Attenuated Phenotype of a Recent House Finch-Associated Mycoplasma gallisepticum Isolate in Domestic Poultry.

Mycoplasma gallisepticum, known primarily as a respiratory pathogen of domestic poultry, has emerged since 1994 as a significant pathogen of the house finch (Haemorhousmexicanus) causing severe conjunctivitis and mortality. House finch-associated M. gallisepticum (HFMG) spread rapidly and increased in virulence for the finch host in the eastern United States. In the current study, we assessed virulence in domestic poultry with two temporally distant, and yet geographically consistent, HFMG isolates which differ in virulence for house finches-Virginia 1994 (VA1994), the index isolate of the epidemic, and Virginia 2013 (VA2013), a recent isolate of increased house finch virulence. Here we report a significant difference between VA1994 and VA2013 in their levels of virulence for chickens; notably, this difference correlated inversely to the difference in their levels of virulence for house finches. VA1994, while moderately virulent in house finches, displayed significant virulence in the chicken respiratory tract. VA2013, while highly virulent in the house finch, was significantly attenuated in chickens relative to VA1994, displaying less-severe pathological lesions in, and reduced bacterial recovery from, the respiratory tract. Overall, these data indicate that a recent isolate of HFMG is greatly attenuated in the chicken host relative to the index isolate, notably demonstrating a virulence phenotype in chickens inversely related to that in the finch host.

Evidence of trade-offs shaping virulence evolution in an emerging wildlife pathogen.

In the mid-1990s, the common poultry pathogen Mycoplasma gallisepticum (MG) made a successful species jump to the eastern North American house finch Haemorhous mexicanus (HM). Subsequent strain diversification allows us to directly quantify, in an experimental setting, the transmission dynamics of three sequentially emergent geographic isolates of MG, which differ in the levels of pathogen load they induce. We find significant among-strain variation in rates of transmission as well as recovery. Pathogen strains also differ in their induction of host morbidity, measured as the severity of eye lesions due to infection. Relationships between pathogen traits are also investigated, with transmission and recovery rates being significantly negatively correlated, whereas transmission and virulence, measured as average eye lesion score over the course of infection, are positively correlated. By quantifying these disease-relevant parameters and their relationships, we provide the first analysis of the trade-offs that shape the evolution of this important emerging pathogen.

Pathogen priming alters host transmission potential and predictors of transmissibility in a wild songbird species.

Pathogen reinfections occur widely, but the extent to which reinfected hosts contribute to ongoing transmission is often unknown despite its implications for host-pathogen dynamics. House finches ( Haemorhous mexicanus ) acquire partial protection from initial exposure to the bacterial pathogen Mycoplasma gallisepticum (MG), with hosts readily reinfected with homologous or heterologous strains on short timescales. However, the extent to which reinfected hosts contribute to MG transmission has not been tested. We used three pathogen priming treatments- none, intermediate (repeated low-dose priming), or high (single high-dose priming)- to test how prior pathogen priming alters the likelihood of transmission to a cagemate during index bird reinfection with a homologous or heterologous MG strain. Relative to unprimed control hosts, the highest priming level strongly reduced maximum pathogen loads and transmission success of index birds during reinfections. Reinfections with the heterologous strain, previously shown to be more virulent and transmissible than the homologous strain used, resulted in higher pathogen loads within high-primed index birds, and showed higher overall transmission success regardless of host priming treatment. This suggests that inherent differences in strain transmissibility are maintained in primed hosts, leading to the potential for ongoing transmission during reinfections. Finally, among individuals, transmission was most likely from hosts harboring higher within-host pathogen loads, while associations between disease severity and transmission probability were dependent on a given bird's priming treatment. Overall, our results indicate that reinfections can result in ongoing transmission, particularly where reinfections result from heterologous and highly transmissible strains, with key implications for virulence evolution. Importance: As Covid-19 dramatically illustrated, humans and other animals can become infected with the same pathogen multiple times. Because individuals already have defenses against pathogen their immune systems have encountered before, reinfections are typically less severe, and are thought to be less contagious, but this is rarely directly tested. We used a songbird species and two strains of its common bacterial pathogen to study how contagious hosts are when their immune systems have some degree of prior experience with a pathogen. We found that reinfected hosts are not as contagious as initially infected ones. However, the more transmissible of the two strains, which also causes more harm to its hosts, was able to multiply more readily than the other strain within reinfected hosts, and was more contagious in both reinfected and first-infected hosts. This suggests that reinfections might favor more harmful pathogen strains that are better able to overcome immune defenses.

