Susan Ienne

Genomic organization and transcription analysis of the 195-bp satellite DNA in Trypanosoma cruzi

The 195-bp satellite DNA is the most abundant Trypanosoma cruzi repetitive sequence. Here we show by RNA blotting and RT-PCR that 195 SAT is intensely transcribed. We observed a positive correlation between the level of satellite RNA and the abundance of the satellite copies in the genome of T. cruzi strains and that the satellite expression is not developmentally regulated. By analyzing CL Brener individual reads, we estimated that 195 SAT corresponds to approximately 5% of the CL Brener genome. 195 SAT elements were found in only 37 annotated contigs, indicating that a large number of satellite copies were not incorporated into the assembled data. The assembled satellite units are distributed in non-syntenic regions with Trypanosoma brucei and Leishmania major genomes, enriched with surface proteins, retroelements, RHS and hypothetical proteins. Satellite repeats were not observed in annotated subtelomeric regions. We report that 12 satellite sequences are truncated by the retroelement VIPER.

Network genealogy of 195-bp satellite DNA supports the superimposed hybridization hypothesis of Trypanosoma cruzi evolutionary pattern.

Trypanosoma cruzi is highly diverse genetically and has been partitioned into six discrete typing units (DTUs), recently re-named T. cruzi I-VI. Although T. cruzi reproduces predominantly by binary division, accumulating evidence indicates that particular DTUs are the result of hybridization events. Two major scenarios for the origin of the hybrid lineages have been proposed. It is accepted widely that the most heterozygous TcV and TcVI DTUs are the result of genetic exchange between TcII and TcIII strains. On the other hand, the participation of a TcI parental in the current genome structure of these hybrid strains is a matter of debate. Here, sequences of the T. cruzi-specific 195-bp satellite DNA of TcI, TcII, TcIII, TcV, and TcVI strains have been used for inferring network genealogies. The resulting genealogy showed a high degree of reticulation, which is consistent with more than one event of hybridization between the Tc DTUs. The data also strongly suggest that TcIII is a hybrid with two distinct sets of satellite sequences, and that genetic exchange between TcI and TcII parentals occurred within the pedigree of the TcV and TcVI DTUs. Although satellite DNAs belong to the fast-evolving portion of eukaryotic genomes, in >100 satellite units of nine T. cruzi strains we found regions that display 100% identity. No DTU-specific consensus motifs were identified, inferring species-wide conservation.

Anurofauna de remanescentes de floresta Atlântica do município de São José do Barreiro, estado de São Paulo, Brasil

The municipality of Sao Jose do Barreiro (SP) located in the Atlantic Forest Domain, at elevations between 480 and 2088 m above sea level, exhibit ample topografic and climatic complexity that result in the presence of severals phytophysionomies with High Mountain Grasslands, Seasonal Semideciduous Forest, Tropical Rainforest, and Araucaria Forest. The aim of this study was to verify the anuran species richness in two different forest habitats in this area, one in a Seasonal Semideciduous Forest and the other in a Tropical Rainforest. Animals were collected by active search from April 2004 to December 2006. In the studied period, we registered 35 anuran species in nine families. The anuran richness observed in the studied region is similar to other localities considered preserved, as the Estacao Ecologica Jureia-Itatins (Peruibe, SP) and Serra do Japi (Jundiai-SP). The similarity in species composition among seven localities in Atlantic Forest in Sao Paulo state was associated to the vegetal types of the studied areas. The ample altitude variation, the presence of different types of vegetation, and the little knowledge of the local fauna in this region, make the surveys very important to support future studies on species conservation.

A novel ABCG-like transporter of Trypanosoma cruzi is involved in natural resistance to benznidazole

Benznidazole (BZ) is one of the two drugs used for Chagas disease treatment. Nevertheless therapeutic failures of BZ have been reported, which were mostly attributed to variable drug susceptibility among Trypanosoma cruzi strains. ATP-binding cassette (ABC) transporters are involved in a variety of translocation processes and some members have been implicated in drug resistance. Here we report the characterisation of the first T. cruzi ABCG transporter gene, named TcABCG1, which is over-expressed in parasite strains naturally resistant to BZ. Comparison of TcABCG1 gene sequence of two TcI BZ-resistant strains with CL Brener BZ-susceptible strain showed several single nucleotide polymorphisms, which determined 11 amino acid changes. CL Brener transfected with TcI transporter genes showed 40-47% increased resistance to BZ, whereas no statistical significant increment in drug resistance was observed when CL Brener was transfected with the homologous gene. Only in the parasites transfected with TcI genes there was 2-2.6-fold increased abundance of TcABCG1 transporter protein. The analysis in wild type strains also suggests that the level of TcABCG1 transporter is related to BZ natural resistance. The characteristics of untranslated regions of TcABCG1 genes of BZ-susceptible and resistant strains were investigated by computational tools.

