The resistance to Paracoccidioides brasiliensis is controlled by a single dominant autosomal gene

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The resistance to Paracoccidioides brasiliensis is controlled by a single dominant autosomal gene ARTICLE in INFECTION AND IMMUNITY · SEPTEMBER 1987 Impact Factor: 3.73 · Source: PubMed

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Vol. 55, No. 8

INFECTION AND IMMUNITY, Aug. 1987, p. 1919-1923 0019-9567/87/081919-05 $02.00/0 Copyright © 1987. American Society for Microbiology

Resistance to Paracoccidioides brasiliensis in Mice Is Controlled by a Single Dominant Autosomal Gene VERA L. G. CALICH,* EVA BURGER, SUELY S. KASHINO, RAQUEL A. FAZIOLI, AND LUCIA M. SINGER-VERMES Departanmento de Imuinologia, Instituito de Chen(ias BiomMdicas, Universidade de Sao Paiulo, Sdo Pauilo, Brazil CP 4365 Received 30 January 1987/Accepted 8 May 1987

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 106 P. brasiliensis yeast cells, and mortality was scored daily. Analysis of susceptible and resistant parental strains and of Fl, 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 F, 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.

Genetically determined resistance or susceptibility to infectious disease has been recognized for many years and has been extensively investigated in a number of experimental models. Although in most cases it appears that there are multigenic factors which influence the susceptibility or resistance to these infectious agents (1, 13, 26, 30), in several studies host-specific loci governing resistance to infection were mapped (3, 6, 16, 28, 29). Inbred mice strains have been used in studies on the influence of the genetic pattern on the development of fungal diseases. Susceptibility differences among isogenic strains of mice were determined by using Blastomyces derinatitidis (24, 25), Cryptococcus neoformans (12, 20, 21, 29), Coccidioides iminitis (15, 16), Histoplasina capsulatam (7), and Candida albicans (9, 18) as infectious agents. Our previous studies on the genetics of resistance to infection with the fungal parasite Paracoccidioides brasiliensis showed that inbred strains of mice can be divided into four groups on the basis of the mortality data obtained after intraperitoneal infection. A/SN mice are very resistant, C3H/He mice are resistant, BALB/c, CBA, C57BL/10, and C3HeB/Fe mice are intermediate, and B10.A, B10.D2/oSn, and B10.D2/nSn mice are susceptible (5). The same study also suggested that resistance to P. brasiliensis is not H-2 linked. To determine the type of inheritance as well as the number of genes that confer resistance to paracoccidioidomycosis in the most susceptible and in the most resistant mouse strains, we tested the progeny of these mice for resistance to infection. In this work, we present data supporting the hypothesis that a single autosomal dominant gene has a major influence on resistance to paracoccidioidomyco-

tained from the Jackson Laboratory, Bar Harbor, Maine. (A/SN x B10.A)F1,, (B10.A x A/SN)Flb, (Fib X Flb)F2, (Flb x A/SN)BXR, and (Flb x B1O.A)BXS were obtained from our animal facilities. The first strain used to designate a hybrid identifies the maternal partner of a cross. Male and female mice were infected with P. briasilienisis yeast cells at 10 to 12 weeks of age. P. brasiliensis. The yeast phase of the virulent "Pb 18" isolate (14), obtained 30 years ago from a patient with paracoccidioidomycosis, was used. It has been maintained by weekly passages on Fava Netto medium (10). Yeast cells were harvested by flooding the surface of agar with sterile phosphate-buffered saline and gently scraping the surface. Cells were washed three times in phosphate-buffered saline, and concentrations were adjusted to 10 x 106 cells per ml (mortality analyses) or 2 x 106 yeast cells per ml (pathological studies) after being counted in a hemacytometric chamber. Viability was determined with the Janus Green B vital stain (2) and was always higher than 85%. Infection model. Age-matched female and male mice were inoculated intraperitoneally with 5 x 106 P. br-asiliensis yeast cells contained in 0.5 ml. This dose was chosen as an adequate inoculum to discriminate susceptible from resistant mice on the basis of our previous studies (5). Then, after infection, parental and hybrid mice were housed in our laboratory and mortality was scored daily. Classification of mice according to survival pattern. The criterion used to separate mice into susceptible or resistant was the day of death. As the mortality of the parental B10.A and A/SN mice presented Gaussian but overlapping distributions, the day postinfection which leads to equivalent and low misclassification of resistant and susceptible mice was estimated. Male mice were categorized as susceptible if they died on or before day 150 postinfection. By using the same criterion, day 195 postinfection was used to classify resistant and susceptible female mice. Segregation analysis. Our null hypothesis was that a single dominant gene controls resistance to murine paracoccidioidomycosis. According to the laws of Mendel, the F1 population obtained from resistant (A/SN) and susceptible

