An outbreak of severe or lethal infections by a multidrug-resistant Acinetobacter baumannii ST126 strain carrying a plasmid with blaNDM-1 and blaOXA-58 carbapenemases.

Acinetobacter baumannii poses a significant health threat because of its frequent implications in hospital outbreaks and multidrug resistance (MDR). Here, we studied four A. baumannii isolates recovered during a hospital outbreak of severe or fatal cases to elucidate their diversity and factors contributing to their increased virulence and antibiotic resistance. The isolates were identified using MALDI-ToF and characterized using comparative genomics, PCR, and antimicrobial susceptibility tests. They were classified as ST126 and exhibited fewer than five chromosomal single-nucleotide variants and the same extrachromosomal content, indicating that they are a single strain (A. baumannii AB01). A. baumannii AB01 showed an MDR phenotype that could be linked to the carriage of parC and gyrA mutations, efflux transporters, aminoglycoside resistance genes, a class C beta-lactamase, and three carbapenemases, some of which are encoded on a 72 kb plasmid. ST126 is infrequent and has not been reported in Latin America, and our genomic data indicate a plausible origin for A. baumannii AB01 within the Pan Pacific region.

Gut colonization by extended-spectrum ß-lactamase-producing Escherichia coli in dairy herd in Brazil: successful dissemination of a One Health clone.

The overuse of antimicrobials in livestock has contributed to the emergence and selection of clinically relevant multidrug-resistant bacteria. In Brazil, there is no conclusive information on the occurrence of Escherichia coli producing extended-spectrum ß-lactamase (ESßL) in cattle breeding, which is an important sector of agribusiness in this country. Herein, we investigated the presence of ESßL-positive E. coli strains in dairy cattle from a commercial farm with routine practice of therapeutic cephalosporins. Ninety-five rectal swab samples were collected from healthy dairy calves and cows under treatment with ceftiofur. Samples were screened for the presence of ESßL producers, and positive isolates were identified by MALDI-TOF, with subsequent screening for genes encoding ESßL variants by PCR and sequencing. The presence of ESßL (CTX-M-15)-producing E. coli was confirmed in calves, and lactating and dry cows. Most ESßL strains with genetic homologies ≥ 90% were grouped into two major PFGE clusters, confirming the suscessful expansion of clonally related lineages in animals from different lactating cycles, on the same property. Four representatives CTX-M-15-positive E. coli strains had their genomes sequenced, belonging to the clonal complex (CC) 23 and sequence type (ST) 90. A phylogeographical landscape of ST90 was performed revealing a global One Health linkage. Our results highlight the intestinal microbiota of dairy cattle as a hotspot for the spread of critical priority ESßL-producing E. coli and demonstrate that ST90 is an international clone genomically adapted to human and animal hosts, which deserve additional investigation to determine its zoonotic potential and impact in food chain.

Unravelling the genomic characteristics of a Klebsiella quasipneumoniae clinical isolate carrying blaNDM-1.