Common garden experiment reveals pathogen isolate but no host genetic diversity effect on the dynamics of an emerging wildlife disease.

Host genetic diversity can mediate pathogen resistance within and among populations. Here we test whether the lower prevalence of Mycoplasmal conjunctivitis in native North American house finch populations results from greater resistance to the causative agent, Mycoplasma gallisepticum (MG), than introduced, recently-bottlenecked populations that lack genetic diversity. In a common garden experiment, we challenged wild-caught western (native) and eastern (introduced) North American finches with a representative eastern or western MG isolate. Although introduced finches in our study had lower neutral genetic diversity than native finches, we found no support for a population-level genetic diversity effect on host resistance. Instead we detected strong support for isolate differences: the MG isolate circulating in western house finch populations produced lower virulence, but higher pathogen loads, in both native and introduced hosts. Our results indicate that contemporary differences in host genetic diversity likely do not explain the lower conjunctivitis prevalence in native house finches, but isolate-level differences in virulence may play an important role.

Regulation of insulin-receptor mRNA levels by glucocorticoids.

We found with IM-9 human cultured lymphocytes, that the glucocorticoid dexamethasone increased insulin-receptor mRNA levels. This increase correlated in a time- and dose-dependent manner with the increase in the biosynthesis of the insulin-receptor precursor. In addition, in AR42J cultured rat pancreatic acinar cells, dexamethasone increased insulin-receptor mRNA levels. These studies suggest, therefore, that an increase in mRNA levels is an early step in the regulation of the insulin receptor by glucocorticoids in several cell types.

Deletion of residues 485-599 from the human insulin receptor abolishes antireceptor antibody binding and influences tyrosine kinase activation.

We have studied insulin and antireceptor antibody binding to mutated human insulin receptors deleted of residues 485-599 in the alpha-subunit by site-directed mutagenesis. Both normal and mutated receptors were expressed in rat HTC hepatoma cells. Cells expressing either the normal receptor or the mutated receptor retained the ability to bind insulin. In contrast to the normal receptor, however, the mutated receptor failed to interact with antireceptor alpha-subunit antibodies. The inability of the mutated receptor to interact with various antireceptor antibodies was further documented by photoaffinity labeling studies. In intact HTC cells expressing mutated receptors, basal insulin receptor tyrosine autophosphorylation was 2-fold elevated when compared to cells expressing normal receptors. In these cells, however, the response of this function to insulin was blunted. When receptors were isolated from these cells and assayed for both autophosphorylation and phosphotransferase activities toward the synthetic substrate poly(Glu, Tyr), the response to insulin was also blunted. To study the ability of the mutated receptor to transmembrane signal, insulin stimulation of S6 kinase activity was measured. In cells with mutated receptors, in concert with the insulin receptor kinase data, basal S6 kinase activity was elevated, and the response to insulin was blunted. The data suggest, therefore, that residues 485-599 in the alpha-subunit of the insulin receptor are critical for antireceptor antibody binding, but not for insulin binding. Moreover, these data suggest that residues 485-599 contain a regulatory domain for insulin regulation of receptor beta-subunit functions.

Glucocorticoid-regulated and constitutive trafficking of proteolytically processed cell surface-associated glycoproteins in wild type and variant rat hepatoma cells.