SiS.

MATERIALS AND METHODS Mice. On the basis of our previous work, B10.A and A/SN strains of mice were chosen as susceptible and resistant animals, respectively (5). These strains were originally ob*

Corresponding author. 1919

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INFECT. IMMUN.

CALICH ET AL.

1920

60Q

400o . z

0

3001

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nment. Since there was little difference in the patterns of survival for individual groups, results were pooled. The results obtained with male offspring are shown in Fig. 1. Equivalent results were obtained with female animals: 92% of the (B10.A x A/SN)Flb and 100% of the (A/SN x B10.A)F1a mice behaved as the resistant parental mice. Almost all

F1 segregants were shown to be resistant to P.

brasiliensis infection. z Linkage test between the resistance gene and X chromosome }in F, mice. Because male mice are more susceptible to || 4 am I,U, r infection than female mice (5), the question of X linkage was c * s ^ | 200. r investigated. (B10.A x A/SN)Flb and (A/SN x B10.A)F1a of both sexes showed an equivalent pattern of resistance to P. L4 .0 brasiliensis infection, indicating that the dominant resistance * r~~~~~~.~~~~~~" ~~~ gene is located in an autosomal chromosome (Table 1). 150~~~~~=~~~~~~i--~~~~~~~~~~~~ L Resistance of backcross progeny. (BlO.A x A/SN)Flb hyU) 0 * -p.gpi brids were backcrossed to both the susceptible and the 0. 1201 parental strains. Backcrosses of Flb mice to the I susceptible B10.A parent produced 53.2% resistant progeny 90] and 46.8% susceptible progeny. This result indicated a 1:1 r La so. distribution of resistant and susceptible mice. Backcrosses * t to the resistant parental strain produced 95.5% of progeny 00. with the resistant phenotype. These results are consistent 0 60. with resistance determined by a single gene (see Fig. 1 and Table 2). Furthermore, this locus is not linked to the albino 50 * gene. Resistance of F2 progeny. F2 mice (137 male and 144 40Q _ female) were tested for resistance after P. brasiliensis infecBXS F2 BXR Fib Fla A/SN B1OA tion. A total of 72.8% of male and 72.2% of female F2 animals 72.8 955 979 512 100 e I.- 8a2 lQ5 were classed in the resistant category. These data agree with the 75%:25% (resistant:susceptible) proportion expected ] 100 0 100 75 50 100 100 with a single dominant gene. Statistical analysis confirmed this assumption (Fig. 1 and Table 2). Comparison among MSTs of susceptible mice. The mean F:IG. 1. Mortality data for parental (A/SN and B10.A) and hybrid survival times (MSTs) of female and male F2 and backcross [(B10.A x A/SN)Flb, (A/SN x B1O.A)Fia, (Flb x Flb)F2, (Flb X x BlO.A)BXS subpopulations that behaved as suscepA/SIN)BXR, and (Flb x B10.A)BXS] male mice infected intraperi(Flb tible animals were not statistically different from the MST of tone-ally with 5 x 106 P. brasiliensis yeast cells. The results for the parental susceptible mice (BlO.A). Data are shown in mortality (using a log scale) were plotted by individual values (O Syrribol: 0, Individual animals sacrificed 230 days postinfection Table 3. whe-n they could clearly be classified as resistant. - - -, Upper limit Pathological studies. The surveys of A/SN, B1O.A, and F of s,usceptibility. Percentages of mice resistant to P. brasiliensis mice were done 6 months after P. brasiliensis inoculation. (%riesist): Pred, predicted; Obs, observed. The percentages of lesions found on various organs are shown in Table 4. It was observed that in the chronic phase (BiO.A) progenitors is expected to be 100% resistant. The F2 of infection all B1O.A mice presented an intense peritonitis with large lesions affecting most of the abdominal organs; on genieration is expected to be 25% susceptible and 75% the other hand, only a small percentage of A/SN and F1 mice resiistant. Backcrosses of the F1 progeny to the susceptible presented a few circumscribed lesions. These data support par ental mice (B1O.A) would derive 50% susceptible and the hypothesis that a dominant gene plays a major influence 509kto resistant mice. When the backcross is done by using the in resistance to paracoccidioidomycosis. resiistant parental mice (A/SN), 100% resistant progeny would be expected. Observed and expected values were compared by using the log likelihood ratio goodness-of-fit DISCUSSION test (31). studies Clinical that susceptibility to P. suggested Pathological study. Female B10.A, A/SN, and (B10.A x is several brasiliensis related to factors, including genetic were with P. brasiliensis mice infected 106 yeast A/SN)Flb background (11) and hormonal factors of the host (17). Our cells. Animals were sacrificed 180 days postinfection, and previous studies showed that inbred strains of mice have their organs were examined for the presence of gross granulomata. Statistics. The Lilliefors test was used for normality testing TABLE 1. Absence of linkage between the X chromosome and of parental mice survival times (8). Besides the log likelihood the gene that confers resistance to P. brasiliensis infection ratio test applied for segregation analysis, single-factor analSex No. resistant n No. susceptible Offspringa ysis of variance was used to compare means; P values M 25 0 25 Fla greater than 0.05 were assumed not to be significant. LiL

