Antonio Mauro Rezende

Plasmodium falciparum malaria remains a public health concern in the Brazilian Amazon, particularly among mobile and remote populations. Antimalarial drug resistance threatens elimination efforts, particularly in the Guiana Shield, which has historically been linked to the spread of resistant parasites. We analyzed 99 P . falciparum isolates collected in Roraima State, northern Brazil (2016–2020), using targeted deep sequencing. Variants were assessed in pfcrt , pfmdr1 , and pfk13 , as well as copy number variation in pfpm2 and pfpm3 , including detection of the pfpm2/3 hybrid. High coverage enabled the detection of both dominant and minority alleles. Among the 99 isolates, the pfmdr1 -NFD haplotype was found in 6% (6/99, all likely originating from Venezuela), pfcrt -C350R in 7% (7/99), and pfpm2 and pfpm3 amplifications in 5% (5/94) and 6% (6/97), respectively, and the pfpm2/3 hybrid in 4% (4/99). No validated or candidate pfk13 mutations associated with artemisinin partial resistance were detected. This study provides the first report of the pfpm2/3 hybrid and the pfmdr1 -NFD haplotype in Brazil and confirms the presence of pfcrt -C350R in isolates from the tri-border area. These variants, associated with reduced susceptibility to lumefantrine and piperaquine, suggest that parasites in northern Brazil already carry resistance markers that are prevalent in neighboring countries. These results highlight the need for continued genomic surveillance to detect early warning signals, guide treatment policies, and contain the spread of parasites with reduced susceptibility to artemisinin-based combination therapy partner drugs.

Plasmodium falciparum malaria remains a public health concern in the Brazilian Amazon, particularly among mobile and remote populations. Antimalarial drug resistance threatens elimination efforts, particularly in the Guiana Shield, which has historically been linked to the spread of resistant parasites. We analyzed 99 P. falciparum isolates collected in Roraima State, northern Brazil (2016–2020), using targeted deep sequencing. Variants were assessed in pfcrt , pfmdr1 , and pfk13 , and copy number variation in pfpm2 and pfpm3 , including detection of the pfpm2/3 hybrid. High coverage enabled the detection of both dominant and minority alleles. Among the 99 isolates, the pfmdr1 -NFD haplotype was found in 6% (6/99, all likely originating from Venezuela), pfcrt -C350R in 7% (7/99), and pfpm2 and pfpm3 amplifications in 5% (5/94) and 6% (6/97), respectively, and the pfpm2/3 hybrid in 4% (4/99). No validated or candidate pfk13 mutations associated with artemisinin partial resistance were detected. This study provides the first report of the pfpm2/3 hybrid and the pfmdr1 -NFD haplotype in Brazil, and confirms the presence of pfcrt -C350R in isolates from the tri-border area. These variants, associated with reduced susceptibility to lumefantrine and piperaquine, suggest that parasites in northern Brazil already carry resistance markers that are prevalent in neighboring countries. These results highlight the need for continued genomic surveillance to detect early warning signals, guide treatment policies, and contain the spread of parasites with reduced susceptibility to ACT partner drugs.

Protein acetylation regulates essential processes across eukaryotes. In trypanosomatids, stage-specific acetylation suggests roles in parasite differentiation. Here, we functionally characterized zinc-dependent lysine deacetylases (DAC1, DAC3, DAC4, and DAC5) in Leishmania mexicana. CRISPR-Cas9-mediated disruption revealed that DAC1 and DAC3 are essential for procyclics, while DAC4 and DAC5 are dispensable. DAC1 and DAC5 are localized in the cytoplasm, and DAC3 and DAC4 in the nucleus. Functional analysis implicates DAC1, DAC3, and DAC5 in procyclic proliferation, whereas DAC1 and DAC5 drive promastigote-to-metacyclic differentiation. DAC5 was required for metacyclogenesis in the sand flies, the promastigote–amastigote transition, and amastigote intracellular replication. Notably, DAC5-null parasites failed to induce lesions in mice, displaying an attenuated phenotype. Proteomic profiling uncovered altered acetylation patterns in DAC mutants, linking DAC5 to cytoskeleton regulation and cell cycle control. These findings identify acetylation as a central regulator of Leishmania stage differentiation and highlight DAC5 as a key factor in parasite virulence.

Background: Chemical modifications of mRNAs constitute an alternative mechanism for gene expression regulation, which involves proteins responsible for adding, recognizing and removing these modifications. While orthologs of enzymes involved in adding m1A (TRMT6/TRMT61A) and m5C (NSUN2) modifications are present in trypanosomatid species, a clear understanding of their biological role in these parasites is necessary. Methods: To shed light on this, we genetically manipulated the TRMT61A and NSUN2 protein-encoding genes in the Leishmania mexicana species using the CRISPR-Cas9 editing technique and analyzed the impact on cell growth and differentiation as well as the global gene expression profile. Results: Deletion of the genes investigated here caused changes in the normal pattern of L. mexicana differentiation, and functional analyses of differentially expressed genes in the mutants unveiled significant biological effects. For the TRMT61A gene, transcripts related to nucleotide metabolism, translation, protein folding and refolding were affected. For the NSUN2 genes, enrichment analysis indicated impacts on biological processes mostly related to nucleotide metabolism and DNA binding. Conclusions: Our findings provide insights into the role of these methyltransferases orthologs in the regulation of trypanosomatid transcriptome, contributing to our understanding of gene expression control in this parasite. Graphical

