Across Latin America, an estimated 6 million people suffer from Chagas disease (CD), a chronic debilitating illness that has a high (~30%) mortality rate. This infection primarily afflicts poor rural communities, having a major impact on the socio-economic development of regions least able to deal with the associated economic burden, often trapping sufferers and their families in a disease-poverty cycle. The causative agent of CD is Trypanosoma cruzi, a protozoan that is spread between mammalian populations through the spread hematophagous feeding behaviour of triatomine insects. When a triatomine takes a blood meal, it defecates on the mammalian host. Infectious, non-dividing metacyclic trypomastigote parasites present in the insect’s faeces then enters the host through the bite site or mucus membranes, such as the conjunctiva. In the host, trypomastigotes invade cells near the site of entry, where they transform into intracellular amastigotes. The amastigotes divide by binary fission, differentiate into infectious, non-dividing bloodstream trypomastigotes that upon rupturing of the mammalian cell, are released into the host’s circulatory systems. The trypomastigotes can then infect cells at sites throughout the mammalian host thereby propagating the intracellular phase of the life cycle, or can be taken up by a triatomine as it feeds. In the insect’s midgut, the trypomastigotes differentiate into replicative epimastigotes that can enter the hindgut where they transform into metacyclic trypomastigotes ready for transmission to the next victim. Through the implementation of vector control strategies and improved housing, disease prevalence at endemic sites has dramatically fallen over the past 25 years such that CD has now been eliminated from Chile, Uruguay and several regions of Argentina and Brazil. However, because this disease is a zoonosis (T. cruzi can infect all mammals), it is imperative that these insect control/housing programmes are maintained as CD disease could re-emerge as a problem in areas where it had been thought of as being eradicated. Worryingly, and due to congenital and sexual transmission, blood transfusion and organ transplantation coupled with population migration and autochthonous transmissions cycles, CD has started to emerge as a global public health problem. The Centers for Disease Control and Prevention estimate that up to 300,000 people living in the USA are infected with T. cruzi while it has been proposed that up to 4.2% of Latin American born nationals now residing in Europe harbour the parasite, a value that rises to 18.0% for people born in Bolivia [1], [2].

Current treatment of CD is based on the two nitroheterocylic prodrugs benznidazole and nifurtimox with their anti-parasitic selectivities dependent on their activation mechanism. In a reaction catalysed by a NADH-dependent, FMN-containing mitochondrial type I nitroreductase (NTR) [3], a conserved nitro group linked to an imidazole (in the case of benznidazole) or furan (in the case of nifurtimox) ring undergoes two sequential 2 electron reduction events to form nitroso and then hydroxylamine forms [4], [5]. The latter derivatives are unstable and can undergo further, non-enzymatic processing to generate highly reactive end products via a series of adduct forming intermediates [4], [5], [6], [7]. Based on the mutagenic properties the two prodrugs display to NTR competent bacterial and/or trypanosomal cells, DNA appears to constitute a major target [8], [9], [10], [11], [12]. Although the precise nature of this interaction remains unclear, it has been reported that both compounds can lead to base mismatch pairing [10] with, in the case of benznidazole, this potentially arising from incorporation of oxidised nucleotides into the parasite genome [13]. Such lesions can then promote DNA damage through formation of point mutations and/or double-strand DNA breaks (DSBs) [8], [11], [14]. As humans lack orthologues of the trypanosomal type I NTR, this parasite activity has been exploited to screen distinct groups of nitroaromatic and quinone-based compounds for anti-T. cruzi properties. From this, several nitrobenzyl-based prodrugs that contain functional groups which have potential to promote DNA interstrand crosslinks (ICLs) were shown to display significant anti-parasitic effects while exhibiting low mammalian cell toxicity [15], [16], [17].