Glucocorticoids regulate the trafficking of mouse mammary tumor virus (MMTV) glycoproteins to the cell surface in the rat hepatoma cell line M1.54, but not in the immunoselected sorting variant CR4. To compare the localization of MMTV glycoproteins to another proteolytically processed glycoprotein, both wild type M1.54 cells and variant CR4 cells were transfected with a human insulin receptor (hIR) expression vector, pRSVhIR. The production of cell surface hIR was monitored in dexamethasone-treated and -untreated wild type M1.54 and variant CR4 cells by indirect immunofluorescence, direct plasma membrane immunoprecipitation, and by [125I] insulin binding. In both wild type and variant rat hepatoma cells, hIR were localized at the cell surface in the presence or in the absence of 1 microM dexamethasone. In contrast, the glucocorticoid-regulated trafficking of cell surface MMTV glycoproteins occurred only in wild type M1.54 cells. We conclude that the hIR, which undergoes posttranslational processing reactions similar to MMTV glycoproteins, does not require glucocorticoids to be transported to the plasma membrane and is representative of a subset of cell surface glycoproteins whose trafficking is constitutive in rat hepatoma cells. Thus, MMTV glycoproteins and hIR provide specific cell surface markers to characterize the glucocorticoid-regulated and constitutive sorting pathways.

Distribution of phosphodiesterase I in normal human tissues.

Phosphodiesterase I (PDE I) is an exonuclease capable of hydrolyzing a variety of phosphate ester and pyrophosphate bonds. Cell fractionation and histochemical studies in animal tissues have localized PDE I in the plasma membrane of various epithelia. This suggests a role for the enzyme in active transport. Distribution of PDE I in human tissues has not previously been studied. We have produced a polyclonal antiserum to bovine intestinal PDE I and have demonstrated crossreactivity with the human intestinal enzyme. This polyclonal antiserum was used in PAP immunocytochemistry to localize immunoreactive PDE I in a variety of human tissues. Localization was prominent in the gastrointestinal tract, including the cytoplasm of gastric mucosa parietal cells, cytoplasm of surface epithelium and isolated crypt cells in small intestine, and the colonic epithelial cytoplasm and brush border. Parotid gland acinar cells and scattered ductal cells showed positive cytoplasmic staining. Acinar and scattered pancreatic islet cells contained immunoreactive PDE I, as did Kupffer cells of the liver sinusoids. Immunoreactive PDE I was found in all vascular endothelia. The epithelium of the urinary tract showed extensive immunoreactivity. This included the distal convoluted and collecting tubules of the kidney, and ureteral and bladder urothelium. In previous histochemical studies of animal tissues, no evidence of PDE I activity was noted in male or female reproductive tract. In this study, immunoreactive PDE I was localized to human Sertoli cells and to basal epithelium of the epididymis and prostate acini. Fallopian tube epithelium of female reproductive tract also demonstrated immunoreactive PDI I, as did several cell types in term placenta. Our immunocytochemical results with human tissues differ significantly from previous histochemical studies in animal tissues, principally in the genitourinary system. This may be due in part to the different detection systems employed as well as the higher sensitivity of the immunoperoxidase technique. This underscores the importance of adjunct techniques in tissue surveys. The widespread epithelial distribution of immunoreactive PDE I detected by this polyclonal antibody implies an integral role in cell function, probably in active transport.

Tissue specificity of human phosphodiesterase. I. Blood serum.

Human blood serum was found to contain two enzymes which hydrolyze various phosphate diester and phosphonate ester bonds. The enzymes were isolated by butanol extraction, ammonium sulfate precipitation, column chromatography and gel electrophoresis. Zymograms showed that one of these enzymes is serum alkaline phosphatase and the other is a 'true' phosphodiesterase I. Serums of 25 persons showed no polymorphisms for either activity. Alkaline phosphatase hydrolyzes phenolic thymidine 5'-nucleotide esters readily, but phenylphosphonate esters very poorly. Serum phosphodiesterase I prefers phenylphosphonate esters to nucleotide diesters, and has no detectable monoesterase activity.

Tissue specificity of human phosphodiesterase. II. Intestinal mucosa.

Extracts of human intestinal mucosa were examined for their ability to hydrolyze various phosphodiester, phosphomonoester and phenylphosphonate ester linkages. Enzymes carrying out these reactions were partially purified by butanol extraction, ammonium sulfate precipitation and DEAE-cellulose chromatography, and examined for polymorphism on polyacrylamide gels. Two species of alkaline phosphatase and at least five species of PDE I were identified. Antibodies to purified bovine intestinal phosphatase and phosphodiesterase were found specific for the respective human enzymes.

Quantitation of the common components of deoxyribonucleic acids by mass spectrometry: application to the analysis of DNAs of unusual composition.