OBJECTIVE: Despite the increasing reports of blaNDM in Enterobacterales in Brazil, comprehensive whole genome sequencing (WGS) data remain scarce. To address this knowledge gap, our study focuses on the characterization of the genome of an New Delhi Metallo-ß-lactamase (NDM)-1-producing Klebsiella quasipneumoniae subsp. quasipneumoniae (KQPN) clinical strain isolated in Brazil. METHODS: The antimicrobial susceptibility profile of the A-73.113 strain was performed by agar dilution or broth microdilution following the Brazilian Antimicrobial Susceptibility Testing Committee/European Committee on Antimicrobial Susceptibility Testing recommendations. WGS was performed using the Illumina® NextSeq platform and the generated reads were assembled using the SPAdes software. The sequences obtained were submitted to the bioinformatics pipelines to determine the sequence type, resistome, plasmidome, and virulome. RESULTS: The A-73.113 strain was identified as KQPN and was susceptible to polymyxins (MICs, ≤0.25 µg/mL), tigecycline (MIC, 0.5 µg/mL), ciprofloxacin (MIC, 0.5 µg/mL), and levofloxacin (MIC, 1 µg/mL). WGS analysis revealed the presence of genes conferring resistance to ß-lactams (blaNDM-1, blaCTX-M-15, blaOXA-9, blaOKP-A-5, blaTEM-1), aminoglycosides [aph(3')-VI, aadA1, aac(6')-Ib], and fluoroquinolones (oqxAB, qnrS1, aac(6')-Ib-cr]. Additionally, the presence of the plasmid replicons Col(pHAD28), IncFIA(HI1), IncFIB(K) (pCAV1099-114), IncFIB(pQil), and IncFII(K), as well as virulence-encoding genes fimABCDEFGHIK (type 1 fimbria), pilW (type IV pili), iutA (aerobactin), entABCDEFS/fepABCDG/fes (Ent siderophores), iroE (salmochelin), and allABCDRS (allantoin utilization) was verified. Furthermore, we found that the A-73.113 strain belongs to ST1040. CONCLUSIONS: Here we report the genomic characteristics of an NDM-1-producing KQPN ST1040 strain isolated from blood cultures in Brazil. These data will enhance our comprehension of how this species contributes to the acquisition and dissemination of blaNDM-1 in Brazilian nosocomial settings.

The detection of KPC-2, NDM-1, and VIM-2 carbapenemases in international clones isolated from fresh vegetables highlights an emerging food safety issue.

Resistance to carbapenems emerged in clinical settings and has rapidly spread to other sectors, such as food and the environment, representing a One Health problem. In this regard, vegetables contaminated by critical priority pathogens have raised global concerns. Here, we have performed a whole-genome sequence-based analysis of extensively drug-resistant Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa strains isolated from cabbage, spinach, and lettuce, respectively. Genomic analysis revealed the emergence of international and high-risk clones belonging to ST340, ST155, and ST233, harboring a broad resistome to clinically important antimicrobials. In this context, K. pneumoniae, E. coli, and P. aeruginosa strains carried blaKPC-2, blaNDM-1, and blaVIM-2, respectively. The blaKPC-2 gene with a non-Tn4401 element (NTEKPC-Ic) was located on an IncX3-IncU plasmid, while the blaVIM-2 gene was associated with a Tn402-like class 1 integron, In559, on the chromosome. Curiously, the blaNDM-1 gene coexisted with the blaPER-2 gene on an IncC plasmid and the regions harboring both genes contained sequences of Tn3-like element ISKox2-like family transposase. Comparative genomic analysis showed interspecies and clonal transmission of carbapenemase-encoding genes at the human-animal-environmental interface. These findings raise a food safety alert about hospital-associated carbapenemase producers, supporting that fresh vegetables can act as a vehicle for the spread of high-risk clones.

Temozolomide resistance mechanisms: unveiling the role of translesion DNA polymerase kappa in glioblastoma spheroids in vitro.

Temozolomide (TMZ) is the leading therapeutic agent for combating Glioblastoma Multiforme (GBM). Nonetheless, the persistence of chemotherapy-resistant GBM cells remains an ongoing challenge, attributed to various factors, including the translesion synthesis (TLS) mechanism. TLS enables tumor cells to endure genomic damage by utilizing specialized DNA polymerases to bypass DNA lesions. Specifically, TLS polymerase Kappa (Polκ) has been implicated in facilitating DNA damage tolerance against TMZ-induced damage, contributing to a worse prognosis in GBM patients. To better understand the roles of Polκ in TMZ resistance, we conducted a comprehensive assessment of the cytotoxic, antiproliferative, antimetastatic, and genotoxic effects of TMZ on GBM (U251MG) wild-type (WTE) and TLS Polκ knockout (KO) cells, cultivated as three-dimensional (3D) tumor spheroids in vitro. Initial results revealed that TMZ: (i) induces reductions in GBM spheroid diameter (10-200 µM); (ii) demonstrates significant cytotoxicity (25-200 µM); (iii) exerts antiproliferative effects (≤25 µM) and promotes cell cycle arrest (G2/M phase) in Polκ KO spheroids when compared with WTE counterparts. Furthermore, Polκ KO spheroids exhibit elevated levels of cell death (Caspase 3/7) and display greater genotoxicity (53BP1) than WTE following TMZ exposure. Concerning antimetastatic effects, TMZ impedes invadopodia (3D invasion) more effectively in Polκ KO than in WTE spheroids. Collectively, the results suggest that TLS Polκ plays a vital role in the survival, cell death, genotoxicity, and metastatic potential of GBM spheroids in vitro when subjected to TMZ treatment. While the precise mechanisms underpinning this resistance remain elusive, TLS Polκ emerges as a potential therapeutic target for GBM patients.

