Susceptible Parent Strain Research Articles

Susceptibility to tuberculosis as a complex genetic trait: analysis using recombinant congenic strains of mice.

Previous advances in the genetics of infectious diseases derived principally from identification of single genes and their isolated effects on the progression of infection. Modern genetic analysis represents a powerful means of understanding the interplay among different pathways activated in the course of infection, their hierarchy and interactions in terms of the development of an optimal protective strategy. By utilizing both whole-genome scanning of (C3HxC57BL/6)F2 and a set of the recombinant congenic strains, produced by backcrossing B10 onto a C3H background, we demonstrated that susceptibility to tuberculosis is a multigenic trait. We have identified two distinct groups of susceptible mice: one that dies within four to six weeks of infection (supersusceptible) and another that dies within seven to 10 weeks (comparable to the susceptible parental strain). Our preliminary genetic analysis suggests that the susceptibilty of those groups is controlled by different genetic factors. Supersusceptible mice exhibit dramatic lung pathology, not observed in either parental strain, and their survival after infection with virulent Mycobacterium tuberculosis is comparable to that of mice rendered immunodeficient by disruption of essential immune genes. Further genetic and functional analyses of these strains offer possibilities for understanding the control of transmission, preferential growth of the pathogen in the lung, and mechanisms of local and systemic protective immune responses.

Inheritance of resistance to pyrethroid insecticides in Tribolium castaneum (Herbst)

A strain of Tribolium castaneum, highly resistant to pyrethroids, was isolated (single-pair method) from a pyrethroid-resistant field strain and made isogenic with a susceptible laboratory strain. Insecticide assays were used to determine the mode of inheritance and linkage of pyrethroid resistance. Resistance to deltamethrin could not be measured directly but at a 2% mortality response was about 1600-fold. The responses of SR and RS hybrids to deltamethrin were intermediate between the resistant and susceptible parent strains; there was no significant difference between the SR and RS hybrids ( P>0.05). Chi-square analysis of the observed responses of the F 1–backcross and F 2 progenies rejected the null hypothesis that a single gene was responsible for resistance. Successive backcrosses with selection indicated the involvement of more than one gene. Test crosses with visible mutant markers suggested that resistance was associated with linkage groups VIII and IX. Assays with deltamethrin applied to grain indicated that this resistance will be rapidly selected in the field resulting in control failures.

Loss of Endogenous Mouse Mammary Tumor Virus Superantigen Increases Tumor Resistance

From a cross between a tumor-susceptible mouse strain (DBA/2; D) and a tumor-resistant MHC-identical strain (B10.D2; D2) new recombinant inbred mouse strains were established over many generations of inbreeding and tumor resistance selection. Since resistance to the highly metastatic DBA/2 lymphoma variant ESb had an immunologic basis, and the two parental strains differed in endogenous viral superantigens (vSAGs), DNA of three D2 x D recombinant inbred mouse lines was typed for endogenous mouse mammary tumor viruses using mouse mammary tumor virus long terminal repeat- and env gene-specific probes. The resistant D2 x D mice were very similar to the susceptible parental strain D in their Mtv Southern blots, except for the lack of a single band corresponding to Mtv-7, the provirus coding for the strong DBA/2 superantigen Mls-1a. A backcross analysis revealed that Mtv-7-negative F2 mice were significantly more resistant than Mtv-7-positive F2 mice. When Mtv-7 was reintroduced into the resistant lines by crossing them with either CBA/J or BALB/D2.Mls-1a, the mice became again more tumor susceptible. Finally, we demonstrate the ability to transfer immunoresistance and graft-vs-leukemia reactivity from tumor-resistant to tumor-susceptible mice.

In-vitro isolation and antifungal susceptibility of amphotericin B-resistant mutants of Aspergillus fumigatus.