A mass spectral method for the quantitation of the percentages of deoxyadenosine, deoxyguanosine, deoxycytidine, and thymidine in intact DNAs has been devised. Standard curves for each nucleoside have been constructed which are based upon the observation that a direct correlation exists between the heights (% deflection) of diagnostic peaks from these nucleosides in a mass spectrum and the published percent composition of specific DNAs. Analyses of DNA from Clostridiumperfringens, Micrococcusluteus, Escherichiacoli, Bacillussubtilis, Pseudomonasfluorescens, Drosophilamelanogaster, salmon sperm, and bacteriophage lambda were used to determine standard curves. The validity of the method was demonstrated by comparison of the results from the mass spectral procedure with results from the chemical analyses of the DNAs from calf thymus and wheat germ. Analysis of ØX-174 DNA yielded values consistent with the published values obtained via sequence analysis and indicated that the method is applicable to both single and double-stranded DNAs. Results from T2 DNA, which contains no cytidine, exhibited artificially high values for adenosine, guanosine and thymidine with concomitant alteration in the A/T and G/C molar ratios. Such skewed results are useful in predicting the presence of modified nucleosides. The extreme sensitivity of the method has been exploited in the analysis of subnanogram quantities of restriction endonuclease fragments from DNA.

A comparative kinetic analysis of six substrates widely used for the detection and quantitation of phosphodiesterase I.

Six of the substrates currently used for the detection and quantitation of phosphodiesterase I (PDE I) were examined. Michaelis-Menten kinetic analysis of the hydrolysis of each of these by venom and bovine intestinal PDE I was performed to allow a comparison of the sensitivity of detection for each substrate. The results confirm that phenolic esters of phenylphosphonate are hydrolyzed 2- to 4-fold more rapidly than are the same esters of thymidine 5'-phosphate when both are present in saturating concentrations. The identification of the bovine intestinal enzyme as a PDE I is verified by its extensive similarity to the paradigmatical PDE I from rattlesnake venom. A new definition for PDE I activity is proposed which takes into account its ability to hydrolyze several types of substrates.

The synthesis of 4-methylumbelliferyl phenylphosphonate and its used in an improved method for the zymogram analysis of phosphodiesterase I.

The 4-methylumbelliferyl analog of p-nitrophenyl phenylphosphonate was prepared and its cleavage by different phosphohydrolases was studied. The new compound, 4-methylumbelliferyl phenylphosphonate, is hydrolyzed by purified phosphodiesterase I (EC 3.1.4.1) from snake venom or bovine intestinal mucosa. This fact has been exploited to develop a zymogram method which allows the detection of these phosphohydrolases on gels, even when the enzymes are present in small amounts in crude extracts. This method is superior to chromogenic methods evolving p-nitrophenol in that it is far more sensitive and is superior to the 4-methylumbelliferyl thymidine 5'-phosphate method in that the substrate is easier to prepare and can be isolated in large quantity.

Mutation of the high cysteine region of the human insulin receptor alpha-subunit increases insulin receptor binding affinity and transmembrane signaling.

Our previous studies indicated that amino acid residues 240-250 in the cysteine-rich region of the human insulin receptor alpha-subunit constitute a site in which insulin binds (Yip, C. C., Hsu, H., Patel, R. G., Hawley, D. M., Maddux, B. A., and Goldfine, I. D. (1988) Biochem. Biophys. Res. Commun. 157, 321-329). We have now constructed a human insulin receptor mutant in which 3 residues in this sequence were altered (Thr-Cys-Pro-Pro-Pro-Tyr-Tyr-His-Phe-Gln-Asp to Thr-Cys-Pro-Arg-Arg-Tyr-Tyr-Asp-Phe-Gln-Asp) and have expressed this mutant in rat hepatoma (HTC) cells. When compared with cells transfected with normal insulin receptors, cells transfected with mutant receptors had an increase in insulin-binding affinity and a decrease in the dissociation of bound 125I-insulin. Studies using solubilized receptors also demonstrated that mutant receptors had a higher binding affinity than normal receptors. In contrast, cells transfected with either mutant or normal receptors bound monoclonal antibodies against the receptor alpha-subunit with equal affinity. When receptor tyrosine kinase activity and alpha-aminoisobutyric acid uptake were measured, cells transfected with mutant insulin receptors were more sensitive to insulin than cells transfected with normal receptors. These findings lend further support therefore to the hypothesis that amino acid sequence 240-250 of the human insulin receptor alpha-subunit constitutes one site that interacts with insulin, and they indicate that mutations in this site can influence insulin receptor binding and transmembrane signaling.