Genomic analysis of the first multidrug-resistant ESBL-producing high-risk clonal lineage Klebsiella pneumoniae ST147 isolated from a cat with urinary tract infection.

Urinary tract infections (UTIs) are pervasive in human and veterinary medicine, notably affecting companion animals. These infections frequently lead to the prescription of antibiotics, contributing to the rise of antimicrobial-resistant bacteria. This escalating concern is underscored by the emergence of a previously undocumented case: a high-risk clone, broad-spectrum cephalosporin-resistant K. pneumoniae ST147 strain, denoted USP-275675, isolated from a cat with UTI. Characterized by a multidrug-resistant (MDR) profile, whole genome sequencing exposed several antimicrobial-resistance genes, notably blaCTX-M-15, blaTEM-1B, blaSHV-11, and blaOXA-1. ST147, recognized as a high-risk clone, has historically disseminated globally and is frequently associated with carbapenemases and extended-spectrum ß-lactamases. Notably, the core-genome phylogeny of K. pneumoniae ST147 strains isolated from urine samples revealed a unique aspect of the USP-276575 strain. Unlike its counterparts, it did not cluster with other isolates. However, a broader examination incorporating strains from both human and animal sources unveiled a connection between USP-276575 and a Portuguese strain from chicken meat. Both were part of a larger cluster of ST147 strains spanning various geographic locations and sample types, sharing commonalities such as IncFIB or IncR plasmids. This elucidates the MDR signature inherent in widespread K. pneumoniae ST147 strains carrying these plasmids, highlighting their pivotal role in disseminating antimicrobial resistance (AMR). Finally, discovering the high-risk clone K. pneumoniae ST147 in a domestic feline with a UTI in Brazil highlights the urgent need for thorough AMR surveillance through a One Health approach.

Exploring the cellulolytic activity of environmental mycobacteria.

Although studies on non-tuberculous mycobacteria have increased in recent years because they cause a considerable proportion of infections, their cellulolytic system is still poorly studied. This study presents a characterization of the cellulolytic activities of environmental mycobacterial isolates derived from soil and water samples from the central region of Argentina, aimed to evaluate the conservation of the mechanism for the degradation of cellulose in this group of bacteria. The molecular and genomic identification revealed identity with Mycolicibacterium septicum. The endoglucanase and total cellulase activities were assessed both qualitatively and quantitatively and the optimal enzymatic conditions were characterized. A specific protein of around 56 kDa with cellulolytic activity was detected in a zymogram. Protein sequences possibly arising from a cellulase were identified by mass spectrometry-based shotgun proteomics. Results showed that M. septicum encodes for cellulose- and hemicellulose-related degrading enzymes, including at least an active ß-1,4 endoglucanase enzyme that could be useful to improve its survival in the environment. Given the important health issues related to mycobacteria, the results of the present study may contribute to the knowledge of their cellulolytic system, which could be important for their ability to survive in many different types of environments.

Emergence of carbapenem-resistant Klebsiella pneumoniae species complex from agrifood systems: detection of ST6326 co-producing KPC-2 and NDM-1.

