined stereologically, following serial slicing of lungs fixed by vascular perfusion. Twenty-four hours after inoculation, consistent findings were: neutrophilic to fibrinoid vasculitis, degeneration of alveolar macrophages, necrotizing bronchiolitis, suppurative bronchopneumonia, lobular necrosis, and dilation and thrombosis of lymphatics. Bacteria were identified histologically by an immunoperoxidase technique and were either free in alveoli or associated with degenerate alveolar macrophages. The latter suggests that macrophage degeneration may be a result of bacteria/macrophage interaction. Immune complex deposition is unlikely to be the principal mechanism for the vasculitis because bacterial antigen was not generally found in necrotic vessel walls, and two colostrum-deprived, H. somnus antibody-negative calves also had neutrophilic vasculitis 12 to 24 hours after inoculation with the lowest dose of H. somnus used in the above experiment.

Isolation and characterization of Fc receptors from Haemophilus somnus.

Receptors that bind the Fc region of bovine immunoglobulin (Ig) have been isolated from the culture supernatant of Haemophilus somnus by chromatography on a Sepharose 4B column. One receptor with a relative molecular weight of 41,000 weakly binds both bovine IgG subclasses, IgA and IgM, while three high molecular weight receptors (350,000, 270,000, and 120,000) strongly bind bovine IgG2, FgA, and IgM. All four Fc receptors are antigenically related and the 41,000 receptor appears to be a subunit of the high molecular weight receptors. In addition to bovine Ig, the purified 270,000 Fc receptor strongly binds horse IgG, rabbit IgG, pig IgG, cat IgG, dog IgG, and sheep IgG. The receptor also reacts weakly with mouse, rat, chicken, human, and guinea pig IgG and does not bind goal IgG. Fc receptors from 19 H. somnus isolates were compared. Variations in the molecular weight of the 41,000 protein were demonstrated among preputial isolates from asymptomatic carriers, but ad other Isolates appeared to have identically migrating proteins.

Haemophilus somnus Induces Apoptosis in Bovine Endothelial Cells In Vitro

ABSTRACT Haemophilus somnus causes pneumonia, reproductive failure, infectious myocarditis, thrombotic meningoencephalitis, and other diseases in cattle. Although vasculitis is commonly seen as a result of systemic H. somnus infections, the pathogenesis of vascular damage is poorly characterized. In this study, we demonstrated that H. somnus (pathogenic isolates 649, 2336, and 8025 and asymptomatic carrier isolates 127P and 129Pt) induce apoptosis of bovine endothelial cells in a time- and dose-dependent manner, as determined by Hoechst 33342 staining, terminal deoxynucleotidyl transferase-mediated dUTP-FITC nick end labeling, DNA fragmentation, and transmission electron microscopy. H. somnus induced endothelial cell apoptosis in as little as 1 h of incubation and did not require extracellular growth of the bacteria. Viable H. somnus organisms induced greater endothelial cell apoptosis than heat-killed organisms. Since viableH. somnus cells release membrane fibrils and blebs, which contain lipooligosaccharide (LOS) and immunoglobulin binding proteins, we examined culture filtrates for their ability to induce endothelial cell apoptosis. Culture filtrates induced similar levels of endothelial cell apoptosis, as did viable H. somnus organisms. Heat inactivation of H. somnus culture filtrates partially reduced the apoptotic effect on endothelial cells, which suggested the presence of both heat-labile and heat-stable factors. We found thatH. somnus LOS, which is heat stable, induced endothelial cell apoptosis in a time- and dose-dependent manner and was inhibited by the addition of polymyxin B. These data demonstrate that H. somnus and its LOS induce endothelial cell apoptosis, which may play a role in producing vasculitis in vivo.