Monoclonal antibodies mimic insulin activation of ribosomal protein S6 kinase without activation of insulin receptor tyrosine kinase. Studies in cells transfected with normal and mutant human insulin receptors.

The effects of species-specific monoclonal antibodies to the human insulin receptor on ribosomal protein S6 phosphorylation were studied in rodent cell lines transfected with human insulin receptors. First, Swiss mouse 3T3 fibroblasts expressing normal human insulin receptors (3T3/HIR cells) were studied. Three monoclonal antibodies, MA-5, MA-20, and MA-51, activated S6 kinase in these cells but had no effects in untransfected 3T3 cells. Both insulin and MA-5, the most potent antibody, activated S6 kinase in a similar time- and dose-dependent manner. To measure S6 phosphorylation in vivo, 3T3/HIR cells were preincubated with [32P]Pi and treated with insulin and MA-5. Both agents increased S6 phosphorylation, and their tryptic phosphopeptide maps were similar. MA-5 and the other monoclonal antibodies, unlike insulin, failed to stimulate insulin receptor tyrosine kinase activity either in vitro or in vivo. Moreover, unlike insulin, they failed to increase the tyrosine phosphorylation of the endogenous cytoplasmic protein, pp 185. Next, HTC rat hepatoma cells, expressing a human insulin receptor mutant that had three key tyrosine autophosphorylation sites in the beta-subunit changed to phenylalanines (HTC-IR-F3 cells), were studied. In this cell line but not in untransfected HTC cells, monoclonal antibodies activated S6 kinase without stimulating either insulin receptor autophosphorylation or the tyrosine phosphorylation of pp 185. These data indicate, therefore, that monoclonal antibodies can activate S6 kinase and then increase S6 phosphorylation. Moreover, they suggest that activation of receptor tyrosine kinase and subsequent tyrosine phosphorylation of cellular proteins may not be crucial for activation of S6 kinase by the insulin receptor.

Insulin down-regulates insulin receptor number and up-regulates insulin receptor affinity in cells expressing a tyrosine kinase-defective insulin receptor.

We examined the effect of insulin treatment on HTC cells transfected with large numbers of either normal insulin receptors (HTC-IR) or insulin receptors defective in tyrosine kinase (HTC-IR/M-1030). In both HTC-IR and HTC-IR/M-1030 cells, 20 h of insulin treatment (1 microM) at 37 degrees C resulted in a 65% decrease in the number of binding sites with a reciprocal 6-fold increase in affinity. In contrast, treatment with 10 nM insulin (20 h, 37 degrees C) also increased receptor affinity but had a smaller effect on the number of binding sites. 125I-Insulin binding to soluble receptors from HTC-IR and HTC-IR/M-1030 cells pretreated with insulin showed results similar to those obtained in intact cells. In both HTC-IR and HTC-IR/M-1030 cells, insulin enhanced insulin receptor degradation. In HTC-IR/M-1030 cells a 1-h incubation with insulin did not change receptor number and had only a small effect on receptor affinity; also there was no effect of insulin after a 20-h incubation at 15 degrees C. Inhibiting protein synthesis by pretreatment with cycloheximide (100 microM) did not block either the decrease in receptor number or the increase in receptor affinity. Both HTC-IR and HTC-IR/M-1030 cells exhibited a very slow rate of insulin and insulin receptor internalization and no differences were seen in this parameter when HTC-IR cells were compared to HTC-IR/M-1030 cells. These studies indicate, therefore, that in cells expressing kinase-defective insulin receptors, insulin down-regulates insulin receptor number via enhanced receptor degradation, and up-regulates receptor affinity. These effects were time- and temperature-dependent, but not dependent on new protein synthesis, and suggest that activation of tyrosine kinase may not be a prerequisite for certain mechanisms whereby insulin regulates its receptor.