BACKGROUND: Klebsiella pneumoniae species complex (KpSC) is an important disseminator of carbapenemase-encoding genes, mainly blaKPC-2 and blaNDM-1, from hospitals to the environment. Consequently, carbapenem-resistant strains can be spread through the agrifood system, raising concerns about food safety. This study therefore aimed to isolate carbapenem-resistant KpSC strains from the agricultural and environmental sectors and characterize them using phenotypic, molecular, and genomic analyses. RESULTS: Klebsiella pneumoniae and Klebsiella quasipneumoniae strains isolated from soils used for lemon, guava, and fig cultivation, and from surface waters, displayed an extensive drug-resistance profile and carried blaKPC-2, blaNDM-1, or both. In addition to carbapenemase-encoding genes, KpSC strains harbor a broad resistome (antimicrobial resistance and metal tolerance) and present putative hypervirulence. Soil-derived K. pneumoniae strains were assigned as high-risk clones (ST11 and ST307) and harbored the blaKPC-2 gene associated with Tn4401b and Tn3-like elements on IncN-pST15 and IncX5 plasmids. In surface waters, the coexistence of blaKPC-2 and blaNDM-1 genes was identified in K. pneumoniae ST6326, a new carbapenem-resistant regional Brazilian clone. In this case, blaKPC-2 with Tn4401a isoform and blaNDM-1 associated with a Tn125-like transposon were located on different plasmids. Klebsiella quasipneumoniae ST526 also presented the blaNDM-1 gene associated with a Tn3000 transposon on an IncX3 plasmid. CONCLUSION: These findings provide a warning regarding the transmission of carbapenemase-positive KpSC across the agricultural and environmental sectors, raising critical food safety and environmental issues. © 2024 Society of Chemical Industry.

Genomic characterization of carbapenemase-producing Klebsiella pneumoniae ST307 revealed multiple introductions in Buenos Aires, Argentina.

OBJECTIVES: To describe at genomic level nine carbapenemase-producing Klebsiella pneumoniae ST307 (Kp-ST307) clinical isolates recovered in Buenos Aires during 2017 to 2021, investigating their resistome, virulome, and phylogeny. METHODS: Antimicrobial susceptibility was determined according to Clinical and Laboratory Standards Intitute (CLSI). Genomic DNA was sequenced by Illumina MiSeq and analysed using SPAdes, PROKKA, and Kleborate. Phylogeny of 355 randomly selected Kp-ST307 genomes and those from nine local isolates was inferred by a maximum-likelihood approach. The tree was visualized using Microreact. RESULTS: Besides resistance to ß-lactams and fluoroquinolones, six out of nine Kp-ST307 were also resistant to ceftazidime/avibactam (CZA). This difficult-to-treat resvistance phenotype was mediated by blaSHV-28 and GyrA-83I/ParC-80I mutations in addition to carbapenemase coding genes. Among CZA susceptible isolates, two of them harboured blaKPC-3 while the other harboured blaKPC-2+blaCTX-M-15. Regarding CZA-resistant isolates, three harboured blaKPC-3+blaNDM-1+blaCMY-6, two carried blaKPC-2+blaNDM-5+blaCTX-M-15, and blaNDM-5+blaCTX-M-15 were detected in the remaining isolate. Furthermore, five colistin-resistant isolates presented a nonsense mutation in mgrB. Global Kp-ST307 isolates were distributed in two deep-branching lineages while local isolates were set in the main clade of the phylogenetic tree. The five isolates from the same hospital, harbouring blaKPC-3 or blaKPC-3+blaNDM-1+blaCMY-6, clustered in a monophyletic subclade with Italian isolates. Also, an isolate harbouring blaKPC-2+blaNDM-5+blaCTX-M-15 recovered in another hospital was closed to this group. The remaining local Kp-ST307 were grouped in other subclades containing isolates of diverse geographical origin. CONCLUSION: The inferred resistome was consistent with the resistant phenotype. Phylogeny suggested multiple introduction events in our region and a single major introduction in one hospital followed by local spread.

Genetic diversity of KPC-2-producing Klebsiella pneumoniae complex from aquatic ecosystems.