Quorum sensing (QS) is a microbial communication mechanism that can modulate the expression of specific genes related to virulence, biofilm formation, antimicrobial resistance, enzyme production, adaptability to adverse conditions, among others. Various QS systems have been described, including the QseB/QseC system. In Klebsiella aerogenes, a nosocomial pathogen, this system remains poorly understood. This study aimed to identify the qseB and qseC genes in the genome of a clinical isolate of K. aerogenes, as well as to characterize the functions and structures of the QseB and QseC proteins. We utilized a suite of bioinformatics tools to assess the physicochemical and structural properties of these proteins, predict signal peptide presence, secretion pathways, subcellular localization, antigenic sites, and functional roles. Additionally, we modeled the three-dimensional structures and determined the active sites of QseB and QseC. Our results confirm the presence of qseB and qseC genes in the K. aerogenes. In silico analyses revealed that both proteins are stable, hydrophilic, and exhibit similar instability indices. Neither protein contains signal peptides for secretion; QseB is localized in the cytoplasm, while QseC is situated in the cellular membrane. Both proteins possess antigenic determinants. Functionally, QseB acts as a DNA-binding response regulator, whereas QseC functions as a histidine kinase. Structural models validated exhibit high-quality predictions for both proteins. Active site analysis pinpointed crucial amino acid residues involved in the biochemical activities of both proteins. These data show that the isolate has a complete QseB/QseC system that recognizes AI-3 and catecholamines and may modulate genes essential to pathogenicity.

A large proportion of HIV-coinfected visceral leishmaniasis (VL-HIV) patients exhibit chronic disease with frequent VL recurrence. However, knowledge on immunological determinants underlying the disease course is scarce. We longitudinally profiled the circulatory cellular immunity of an Ethiopian HIV cohort that included VL developers. We show that chronic VL-HIV patients exhibit high and persistent levels of TIGIT and PD-1 on CD8⁺/CD8^- T cells, in addition to a lower frequency of IFN-γ⁺ TIGIT^- CD8⁺/CD8^- T cells, suggestive of impaired T cell functionality. At single T cell transcriptome and clonal resolution, the patients show CD4⁺ T cell anergy, characterised by a lack of T cell activation and lymphoproliferative response. These findings suggest that PD-1 and TIGIT play a pivotal role in VL-HIV chronicity, and may be further explored for patient risk stratification. Our findings provide a strong rationale for adjunctive immunotherapy for the treatment of chronic VL-HIV patients to break the recurrent disease cycle.

Culex quinquefasciatus resistance to the binary (Bin) toxin, the major larvicidal component from Lysinibacillus sphaericus, is associated with mutations in the cqm1 gene, encoding the Bin-toxin receptor. Downregulation of the cqm1 transcript was found in the transcriptome of larvae resistant to the L. sphaericus IAB59 strain, which produces both the Bin toxin and a second binary toxin, Cry48Aa/Cry49Aa. Here, we investigated the transcription profiles of two other mosquito colonies having Bin resistance only. These confirmed the cqm1 downregulation and identified transcripts encoding the enzyme pantetheinase as the most downregulated mRNAs in both resistant colonies. Further quantification of these transcripts reinforced their strong downregulation in Bin-resistant larvae. Multiple genes were found encoding this enzyme in Cx. quinquefasciatus and a recombinant pantetheinase was then expressed in Escherichia coli and Sf9 cells, with its presence assessed in the midgut brush border membrane of susceptible larvae. The pantetheinase was expressed as a ~70 kDa protein, potentially membrane-bound, which does not seem to be significantly targeted by glycosylation. This is the first pantetheinase characterization in mosquitoes, and its remarkable downregulation might reflect features impacted by co-selection with the Bin-resistant phenotype or potential roles in the Bin-toxin mode of action that deserve to be investigated.

Yersinia pestis, the etiological agent of the plague, is considered a genetically homogeneous species. Brazil is currently in a period of epidemiological silence but plague antibodies are still detected in sentinel animals, suggesting disease activity in the sylvatic cycle. The present study deployed an in silico approach to analyze virulence factors among 407 Brazilian genomes of Y. pestis belonging to the Fiocruz Collection (1966–1997). The pangenome analysis associated several known virulence factors of Y. pestis in clades according to the presence or absence of genes. Four main strain clades (C, E, G, and H) exhibited the absence of various virulence genes. Notably, clade G displayed the highest number of absent genes, while clade E showed a significant absence of genes related to the T6SS secretion system and clade H predominantly demonstrated the absence of plasmid-related genes. These results suggest attenuation of virulence in these strains over time. The cgMLST analysis associated genomic and epidemiological data highlighting evolutionary patterns related to the isolation years and outbreaks of Y. pestis in Brazil. Thus, the results contribute to the understanding of the genetic diversity and virulence within Y. pestis and the potential for utilizing genomic data in epidemiological investigations.

Enterobacter aerogenes represents a non-spore-forming, facultative anaerobic, gram-negative bacterium. It presents itself as a multiresistant microorganism that is directly related to opportunistic infections, especially in the hospital environment. This bacterium has several mechanisms to remain active, we can highlight among these, the use of the CRISPR-Cas system to immunize them from infection by mobile genetic elements. The CRISPR system (short palindromic repeats at regular intervals in cluster) is a genetic tool responsible for cleaving the double strand of DNA at specific loci through Cas endonucleases. In view of the above, the present study aimed to characterize a sample of Enterobacter aerogenes from colonization regarding the CRISPR-Cas systems and compare it with a sample of infection. When analyzing the Colonization isolate (Ea5A), 31 genes were identified that were related to the CRISPR system, among these, only 4 were associated with Cas endonucleases, numerous spacers were found in the sample. Furthermore, the comparison between the colonization and infection isolates proposed that the genes are independent of the source of bacterial isolation. The CRISPR-Cas system is a new subject, but it is already considered an important tool for genetic engineering.