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Exploring the Evolutionary Origin and Biological Role of the Trypanosoma cruzi Ecotin-Like Molecule in Chagas’ Disease
Written By
Carla Moreira Santana, Max Mario Fuhlendorf, Felipe Baena Garcia, Edmar Silva Santos, Ângela Maria Lourenço, Cristiane Castro Faccini, Abílio Augusto Fragata Filho, Aline Diniz Cabral and Márcia Aparecida Sperança
Submitted: 10 October 2022Reviewed: 09 January 2023Published: 24 January 2023
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Enzymes called proteases play important roles in the physiology of all living organisms and in the interaction of a parasite/symbiont with its host. Different types of peptidases act on specific substrates and are regulated by specific inhibitors. Ecotins, described firstly in Eschericchia coli, are inhibitors of serine peptidases (ISP) from S1A family including trypsin, chymotrypsin, neutrophil elastase, and cathepsin G. Ecotin-like inhibitors are present in parasites from Trypanosomatidae family, including Trypanosoma cruzi, the causative agent of Chagas’ disease. This chapter explores the evolutive origin of the T. cruzi TcISP2 and its possible interactions with proteins of the human immune system and in Chagas’ disease. The phylogenetic relationship of TcISP2 with trypanosomatids ISPs, comparative loci analysis among trypanosomatids, and the occurrence of bacteria endosymbionts in the group strongly suggest horizontal transfer as the main origin mechanism for trypanosomatids ISPs, followed by duplication events and losses that could explain its current genomic pattern. The relationship of TcISP2 with the vertebrate host immune system can be inferred by its antigenicity in Chaga’s disease murine model, presenting high antibody titer after 60 days post-infection, which could indicate the inhibition of TcISP2 activity associated with chronic phase of the Chaga’s disease.
Center for Natural and Human Sciences, Federal University of ABC, Brazil
Max Mario Fuhlendorf
Center for Natural and Human Sciences, Federal University of ABC, Brazil
Felipe Baena Garcia
Center for Natural and Human Sciences, Federal University of ABC, Brazil
Edmar Silva Santos
Center for Natural and Human Sciences, Federal University of ABC, Brazil
Ângela Maria Lourenço
Outpatient Clinic for Chagas Disease, Dante Pazzanese Cardiology Institute, Brazil
Cristiane Castro Faccini
Outpatient Clinic for Chagas Disease, Dante Pazzanese Cardiology Institute, Brazil
Abílio Augusto Fragata Filho
Outpatient Clinic for Chagas Disease, Dante Pazzanese Cardiology Institute, Brazil
Aline Diniz Cabral
Department of Parasitology, Federal University of Uberlandia, Brazil
Márcia Aparecida Sperança*
Center for Natural and Human Sciences, Federal University of ABC, Brazil
*Address all correspondence to: marcia.speranca@ufabc.edu.br
1. Introduction
Chagas disease, despite the success of public policies for vector control, still has a worrying annual incidence in Brazil, especially in the precarious and growing frontiers of the legal Amazon [1]. The etiologic agent of Chagas’ disease is the protozoan Trypanosoma cruzi, transmitted through the bites of insects of the Triatominae subfamily, in blood transfusions, or orally through the ingestion of contaminated food with the waste of vector insects [2]. The incidence of the disease through classical transmission by triatomines cohabiting human residences has been significantly reduced as a result of the effectiveness of public vector control and housing improvement programs [3]. However, there is a growing number of cases in the states of the Legal Amazon, where there are wild triatomines that, as they do not invade human dwellings, cannot be easily controlled by traditional containment measures [4]. The frequent result is oral contamination through ingestion of products contaminated by vectors infected with Trypanosoma cruzi, such as açaí pulp [5], in addition to the expansion of vector transmission by wild triatomines, whose incidence only should grow as deforestation continues in the region [6]: between 2000 and 2016, more than 2000 confirmed acute cases in Belém in the State of Pará, Brazil, Amazon region [7].
T. cruzi belongs to the order Trypanosomatida, which also harbors the genus Leishmania, another widely distributed human parasite that causes leishmaniasis, also, an important neglected disease. Genetic analysis of members of this order revealed the presence of genes that encode serine peptidase inhibitors (ISPs), in the genera Leishmania and Trypanosoma. The ISPs observed in these trypanosomatids are orthologs of ecotin, an inhibitor found only in gram-negative bacteria [8], suggesting a possible lateral genetic transfer at some point in the evolution of these taxa. This fact, quite interesting in itself, becomes more intriguing when we consider that the sequencing did not reveal the presence, in the genome of the studied parasites, of serine peptidases of the types that ecotin inhibits, as it indicates that trypanosomatid ISPs have exogenous targets [9].
In bacteria, especially those that invade arthropod and vertebrate tissue, a growing variety of studies indicates that ecotin is a key factor in defense against the immunological barriers of infected organisms. Recent evidence indicates that ISPs synthesized by Leishmania major, which are also ecotin orthologs, have an exogenous function, facilitating the parasite’s survival against the host immune system. Drawing a parallel between studies with ecotin-bearing bacteria and ISPs of L. major —a parasite that belongs to a sister group of T. cruzi—it is clear the fundamental role that these serine peptidase inhibitor proteins play in modulating the immune response of these parasite hosts. Next, we compared the position of ecotin in various members of the Kinetoplastida group to reinforce the hypothesis of its possible common origin from horizontal transfer. Besides, the interaction of ISP2 from T. cruzi (TcISP2) with the vertebrate host immune system will be demonstrated by its antigenicity property in the murine model of Chagas’ disease.
1.1 General taxonomic aspects and life cycle of Trypanosoma cruzi
Kinetoplastida is a class of unicellular eukaryotes characterized by the presence of the kinetoplast, a feature formed by circular concatenated DNA molecules (kDNA) inside a solitary mitochondrion [10]. Trypanosomatida is an order of kinetoplastids composed of obligatory parasites of invertebrates, plants, and all classes of vertebrates [11, 12]. This order includes genera that are exclusive arthropod parasites, such as Crithidia [13, 14] and Herpetomonas [11], and also genera with heteroxenous life cycles (involving more than one host), such as Leishmania and Trypanosoma. These two genera are of great medical importance as they contain species that are etiological agents for serious human diseases, such as sleeping sickness, Chagas’ disease, and visceral and cutaneous leishmaniasis. Some Trypanosomatida genera have obligate endosymbiont bacteria, which have been used as model organisms in studies on unicellular symbiosis [15, 16].
