Characterization and Seasonal Dynamics of Tick Populations in Dairy Cattle Production Systems of Northwestern Colombian Amazon.

Cattle ticks are a significant health concern in tropical livestock production due to their hematophagous behavior and potential as vectors for human and animal pathogens. In this study, we investigated the tick population present in dairy cattle production, calves, and grazing areas of livestock systems in the northwestern Colombian Amazon. Identification was based on taxonomic keys and molecular markers. Phylogenetic relationships were established using mitochondrial COX1 and 16S genes. Population structure analysis was performed considering age, racial type (B. indicus vs. B. taurus), and the influence of environmental factors and the geomorphological landscape on tick population dynamics. Our findings revealed the presence of a single tick species, with a unique haplotype identified for each mitochondrial gene assessed. Phylogenetic analysis classified the found species within Clade A of the Rhipicephalus microplus complex. Ticks were more prevalent during periods of low rainfall and high temperature, and B. taurus cows exhibited the highest tick abundance. Thus, these results provide insights into the population characteristics and distribution of the tick species present in dairy cattle production systems in the northwestern part of the Colombian Amazon. This information is fundamental for developing targeted strategies based on seasonal variation and host characteristics to mitigate tick infestation severity in the region.

RPA-1 from Leishmania sp.: Recombinant Protein Expression and Purification, Molecular Modeling, and Molecular Dynamics Simulations Protocols.

RPA is a conserved heterotrimeric complex and the major single-stranded DNA (ssDNA)-binding protein heterotrimeric complex, which in eukaryotes is formed by the RPA-1, RPA-2, and RPA-3 subunits. The main structural feature of RPA is the presence of the oligonucleotide/oligosaccharide-binding fold (OB-fold) domains, responsible for ssDNA binding and protein:protein interactions. Among the RPA subunits, RPA-1 bears three of the four OB folds involved with RPA-ssDNA binding, although in some organisms RPA-2 can also bind ssDNA. The OB-fold domains are also present in telomere end-binding proteins (TEBP), essential for chromosome end protection. RPA-1 from Leishmania sp., as well as RPA-1 from trypanosomatids, a group of early-divergent protozoa, shows some structural differences compared to higher eukaryote RPA-1. Also, RPA-1 from Leishmania sp., similar to TEBPs, may exert telomeric protective functions. Remarkably, different pieces of evidence have pointed out that trypanosomatids may not have OB fold-containing TEBPs. Moreover, recent data indicate that trypanosomatid RPA-1 may be considered a TEBP since it shares with TEBPs conserved functional and structural features. However, it is still unknown whether the RPA-1 protective telomeric role is exclusive to trypanosomatids or is also present in other primitive eukaryotes. Here, we describe a protocol to obtain highly purified and biologically active Leishmania amazonensis recombinant RPA-1, and to perform molecular modeling and molecular dynamics simulations methods which could be probably applied to functional and structural studies of homologous proteins in other primitive eukaryotes.

Leishmania major RUVBL1 has a hexameric conformation in solution and, in the presence of RUVBL2, forms a heterodimer with ATPase activity.

ATPases belonging to the AAA+ superfamily are associated with diverse cellular activities and are mainly characterized by a nucleotide-binding domain (NBD) containing the Walker A and Walker B motifs. AAA+ proteins have a range of functions, from DNA replication to protein degradation. Rvbs, also known as RUVBLs, are AAA+ ATPases with one NBD domain and were described from human to yeast as participants of the R2TP (Rvb1-Rvb2-Tah1-Pih1) complex. Although essential for the assembly of multiprotein complexes-containing DNA and RNA, the protozoa Rvb orthologs are less studied. For the first time, this work describes the Rvbs from Leishmania major, one of the causative agents of Tegumentar leishmaniasis in human. Recombinant LmRUVBL1 and LmRUVBL2 his-tagged proteins were successfully purified and investigated using biophysical tools. LmRUVBL1 was able to form a well-folded elongated hexamer in solution, while LmRUVBL2 formed a large aggregate. However, the co-expression of LmRUVBL1 and LmRUVBL2 assembled the proteins into an elongated heterodimer in solution. Thermo-stability and fluorescence experiments indicated that the LmRUVBL1/2 heterodimer had ATPase activity in vitro. This is an interesting result because hexameric LmRUVBL1 alone had low ATPase activity. Additionally, using independent SL-RNAseq libraries, it was possible to show that both proteins are expressed in all L. major life stages. Specific antibodies obtained against LmRUVBLs identified the proteins in promastigotes and metacyclics cell extracts. Together, the results here presented are the first step towards the characterization of Leishmania Rvbs, and may contribute to the development of possible strategies to intervene against leishmaniasis, a neglected tropical disease of great medical importance.