Aspergillus fumigatus mutants resistant to amphotericin B were selected in the laboratory following UV irradiation. A total of 18 colonies (frequency 1.8 x 10(-7)) that grew in the presence of amphotericin B (8 mg/L and 16 mg/L) on peptone yeast extract glucose agar were tested for their susceptibility to amphotericin B, nystatin, azoles, and the echinocandin L-743872. Ten of the 18 isolates showed an eight-fold rise in amphotericin B MIC (4 mg/L) compared with the susceptible parent whereas the remaining isolates showed a 16 to 32-fold rise in amphotericin B MIC (8-16 mg/L). Subculturing of three representatives from each of the groups that had MIC values of 4 mg/L and 8-16 mg/L for six cycles revealed that the resistance trait was stably expressed. All amphotericin B-resistant isolates showed a significant level of cross-resistance to nystatin but not to azoles and L-743872. Kill-curve studies with amphotericin B revealed that the killing of the resistant isolates was significantly less than that of the susceptible parent strain. These results show that amphotericin B-resistant mutants of A. fumigatus can be isolated in the laboratory following a single-step UV-induced mutagenesis and suggest that similar mechanisms could operate in nature for the emergence of resistance in clinical isolates.

A new specific gene for wasp cellular immune resistance in Drosophila

Larvae of Drosophila melanogaster produce a haemocytic reaction against eggs of the parasitoid, Asobara tabida, which leads to the formation of a multicellular capsule surrounding the foreign object. The same phenomenon was observed with the parasitoid, Leptopilina boulardi. Concerning the resistance of D. melanogaster to L. boulardi, a single major segregating locus with the resistant allele dominant to the susceptible one was found. The host strain susceptible to this parasitoid species was found to be highly immune reactive against the eggs of A. tabida. The inheritance of the capacity to encapsulate A. tabida was analysed by comparing reciprocal crosses made using inbred resistant and susceptible parental strains. We conclude that differences in the encapsulation capacity are inherited autosomally, with the reactive phenotype showing complete dominance over the non-reactive one. These data suggest the existence of two independent gene systems, each being concerned with the recognition of one species only.

A new specific gene for wasp cellular immune resistance in Drosophila.

Larvae of Drosophila melanogaster produce a haemocytic reaction against eggs of the parasitoid, Asobara tabida, which leads to the formation of a multicellular capsule surrounding the foreign object. The same phenomenon was observed with the parasitoid, Leptopilina boulardi. Concerning the resistance of D. melanogaster to L. boulardi, a single major segregating locus with the resistant allele dominant to the susceptible one was found. The host strain susceptible to this parasitoid species was found to be highly immune reactive against the eggs of A. tabida. The inheritance of the capacity to encapsulate A. tabida was analysed by comparing reciprocal crosses made using inbred resistant and susceptible parental strains. We conclude that differences in the encapsulation capacity are inherited autosomally, with the reactive phenotype showing complete dominance over the non-reactive one. These data suggest the existence of two independent gene systems, each being concerned with the recognition of one species only.

In vivo alterations in cytokine production following interleukin-12 (IL-12) and anti-IL-4 antibody treatment of CB6F1 mice with chronic cutaneous leishmaniasis.

CB6F1 mice exhibit intermediate resistance to infection with Leishmania major compared to their highly susceptible (BALB/c) and resistant (C57BL/6) parental strains. Unlike the C57BL/6 and BALB/c strains, which rapidly develop dominant Th1- or Th2-type responses, respectively, after infection, CB6F1 mice develop responses in which both Th1- and Th2-type cytokines are elevated through at least the first month of infection before Th1 responses become dominant as cutaneous lesions gradually heal. We have examined the effects of interleukin-12 (IL-12) and/or anti-IL-4 antibody treatment on cytokine production and the course of disease in CB6F1 mice with chronic L. major infections. When administered at 1 month of infection, IL-12 treatment led to a rapid decrease in mRNA levels for IL-4 within parasitized lesions and a moderate increase in gamma interferon (IFN-gamma) transcript levels in lymph nodes draining the site of infection. When IL-12 and anti-IL-4 antibody were administered together, they induced a marked decrease in IL-4 and transforming growth factor beta mRNA expression within lesions and a more dramatic increase in lymph node IFN-gamma transcript levels within 4 days after treatment. In comparison, similar treatment of infected BALB/c mice led to only a moderate increase in IFN-gamma transcripts but no decrease in mRNA levels for Th2-type cytokines. Treatment of CB6F1 mice with either IL-12 or anti-IL-4 antibody had no significant effect on the subsequent course of infection, whereas combined IL-12 plus anti-IL-4 treatment resulted in a decrease in lesion size and parasite numbers and a shift towards a Th1-dominant response. These results suggest that the immediate effects of cytokine or anti-cytokine therapy may be predictive of the long-term effects on the course of infection and that down-regulation of Th-2 type cytokines may be critical to the development of a Th1-dominant response.

