Biotransformation of 2,4,6-Trinitrotoluene by a cocktail of native laccases from Pycnoporus sanguineus CS43 under oxygenic and non-oxygenic atmospheres.

2,4,6-Trinitrotoluene (TNT) is a highly toxic nitroaromatic explosive known for its environmental consequences, contaminating soil and groundwater throughout its life cycle, from production to disposal. Therefore, the urgency of developing innovative and ecological strategies to remedy the affected areas is recognized. This study reports, for the first time, the enzymatic biotransformation of TNT by a cocktail of native laccases from Pycnoporus sanguineus CS43. The laccases displayed efficient TNT conversion under both oxygenic and non-oxygenic conditions, achieving biotransformation rates of 80% and 87% within 48 h at a temperature of 60 °C and pH 7. Preliminary kinetic constants were calculated with the laccase cocktail, being a Vmax of 1.133 µM min-1 and 0.2984 µM min-1, and the Km values were 1586 µM and 458 µM, in an oxygenic and non-oxygenic atmosphere, respectively. High-performance liquid chromatography-mass spectrometry (HPLC/MS) confirmed the formation of amino dinitrotoluene isomers and hydroxylamine isomers as biotransformation products. In summary, this study suggests the potential application of laccases for the direct biotransformation of recalcitrant compounds like TNT, offering an environmentally friendly approach to address contamination issues.

ESX-3 secretion system in Mycobacterium: An overview.

Mycobacteria are microorganisms distributed in the environment worldwide, and some of them, such as Mycobacterium tuberculosis or M. leprae, are pathogenic. The hydrophobic mycobacterial cell envelope has low permeation and bacteria need to export products across their structure. Mycobacteria possess specialized protein secretion systems, such as the Early Secretory Antigenic Target 6 secretion (ESX) system. Five ESX loci have been described in M. tuberculosis, called ESX-1 to ESX-5. The ESX-3 secretion system has been associated with mycobacterial metabolism and growth. The locus of this system is highly conserved across mycobacterial species. Metallo-proteins regulate negative ESX-3 transcription in high conditions of iron and zinc. Moreover, this secretion system is part of an antioxidant regulatory pathway linked to Zinc. EccA3, EccB3, EccC3, EccD3, and EccE3 are components of the ESX-3 secretion machinery, whereas EsxG-EsxH, PE5-PPE4, and PE15-PPE20 are proteins secreted by this system. In addition, EspG3 and MycP3 are complementary proteins involved in transport and proteolysis respectively. This system is associated to mycobacterial virulence by releasing the bacteria from the phagosome and inhibiting endomembrane damage response. Furthermore, components of this system inhibit the host immune response by reducing the recognition of M. tuberculosis-infected cells. The components of the ESX-3 secretion system play a role in drug resistance and cell wall integrity. Moreover, the expression data of this system indicated that external and internal factors affect ESX-3 locus expression. This review provides an overview of new findings on the ESX-3 secretion system, its regulation, expression, and functions.

Role of esxG and esxH Genes in Drug-Resistant Mycobacterium.

Tuberculosis drug resistance (DR) is a global problem that is not fully elucidated. Previously, overexpression of esxG and esxH genes was reported in a multidrug-resistant (MDR) Mycobacterium tuberculosis isolate compared with a reference H37Rv strain. To evaluate the roles of esxG and esxH in DR, analysis of their regulatory and coding sequences in sensitive and resistant strains was performed, and the expression levels of their transcriptional regulators IdeR, Zur, and MntR were evaluated. esxG and esxH were expressed heterologously using mycobacterial constructs, and the orthologs Msmeg_0620 and Msmeg_0621 were attenuated in Mycobacterium smegmatis by antisense knockdown. We found no differences in the regulatory and coding sequences of esxG and esxH between the sensitive strain and the MDR isolate. Expression analysis of transcriptional regulators showed that ideR was upregulated in isoniazid (INH)-resistant isolates; in addition, growth inhibition of the M. smegmatis strain was observed in the presence of rifampicin (RIF) and INH when esxG and esxH were expressed heterologously, while faster growth in the presence of RIF was observed when the orthologs were attenuated. In conclusion, the expression of esxG and esxH altered the growth of Mycobacterium in the presence of INH and RIF, suggesting a potential association with DR.

Laccases: structure, function, and potential application in water bioremediation.