During the COVID-19 pandemic, the occurrence of carbapenem-resistant Klebsiella pneumoniae increased in human clinical settings worldwide. Impacted by this increase, international high-risk clones harboring carbapenemase-encoding genes have been circulating in different sources, including the environment. The blaKPC gene is the most commonly disseminated carbapenemase-encoding gene worldwide, whose transmission is carried out by different mobile genetic elements. In this study, blaKPC-2-positive Klebsiella pneumoniae complex strains were isolated from different anthropogenically affected aquatic ecosystems and characterized using phenotypic, molecular, and genomic methods. K. pneumoniae complex strains exhibited multidrug-resistant and extensively drug-resistant profiles, spotlighting the resistance to carbapenems, ceftazidime-avibactam, colistin, and tigecycline, which are recognized as last-line antimicrobial treatment options. Molecular analysis showed the presence of several antimicrobial resistance, virulence, and metal tolerance genes. In-depth analysis showed that the blaKPC-2 gene was associated with three different Tn4401 isoforms (i.e., Tn4401a, Tn4401b, and Tn4401i) and NTEKPC elements. Different plasmid replicons were detected and a conjugative IncN-pST15 plasmid harboring the blaKPC-2 gene associated with Tn4401i was highlighted. K. pneumoniae complex strains belonging to international high-risk (e.g., ST11 and ST340) and unusual clones (e.g., ST323, ST526, and ST4216) previously linked to clinical settings. In this context, some clones were reported for the first time in the environmental sector. Therefore, these findings evidence the occurrence of carbapenemase-producing K. pneumoniae complex strains in aquatic ecosystems and contribute to the monitoring of carbapenem resistance worldwide.

Identification, Classification, and Transcriptional Analysis of Rab GTPase Genes from Tomato (Solanum lycopersicum) Reveals Salt Stress Response Genes.

Salinity in plants generates an osmotic and ionic imbalance inside cells that compromises the viability of the plant. Rab GTPases, the largest family within the small GTPase superfamily, play pivotal roles as regulators of vesicular trafficking in plants, including the economically important and globally cultivated tomato (Solanum lycopersicum). Despite their significance, the specific involvement of these small GTPases in tomato vesicular trafficking and their role under saline stress remains poorly understood. In this work, we identified and classified 54 genes encoding Rab GTPases in cultivated tomato, elucidating their genomic distribution and structural characteristics. We conducted an analysis of duplication events within the S. lycopersicum genome, as well as an examination of gene structure and conserved motifs. In addition, we investigated the transcriptional profiles for these Rab GTPases in various tissues of cultivated and wild tomato species using microarray-based analysis. The results showed predominantly low expression in most of the genes in both leaves and vegetative meristem, contrasting with notably high expression levels observed in seedling roots. Also, a greater increase in gene expression in shoots from salt-tolerant wild tomato species was observed under normal conditions when comparing Solanum habrochaites, Solanum pennellii, and Solanum pimpinellifolium with S. lycopersicum. Furthermore, an expression analysis of Rab GTPases from Solanum chilense in leaves and roots under salt stress treatment were also carried out for their characterization. These findings revealed that specific Rab GTPases from the endocytic pathway and the trans-Golgi network (TGN) showed higher induction in plants exposed to saline stress conditions. Likewise, disparities in gene expression were observed both among members of the same Rab GTPase subfamily and between different subfamilies. Overall, this work emphasizes the high degree of conservation of Rab GTPases, their high functional diversification in higher plants, and the essential role in mediating salt stress tolerance and suggests their potential for further exploration of vesicular trafficking mechanisms in response to abiotic stress conditions.

Evaluation of the rs35996865 polymorphism of the ROCK1 gene in sepsis.

OBJECTIVE: Sepsis is a complex and serious medical condition resulting from the activation of an innate host response to infections. The etiology of sepsis is complex and can be influenced by genetic susceptibility. The purpose of the present study was to investigate a possible association of Rho-kinase 1 (ROCK1) gene polymorphism with sepsis in a Turkish population. METHODS: The study group consisted of 100 unrelated patients with sepsis and 100 healthy controls. Genomic DNA was isolated from peripheral leukocytes from EDTA-containing blood using the QIAamp DNA Blood Mini Kit. ROCK1 gene rs35996865 and rs112130712 (Lys1054Arg) polymorphisms were analyzed in genomic DNA using the LightCycler 480 II real-time polymerase chain reaction. RESULTS: There were no significant differences in allele and genotype frequencies for ROCK1 gene rs35996865 polymorphism between the patients with sepsis and control group (p>0.05). Additionally, no association was detected between the rs35996865 polymorphism and mortality in the patient group. No polymorphism was detected with ROCK1 gene rs112130712 (Lys1054Arg) in our study groups. CONCLUSIONS: Our data showed that there is no marked association between the rs35996865 polymorphism and sepsis. Therefore, these results suggest that ROCK1 gene rs35996865 polymorphism is not risk factor for the development of sepsis in the Turkish population.