An electron microscopic study of clinical and laboratory-derived strains of teicoplanin-resistant Staphylococcus haemolyticus.

Staphylococcal resistance to glycopeptides (which involves more teicoplanin than vancomycin) is uncommon and largely confined to Staphylococcus haemolyticus, an emerging nosocomial pathogen with a tendency to develop antibiotic resistance. In this study, six S. haemolyticus strains, including two isogenic pairs of teicoplanin-susceptible/-resistant strains and two resistant clinical isolates, were used in a morphologic and morphometric electron microscope investigation. Cells from both clinical and laboratory-derived teicoplanin-resistant strains exhibited abnormally roughened, irregular outlines when observed by transmission electron microscopy. However, no significant differences in cell wall thickness resulted from morphometric analysis when the susceptible/resistant cells of the two isogenic pairs were compared. By scanning electron microscopy, an abnormally roughened, blistered surface was associated with teicoplanin-resistant cocci. A certain variability was noted between strains, not clearly related to the resistance level. In freeze-fracture investigations, a higher number per square micrometer of intramembrane particles, more significant in the E than in the P membrane fracture face, was observed in the laboratory-derived resistant clones as compared to susceptible parent strains. Further studies are needed to understand the cause-effect relation between these ultrastructural alterations and staphylococcal resistance to teicoplanin (but not to vancomycin).

Effects of penicillin-binding protein 4 overproduction in Staphylococcus aureus.

The pbp4 gene of a Staphylococcus aureus strain selected stepwise in vitro for growth on increasing concentrations of penicillin and of its susceptible parent strain showed overall identity except in the promoter region. In the mutant a deletion upstream of the pbp4 structural gene removed 90 nucleotides (nt) that were framed by a 12 nt inverted repeat. This deletion occurred in step 4 of the in vitro selection procedure and was paralleled by a significant increase in the penicillin-binding protein 4 (PBP4) production. The in vitro step selected mutant showed a remarkable increase in the cross-linking of the peptidoglycan compared to its parent. This was linked to morphological changes in the appearance of the cells, which were surrounded by a very thick and fuzzy cell wall.

Activity of carbapenem BMS-181139 against Pseudomonas aeruginosa is not dependent on porin protein D2.

The broad antipseudomonal spectrum of the carbapenem BMS-181139 includes clinical strains and laboratory mutants of Pseudomonas aeruginosa that are resistant to imipenem. Unlike other known carbapenems (meropenem, panipenem, biapenem, and BO-2727), which have reduced activity against imipenem-resistant strains of P. aeruginosa, BMS-181139 was equally active against imipenem-susceptible (D2-sufficient) and imipenem-resistant (D2-deficient) strains. Conversely, imipenem and meropenem activities were the same against the susceptible parental strains and their BMS-181139-resistant mutants. Whereas basic amino acids antagonized the antipseudomonal activities of imipenem and meropenem, they had no effect on the activity of BMS-181139. These results suggest that the uptake of BMS-181139 into pseudomonal cells occurs by a non-D2 pathway. Compared with imipenem and meropenem, BMS-181139 may have a slightly higher affinity for penicillin-binding protein 2 (PBP-2) of P. aeruginosa. The rates of resistance development to imipenem, meropenem, and BMS-181139 in P. aeruginosa strains were similar; resistance occurred at frequencies of approximately 10(-7) to 10(-8). Resistance to BMS-181139 in P. aeruginosa is presumed to be caused by its diminished permeability since no change in their penicillin-binding protein affinities or beta-lactamase levels could be detected. In summary, BMS-181139 is a new carbapenem which differs from other known carbapenems in its lack of cross-resistance with imipenem. This difference could be explained by the permeation of BMS-181139 through a non-D2 channel, compared to the preferential uptake of other carbapenems by the D2 porin.