The global rise in urbanization and industrial activity has led to the production and incorporation of foreign contaminant molecules into ecosystems, distorting them and impacting human and animal health. Physical, chemical, and biological strategies have been adopted to eliminate these contaminants from water bodies under anthropogenic stress. Biotechnological processes involving microorganisms and enzymes have been used for this purpose; specifically, laccases, which are broad spectrum biocatalysts, have been used to degrade several compounds, such as those that can be found in the effluents from industries and hospitals. Laccases have shown high potential in the biotransformation of diverse pollutants using crude enzyme extracts or free enzymes. However, their application in bioremediation and water treatment at a large scale is limited by the complex composition and high salt concentration and pH values of contaminated media that affect protein stability, recovery and recycling. These issues are also associated with operational problems and the necessity of large-scale production of laccase. Hence, more knowledge on the molecular characteristics of water bodies is required to identify and develop new laccases that can be used under complex conditions and to develop novel strategies and processes to achieve their efficient application in treating contaminated water. Recently, stability, efficiency, separation and reuse issues have been overcome by the immobilization of enzymes and development of novel biocatalytic materials. This review provides recent information on laccases from different sources, their structures and biochemical properties, mechanisms of action, and application in the bioremediation and biotransformation of contaminant molecules in water. Moreover, we discuss a series of improvements that have been attempted for better organic solvent tolerance, thermo-tolerance, and operational stability of laccases, as per process requirements.

EVALUACIÓN DE ANTICUERPOS ANTI-Trichinella spiralis OBTENIDOS POR INMUNIZACIONES SUBLINGUALES Y CONVENCIONALES CON LA PROTEÍNA 45kDa / Evaluation of Anti-Trichinella spiralis Obtained by Sublingual and Conventional Immunizations with the 45kDa Protein

RESUMEN La trichinellosis es una enfermedad parasitaria zoonótica y cosmopolita, se debe al consumo de carne deficientemente cocida, principalmente proveniente del cerdo, diversos estudios avalan la eficacia de la administración de inmunoterapia. Se ha caracterizado un antígeno inmunodominante de 45 kDa y se plantea como objetivo evaluar la presencia de anticuerpos IgA, IgM e IgG antti-Trichinella spiralis a lo largo de la infección, así como el comportamiento en la administración de la inmunización de 45 kDa de Trichinella spiralis (T. spiralis) administrado por vía sublingual y vía parenteral. Se utilizaron 36 murinos (Long Evans), seis para la infección y purificación del antígeno de 45 kDa, 30 para formar los grupos de trabajo, control sano (cinco murinos), control infectado (cinco murinos), y 20 para los grupos experimentales, se inmunizaron dos grupos con cuatro dosis (0, 7, 14 y 21 días) del inmunógeno de 45 KDa de T. spiralis, uno por vía sublingual y otro por vía parenteral y se retaron con 500 larvas infectantes (LI) de T. spiralis siete días después de la ultima inmunización y dos grupos más se infectaron con 500 LI y se inmunizaron a las cuatro semanas postinfección por ambas vías. La respuesta se evaluó por inmunofluorescencia indirecta (IFI) por microscopia confocal para determinar la respuesta humoral con anticuerpos de clase IgG, IgM e IgA.

ABSTRACT Trichinellosis is a cosmopolitan zoonotic disease produced mainly by the consumption of poorly cooked swine meat. Several studies have probed the efficiency of immunotherapy as a method for the treatment of trichinellosis. In this work, a 45 kDa immunodominant antigen was characterized, and the presence of IgA, IgM and IgG antti-Trichinella spiralis antibodies was evaluated during the course of the infection. In addition, the differences between sublingual and parenteral administration of the 45 kDa T. spiralis antigen were determined. Long Evans rats were used both to purify the 45 kDa antigen and to evaluate the immune response produced in six different groups: healthy and infected controls; two groups of immunized murines (sublingually and parenterally) with four doses of the 45 kDa T. spiralis immunogen administered at days 0, 7, 14 and 21 and challenged with 500 T. spiralis infective larvae (IL) 7 days after the last immunization; and finally, two groups of murines infected with 500 IL ofT. spiralis, immunized at week 4 post infection by the same two routes. The humoral response was evaluated by indirect immunofluorescence by confocal microscopyin order to determine the presence of IgA, IgM and IgG antibodies.