Species in the Trypanosoma genus are heteroxenous and usually have complex life cycles [17]. In trypanosomatids belonging to the Stercoraria section, trypanosomes are transmitted to vertebrate hosts by hemipteran insects in the Reduviidae family [18], such as Trypanosoma cruzi, the etiological agent of Chagas’ disease. Motile metacyclic trypomastigote infective form of T. cruzi penetrates the skin or skin lesion of the vertebrate host after being expelled with the feces of the insect. Once inside the vertebrate host, the infective metacyclic trypomastigotes transform into bloodstream trypomastigotes, which invade cells of various tissues and, differentiate into amastigotes, static forms that multiply inside the cells by binary fission. After a number of division cycles, T. cruzi amastigotes transform into bloodstream trypomastigotes and are released into the circulatory system, infecting other cells in the body. When a triatomine insect vector feeds on an infected vertebrate with the motile trypomastigotes present in the blood, the ingested parasite differentiates into epimastigote forms that multiply by binary fission and migrate to the posterior intestine of the insect where it transforms to vertebrate infective metacyclic trypomastigotes, completing the parasite cycle. In members of the Salivaria section of Trypanosoma genera, such as Trypanosoma brucei, the metacyclic trypomastigote is injected directly from the salivary gland of the insect host, flies from Glossina genus, into the bloodstream of the vertebrate host [19, 20]. T brucei trypomastigotes can directly multiply by binary fission in the vertebrate blood. When a hematophagous insect vector feeds on the blood of an infected vertebrate it consumes bloodstream trypomastigotes forms, which differentiate inside the insect into procyclic trypomastigotes and then into epimastigotes, which are capable of multiplying by binary fission. The cycle is closed with the transformation of epimastigote forms into metacyclic trypomastigote forms in the salivary gland of the insect fly that infects a new vertebrate host during blood feeding [18].
The heteroxenous life cycle in trypanosomatids may have an evolutionary history beginning before the start of the tertiary period, but the overall Kinetoplastida phylogeny is still filled with uncertainty, even more so among trypanosomatids [21]. The existence of bacterial endosymbionts in the group is of marked interest to researchers, possibly being related to the transition from free to parasitic life cycles or being involved in lateral gene transfers between bacteria and eukaryotes [22]. Trypanosomatids have polycistronic DNA transcription which tends to keep coding sequences conserved in contiguous groups, resulting in multiple gene loci being preserved between different species [23].
1.2 Serine protease inhibitors: ecotins and Trypanosomatida ISPs
Ecotins are serine protease inhibitors initially described in E. coli bacteria and named for their capacity to inhibit the digestive enzyme trypsin—E. coli trypsin inhibitor [8]. The E. coli ecotin has a molecular weight of 18 kDa and is expressed in the cellular periplasm with a homodimeric active form. It inhibits serine proteases of family S1A, including trypsin, chymotrypsin, neutrophil elastase, and cathepsin G [24, 25]. Ecotin activity protect cells against exogenous serine peptidases involved in various biological processes, including coagulation and fibrinolysis; this capacity for inhibiting a considerable number of different proteins differentiates ecotin from most other serine protease inhibitors, which generally are highly specific [26, 27, 28, 29].
Trypanosomatids are the only eukaryotes with genes coding for ecotin analogs, described for the first time in 2005 by Ivens et al. in L. major, with three variants that were named ISP1, ISP2, and ISP3 [9, 30]. In L. major, the ISP1 and ISP2 ecotins have 16.5 and 17.5 kDa, respectively, and while structurally similar to the E. coli ecotin, their amino acid sequence identity is only 36% [31], and they have different patterns of expression and inhibitory activity in the various stages of the parasite life cycle [30]. In L. major, the ISP1 variant is expressed in larger quantities in the life cycle forms living in the insect host, and knockout studies with this gene suggest that it has endogenous functions, mainly in the flagellar formation process [31]. Also in L. major, ISP2 expression occurs in all life cycle stages and there are evidences that this enzyme participates in the parasite macrophage infection process in hosts, by inhibiting serine proteases such as neutrophil elastase in vertebrates. There is evidence that Leishmania parasites with knocked down ISP2 suffer more intense phagocytosis by host macrophages [30, 32, 33]. E. coli and other bacteria that have periplasmic ecotin use it to evade hosts’ immune systems, and L. major employs its ISP2 inhibitor in a similar fashion [26, 34]. The lack of genes coding for ecotin target enzymes (the S1A family of serine proteases) in both E. coli and L. major is a strong indicator of the probable role of ecotins in these species’ interactions with vertebrate hosts [30].
An ISP2 homolog has been found in T. cruzi with a high degree of sequence similarity to the L. major gene [35]. BLAST searches in the NCBI GenBank database reveal that other members of the genus Trypanosoma also possess ISP2 homologs, as well as close relatives in the order Trypanosomatida such as Leptomonas spp., most papers published on the subject have focused on Leishmania ISPs. It is probable that, due to both its conservation in various species of trypanosomatids and its flexible functional properties, ecotin homologs have offered some fitness gain to trypanosomatids with vertebrate hosts. Also, the conservation of ISPs in various species indicates an origin in the common ancestor of Trypanosomatida. The similarity between trypanosomatid ISPs and bacterial ecotins makes us raise the hypothesis of a lateral gene transfer between E. coli-like bacteria and the common ancestor of the various Trypanosomatida genera as the origin of ISPs [30, 36]. Recent research suggest that this kind of lateral gene transfer has been essential in this group’s evolutionary history [22]. The bacterial endosymbionts in Kinetoplastida are in class Betaproteobacteria [37]. This group contains vertebrate infecting species that not only possess ecotin-encoding genes, but that depends on those ecotins being expressed to maintain their virulence [34], which may be another hint of ancestral lateral gene transfers between Betaproteobacteria and Trypanosomatida.
Leishmania and Trypanosoma parasites are responsible for a number of severely neglected tropical diseases, as officially listed by the World Health Organization [38]. Multiple sources indicate that ecotin and its homologs are connected to these parasites’ infective capacity, but research in this specific subject is still timid, especially in the Trypanosoma genus. Neglected tropical diseases like Chagas’ disease (caused by T. cruzi) and African sleeping sickness (caused by T. brucei) are neglected for socio-historical reasons, as these afflictive diseases rarely, if ever, occur in developed countries.