Altered Protoxin Activation by Midgut Enzymes from a Bacillus thuringiensis Resistant Strain of Plodia interpunctella

Processing of Bacillus thuringiensis protoxins to toxins by midgut proteinases from a strain of the Indianmeal moth, Plodia interpunctella (Hubner), resistant to B. thuringiensis subspecies entomocidus (HD-198) was slower than that by midgut proteinases from the susceptible parent strain or a strain resistant to B. thuringiensis subspecies kurstaki (HD-1, Dipel). Midgut extracts from entomocidus-resistant insects exhibited five-fold lower activity toward the synthetic substrate α-N-benzoyl-DL-arginine ρ-nitroanilide than extracts from susceptible or kurstaki-resistant insects. Midgut enzymes from susceptible or kurstaki-resistant insects converted the 133 kDa CryIA(c) protoxin to 61-63 kDa proteins, while incubations with entomocidus-resistant enzymes resulted in predominantly products of intermediate size, even with increased amounts of midgut extract. The 61-63 kDa proteins were only produced by entomocidus-resistant midgut extracts after long term incubations with the protoxin. The data suggest that altered protoxin activation by midgut proteinases is involved in some types of insect resistance to B. thuringiensis.

Genetic determinism of the cellular immune reaction in Drosophila melanogaster.

Larvae of Drosophila melanogaster produce a haemocytic reaction against eggs of the parasitoid Leptopilina boulardi, which leads to the formation of a multicellular capsule surrounding the foreign object. Melanization resulting from the conversion of phenol to o-quinones frequently accompanies the cellular reaction. Although various cytological and biochemical aspects of this reaction have been investigated, very little is known about genetic determinism of the insect immune response. The heredity of the capacity to encapsulate was analysed by comparing 16 reciprocal crosses made using inbred resistant and susceptible parental strains. We conclude that differences in the encapsulation capacity of D. melanogaster are inherited autosomally, with the reactive phenotype showing complete dominance over the non-reactive one. There were neither sex-linked nor maternal effects. The results of all crosses suggest a single major segregating locus with two alleles and complete dominance of the resistant allele, with cytoplasmic factors and minor modifying genes acting on the major locus.

Five independent combinations of mutations can result in low-affinity penicillin-binding protein 2x of Streptococcus pneumoniae

Penicillin-binding protein 2x (PBP 2x) of Streptococcus pneumoniae is one of the high-molecular-weight PBPs involved in the development of intrinsic beta-lactam resistance. Point mutations in the PBP 2x genes (pbpX) have now been characterized in five independent spontaneous laboratory mutants in order to identify protein regions which are important for interaction with beta-lactam antibiotics. All mutant genes contained two to four mutations resulting in amino acid substitutions within the penicillin-binding domain of PBP 2x, and none of the mutants carried an identical set of mutations. For one particular mutant, C606, carrying four mutations in pbpX, the mutations at positions 601 and 597 conferred first- and second-level resistance when introduced into the susceptible parent strain S. pneumoniae R6. However, the other two mutations, at amino acid positions 289 and 422, which were originally selected at the fifth and sixth isolation steps, did not contribute at all to resistance in similar experiments. This suggests that they are phenotypically expressed only in combination with mutations in other genes. Three PBP 2x regions were mutated in from two to all four mutants carrying a low-affinity PBP 2x. However, in a fifth mutant containing a PBP 2x with apparent zero affinity for beta-lactams, the three mutations in pbpX mapped at entirely different positions. This demonstrates that different mutational pathways exist for remodeling this PBP during resistance development.