Horizontal gene transfer confers fermentative metabolism in the respiratory-deficient plant trypanosomatid Phytomonas serpens

Among trypanosomatids, the genus Phytomonas is the only one specifically adapted to infect plants. These hosts provide a particular habitat with a plentiful supply of carbohydrates. Phytomonas sp. lacks a cytochrome-mediated respiratory chain and Krebs cycle, and ATP production relies predominantly on glycolysis. We have characterised the complete gene encoding a putative pyruvate/indolepyruvate decarboxylase (PDC/IPDC) (548 amino acids) of P. serpens, that displays high amino acid sequence similarity with phytobacteria and Leishmania enzymes. No orthologous PDC/IPDC genes were found in Trypanosoma cruzi or T. brucei. Conservation of the PDC/IPDC gene sequence was verified in 14 Phytomonas isolates. A phylogenetic analysis shows that Phytomonas protein is robustly monophyletic with Leishmania spp. and C. fasciculata enzymes. In the trees this clade appears as a sister group of indolepyruvate decarboxylases of γ-proteobacteria. This supports the proposition that a horizontal gene transfer event from a donor phytobacteria to a recipient ancestral trypanosome has occurred prior to the separation between Phytomonas, Leishmania and Crithidia. We have measured the PDC activity in P. serpens cell extracts. The enzyme has a Km value for pyruvate of 1.4mM. The acquisition of a PDC, a key enzyme in alcoholic fermentation, explains earlier observations that ethanol is one of the major end-products of glucose catabolism under aerobic and anaerobic conditions. This represents an alternative and necessary route to reoxidise part of the NADH produced in the highly demanding glycolytic pathway and highlights the importance of this type of event in metabolic adaptation.

A genetic cluster of xeroderma pigmentosum variant patients with two different founder mutations

Xeroderma pigmentosum (XP) is a rare human syndrome associated with hypersensitivity to sunlight and a high frequency of skin tumours at an early age. We identified a community in the state of Goias (central Brazil), a sunny and tropical region, with a high incidence of XP (17 patients among approximately 1000 inhabitants).

Draft genome sequence of a CTX-M-15-producing Klebsiella pneumoniae sequence type 340 (clonal complex 258) isolate from a food-producing animal

Klebsiella pneumoniae carrying blaCTX-M-15 have been widely disseminated in hospital settings. In this regard, most clinically important strains belong to clonal complex 28 (CC258), which includes sequence type 340 (ST340). In this study, we present the draft genome sequence of a CTX-M-15-producing ST340 K. pneumoniae strain isolated from a food-producing animal in Brazil.

Draft Genome Sequence of a Hospital-Associated Clone of Klebsiella pneumoniae ST340/CC258 Coproducing RmtG and KPC-2 Isolated from a Pediatric Patient

ABSTRACT We report here the draft genome sequence of a Klebsiella pneumoniae strain 1194/11, belonging to the hospital-associated sequence type 340 (ST340; clonal complex CC258), isolated from a catheter tip culture from a pediatric patient. The multidrug-resistant strain coproduced the 16S rRNA methyltransferase rRNA RmtG and β-lactamases KPC-2 and CTX-M-15.

Draft genome sequence of an aminoglycoside-resistant RmtG-producing Pseudomonas aeruginosa ST235 isolated from a cystic fibrosis patient

Cystic fibrosis (CF) patients are often chronically colonised or infected by non-fermenting Gram-negative bacilli, with Pseudomonas aeruginosa being the most prevalent. In this study, we report the draft genome sequence of a multidrug-resistant P. aeruginosa strain belonging to sequence type ST235, isolated from the respiratory tract of a CF patient with chronic colonisation. Whole-genome sequencing analysis revealed a 6.7Mb genome size and the presence of 12 antibiotic resistance genes, including the rmtG gene conferring high-level aminoglycoside resistance, located on the chromosome.

Auxin production by the plant trypanosomatid Phytomonas serpens and auxin homoeostasis in infected tomato fruits.

Previously we have characterized the complete gene encoding a pyruvate decarboxylase (PDC)/indolepyruvate decarboxylase (IPDC) of Phytomonas serpens, a trypanosomatid highly abundant in tomato fruits. Phylogenetic analyses indicated that the clade that contains the trypanosomatid protein behaves as a sister group of IPDCs of γ-proteobacteria. Since IPDCs are key enzymes in the biosynthesis of the plant hormone indole-3-acetic acid (IAA), the ability for IAA production by P. serpens was investigated. Similar to many microorganisms, the production of IAA and related indolic compounds, quantified by high performance liquid chromatography, increased in P. serpens media in response to amounts of tryptophan. The auxin functionality was confirmed in the hypocotyl elongation assay. In tomato fruits inoculated with P. serpens the concentration of free IAA had no significant variation, whereas increased levels of IAA-amide and IAA-ester conjugates were observed. The data suggest that the auxin produced by the flagellate is converted to IAA conjugates, keeping unaltered the concentration of free IAA. Ethanol also accumulated in P. serpens-conditioned media, as the result of a PDC activity. In the article we discuss the hypothesis of the bifunctionality of P. serpens PDC/IPDC and provide a three-dimensional model of the enzyme.