Impairment of infectivity and immunoprotective effect of a LYT1 null mutant of Trypanosoma cruzi

Abstract

Trypanosoma cruzi infection of host cells is a complex process in which many proteins participate but only a few of these proteins have been identified experimentally. One parasite factor likely to be involved is the protein product of LYT1, a single-copy gene cloned, sequenced, and characterized by Manning-Cela et al. (Infect. Immun. 69:3916-3923, 2001). This gene was potentially associated with infectivity, since the deletion of both LYT1 alleles in the CL Brenner strain (the wild type [WT]) resulted in a null mutant T. cruzi clone (L16) that shows an attenuated phenotype in cell culture models. The aim of this work was to characterize the infective behavior of L16 in the insect vector and murine models. The infection of adult Swiss mice with 10(3) trypomastigotes of both clones revealed a significant reduction in infective behavior of L16, as shown by direct parasitemia, spleen index, and quantitation of tissue parasite burden, suggesting the loss of virulence in the null mutant clone. Although L16 blood counts were almost undetectable, blood-based PCRs indicated the presence of latent and persistent infection during all of the study period and epimastigotes were reisolated from hemocultures until 12 months postinfection. Nevertheless, virulence was not restored in L16 by serial passages in mice, and reisolated parasites lacking the LYT1 gene and bearing the antibiotic resistance genes revealed the stability of the genetic manipulation. Histopathological studies showed a strong diminution in the muscle inflammatory response triggered by L16 compared to that triggered by the WT group, consistent with a lower tissue parasite load. A strong protection against a virulent challenge in both L16- and WT-infected mice was observed; however, the immunizing infection by the genetically modified parasite was highly attenuated.

FIG. 1.

Stability of the mutation after long-term culture. PCR to discard contamination or instability of the gene-targeted mutation directed towards the LYT1 gene. The bar represents the gene, with its initiation and termination codons (black dots), and the approximate location and orientation of primers. The reaction was performed using genomic DNA isolated from either the L16 clone (LYT1 null mutant) or the WT CL Brenner strain as the template. Three sets of primers were used. The absence of amplification products on the L16 clone demonstrated that both alleles of LYT1 were completely replaced. M, DNA size marker.

FIG. 2.

Double LYT1 allele replacement decreases in vivo infectivity of CL Brenner T. cruzi. Adult Swiss mice (2 months old) were infected with 10³ CRF of either the WT CL Brenner or the L16 clone. Parasite number and infections were monitored as described in Materials and Methods. (A) Parasitemia levels show a significant decrease in blood counts of L16 parasites (P < 0.001) during the entire time course studied. (B) Spleen index, an indirect measurement of infection severity, displayed a significant reduction (P < 0.0001). A representative experiment of five different independent repeats is shown. (C) Parasitic loads in skeletal and cardiac muscles at 28 days postinfection (d.p.i.). Data are expressed as number of parasites for 10⁶ host cells. Values are given as means; error bars indicate standard errors of the means.

FIG. 3.

Stability of the mutation after long-term infection. PCR to discard that the in vivo persistence of the infection was due to the recovery of the wild-type phenotype by redundancy or selection of revertants. The reaction was performed using genomic DNA isolated from parasites recovered by hemoculture from chronically infected mice, with the L16 clone or the WT strain as the template. Four sets of primers were used: (i) 121F-122R, to check that the isolated DNA belonged to T. cruzi, (ii) N1N2 to amplify the neomycin gene (850 bp), (iii) H1H2 to amplify the hygromycin gene (960 bp), and (iv) LYT1LYT2 to amplify the complete CDS of LYT1 (1,659 bp). The presence of amplification products for the antibiotic resistance genes as well as the absence of amplification for the LYT1 gene on the L16 clone demonstrated that the genetic replacement kept stable. M, DNA size marker.

FIG. 4.

In vitro transformation experiments. By their abilities to convert to metacyclic trypomastigotes, we determined the transition efficiencies of WT and L16 clone epimastigotes by adding 1% triatomine gut homogenate to the cultures. The L16 clone showed a significant increase in the percentage of trypomastigotes compared to that of the WT (P < 0.05). The results plotted are the means ± standard errors of the means (error bars) of two different experiments.

FIG. 5.

Levels of T. cruzi antibodies elicited in mice inoculated with either L16 clone or CL Brenner WT T. cruzi. Dispersion diagrams of antibody levels in control (untreated) animals and those inoculated with 10³ CRF of the L16 clone or the WT strain. Serum samples were taken at 6 (A) and 12 (B) months postinoculation. The results are expressed as the ratio of the absorbance of each serum sample at a 490-nm optical density (OD) to the cutoff value. Dotted lines indicate the cutoff adopted for positivity, calculated as the mean of the values determined for the controls (Cont.) plus three times the standard deviation. Each symbol represents a mouse. Neg, negative serum standard; Pos, positive serum standard. L16-inoculated mice showed significantly reduced antibody titers compared to those of mice inoculated with WT strain. (P values of L16 versus WT mice were 0.0031 [A] and 0.004 [B]).

FIG. 6.

Inflammatory infiltrates and tissue parasitism in the heart and skeletal muscles of mice chronically infected with either the L16 clone or the WT CL Brenner strain. (A) Histopathological analysis was plotted as dispersion diagrams indicating the intensities of inflammatory infiltrates graded as absent (−), slight (+), moderate (++), and severe (+++). Each dot represents a mouse. Mice inoculated with 10³ CRF were autopsied at 7 months postinfection. The L16 group showed a significantly low inflammatory response in muscle tissue (P < 0.05) and a low degree of inflammation in heart tissue compared to the levels of the WT group. (B) Parasite burden in skeletal and cardiac muscles at the chronic phase of infection. Data are expressed as the number of parasites for 10⁶ or 10³ host cells. (C and D) Microphotograph of muscle from mice infected with WT strain; note the amastigote nest inside the inflammatory infiltrates (arrow) (magnification, ×25). Values are given as means; error bars indicate standard errors of the means. (E) Muscle of L16-infected mouse, showing the absence of dense inflammatory infiltrates relative to the WT group.

FIG. 7.

Long-term protective immunization with WT or L16 clone epimastigotes against challenge with virulent T. cruzi Tulahuen. Adult Swiss mice (n = 15) were primed with 10³ CRF of either the L16 clone or the WT strain. Fourteen months later, the mice were challenged, together with naive controls (n = 5), with 10⁴ bloodstream forms of the highly virulent T. cruzi Tulahuen strain. (A) Parasitemia levels periodically measured during 60 days. Note the significant protection (P < 0.001) in L16-preinoculated mice. Values are given as means; error bars indicate standard errors of the means. (B) Spleen indexes on day 65 postchallenge. Note the significant protective effect of the genetically altered parasites (P < 0.002). A representative experiment of three independent assays is shown.

FIG. 8.

Histopathological observations in muscles of mice infected for 2 months with the virulent T. cruzi Tulahuen strain. The animals had been primed 14 months before the Tulahuen infection, with either WT, L16, or saline (see the text). Each dot represents a mouse. Note the significant degree of protection against necrosis and calcium deposits (P = 0.018) (left panel) or lymphomonocytic infiltrates (P = 0.0085) (right panel) bestowed by previous L16 infection, but not by WT infection. −, absent; +, slight; ++, moderate; +++, severe.