The evolution of the genes encoding for trypanosomatid ISPs can shed light not only on the group’s evolutionary history but also on the overall importance of this enzyme for future researchers. In the next few pages, we show evidence for a common ancestry of ISPs in extant trypanosomatids using both phylogenetic inferences and a novel method for gene loci analysis. Also, we demonstrate the antigenicity of T. cruzi ISP2 in the murine model of Chagas’ disease and the presence of a higher concentration of antibodies against TcISP2 in the chronic phase of infection.
Amino acid sequences of ISPs were obtained from the NCBI database (National Center for Biotechnology Information, U.S. National Library of Medicine) using their BLAST search package using E. coli ecotin as a BLAST target [39]. Sequence alignment using the MUSCLE algorithm and phylogenetic maximum likelihood analysis was done in SeaView [40], with the selection of best-fit amino acid substitution matrices done with PROTTEST 3 [41]. Resulting tree files were manually edited to standardize terminal labels, and cladogram image files were exported using the iTol web tree tool (Figure 1) [42].
Figure 1.
Scheme on methodology employed in the phylogenetic reconstruction of ISPs from trypanosomatids.
For the loci viewer project database input, complete genomic sequences for loci analysis were obtained from the same database using tBLASTn, a tool that takes amino acid sequences as input and searches for corresponding nucleotide sequences, with the search limited to RefSeq annotated genomes [39]. Data was input and manipulated into custom software developed for the author’s master thesis [43]. The complete list of sequences with respective web links and NCBI GenBank IDs is listed in Table 1. After data input was completed, the database was manually manipulated using SQL queries to identify close genes to the left or right of the ecotin homologs in various species, aided by the visual map generated showing that ISPs occurred in two different loci around 50 kbp apart in most species. One of these genes is a putative katanin-encoding gene, and the other three are conserved hypothetical protein-encoding genes that were called CHP1, 2, and 3. Using the L. braziliensis genome as a reference, these four amino acid sequences were run through the NCBI tBLASTn tool using the same settings used for the ecotin homologs, and resulting CDSs were manually labeled CHP1, CHP2, CHP3, and katanin-like in the loci viewer database.
List of RefSeq genomic records inserted into the loci image generator database.
2.2 Purification of recombinant TcISP2 (TcISP2r) from Escherichia coli
The construction of recombinant plasmid to express TcISP2r in E. coli and extraction of TcISP2r were previously described by our research group [44]. Initially, a culture of E. coli from DH5α strain was transformed with pET28a plasmid containing TcISP2r construction with a histidine tag at N-terminus, in the presence of 50 ug/mL of Kanamycin at 600 nm optical density of 0,5, following expression induction with 1 mM of IPTG and incubation for more 16 hours in the shaker at 37⁰C. For protein extraction, 50 mL of bacteria culture was precipitated by centrifugation and the pellet was dissolved in 6 mL of 20 mM of phosphate buffer pH 7.4 and 0.024 g of lysozyme (Sigma, USA), and incubated at room temperature for 15 minutes. Then 6 ml of lysis buffer (20 mM phosphate buffer pH 7.4 and 0.5% Tween 20) was added and incubated for 30 minutes at 37°C. Finally, the bacterial lysate was centrifuged at 8000 x g for 15 minutes at 4°C to precipitate cell debris. The protein present in the supernatant was purified on an affinity chromatography column containing nickel, HisPur Ni-NTA Spin 0.2 ml (Thermo Fisher Scientific, USA), following the supplier’s instructions. After passing the entire supernatant through the column by successive centrifugations at low speed (4000 x g) for 1 min, the column was washed with 10 volumes of 20 mM phosphate buffer pH 7.4 with 20 mM Imidazole. Subsequently, the protein was eluted from the column with 150 mM Imidazole. The purified TcISP2r protein was subjected to concentration by filtration on 10 KDa cellulose filters to replace the 20 mM phosphate buffer pH 7.4 containing 150 mM imidazole with 20 mM phosphate buffer pH 7.4 without imidazole. The final protein concentration was determined by absorbance at 280 nm in a UV light spectrophotometer. The quality and quantity of protein obtained were also verified by protein electrophoresis in 12% acrylamide gel. Protein specificity was verified by Western Blotting using an anti-histidine antibody.
2.3 Obtention of sera from murine model of acute and chronic Chagas’ disease
The murine model of acute and chronic Chagas’ disease is already established in the Chagas Disease laboratory of the Instituto Dante Pazzanese. For the proposed study, a total of 8 male mice, Black-C57, 20 days old, were inoculated intraperitoneally with 1x104 parasites per milliliter (mL) in 0.9% saline, pH 6.0, and three animals were used as negative controls.
After infection, 200 to 400 uL of blood samples were collected from the mice on days 7, 15 (acute phase of infection), 30, 45, and 60 (chronic phase of infection) after infection, through submandibular vein puncture, and centrifuged at 1200 x g for 10 minutes at 25°C, to obtain the serum, which was stored at −20°C until analysis. On day 60, the mice were euthanized in a chamber with cotton soaked in Sevoflurane (C4H3F7O), following cervical dislocation to ensure death. After euthanasia, a necropsy of brain tissue, liver, spleen, kidneys, and heart was performed. The heart was washed with saline and cardiac tissue and saline lavage were stored at −20°C. The euthanasia method was performed according to the recommendations of the Brazilian guide to good practices for the euthanasia of animals established by the consultants of the Ethics, Bioethics and Animal Welfare Committee of the Federal Council of Veterinary Medicine (CEBEA/CFMV). All handling of the animals was carried out in accordance with the Brazilian guideline for the care and use of animals for scientific and educational purposes (DBCA), of the National Council for the Control of Animal Experimentation (CONCEA) (https://www.sbcal.org.br/download/download?ID_DOWNLOAD=58). The protocol was approved by the Animal Research Ethics Committee of the Instituto Dante Pazzanese (Registration number 020/2019).
T. cruzi infection was confirmed by reading slides containing blood smears and by polymerase chain reaction (PCR) with the oligonucleotides Tc121 (5′ AAATAATGTACGGGKGAGATGCATGA 3′) and Tc122 (5′ GGTTCGATTGGGGTTGGTGTAATATA 3′), which amplifies a 330 bp fragment corresponding to T. cruzi kDNA [45, 46]. The PCR reaction was performed with the enzyme Platinum Taq DNA polymerase (ThermoFischer Scientific), according to the supplier’s instructions. The cycles used for amplification were 3 min 94°C/40x 94°C 1 min; 62°C 1 min; 72°C 1 min/7 min 72°C. PCR products were analyzed on 2% agarose gel stained with ethidium bromide.