Spread of Gamma (P.1) Sub-Lineages Carrying Spike Mutations Close to the Furin Cleavage Site and Deletions in the N-Terminal Domain Drives Ongoing Transmission of SARS-CoV-2 in Amazonas, Brazil.

The Amazonas was one of the most heavily affected Brazilian states by the COVID-19 epidemic. Despite a large number of infected people, particularly during the second wave associated with the spread of the Variant of Concern (VOC) Gamma (lineage P.1), SARS-CoV-2 continues to circulate in the Amazonas. To understand how SARS-CoV-2 persisted in a human population with a high immunity barrier, we generated 1,188 SARS-CoV-2 whole-genome sequences from individuals diagnosed in the Amazonas state from 1st January to 6th July 2021, of which 38 were vaccine breakthrough infections. Our study reveals a sharp increase in the relative prevalence of Gamma plus (P.1+) variants, designated Pango Lineages P.1.3 to P.1.6, harboring two types of additional Spike changes: deletions in the N-terminal (NTD) domain (particularly Δ144 or Δ141-144) associated with resistance to anti-NTD neutralizing antibodies or mutations at the S1/S2 junction (N679K or P681H) that probably enhance the binding affinity to the furin cleavage site, as suggested by our molecular dynamics simulations. As lineages P.1.4 (S:N679K) and P.1.6 (S:P681H) expanded (Re > 1) from March to July 2021, the lineage P.1 declined (Re < 1) and the median Ct value of SARS-CoV-2 positive cases in Amazonas significantly decreases. Still, we did not find an increased incidence of P.1+ variants among breakthrough cases of fully vaccinated patients (71%) in comparison to unvaccinated individuals (93%). This evidence supports that the ongoing endemic transmission of SARS-CoV-2 in the Amazonas is driven by the spread of new local Gamma/P.1 sublineages that are more transmissible, although not more efficient to evade vaccine-elicited immunity than the parental VOC. Finally, as SARS-CoV-2 continues to spread in human populations with a declining density of susceptible hosts, the risk of selecting more infectious variants or antibody evasion mutations is expected to increase. IMPORTANCE The continuous evolution of SARS-CoV-2 is an expected phenomenon that will continue to happen due to the high number of cases worldwide. The present study analyzed how a Variant of Concern (VOC) could still circulate in a population hardly affected by two COVID-19 waves and with vaccination in progress. Our results showed that the answer behind that was a new generation of Gamma-like viruses, which emerged locally carrying mutations that made it more transmissible and more capable of spreading, partially evading prior immunity triggered by natural infections or vaccines. With thousands of new cases daily, the current pandemics scenario suggests that SARS-CoV-2 will continue to evolve and efforts to reduce the number of infected subjects, including global equitable access to COVID-19 vaccines, are mandatory. Thus, until the end of pandemics, the SARS-CoV-2 genomic surveillance will be an essential tool to better understand the drivers of the viral evolutionary process.

Genomic epidemiology of rifampicin ADP-ribosyltransferase (Arr) in the Bacteria domain.