Pristane Induced Arthritis. II. Genetic Regulation in Fl Hybrid Mice and Cellular Immune Abnormalities Following Pristane Injection

Pristane-induced arthritis was investigated in DBA/1, DBA/2, and BALB/c mice, and F1 hybrid mice generated from inter-crosses between these strains. The incidence of disease in F1 hybrid mice was significantly lower than the susceptible parental strains (DBA/1 and BALB/c), and resistance to arthritis was observed in both DBA/2 mice and the (DBA/2 x BALB/c) F1 hybrid mice. Several cellular immune abnormalities were observed in pristane-injected DBA/1 mice. Con A mitogen responses were depressed following pristane injection, and a functional suppressor cell population was detected. Delayed type hypersensitivity responses to type II collagen were observed in pristane injected mice. The intraperitoneal injection of pristane appears to alter immune regulation and induce autoimmune responses to connective tissue components.

Genetic influences upon eosinophilia and resistance in mice infected withMesocestoides corti

The genetic influences upon host variation in eosinophilia and resistance to helminth infection, and the relationship between these parameters, was investigated in 9 inbred and 3 hybrid strains of mice infected with Mesocestoides corti. Blood, bone marrow, spleen and peritoneal fluid eosinophilia were far higher in SJL mice than in any other inbred strain. SWR, NIH, C3H and BALB/c mice were high responders to M. corti whereas CBA and 3 congenic strains sharing the B10 background (C57BL/10, B10.S, B10.G) were low responders. Some of the genes for high eosinophil responsiveness appeared to be dominant, as F1 hybrids from high and low response parental strains were intermediate to high in response to infection. SJL and NIH strains were highly susceptible to infection with M. corti, larval burdens at 21 days after infection with 100 tetrathyridia being considerably higher (greater than 1000) than all other strains. BALB/c (congruent to 700 larvae) were designated susceptible, SWR (greater than 400 larvae) were resistant and the B10 congenics (less than 400 larvae) were highly resistant. Genes influencing resistance also appeared to be dominant, as F1 hybrids between resistant and susceptible parental strains were intermediate to resistant on infection. The overall response patterns indicate a direct correlation between susceptibility to infection and high eosinophil responsiveness, but this relationship is not consistent in all strains.

Emergence of Teicoplanin Resistance During Therapy of Staphylococcus aureus Endocarditis

Serial isolates of Staphylococcus aureus showing two- to eightfold increases in teicoplanin minimum inhibitory concentrations (MICs) and twofold or less increases in MICs of other glycopeptides were recovered from the blood of a patient with endocarditis in whom drug therapy was unsuccessful. Comparable resistance emerged during teicoplanin treatment of rabbits with endocarditis caused by the original susceptible parent strain. For the parent strain, spontaneous resistance to teicoplanin at concentrations of 2-10 times the MIC was detected in vitro at frequencies of 10(-7) to 10(-9). Similar results were found for isolates of S. aureus from other geographic locations. Resistance was constitutive and not plasmid mediated, and its acquisition was not associated with changes in cytoplasmic membrane proteins. Teicoplanin was less effective than vancomycin at inhibiting peptidoglycan synthesis in resistant strains, suggesting that there is differential interference with the access of teicoplanin to or interaction with its target(s). Alternatively, teicoplanin and vancomycin may differ in some detail(s) of their mechanism of action against S. aureus.

Cmv-1, a genetic locus that controls murine cytomegalovirus replication in the spleen.