2.4 Standardization of TcISP2 enzyme-linked immunosorbent assay (ELISA)
To perform the indirect ELISA test for antibodies against TcISP2r, the binding efficiency of the recombinant protein was initially verified in 96-well plates with low, medium, and high binding capacity (Figure 2). Each of the plaque types was sensitized with 1.0, 2.0, and 5 μg of TcISP2r protein diluted in 0.2 M carbonate-bicarbonate buffer, pH 9.2. The plates were filled with 100 μL of buffer containing 1, 2, or 5 μg of protein and kept in the refrigerator for 16 hours. Subsequently, the plates were washed with 20 mM of phosphate buffer pH 7.4 and 0.05% of tween 20 and subsequently incubated with blocking solution (5% skim milk, 20 mM of phosphate buffer pH 7.4 and 0, 05% tween 20) for 30 minutes. Then, the blocking solution was removed and different dilutions of the anti-histidine monoclonal antibody (1 ug, 0.1 ug, and 0.05 ug/mL) produced in mice (Invitrogen) in 50 uL of blocking solution were added per well. Serial dilutions in the order of 2 of the anti-histidine antibodies were also made in columns A to H, followed by incubation for 2 hours at room temperature. After washing three times with 20 mM of phosphate buffer pH 7.4 and 0.05% tween 20, 0.1 ug/mL of the anti-mouse IgG second antibody, bound with alkaline phosphatase, was added to 50 uL of blocking solution and incubated for 1 hour at room temperature. The plate was then washed 3 times with 20 mM phosphate buffer pH 7.4 and 0.05% tween 20 and 2 times with 20 mM phosphate buffer pH 7.4 without tween 20. Reactions were developed with PNPP substrate for alkaline phosphatase (Invitrogen), according to the manufacturer’s instructions and the plates were incubated for 30 minutes at room temperature and read in a spectrophotometer at a wavelength of 405 nm.
Figure 2.
Indirect ELISA to standardize the ligation of TcISP2r in 96 wells plates with different binding capabilities using anti-histidine antibody. TcISP2r histidine tailed protein, purified by histidine affinity chromatography, were ligated to plates of high, medium, and low binding capabilities as indicated in the figure. Columns 1 to 4 contain 1 ug of protein per well; 5 to 8, 2 ug; and 9 to 12, 5 ug. Columns 1, 5, and 9 started with 1 ug of anti-his antibody; columns 2, 6 e 10, started with 0,1 ug of anti-his antibody; colunms 3, 7, and 11, started with 0,05 ug of anti-his antibody; in columns 4, 8, and 12 no anti-his antibody were used. From a to H, anti-his antibodies were serially diluted in order of 2 (1:2; 1:4; 1:6; 1:8; 1:10; 1:12: 1:14; 1:16).
ELISA tests were performed in 96-well plates with medium binding capacity and three concentrations of TcISP2r protein (2, 5, and 10 μg). Recombinant protein binding was carried out for 16 hours in 100 uL of 0.1 M carbonate-bicarbonate buffer pH 9.2, in the refrigerator. Subsequently, the plates were washed with 20 mM of phosphate buffer pH 7.4 and 0.05% of tween 20 and subsequently incubated with blocking solution (5% skim milk, 20 mM of phosphate buffer pH 7.4 and 0, 05% tween 20) for 30 minutes. Then, the blocking solution was removed and 50 uL of serial dilutions of serum from 3 mice collected at 7-, 15-, and 60-days post-infection were added to the wells and the plate incubated for 2 hours at room temperature. After three washes with 20 mM of phosphate buffer pH 7.4 and 0.05% tween 20, 0.1 ug/mL of the anti-mouse IgG second antibody, bound with alkaline phosphatase, was added to 50 uL of blocking solution and incubated for 1 hour at room temperature. The plate was then washed 3 times with 20 mM phosphate buffer pH 7.4 and 0.05% tween 20 and 2 times with 20 mM phosphate buffer pH 7.4 without tween 20. Reactions were developed with PNPP substrate for alkaline phosphatase (Invitrogen), according to the manufacturer’s instructions and the plates were incubated for 30 minutes at room temperature and read in a spectrophotometer at a wavelength of 405 nm.
3.1 Phylogenetic inferences and TcISP2 origin and role in Chagas’ disease
Maximum likelihood phylogenetic inference resulted in the tree represented in Figure 3, with E. coli ecotin as the outgroup and color-coded ISPs 1, 2, and 3. The tree topology strongly indicates that the ISP ecotin homologs have differentiated from each other a long time ago, and it is probable that at least ISP1 and ISP2 have been with these organism’s genome at least since the split between the Leishmania and Trypanosoma genera.
Figure 3.
Maximum likelihood cladogram of ecotin homologs (ISPs), with ISP1, labeled in orange, ISP2 labeled in green, and ISP3 labeled in blue.
Using the loci image generation software described in detail in the author’s master thesis [43] resulted in the images in Figures 4 and 5, showing the full chromosomes and a zoomed-in area of interest respectively. Figure 4 is only useful for comparing Leishmania records as to the overall position in the chromosome: ecotin homologs occur only in chromosome 15 of these species, and the data for other species is either incomplete or badly annotated, resulting in huge contig sequences. Figure 5 clears the image a little, but data for T. grayi and Leptomonas pyrrhocoris sequences is still fragmentary.
Figure 4.
Loci image generator result showing full GenBank records and all selected labels. Conserved hypothetical protein (CHP) 1 to 3, presenting highly similar amino acid sequences are represented by the same color. Seta direction indicates the orientation of the encoding gene according to genome annotation in GenBank.
Figure 5.
Loci image generator result created with the same data as Figure 4 with a higher resolution and individual ISPs identified as ISP1, ISP2, and ISP3 with numbers; conserved hypothetical protein (CHP) 1 to 3, presenting highly similar amino acid sequences are represented by the same color; seta direction indicates the orientation of the encoding gene according to genome annotation in GenBank.
The images show up to five ecotin homologs in various Leishmania species. This duplication was not reported in previous papers. Sequence data analysis and visual genomic context inspection give strong support to the idea that ecotin homologs suffered various duplications and/or multiple events of lateral gene transference before the differentiation of modern Trypanosomatida genera. Closely examining Figure 5, it can be seen that Leishmania spp. mostly retained all five ISP copies, while trypanosomes lost at least a few of them. In these figures, the conserved hypothetical coding sequences (CHP1-3) and katanin-like labels are accessory labels: they serve to identify the complete ecotin loci and were helpful to identify possible genomic evolutionary events involving ISP1, ISP2, and ISP3.