Characterization of phosphohydrolase activity in bovine spleen extracts: identification of a bis(p-nitrophenyl)phosphate-hydrolyzing activity (phosphodiesterase IV) which also acts on adenosine triphosphate.

Several bovine spleen enzymes with acid pH optima, some of which hydrolyze bis(p-nitrophenyl)phosphate and therefore fit the definition of "phosphodiesterase IV," were partially separated by isoelectric focusing and ion-exchange techniques. The activities were characterized by zymogram analysis with the aid of p-nitrophenyl and 4-methylumbelliferyl phosphate and phosphonate substrates. A number of these enzymes meet the criteria for phosphodiesterase I or other phosphodiesterases. However, the predominant phosphodiesterase I hydrolyzes the bis(p-nitrophenyl)-and 4-methylumbelliferyl phosphates, p-nitrophenyl and 4-methylumbelliferyl phenylphosphonate, and ATP at the beta-gamma bond, but not p-nitrophenyl or 4-methylumbelliferyl 5'-thymidylate (the usual PDE I substrates). These properties, as well as the pH optimum, distinguish the activity from the previously described, alkaline pH optimum PDE I. A second phosphodiesterase hydrolyzes only the phenylphosphonates. Several other activities, less well described, are apparent on zymograms. None of the phosphodiesterases IV was also a phosphodiesterase II (no hydrolysis of 4-methylumbelliferyl 3'-thymidylate).

Localization of the insulin-binding site to the cysteine-rich region of the insulin receptor alpha-subunit.

Affinity-purified insulin receptor was photoaffinity labeled with a cleavable radioactive insulin photoprobe. Exhaustive digestion of the labeled alpha-subunit with endoproteinase Glu-C produced a major radioactive fragment of 23 kDa as a part of the putative insulin-binding domain. This fragment could contain either residues 205-316 or 518-633 of the alpha-subunit. Rat hepatoma cells and Chinese hamster ovary cells were transfected with cDNA encoding a human insulin receptor mutant with a deletion of the cysteine-rich region spanning amino acid residues 124-319. Insulin binding by these cells was not increased in spite of high numbers of the mutant insulin receptors being expressed. A panel of monoclonal antibodies which was specific for the receptor alpha-subunit and inhibited insulin binding immunoprecipitated the photolabeled 23-kDa receptor fragment but not the receptor mutant. A synthetic peptide containing residues 243-251 was specifically bound by agarose-insulin beads. We therefore suggest that the 23-kDa fragment contains residues 205-316, and that insulin binding occurs, in part, in the cysteine-rich region of the alpha-subunit.

Transmembrane signalling by insulin via an insulin receptor mutated at tyrosines 1158, 1162, and 1163.

In order to study the role of tyrosine autophosphorylation in insulin receptor signalling, we investigated a mutant human insulin receptor whereby the three major tyrosine autophosphorylation sites at positions 1158, 1162, and 1163 in the receptor beta-subunit were mutated to phenylalanines. When these mutant receptors were expressed in HTC rat hepatoma cells, there was no enhanced beta-subunit autophosphorylation and tyrosine kinase activity. In these cells there was enhanced insulin stimulation of [3H]AIB uptake and [3H]thymidine incorporation when compared to wild type HTC cells. The present study suggests therefore that the presence of the major insulin autophosphorylation sites is not a requirement for insulin stimulation of amino acid transport and mitogenesis.

Insulin receptor monoclonal antibodies that mimic insulin action without activating tyrosine kinase.

HTC rat hepatoma cells were transfected with human insulin receptor cDNA to a level of 40,000 receptors/cell. In these cells, as well as in nontransfected cells, insulin stimulated the uptake of alpha-aminoisobutyric acid. Two monoclonal antibodies directed against the human insulin receptor alpha subunit, like insulin, stimulated amino acid uptake in transfected HTC cells, but not in nontransfected HTC cells. The antibodies, in contrast to insulin, failed to stimulate insulin receptor tyrosine kinase activity, both in intact transfected cells and in cell free extracts prepared from them. These data suggest, therefore, that activation of insulin receptor tyrosine kinase may not be an obligatory step in all of the transmembrane signaling mechanisms of the insulin receptor.