E

2

To 70.

I

RESULTS Resistance of F1 progeny. Over 2 years, a series of experiments was done. Controls were included in each experi-

Fib

FIa

Flb " Fia, (A/SN x

M F F

49 19 50

B10.A)Fj; Fjb, (B13.A

1 0 4 x

A/SN)F,.

48 19 46

1921

GENETIC CONTROL OF P. BRASILIENSIS INFECTION

VOL. 55, 1987

TABLE 2. Genetic analysis in F1, F2, BXR (backcross to the resistant parent), and BXS (backcross to the susceptible parent) offspring of the susceptibility to P. brasiliensis infection

No. susceptible

Male offspring"

Fla

25 49 136 66 47

Fib F2 BXR BXS

No. resistant

Observed

Expected

Observed

0 1 37 3 22

0 0 34 0 23.50

25 48 99 63 25

Expected

Gb

P

0.346

0.50 < P < 0.75

0.192

0.50 < P < 0.75

25 49

102 66 23.50

" Fla, (A/SN x B1O.A)FI; Fib, (B10.A x A/SN)FI; F,, (Fib x Fib); BXR, (Fib x A/SN)BX; BXS, (F,b x B1O.A)BX. b G, Log likelihood goodness-of-fit tests were used.

different susceptibility patterns to P. brasiliensis. The A/SN and B1O.A strains were found to be, respectively, the most resistant and the most susceptible mice to paracoccidioidomycosis (5). This fact demonstrates a genetic basis for natural resistance to P. brasiliensis. To study the type of inheritance and the number of genes determining resistance to paracoccidioidomycosis, we tested progeny of resistant (A/SN) and susceptible (B10.A) mice for resistance to intraperitoneal infection. F1 mice were found to be as resistant as the A/SN parent, indicating that resistance is a dominant trait. Among F2 mice, resistance was observed in 72.8% of the animals, near the 75% that would be expected if a single gene were governing resistance. When (B10.A x A/SN)Flb mice were backcrossed to the resistant parent, 95.5% of the progeny was found to be resistant. Confirming the expected 1:1 distribution among backcrosses to the susceptible parent, 53.2% of (Flb B10.A)BXS mice behaved as resistant animals. Although most of the presented data were obtained with male mice, large numbers of F1, F2, BXR, and BXS mice and parental A/SN and B1O.A female mice were also analyzed, confirming the unigenic control of resistance. However, since our previous work (5) showed that some strains of mice have an intermediate pattern of resistance, further studies with segregants of other inbred strains of mice are advisable to rule out the existence of other alleles influencing resistance to paracoccidioidomycosis. As the mortality data of these intermediate resistant strains presented extensive overlapping with both susceptible and resistant inbred mice, precluding segregation analysis, another discriminant characteristic must be found. A trait which might influence resistance is sex. Female mice were consistently more resistant than their male counterparts (5). In the present work, we investigated the effect of sex on resistance to paracoccidioidomycosis and found that the resistance gene was not X linked. Analyzing F1 descendants obtained from (A/SN x B1O.A) and (B10.A x X