Arr is an ADP-ribosyltransferase enzyme primarily reported in association with rifamycin resistance, which has been used to treat tuberculosis in addition to Gram-positive infections and, recently, pan-resistant Gram-negative bacteria. The arr gene was initially identified on the Mycolicibacterium smegmatis chromosome and later on Proteobacteria plasmids. This scenario raised concerns on the distribution and spread of arr, considering the Bacteria domain. Based on 198,082 bacterial genomes/metagenomes, we performed in silico analysis, including phylogenetic reconstruction of Arr in different genomic contexts. Besides, new arr alleles were evaluated by in vitro analysis to assess their association with rifampin resistance phenotype. The arr gene was prevalent in thousands of chromosomes and in hundreds of plasmids from environmental and clinical bacteria, mainly from the phyla Actinobacteria, Proteobacteria, Firmicutes, and Bacteroidetes. Furthermore, this gene was identified in other and new genomic contexts. Interestingly, Arr sequences associated with rifampin resistance were distributed across all phylogeny, indicating that, despite the diversity, their association with rifampin resistance phenotype were maintained. In fact, we found that the key residues were highly conserved. In addition, other analyzes have raised evidence of another Arr function, which is related to guanidine metabolism. Finally, this scenario as a whole also suggested the Actinobacteria phylum as a potential ancestral source of arr within the Bacteria domain.

A clinical Pseudomonas chlororaphis isolate harboring a new blaVIM-2 borne integron, In2088, isolated from bloodstream infection in a newborn patient.

We report the isolation and genomic characterization of a VIM-2 producing Pseudomonas chlororaphis causing bloodstream infection in a newborn in Brazil. A new integron, In2088 (intI1-blaVIM-2-aacA7-aacA27-gcu241), was identified and the first P. chlororaphis genome from a clinical isolate was deposited in public databases.

The gene repertoire of the main cysteine protease of Trypanosoma cruzi, cruzipain, reveals four sub-types with distinct active sites.

Cruzipains are the main papain-like cysteine proteases of Trypanosoma cruzi, the protozoan parasite that causes Chagas disease. Encoded by a multigenic family, previous studies have estimated the presence of dozens of copies spread over multiple chromosomes in different parasite strains. Here, we describe the complete gene repertoire of cruzipain in three parasite strains, their genomic organization, and expression pattern throughout the parasite life cycle. Furthermore, we have analyzed primary sequence variations among distinct family members as well as structural differences between the main groups of cruzipains. Based on phylogenetic inferences and residue positions crucial for enzyme function and specificity, we propose the classification of cruzipains into two families (I and II), whose genes are distributed in two or three separate clusters in the parasite genome, according with the strain. Family I comprises nearly identical copies to the previously characterized cruzipain 1/cruzain, whereas Family II encompasses three structurally distinct sub-types, named cruzipain 2, cruzipain 3, and cruzipain 4. RNA-seq data derived from the CL Brener strain indicates that Family I genes are mainly expressed by epimastigotes, whereas trypomastigotes mainly express Family II genes. Significant differences in the active sites among the enzyme sub-types were also identified, which may play a role in their substrate selectivity and impact their inhibition by small molecules.

LOXL3 Silencing Affected Cell Adhesion and Invasion in U87MG Glioma Cells.

Lysyl oxidase-like 3 (LOXL3), belonging to the lysyl oxidase family, is responsible for the crosslinking in collagen or elastin. The cellular localization of LOXL3 is in the extracellular space by reason of its canonical function. In tumors, the presence of LOXL3 has been associated with genomic stability, cell proliferation, and metastasis. In silico analysis has shown that glioblastoma was among tumors with the highest LOXL3 expression levels. LOXL3 silencing of U87MG cells by siRNA led to the spreading of the tumor cell surface, and the transcriptome analysis of these cells revealed an upregulation of genes coding for extracellular matrix, cell adhesion, and cytoskeleton components, convergent to an increase in cell adhesion and a decrease in cell invasion observed in functional assays. Significant correlations of LOXL3 expression with genes coding for tubulins were observed in the mesenchymal subtype in the TCGA RNA-seq dataset of glioblastoma (GBM). Conversely, genes involved in endocytosis and lysosome formation, along with MAPK-binding proteins related to focal adhesion turnover, were downregulated, which may corroborate the observed decrease in cell viability and increase in the rate of cell death. Invasiveness is a major determinant of the recurrence and poor outcome of GBM patients, and downregulation of LOXL3 may contribute to halting the tumor cell invasion.