We know ISPs came from bacteria by horizontal gene transfer in Kinetoplastida because they appear in no other eukaryotes. The unanswered question is “how.” Looking at Figures 4 and 5 and keeping the cladogram in Figure 3 in mind, we can form a hypothesis for how trypanosomatids acquired ecotin homologs. The next paragraph is speculative, but given the evidence, it probably is not too far off-base.
The ancestor of all trypanosomatids either participated in multiple lateral gene transfers with ecotin-possessing bacteria or this event occurred only once and was followed by multiple gene duplications. If multiple gene transfers occurred, they probably happened no more than three times for ISPs 1, 2, and 3, and the additional ISP2 and ISP3 copies carried by Leishmania spp. are the result of a subsequent duplication. The positions of ISP1 and ISP2 in T. brucei, in the first and second ecotin loci respectively, with the ISP2 being probably homologous to T. cruzi ISP2, points to an early locus duplication, occurring before the two genera split. In this scenario, Trypanosomaspp. subsequently lost copies of the gene. Their sequences show a much more compact genome when compared to Leishmania spp. in the images presented, leading to the suspicion that more deletions occurred in Trypanosoma species than in Leishmania, which would lend credence to the idea that T. cruzi and T. brucei lost some of their ISP copies. A possible sequence of events based on this limited dataset is this: the common Trypanosomatida ancestor had three ISP copies in the first locus (at around position 120 kbp in chromosome 15), get either via lateral transfers with bacteria or via a single lateral transfer followed by contiguous duplication. The ISP2 and ISP3 ancestors in this locus then suffered a simultaneous duplication event, creating the second locus at around 190 kbp. Subsequently, various species lost some of these copies.
The preservation of ISP2 in almost all species is an interesting fact and makes sense given the ample evidence of its importance against hosts’ immune systems. Another interesting fact is that T. brucei parasites preserved the ISP1 variant in all cases, while T. cruzi lost the ISP1 gene. Since ISP1 seems to be involved in the development of motility and flagellar development in promastigotes inside the insect vector in Leishmania species [31], this could be a reason for its preservation in T. brucei and loss in T. cruzi. These species are members of section Salivaria and Stercoraria, respectively, with different life cycles and methods of transmission. While T. cruzi is transmitted by hemipterans, with infecting parasites deposited with their feces on the vertebrate host, T. brucei lives in the salivary gland of dipteran insects and is injected by their proboscis like the Leishmania species. It could be that the ISP1 ecotin variant gives some advantages to trypanosomatids with dipterans as their arthropod hosts. This association needs further investigation, resulting in data with potential public health applications.
These speculations are given to encourage further research. As tempting as it is to affirm their validity, our data set is very limited in scope and of very poor quality in some cases making bold affirmations. Automated genomic annotation can only go so far, and some of these sequences have errors, omissions, and other problems. Looking closely at the L. mexicana data in Figure 5, for example, it seems that the coding sequences between the first ISP occurrence and the CHP2 labeled gene should clearly be labeled as ISP2 and ISP3, but in the automated annotation, they appear as “unknown proteins.” Nevertheless, since the amount of available genomic data grows so fast, these speculations can be further developed as more data becomes available.
One thing this work clearly shows without a doubt is the ubiquity of large amounts of unreviewed genomic data online. The amount of retrievable information at very little monetary cost and using free-to-use bioinformatics tools is huge, and in this world of big data and exponentially falling sequencing costs, this fact will become more obvious as time passes. The next generation of budding biologists may well have to learn to program before they learn the names of all the plant and animal families.
T. cruzi presents only ISP2 in its chromosome 15, and its structural and biochemical characterization revealed high structural similarity with ecotin and strong inhibitory activity on human neutrophil elastase (NE) [44]. Ecotin appears to be a potent prokaryotic tool in evading the immune system. A classic example is that of the inhibition of NE: in humans, a serine peptidase produced in neutrophil granulocytes is one of the main immune defenses in combating pathogenic invaders. In bacteria, one of the ways of action of NE is the cleavage of outer membrane protein A (OmpA, from English outer membrane protein A) in phagocytosed gram-negative bacteria, hindering their multiplication [47, 48]. This makes ecotin an important target for pharmacological research [49].
3.2 TcISP2 is immunogenic in mice with chronic infection
To verify the immunogenicity of the recombinant form of the TcISP2 protein produced in E. coli, the indirect ELISA method was initially standardized. Recombinant proteins have different biochemical properties that determine the best conditions for coating ELISA plates with histidine-tagged TcISP2. Three different types of microplates were tested, each with a binding capacity and with different concentrations of recombinant protein. To assess the distribution of the protein in the wells, serial dilutions of mouse-produced anti-histidine antibodies obtained from Invitrogen were also analyzed. The results obtained showed that the medium binding capacity plate with 2 ug of recombinant protein per well presented the best results.
To confirm the infection of mice with the Y strain of T. cruzi, after 7 and 15 days, PCR was performed using the Tc121/122 oligos from a fraction of the clot obtained by puncturing the submandibular vein. The four mice infected after 7 days with the Y strain of T. cruzi were PCR negative, while the same mice were PCR positive after 15 days (Figure 6). After 60 days of infection, nucleic acids extracted from the cardiac lavage of two euthanized mice with infection confirmed by PCR and light microscopy were positive by PCR (Figure 6). These results indicate that blood parasites can only be detected 15 days post-infection in Black mice.
Figure 6.
Electrophoresis in 2% agarose gel stained with ethidium bromide containing the PCR products of the Trigonoscuta cruzi molecular test with the Tc121/122 oligonucleotides that amplify a 330 bp fragment corresponding to the gene encoding the parasite’s kDNA. Blood clots of 4 infected mice after 7 and 15 days and the washing of cardiac tissue from 2 mice euthanized after 60 days of infection, were evaluated as indicated in the wells. C-, corresponds to the negative control of the PCR reaction; C+, corresponds to the positive control with genomic DNA obtained from T. cruzi of the Y strain; M – 100 bp ladder from synapse biotechnology.
Serum from mice infected by T. cruzi strain Y, and collected after 7, 15, and 60 days, with results confirmed by PCR and light microscopy, was used in an ELISA experiment to evaluate the immunogenicity of TcISP2r. The results are shown in (Figure 7). The identification of TcISP2r was observed in the serum samples of mice 60 days post-infection, suggesting an immune response against ISP2 from T. cruzi in the chronic phase of the disease. In ELISA tests, using mouse serum, the best protein concentration for antibody detection was also 2 ug.