Histophilus somni host–parasite relationships

Abstract Histophilus somni (Haemophilus somnus) is one of the key bacterial pathogens involved in the multifactorial etiology of the Bovine Respiratory Disease Complex. This Gram negative pleomorphic rod also causes bovine septicemia, thrombotic meningencephalitis, myocarditis, arthritis, abortion and infertility, as well as disease in sheep, bison and bighorn sheep. Virulence factors include lipooligosaccharide, immunoglobulin binding proteins (as a surface fibrillar network), a major outer membrane protein (MOMP), other outer membrane proteins (OMPs) and exopolysaccharide. Histamine production, biofilm formation and quorum sensing may also contribute to pathogenesis. Antibodies are very important in protection as shown in passive protection studies. The lack of long-term survival of the organism in macrophages, unlike facultative intracellular bacteria, also suggests that antibodies should be critical in protection. Of the immunoglobulin classes, IgG2 antibodies are most implicated in protection and IgE antibodies in immunopathogenesis. The immunodominant antigen recognized by IgE is the MOMP and by IgG2 is a 40 kDa OMP. Pathogenetic synergy of bovine respiratory syncytial virus (BRSV) and H. somni in calves can be attributed, in part at least, to the higher IgE anti-MOMP antibody responses in dually infected calves. Other antigens are probably involved in stimulating host defense or immunopathology as well.

Female reproductive tract immunity in bovine trichomoniasis

PROBLEM: Mechanisms of protective immunity in the female reproductive tract are poorly understood. For sexually transmitted diseases, bovine trichomoniasis is a useful model because il resembles human trichomoniasis to some extent, and antibodies play an important role in protection against these extracellular parasites. Protective efficacy was compared in animals with genital responses of predominantly immunoglobulin G (IgG) or predominantly IgA antibodies to a purified surface antigen of Tritrichomonas foetus.

Characterization of extracellular proteinases of Tritrichomonas foetus.

Proteinases released from Tritrichomonas foetus into a reducing buffer were characterized because we previously showed that they digested host proteins important in defense of the reproductive tract. These proteinases were shown to be extracellular because cell numbers did not decrease and trichomonads remained motile during their 3.5-hr incubation. Detectable proteinase activity was attributable to enzymes of the cysteine class by spectrophotometric and fluorometric automated assays for peptide substrate specificity. Proteinases from the trichomonad-conditioned buffer were partially purified by bacitracin affinity chromatography. Separation of the eluate on nondenaturing SDS-PAGE gels copolymerized with gelatin, revealed primarily low molecular weight proteinases. Bacitracin-purified T. foetus extracellular cysteine proteinase (TFECP) was separated in a denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, electroblotted, and a 31-kDa band cut out for amino acid sequencing. Four internal fragments were sequenced, 1 of which contained the highly conserved asparagine region of the cysteine proteinase active site. The combined sequences of these enzyme fragments were 66% and 55% identical to and the corresponding deduced amino acid sequences of 2 T. foetus cysteine proteinase genes (TFCP1 and TFCP2, respectively), which we previously cloned. These results indicate that this enzyme (TFECP) is a distinct cysteine proteinase. The extracellular release of TFECP from T. foetus suggests that it is a potential virulence factor in bovine trichomoniasis.

DIFFERENTIAL COMPLEMENT ACTIVATION BY BOVINE IGG2 ALLOTYPES

Immunoglobulin allotypes and complement (C) are known to be related to susceptibility to infection. Because bovine IgG2 is important in resistance to pyogenic infections and because its two allotypes, IgG2a and IgG2b, differ in sequence in the CH1, hinge, CH2, and CH3 regions, we tested the ability of these allotypes to initiate the bovine C cascade. Bovine IgG2a and IgG2b were standardized according to specific anti guinea pig red blood cell (GPRBC) ELISA activity using anti IgG2 reagents shown essentially unbiased for allotype. Complement activating activity of the allotypes was quantitated in a GPRBC lysis assay. With this system, IgG2b consistently had more than twice the activity in bovine C mediated lysis as compared with IgG2a. The fact that both EDTA and EGTA/Mg almost completely inhibited C mediated lysis of GPRBCs indicated that lysis was due to the classical pathway. Since antibody usually activates C by the classical pathway, this supports the supposition that activation was by the IgG2-GPRBC complexes. Flexibility analyses showed that IgG2b had a more rigid hinge than IgG2a, perhaps partially explaining the greater efficiency of IgG2b in C activation. Other mechanisms may include differences in glycosylation and in the amino acid at position 332. The difference in ability to activate C may mean that animals of the IgG2a allotype could be more susceptible to infection with extracellular pyogenic pathogens which are killed by C or by phagocytes after opsonization with IgG2 and C.