Purification and characterization of a novel and conserved TPR-domain protein that binds both Hsp90 and Hsp70 and is expressed in all developmental stages of Leishmania major.

Heat shock proteins (Hsps) are involved in several important aspects of the cell proteostasis. Hsp90 interacts with at least a tenth of the cell proteome helping a large number of proteins to fold correctly. Hsp90 function is modulated by several co-chaperones having TPR (tetratricopeptide repeat) domains that allow for interaction with the C-terminal MEEVD motif of the chaperone. Another important chaperone, Hsp70, has a C-terminal EEVD motif that binds to TPR. Leishmania is a protozoan that causes leishmaniasis, a neglected disease in humans and other animals. There is still no effective treatment for leishmaniasis, however the study of structure and function of the proteins of the parasite may generate potential targets for future therapeutic intervention studies. In this work, the genome of Leishmania major was searched for a novel TPR-domain gene, which is conserved in Leishmania. The recombinant protein, LmTPR, was produced in pure and folded state and was characterized by biophysical tools as a monomer with an elongated conformation. Studies in Leishmania major were also preformed to complement these in vitro experiments. Splice Leader RNA-seq analysis and Western blot indicated that the protein was expressed in all developmental stages of the parasite. Binding assays confirmed that both Hsp90 and Hsp70 bind specifically to LmTPR. Finally, sequence and structural predictions indicated a C-terminal region as a RPAP3 domain. Altogether, this study identified a novel TPR-domain co-chaperone of Hsp90 that is conserved and expressed in all developmental stages of Leishmania major.

A calmodulin-like protein (LCALA) is a new Leishmania amazonensis candidate for telomere end-binding protein.

BACKGROUND: Leishmania spp. telomeres are composed of 5'-TTAGGG-3' repeats associated with proteins. We have previously identified LaRbp38 and LaRPA-1 as proteins that bind the G-rich telomeric strand. At that time, we had also partially characterized a protein: DNA complex, named LaGT1, but we could not identify its protein component. METHODS AND RESULTS: Using protein-DNA interaction and competition assays, we confirmed that LaGT1 is highly specific to the G-rich telomeric single-stranded DNA. Three protein bands, with LaGT1 activity, were isolated from affinity-purified protein extracts in-gel digested, and sequenced de novo using mass spectrometry analysis. In silico analysis of the digested peptide identified them as a putative calmodulin with sequences identical to the T. cruzi calmodulin. In the Leishmania genome, the calmodulin ortholog is present in three identical copies. We cloned and sequenced one of the gene copies, named it LCalA, and obtained the recombinant protein. Multiple sequence alignment and molecular modeling showed that LCalA shares homology to most eukaryotes calmodulin. In addition, we demonstrated that LCalA is nuclear, partially co-localizes with telomeres and binds in vivo the G-rich telomeric strand. Recombinant LCalA can bind specifically and with relative affinity to the G-rich telomeric single-strand and to a 3'G-overhang, and DNA binding is calcium dependent. CONCLUSIONS: We have described a novel candidate component of Leishmania telomeres, LCalA, a nuclear calmodulin that binds the G-rich telomeric strand with high specificity and relative affinity, in a calcium-dependent manner. GENERAL SIGNIFICANCE: LCalA is the first reported calmodulin that binds in vivo telomeric DNA.

A calmodulin-like protein (LCALA) is a new Leishmania amazonensis candidate for telomere end-binding protein

Background Leishmania spp. telomeres are composed of 5'-TTAGGG-3' repeats associated with proteins. We have previously identified LaRbp38 and LaRPA-1 as proteins that bind the G-rich telomeric strand. At that time, we had also partially characterized a protein: DNA complex, named LaGT1, but we could not identify its protein component. Methods and results Using protein-DNA interaction and competition assays, we confirmed that LaGT1 is highly specific to the G-rich telomeric single-stranded DNA. Three protein bands, with LaGT1 activity, were isolated from affinity-purified protein extracts in-gel digested, and sequenced de novo using mass spectrometry analysis. In silico analysis of the digested peptide identified them as a putative calmodulin with sequences identical to the T. cruzi calmodulin. In the Leishmania genome, the calmodulin ortholog is present in three identical copies. We cloned and sequenced one of the gene copies, named it LCalA, and obtained the recombinant protein. Multiple sequence alignment and molecular modeling showed that LCalA shares homology to most eukaryotes calmodulin. In addition, we demonstrated that LCalA is nuclear, partially co-localizes with telomeres and binds in vivo the G-rich telomeric strand. Recombinant LCalA can bind specifically and with relative affinity to the G-rich telomeric single-strand and to a 3'G-overhang, and DNA binding is calcium dependent. Conclusions We have described a novel candidate component of Leishmania telomeres, LCalA, a nuclear calmodulin that binds the G-rich telomeric strand with high specificity and relative affinity, in a calcium-dependent manner.