A/SN) crossings, we verified that almost 100% of male and female F1 offspring were resistant. If the resistance gene were X chromosome linked, it would be expected that 100% of F1 male mice obtained from (B1O.A x A/SN) matings would be susceptible, since the X chromosome of these animals would come from the susceptible parent. Because we did not find any evidence of linkage between resistance and the X chromosome, an attractive hypothesis to explain the observed differences between male and female animals would be the influence of hormonal factors on the resistance mechanisms to P. brasiliensis. In fact, P. brasiliensis has receptors for steroid hormones which inhibit the myceliumyeast transformation (19) and the growth of the yeast cells (27). Furthermore, sexual differences in immune responses to various infectious agents are widely found. In experimental paracoccidioidomycosis, it was observed that female mice presented a higher delayed hypersensitivity reaction to P. brasiliensis antigens compared with male mice (23). When the MSTs of B1i.A, F2, and BXS susceptible mice were compared, no differences were detected (Table 3). The MST of resistant progeny was not compared with the MST of A/SN mice because hybrid animals were sacrificed at day 230 postinfection, when they were clearly classified as resistant animals.

Pathological studies were done after inoculation of parental and F1 mice with an infecting dose previously characterized as nonlethal to the resistant inbred strain (5). The number and distribution of granulomata lesions found in F1 mice were very similar to those observed in the A/SN strain, and both differed extensively from the susceptible parent. The percentage of F1 and A/SN mice that presented granulomata at 180 days postinfection was low, and these lesions were found mainly on the spleen and kidneys (Table 4). These results suggest that the resistance gene maintains TABLE 4. Pathology in parental inbred mice and F1 offspring in the chronic phase of P. brasiliensis infection

TABLE 3. Comparison of MSTs among susceptible inbred mice (B1O.A) and susceptible segregant F2" and BXSb Strain or MST (days) n Sex ~ (ISx SD) offspring

Organ or site

±

B1O.A

43

F2

F

142.6

43.5

40

142.6

31.2

BXS

18

152.4

32.1

B1O.A

38 37 22

110.2 104.4 116.4

31.5 23.4 20.0

F2 BXS

M

Inoculation site Intestinal mesentery Spleen Epiplon

>0.50

Abdominal muscles 0.20

<

P

<

0.50

aF2, (B1O.A x A/SN)Fib x (B1O.A x A/SN)Fib. BXS, (B1O.A x A/SN)Fib x B1O.A. ' Analysis of variance was done under the hypothesis that MSTs of mice do not differ; this hypothesis was confirmed. b