Figure 7.
Indirect ELISA test with different concentrations of TcISP2r and serum from mice infected with Trigonoscuta cruzi strain Y after 7, 15, and 60 days. A. Experimental design to verify the immunogenicity of TcISP2r in mouse sera collected 7, 15, and 60 days after infection with the Y strain of T. cruzi; negative control serum was obtained from uninfected mice; 2 μg; 5 μg and 10 μg are the amount of TcISP2r per well; B. absorbance results obtained after indirect ELISA test with alkaline phosphatase-labeled second antibody against mouse IgG, developed with P-nitrophenyl phosphate substrate.
The ELISA test revealed antibodies against TcISP2 in serum samples from T. cruzi- infected mice only after 60 days post-infection, during the chronic phase. The high activity of TcISP2 on neutrophil elastase indicates its inhibiting action on the immune system [44, 50]. In this way, inflammation would occur in infected organs and tissues during the chronic phase, when cardiomyopathy and mega syndromes (megaesophagus and megacolon) develop. Thus, we could infer that the activity of TcISP2 is to inhibit inflammatory activity and thus facilitate the colonization of tissues and organs by the parasite.
According to Faria et al. [33], ISPs are responsible for inhibiting the neutrophil elastase present in macrophages, through the toll-like receptor 4. Thus, cells with phagocytic activity can be used by the protozoan for their proliferation and escape from the immune system. In Chagas’ disease, macrophages are infected and carry the parasite into tissues. The phagocytic action of macrophages is more relevant in tissues compared to other professional phagocytic cells, due to their ability to migrate from the bloodstream to different tissues and organs [51]. Thus, the TcISP2 can play an essential role in acute and chronic Chagas’ disease.
Ecotin, a serine peptidase inhibitor (ISP), found first in E. coli, is a key factor in bacterial defense against the immunological barriers of vertebrate host organisms. Genomic studies demonstrated that parasites from the Trypanosomatida taxon are unique eukaryotic organisms harboring different ecotin-like encoding genes. Considering existence of trypanosomatida species with bacterial endosymbionts and the genomic context of ISPs demonstrated in this chapter, the evolutive origin of ecotin-like genes in trypanosomatids probably occurred by horizontal transfer from a symbiont bacterium, in a common ancestor of the clade, and this event was followed by successive duplications and losses that would explain the current pattern. T. cruzi present only one copy of ISP (TcISP) with high structural similarity to ecotin from E. coli and a strong inhibitory activity on human neutrophil elastase, indicating an exogenous activity and interaction with vertebrate host serine peptidases involved in the modulation of the immune system. The recombinant TcISP2 protein was immunogenic in a murine model, allowing the detection of antibodies in the chronic phase of T.cruzi infection, which suggested that this protein can also be tested as a target for serological diagnosis and for investigation of disease prognosis.
This work was funded by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (Grant number 2016/14514-4; and by UFABC. MMF and CMS were the recipient of a Master’s scholarship from CAPES.
Parts of this book chapter were initially presented in the Master thesis of Max Mario Fuhlendorf available on the UFABC library platform, titled Loci image generator and the evolution of trypanosomatid ecotin: customized software as a tool for evolutionary analysis, 2018, Programa de pós-graduação em Evolução e Diversidade. The thesis has not been peer-reviewed and published.
References
1.Aguilar VHM. Reflections on Chagas disease in the Amazon. Memórias do Instituto Oswaldo Cruz. 2022;117:e200409chgsa. Epub 20220520. DOI: 10.1590/0074-02760210409chgsa
2.Alvarez-Hernandez DA, Garcia-Rodriguez-Arana R, Ortiz-Hernandez A, Alvarez-Sanchez M, Wu M, Mejia R, et al. A systematic review of historical and current trends in Chagas disease. Therapeutic Advances in Infectious Disease. 2021;8:1-17. Epub 20210804. DOI: 10.1177/20499361211033715
3.de Sousa PH, Scofield A, Junior PSB, Lira Dos Santos D, de Sousa Siqueira J, Chaves JF, et al. Chagas disease in urban and peri-urban environment in the Amazon: Sentinel hosts, vectors, and the environment. Acta Tropica. 2021;217:105858. Epub 20210212. DOI: 10.1016/j.actatropica.2021.105858
4.Sarquis O, Carvalho-Costa FA, Toma HK, Georg I, Burgoa MR, Lima MM. Eco-epidemiology of Chagas disease in northeastern Brazil: Triatoma brasiliensis, T. pseudomaculata and Rhodnius nasutus in the sylvatic, peridomestic and domestic environments. Parasitology Research. 2012;110(4):1481-1485. Epub 20111007. DOI: 10.1007/s00436-011-2651-6
5.Barbosa RL, Dias VL, Pereira KS, Schmidt FL, Franco RM, Guaraldo AM, et al. Survival in vitro and virulence of Trypanosoma cruzi in acai pulp in experimental acute Chagas disease. Journal of Food Protection. 2012;75(3):601-606. Epub 2012/03/14. DOI: 10.4315/0362-028X.JFP-11-233
6.Brum-Soares LM, Xavier SS, Sousa AS, Borges-Pereira J, Ferreira JM, Costa IR, et al. Morbidity of Chagas disease among autochthonous patients from the Rio Negro microregion, state of Amazonas. Revista da Sociedade Brasileira de Medicina Tropical. 2010;43(2):170-177. Epub 2010/05/14. Morbidade da doenca de Chagas em pacientes autoctones da microrregiao do Rio Negro, Estado do Amazonas. DOI: 10.1590/s0037-86822010000200013