Formed when the complementary strands within the DNA double helix become covalently linked by bifunctional alkylating agents, ICLs represent a particularly dangerous lesion that can block essential cellular processes that require DNA strand separation and can lead to chromosomal breakage, rearrangements, and cell death [18], [19], [20], [21]. To maintain integrity and functionality, all cells have evolved multiple mechanisms to remove such lesions from their genomes [20], [22]. Although the precise factors involved in this process have yet to be fully elucidated different mechanisms are known to operate at various stages in the cell cycle, employing components from the “classical” DNA repair pathways (nucleotide excision repair (NER), mismatch repair (MMR), translesion synthesis (TLS) and homologous recombination (HR)) in conjunction with ICL repair specific proteins (e.g., PSO2/SNM1, FAN1) [23], [24], [25], [26], [27], [28]. In transcriptionally active mammalian cells, ICL formation often results in distortion of the DNA double helix and the stalling of an RNA polymerase complex. These events can be recognised by factors such as by the transcription coupled-nucleotide excision repair (TC-NER) helicase CSB, with these subsequently recruiting other NER enzymes including XPG and XPF-ERCC1, and SNM1A to the lesion site [29], [30], [31]. In a process known as “unhooking” and mediated by the nucleolytic activities of XPF-ERCC1 and XPG, the sugar-phosphate backbone on one of the DNA strands is cleaved at sites upstream and downstream of the ICL with nucleases, including SNM1A, subsequently degrading the released sequence up to and beyond the crosslink. The resultant gap is filled by damage tolerant TLS DNA polymerases such as Pol ζ, Pol η, Pol ι, Pol Θ or Pol κ, and the double strand DNA structure restored by DNA ligase [32], [33], [34]. Once this strand has been repaired, the ICL is completely removed from the DNA by a second round of NER incision, TLS DNA polymerase and DNA ligase activities. A variant of this mechanism can operate in non-transcriptionally activity regions of a genome but here recognition of the ICL-induced chromatin structure/DNA conformation alterations is mediated by components of the MMR and global genome-nucleotide excision repair (GG-NER) pathways [29], [35], [36], [37].

During the S-phase of the mammalian, amphibian and fish cell cycle, the so-called Fanconi Anaemia (FA) pathway predominates to remove ICLs from the nuclear genome [38], [39]. Here, the ICL-mediated collapse of DNA replication forks is recognised by the multi-subunit FA core complex that guides and activates the FA recruitment complex. Once triggered, this recruitment complex directs a range of effector proteins from the “classical” DNA repair pathways that operate to resolve the ICL. In dividing yeast cells, an abridged FA pathway is postulated to operate. Here, stalling of a replisome(s) results in the recruitment of MPH1, MUTSα and ancillary factors to the lesion site [27], [28], [40]. These function to regress the replication fork and direct endonuclease (e.g., XPG and XPF-ERCC1), exonuclease (e.g., EXO1, PSO2), TLS DNA polymerase and DNA ligase activities to remove the ICL from the genome with repair of DSBs that arise from this process occurring via the HR pathway.

Informatic and/or functional studies on Trypanosoma brucei, the causative agent of African trypanosomiasis, has shown this parasite expresses several activities that operate in the ICL repair systems of other organisms [41], [42], [43]. These function across at least two distinct networks with CSB, EXO1 and SNM1 operating to repair ICLs that block transcription complexes while the HR factors MRE11, RAD51 and BRCA2 resolve lesions that cause stalling of DNA replication forks [43]. Here, we assess the susceptibility of T. cruzi to various classes of ICL inducing agent and using a classical genetics-based approach, analyse whether DNA repair enzymes from this parasite’s TC-NER (TcCSB) and HR (TcMRE11) pathways also play a role in resolving ICLs alongside that of the ICL repair specific factor, TcSNM1. As T. cruzi and T. brucei diverged from each other in the mid-Cretaceous period, around 100 million years before present [44], and that many aspects of their biology are distinct, our comparative analysis revealed that the ICL repair networks expressed by these two trypanosomal species are similar but not identical.