Lymphatic tract Kidney

Diaphragm Liver Lungs a

% of observed organs with lesions in mouse strain: A/SN B10.A (B13.A x A/SN)Fib (n = 9) (n = 9) (n' = 12)

16.6 91.3 91.3 66.4 16.6 83.3 91.3 83.3 58.1 24.9

0 11.1 33.3 0 0 0 11.1 0 0 0

0 0 0 11.1 0 0 22.2 0 0 0

n, Number of mice infected intraperitoneally with 106 yeast cells.

1922

CALICH ET AL.

its effects even in a chronic phase of murine paracoccidioidomycosis. Histopathological examinations of B10.A, A/ SN, and hybrid mice are already under investigation in our laboratory and will possibly elucidate the pathogenesis of experimental murine paracoccidioidomycosis. We designated the gene determining resistance to P. brasiliensis as Pbr. This locus is not H-2 linked since B10.A and A/SN strains share the same H-2 haplotype. By analyzing mortality data and the coat colors of segregant offspring, it was concluded that resistance to P. brasiliensis was not linked to the albino gene. Resistance to coccidioidomycosis, another chronic granulomatous systemic disease, was found to be controlled by a single dominant gene (16). The comparison between the strain distribution of the Cms gene and the Pbr gene allows us to conclude that these two loci are not linked. On the basis of our previous work, which showed that the congeneic pair B10.D2/oSn and B10.D2/nSn are equally susceptible to P. brasiliensis infection (4), the possibility of linkage between the Hc and Pbr genes could be excluded. Contrasting with experimental paracoccidioidomycosis, the resistance to murine cryptococcosis was shown to be controlled by the Hc locus (28). Besides this major locus, the nu (from hairless athymic mice) (12), the xid (X-linked immunodeficiency) (20), and the bg (Chediak-Higashi syndrome) (21) loci also have great influence on susceptibility to cryptococcosis. Thus, besides a major gene, other loci controlling different traits may also influence the outcome of an infectious disease. In murine paracoccidioidomycosis, there is evidence that the nu locus influences susceptibility (22), and the effect of the xid locus is under investigation in our laboratory. Preliminary studies to localize the action of the Pbr gene, showed that, although resistant and susceptible mice display an equivalent inflammatory cells influx when infected with P. brasiliensis, the macrophage spreading response was significantly decreased in B10.A mice compared with A/SN animals. Depression in humoral immune response to sheep erythrocytes was also observed in susceptible mice but not in the resistant strain (H. C. Teixeira, V. L. G. Calich, M. R. D'Impdrio Lima, and M. Russo, Abstr. XI Congr. Soc. Bras. Immunol., 1986, p. 86). Finally, although resistance to paracoccidioidomycosis is controlled by a single autosomal dominant gene, the action of this gene deserves further studies. These studies will be helpful in gaining a better understanding of host defenses operating in paracoccidioidomycosis. ACKNOWLEDGMENTS This work was supported by grants from Conselho Nacional de Pesquisas and Financiadora de Estudos e Projetos. This work was performed while S. S. Kashino and R. A. Fazioli held a Fundacdo de Amparo a Pesquisa fellowship. We are grateful for the excellent technical assistance of Dolores Vieira Rodrigues and Zilda Gomes. LITERATURE CITED 1. Anderson, G. W., Jr., and J. V. Osterman. 1980. Host defenses in experimental rickettsialpox: genetics of natural resistance to infection. Infect. Immun. 28:132-136. 2. Berliner, M. D., and M. E. Reca. 1966. Vital staining of Histoplasma capsulatum with Janus Green B. Sabouraudia 5: 26-29. 3. Bradley, D. J. 1977. Regulation of leishmania populations within the host. II. Genetic control of acute susceptibility of mice to Leishmania donovani infection. Clin. Exp. Immunol. 30:

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