The genetic basis of the control of acute splenic MCMV infection was studied after intraperitoneal inoculation of the virus. Classical Mendelian analyses using C57BL/6 (resistant) and BALB/c (susceptible) parental strains disclosed an autosomal dominant non-H-2 gene that regulates splenic virus replication. The probable location of this gene, to which we have assigned the symbol Cmv-1, is on chromosome 6 as defined by the strain distribution pattern of splenic MCMV replication in CXB recombinant inbred mice. Although there is a similar hierarchy of resistance to MCMV and HSV-1 with respect to the C57BL and BALB genetic backgrounds, the strain distribution pattern of HSV-1 replication in recombinant inbred mice suggests that Cmv-1 is not involved in restricting the spread of this virus. This is the first clear identification of a non-H-2 gene regulating the magnitude of MCMV infection. Elucidation of the function of this gene may be a fundamental step towards understanding the control of systemic CMV infection.

Correlation of azinphosmethyl resistance with detoxication enzyme activity in the light brown apple moth Epiphyas postvittana (Lepidoptera: Tortricidae)

Bioassay using diet incorporation of azinphosmethyl showed six strains of light brown apple moth to have varying levels of resistance to azinphosmethyl (3- to 15-fold) compared with three susceptible strains. Two of these strains were products of a selection and backcrossing program providing resistant insects that had a common genome with that of one of the susceptible strains. Individual male moths from each strain were tested for (i) glutathione S-transferase and (ii) non-specific esterase activity. Larvae were assayed for (iii) mixed function oxidase activity. Each enzyme group was assayed with two substrates. Significant positive regressions between LC 50 and enzyme activity were observed for all three groups: (i) r 2 = 0.84 for 3,4-dichloronitrobenzene conjugation, (ii) 0.64 for α-naphthyl acetate hydrolysis, 0.54 for α-naphthyl butyrate hydrolysis, and (iii) 0.88 for p-nitroanisole oxidation and 0.86 for aniline oxidation. No correlation with the LC 50 was found for 1-chloro-2,4-dinitrobenzene conjugation ( r 2 = 0.001). Activities of all three groups were significantly higher in the two backcross resistant strains compared to the susceptible parent strain ( P < 0.05). The gene(s) for high enzyme activity had thus been successfully transferred with the factor for resistance. The results suggest a close relationship between detoxication enzyme activity and azinphosmethyl resistance.

Characterization of a halothane-resistant strain of measles virus.

A strain of measles virus (MVr) whose replication demonstrated increased resistance to halothane (2-bromo-2-chloro-1,1,1-trifluoroethane) exposure compared with the susceptible parental strain (MVs) is described. After exposure to a 1.2% halothane concentration, substantial amounts of the measles virus H protein were detected in MVr-infected Vero cell lysates by immunoprecipitation and polyacrylamide gel electrophoresis or by quantitative immunofluorescence staining. The protein was barely detectable in identically treated MVs-infected lysates, however. The recovery of all other measles virus proteins studied was the same in MVr- and MVs-infected cells at this anesthetic concentration. Thus, the altered expression of a single gene product appears to be responsible for the observed halothane resistance.

Resistance to Paracoccidioides brasiliensis in mice is controlled by a single dominant autosomal gene

In a previous report it was shown that there are resistant, susceptible, and intermediate strains of mice to intraperitoneal Paracoccidioides brasiliensis infection. In the present work, we investigated the type of inheritance and the number of genes that determine resistance to paracoccidioidomycosis. Parental and hybrid mice were inoculated intraperitoneally with 5 X 10(6) P. brasiliensis yeast cells, and mortality was scored daily. Analysis of susceptible and resistant parental strains and of F1, F2, and backcross mice showed that the resistance to P. brasiliensis seems to be controlled genetically by a single dominant gene, which we designated the Pbr locus. The mean survival times of susceptible F2 and backcross hybrids were very similar to that of the susceptible parent. Examination of the pathological changes observed in parental and F1 mice, 6 months after infection, showed that F1 offspring presented a similar number and distribution of lesions to those of the resistant strains. The Pbr gene is not linked to H-2, Hc, and albino genes. Furthermore, resistance to paracoccidioidomycosis is controlled by an autosomal gene.