7.Santos V, Meis J, Savino W, Andrade JAA, Vieira J, Coura JR, et al. Acute Chagas disease in the state of Para, Amazon region: Is it increasing? Memórias do Instituto Oswaldo Cruz. 2018;113(5):e170298. Epub 20180507. DOI: 10.1590/0074-02760170298
8.Chung CH, Goldberg AL. Purification and characterization of protease so, a cytoplasmic serine protease in Escherichia coli. Journal of Bacteriology. 1983;154(1):231-238. Epub 1983/04/01. DOI: 10.1128/jb.154.1.231-238.1983
9.Ivens AC, Peacock CS, Worthey EA, Murphy L, Aggarwal G, Berriman M, et al. The genome of the kinetoplastid parasite, Leishmania major. Science. 2005;309(5733):436-442. Epub 2005/07/16. DOI: 10.1126/science.1112680
10.Kaufer A, Ellis J, Stark D, Barratt J. The evolution of trypanosomatid taxonomy. Parasites & Vectors. 2017;10(1):287. Epub 2017/06/10. DOI: 10.1186/s13071-017-2204-7
11.Borghesan TC, Ferreira RC, Takata CS, Campaner M, Borda CC, Paiva F, et al. Molecular phylogenetic redefinition of Herpetomonas (Kinetoplastea, Trypanosomatidae), a genus of insect parasites associated with flies. Protist. 2013;164(1):129-152. Epub 20120829. DOI: 10.1016/j.protis.2012.06.001
12.Borges AR, Engstler M, Wolf M. 18S rRNA gene sequence-structure phylogeny of the Trypanosomatida (Kinetoplastea, Euglenozoa) with special reference to Trypanosoma. European Journal of Protistology. 2021;81:125824. Epub 2021/08/06. DOI: 10.1016/j.ejop.2021.125824
13.Dollet M, Sturm NR, Campbell DA. The internal transcribed spacer of ribosomal RNA genes in plant trypanosomes (Phytomonas spp.) resolves 10 groups. Infection, Genetics and Evolution. 2012;12(2):299-308. Epub 2011/12/14. DOI: 10.1016/j.meegid.2011.11.010
14.Sturm NR, Dollet M, Lukes J, Campbell DA. Rational sub-division of plant trypanosomes (Phytomonas spp.) based on minicircle conserved region analysis. Infection, Genetics and Evolution. 2007;7(5):570-576. Epub 2007/05/15. DOI: 10.1016/j.meegid.2007.04.002
15.de Souza SSA, Catta-Preta CM, Alves JMP, Cavalcanti DP, Teixeira MMG, Camargo EP, et al. Expanded repertoire of kinetoplast associated proteins and unique mitochondrial DNA arrangement of symbiont-bearing trypanosomatids. PLoS One. 2017;12(11):e0187516. Epub 2017/11/14. DOI: 10.1371/journal.pone.0187516
16.Motta MCM, Catta-Preta CMC. Electron microscopy techniques applied to symbiont-harboring Trypanosomatids: The Association of the Bacterium with host organelles. Methods in Molecular Biology. 2020;2116:425-447. Epub 2020/03/30. DOI: 10.1007/978-1-0716-0294-2_26
17.Fraga J, Fernandez-Calienes A, Montalvo AM, Maes I, Deborggraeve S, Buscher P, et al. Phylogenetic analysis of the Trypanosoma genus based on the heat-shock protein 70 gene. Infection, Genetics and Evolution. 2016;43:165-172. Epub 2016/05/18. DOI: 10.1016/j.meegid.2016.05.016
18.Rey L. Parasitologia. 4th ed. Rio de Janeiro: Guanabara Koogan; 2008
19.Tyler KM, Engman DM. The life cycle of Trypanosoma cruzi revisited. International Journal for Parasitology. 2001;31(5-6):472-481. Epub 2001/05/04. DOI: 10.1016/s0020-7519(01)00153-9
20.Hendriks E, van Deursen FJ, Wilson J, Sarkar M, Timms M, Matthews KR. Life-cycle differentiation in Trypanosoma brucei: Molecules and mutants. Biochemical Society Transactions. 2000;28(5):531-536. Epub 2000/10/25. DOI: 10.1042/bst0280531
21.Votypka J, d'Avila-Levy CM, Grellier P, Maslov DA, Lukes J, Yurchenko V. New approaches to systematics of Trypanosomatidae: Criteria for taxonomic (Re)description. Trends in Parasitology. 2015;31(10):460-469. Epub 2015/10/05. DOI: 10.1016/j.pt.2015.06.015
22.Alves JM, Klein CC, da Silva FM, Costa-Martins AG, Serrano MG, Buck GA, et al. Endosymbiosis in trypanosomatids: The genomic cooperation between bacterium and host in the synthesis of essential amino acids is heavily influenced by multiple horizontal gene transfers. BMC Evolutionary Biology. 2013;13:190. Epub 2013/09/11. DOI: 10.1186/1471-2148-13-190
24.McGrath ME, Gillmor SA, Fletterick RJ. Ecotin: Lessons on survival in a protease-filled world. Protein Science. 1995;4(2):141-148. DOI: 10.1002/pro.5560040201
25.Yang SQ , Wang CI, Gillmor SA, Fletterick RJ, Craik CS. Ecotin: A serine protease inhibitor with two distinct and interacting binding sites. Journal of Molecular Biology. 1998;279(4):945-957. DOI: 10.1006/jmbi.1998.1748
26.Eggers CT, Murray IA, Delmar VA, Day AG, Craik CS. The periplasmic serine protease inhibitor ecotin protects bacteria against neutrophil elastase. The Biochemical Journal. 2004;379(Pt 1):107-118. Epub 2004/01/07. DOI: 10.1042/BJ20031790
27.Eggers CT, Wang SX, Fletterick RJ, Craik CS. The role of ecotin dimerization in protease inhibition. Journal of Molecular Biology. 2001;308(5):975-991. DOI: 10.1006/jmbi.2001.4754
28.Jin L, Pandey P, Babine RE, Gorga JC, Seidl KJ, Gelfand E, et al. Crystal structures of the FXIa catalytic domain in complex with ecotin mutants reveal substrate-like interactions. The Journal of Biological Chemistry. 2005;280(6):4704-4712. Epub 2004/11/17. DOI: 10.1074/jbc.M411309200
29.Seymour JL, Lindquist RN, Dennis MS, Moffat B, Yansura D, Reilly D, et al. Ecotin is a potent anticoagulant and reversible tight-binding inhibitor of factor Xa. Biochemistry. 1994;33(13):3949-3958. DOI: 10.1021/bi00179a022
30.Eschenlauer SC, Faria MS, Morrison LS, Bland N, Ribeiro-Gomes FL, DosReis GA, et al. Influence of parasite encoded inhibitors of serine peptidases in early infection of macrophages with Leishmania major. Cellular Microbiology. 2009;11(1):106-120. Epub 20081029. DOI: 10.1111/j.1462-5822.2008.01243.x
