Differential tissue distribution of diverse clones of Trypanosoma cruzi in infected mice

Abstract

Chagas disease, caused by the protozoan Trypanosoma cruzi, presents variable clinical course but the phenomena underlying this variability remain largely unknown. T. cruzi has a clonal population structure and infecting strains are often multiclonal. T. cruzi genetic variability could be a determinant of differential tissue tropism or distribution and consequently of the clinical forms of the disease. We tested this hypothesis by using low-stringency single specific primer polymerase chain reaction (LSSP-PCR) to type genetically the parasites in tissues of experimental infected mice. BALB/c mice were simultaneously inoculated with two different T. cruzi populations (JG strain and Col1.7G2 clone). Doubly infected animals showed clear differential tissue distribution for the two populations (chronic phase). Our results indicate a significant influence of the genetic polymorphism of infecting T. cruzi populations in the pathogenesis of chronic Chagas disease.

Introduction

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi and afflicts almost 20 million people throughout the Americas, thus representing a major public health problem. The transmission of Chagas disease by insect vector has been controlled in some countries but new cases continue to appear from congenital transmission or transfusions. The latter has become a health concern even in the United States [1], [2]. Although the disease has been known since the beginning of the century [3], little is known about its pathogenesis. The clinical course is quite variable, ranging from asymptomatic cases to severe chronic cardiovascular and/or gastrointestinal involvement [4]. The scarcity of parasites microscopically detected in chronic phase tissue lesions has led to doubts about their direct pathogenic role and together with the presence of autoantibodies, has raised the question of autoimmunity in the pathogenesis of Chagas disease [5], [6]. However, the autoimmune theory has recently been strongly refuted experimentally [7]. Moreover, with the aid of new powerful techniques such as polymerase chain reaction (PCR), a strict correlation has been observed between the presence of parasites, the occurrence of inflammatory processes and tissue lesions [8], [9], [10], thus strengthening the notion that T. cruzi directly determines the pathology of the disease.

Differential tissue tropism of T. cruzi strains has been considered as a possible pathogenic determinant for many years [11], [12], [13], [14]. This idea, however, has received little experimental support, other than clear proof of abundant T. cruzi genomic and biologic variations [15], [16], [17], [18], [19]. Many studies have been unsuccessful in correlating genetic variability of the parasite with the clinical characteristics of the disease. One possible explanation for this is that T. cruzi populations have a clonal structure and that many strains are known to be polyclonal [16], [17], [18]. Since most of the genetic profiling techniques used to characterize T. cruzi require parasite isolation and growth in laboratory animals or in vitro cultures, there is ample opportunity for clonal selection and consequently the trypanosome populations in culture can differ from those present in host blood or infected tissues. Furthermore, because of possible differential tropism of different T. cruzi strains in various tissues, the clones present in blood and available for hemoculture could be different from those actually causing tissue lesions. This scenario constitutes what we have called the ‘clonal-histotropic’ hypothesis of Chagas disease [16]. We have recently begun to use an experimental approach to test this hypothesis. The technique, low-stringency single specific primer-PCR (LSSP-PCR) generates T. cruzi genetic profiles directly from infected tissues [20].

LSSP-PCR detects DNA sequence variation and is a simple and rapid PCR-based technique that is both highly sensitive and informative [21], [22]. It consists of submitting a purified DNA fragment to multiple cycles of PCR amplification in the presence of a single oligonucleotide primer (driver) that is specific to one of the fragment extremities, under conditions of very low stringency. The driver hybridizes with high specificity to its complementary extremity and with low specificity to multiple sites within the fragment, in a sequence-dependent manner. The reaction thus yields a large number of products that can be resolved by polyacrylamide gel electrophoresis to give rise to a multiband DNA fragment ‘signature’ that reflects the underlying sequence. Changes as small as single base mutations can drastically alter the multiband pattern, producing new signatures that are diagnostic of the specific alterations. LSSP-PCR is generally applicable to detect single or multiple mutations in any gene-sized DNA fragment [22]. Recently, we were able to translate the intraspecific polymorphism in the kDNA minicircles nucleotide sequence into a specific and highly reproducible ‘kDNA signature’ which could discriminate T. cruzi populations directly from infected tissues [20].

Here we report our success in applying LSSP-PCR to type the parasites present in affected tissues from chronic experimental infected mice. Animals were inoculated with two different clonal populations of T. cruzi. Clear differential tissue distribution was indicating that parasite genetic variability is one of the major factors influencing the pathogenesis of Chagas disease.