Genetic transformation of a Corynebacterial symbiont from the Chagas disease vector Triatoma infestans

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Abstract

Insect-borne diseases have experienced a troubling resurgence in recent years. Emergence of resistance to pesticides greatly hampers control efforts. Paratransgenesis, or the genetic transformation of bacterial symbionts of disease vectors, is an alternative to traditional approaches. Previously, we developed paratransgenic lines of Rhodnius prolixus, a vector of Chagas disease in Central America. Here, we report identification of a Corynebacterial species as a symbiont of Triatoma infestans, a leading vector of Chagas disease in South America. We have modified this bacterium to produce an immunologically active single chain antibody fragment, termed rDB3. This study establishes the basis for generating paratransgenic T. infestans as a strategy for control of Chagas disease.

Introduction

Insect-borne diseases remain a leading cause of human illness throughout the world. Strategies for control of these diseases rely principally on chemical insecticides that eliminate or reduce numbers of vector insects. Chagas disease and its main vector, Triatoma infestans, are widely prevalent in the Gran Chaco, a semi-arid landscape extending over Argentina, Bolivia, Paraguay and southwestern Brazil. Several vector control programs, such as the Southern Cone Initiative for control of Chagas disease, are mostly based on the residual application of pyrethroid insecticides (Dias et al., 2002; Schofield and Dias, 1999). In the Argentine Chaco, however, symptomatic acute cases of Chagas disease are increasingly reported since 2001. Disorganized decentralization of vector control programs in the early 1980s followed by diminishing operational capacity since the 1990s, further compounded by the Argentine economic crisis in late 2001, contributed to the present scenario of persistent peridomestic infestation with recurrent domestic recolonization by T. infestans and renewed transmission to humans in the most affected regions (Gürtler et al., 2005).

Most insecticides cause environmental toxicity and many classes of these chemicals harm humans. Insects have evolved resistance to many of these agents (World Health Organization, 1992) and insecticide failure is common. Resistance of triatomine populations to a variety of insecticides has been documented in other Chagas endemic regions of the world (Picollo et al., 2005). The poor effects of pyrethroid insecticides against peridomestic T. infestans and other triatomines are mainly caused by their short-lasting residual effects in outdoor sites exposed to sunlight, high temperatures, rain and dust (Gürtler et al., 2004). The suite of several insecticide intervention trials and the background experience of Chagas vector control services in the Gran Chaco clearly demonstrate that current tactics and procedures fail to eliminate populations of T. infestans in rural areas and need to be revised.

Control of vector-borne diseases may potentially be achieved by modifying insects, using foreign genes, to reduce their ability to transmit pathogens. This can involve direct germline transformation (Coates et al., 1998, Jasinskiene et al., 1998, Olson et al., 1996), or paratransgenic manipulation (Richards, 1993, Beard et al., 1993, Conte, 1997, Beard et al., 1998, Beard et al., 2000). In the reduviid bug vector of Chagas disease, Rhodnius prolixus, we have expressed the peptide, cecropin A, via an engineered symbiotic bacterium, Rhodococcus rhodnii, at concentrations that eliminate the parasite, Trypanosoma cruzi (Durvasula et al., 1997). We have demonstrated stable expression of an active antibody fragment in R. prolixus via genetically altered R. rhodnii (Durvasula et al., 1999a, Durvasula et al., 1999b). Furthermore, we have shown under simulated field conditions that engineered bacteria can be delivered to populations of newly emerging nymphs of R. prolixus. These transformed bacteria predominate in the host insect throughout its life cycle (Durvasula et al., 1999a, Durvasula et al., 1999b).

A wide variety of bacteria—including Nocardia, Corynebacteria and Streptococci—have been isolated from the midgut of T. infestans (Figueiro et al., 1995). These organisms play an important role in insect development and survival by providing essential nutrients such as pantothenic acid to nymphs (Dasch et al., 1984). Here we report the identification of a Corynebacterial species as a symbiont of T. infestans (Argentina) strain and the genetic transformation of this bacterium to produce an immunologically active single chain antibody fragment. Our test antibody fragment, termed DB3 VH-Kappa (rDB3), is a murine antibody fragment that binds progesterone with affinity in the order of 1 × 109 l/mol (He et al., 1991). It is used as a model for expression of single chain antibodies that have activity against T. cruzi. This study establishes the basis for generating paratransgenic T. infestans—insects that carry genetically altered symbionts—as a strategy for control of Chagas disease transmission.

Section snippets

Identification of Corynebacterium

Adult T. infestans (Argentina) were kindly provided by Dr. Vaclav Hitsva. These insects were initially collected from the Gran Chaco region and had matured for generations under insectary conditions. Fecal droplets from 10 adult T. infestans were collected. Individual droplets were diluted in 100 μl of PBS and plated on BHI agar (Difco, Detroit, MI) for 24 h at 28 °C. Individual colonies of bacteria from a monoculture were identified by 16S rDNA sequence analysis. A 1.4 kb fragment was amplified

Identification of Corynebacterium

FASTA and NetBlast searches of the GenBank databases were used to find sequences similar to the 1.4 kb sequence fragment (Accession No. AF322369). No sequences with 100% identity were found. The highest sequence identities (99%) observed were with Corynebacterium sp. CNJ954 PL04 (Accession No. DQ448694), with a single gap in the sequence and with Corynebacterium sp. BBH8 (Accession No. AM183334), where three separate gaps in the sequence was observed. This sequence also showed 98.8% sequence

Discussion

This study establishes a Corynebacterial species as a symbiont of the T. infestans (Argentina) strain. This organism was isolated from an insectary colony of T. infestans as a monoculture, and is required for the maturation of the triatomine bug. It is therefore highly likely that this Corynebacterim sp. represents the intrinsic flora of T. infestans. The DNA sequence encoding the 16S ribosomal subunit of this bacterium shares 99% homology to corresponding 16S rRNA gene sequences of

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