31.Morrison LS, Goundry A, Faria MS, Tetley L, Eschenlauer SC, Westrop GD, et al. Ecotin-like serine peptidase inhibitor ISP1 of Leishmania major plays a role in flagellar pocket dynamics and promastigote differentiation. Cellular Microbiology. 2012;14(8):1271-1286. Epub 20120508. DOI: 10.1111/j.1462-5822.2012.01798.x
32.Ribeiro-Gomes FL, Moniz-de-Souza MC, Alexandre-Moreira MS, Dias WB, Lopes MF, Nunes MP, et al. Neutrophils activate macrophages for intracellular killing of Leishmania major through recruitment of TLR4 by neutrophil elastase. Journal of Immunology. 2007;179(6):3988-3994. Epub 2007/09/06. DOI: 10.4049/jimmunol.179.6.3988
33.Faria MS, Reis FC, Azevedo-Pereira RL, Morrison LS, Mottram JC, Lima AP. Leishmania inhibitor of serine peptidase 2 prevents TLR4 activation by neutrophil elastase promoting parasite survival in murine macrophages. Journal of Immunology. 2011;186(1):411-422. Epub 20101122. DOI: 10.4049/jimmunol.1002175
34.Ireland PM, Marshall L, Norville I, Sarkar-Tyson M. The serine protease inhibitor Ecotin is required for full virulence of Burkholderia pseudomallei. Microbial Pathogenesis. 2014;67-68:55-58. Epub 2014/01/28. DOI: 10.1016/j.micpath.2014.01.001
35.El-Sayed NM, Myler PJ, Blandin G, Berriman M, Crabtree J, Aggarwal G, et al. Comparative genomics of trypanosomatid parasitic protozoa. Science. 2005;309(5733):404-409. Epub 2005/07/16. DOI: 10.1126/science.1112181
36.Opperdoes FR, Michels PA. Horizontal gene transfer in trypanosomatids. Trends in Parasitology. 2007;23(10):470-476. Epub 2007/09/11. DOI: 10.1016/j.pt.2007.08.002
37.Andrade IDS, Vianez-Junior JL, Goulart CL, Homble F, Ruysschaert JM, Almeida von Kruger WM, et al. Characterization of a porin channel in the endosymbiont of the trypanosomatid protozoan Crithidia deanei. Microbiology (Reading). 2011;157(Pt 10):2818-2830. Epub 2011/07/16. DOI: 10.1099/mic.0.049247-0
38.Molyneux D. Neglected tropical diseases. Community Eye Health. 2013;26(82):21-24. Epub 2013/09/12
39.Boratyn GM, Camacho C, Cooper PS, Coulouris G, Fong A, Ma N, et al. BLAST: A more efficient report with usability improvements. Nucleic Acids Research. 2013;41(Web Server issue):W29-W33. Epub 2013/04/24. DOI: 10.1093/nar/gkt282
40.Gouy M, Guindon S, Gascuel O. SeaView version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Molecular Biology and Evolution. 2010;27(2):221-224. Epub 2009/10/27. DOI: 10.1093/molbev/msp259
41.Darriba D, Taboada GL, Doallo R, Posada D. ProtTest 3: Fast selection of best-fit models of protein evolution. Bioinformatics. 2011;27(8):1164-1165. Epub 2011/02/22. DOI: 10.1093/bioinformatics/btr088
42.Letunic I, Bork P. Interactive tree of life v2: Online annotation and display of phylogenetic trees made easy. Nucleic Acids Research. 2011;39(Web Server issue):W475-W478. Epub 2011/04/08. DOI: 10.1093/nar/gkr201
43.Fuhlendorf MM. Loci Image Generator and Ecotin Phylogenetics: Customized Software as a Tool for Evolutionary Analysis. Santo André, Brazil: Federal University of ABC; 2018
44.Garcia FB, Cabral AD, Fuhlendorf MM, da Cruz GF, Dos Santos JV, Ferreira GC, et al. Functional and structural characterization of an ecotin-like serine protease inhibitor from Trypanosoma cruzi. International Journal of Biological Macromolecules. 2020;151:459-466. Epub 20200218. DOI: 10.1016/j.ijbiomac.2020.02.186
45.Sturm NR, Degrave W, Morel C, Simpson L. Sensitive detection and schizodeme classification of Trypanosoma cruzi cells by amplification of kinetoplast minicircle DNA sequences: Use in diagnosis of Chagas' disease. Molecular and Biochemical Parasitology. 1989;33(3):205-214. DOI: 10.1016/0166-6851(89)90082-0
46.Telleria J, Lafay B, Virreira M, Barnabe C, Tibayrenc M, Svoboda M. Trypanosoma cruzi: Sequence analysis of the variable region of kinetoplast minicircles. Experimental Parasitology. 2006;114(4):279-288. Epub 20060530. DOI: 10.1016/j.exppara.2006.04.005
47.Belaaouaj A, Kim KS, Shapiro SD. Degradation of outer membrane protein a in Escherichia coli killing by neutrophil elastase. Science. 2000;289(5482):1185-1188. Epub 2000/08/19. DOI: 10.1126/science.289.5482.1185
48.Weinrauch Y, Drujan D, Shapiro SD, Weiss J, Zychlinsky A. Neutrophil elastase targets virulence factors of enterobacteria. Nature. 2002;417(6884):91-94. Epub 2002/05/23. DOI: 10.1038/417091a
49.Myint SL, Zlatkov N, Aung KM, Toh E, Sjostrom A, Nadeem A, et al. Ecotin and LamB in Escherichia coli influence the susceptibility to type VI secretion-mediated interbacterial competition and killing by vibrio cholerae. Biochimica et Biophysica Acta - General Subjects. 1865;2021(7):129912. Epub 2021/04/24. DOI: 10.1016/j.bbagen.2021.129912
50.Levy DJ, Goundry A, Laires RSS, Costa TFR, Novo CM, Grab DJ, et al. Role of the inhibitor of serine peptidase 2 (ISP2) of Trypanosoma brucei rhodesiense in parasite virulence and modulation of the inflammatory responses of the host. PLoS Neglected Tropical Diseases. 2021;15(6):e0009526. Epub 20210621. DOI: 10.1371/journal.pntd.0009526
51.van Furth R, Cohn ZA. The origin and kinetics of mononuclear phagocytes. The Journal of Experimental Medicine. 1968;128(3):415-435. DOI: 10.1084/jem.128.3.415
Written By
Carla Moreira Santana, Max Mario Fuhlendorf, Felipe Baena Garcia, Edmar Silva Santos, Ângela Maria Lourenço, Cristiane Castro Faccini, Abílio Augusto Fragata Filho, Aline Diniz Cabral and Márcia Aparecida Sperança
Submitted: 10 October 2022Reviewed: 09 January 2023Published: 24 January 2023
Open access peer-reviewed chapter
