Meningitis caused by Aeromonas hydrophila in Oreochromis niloticus: proteomics and druggability of virulence factors

Meningitis caused by Gram-negative bacteria is a serious public health problem, causing morbidity and mortality in both children and adults. Here, we propose a novel experimental model using Nile tilapia (Oreochromis niloticus) to study neuroinflammation. The fish were infected with Aeromonas hydrophila, and the course of infection was monitored in the peripheral blood. Septicemia was obvious in the blood, while in the brain tissue, infection of the meninges was present. The histopathological examination showed suppurative meningitis, and the cellular immune response in the brain tissue during infection was mediated by microglia. These cells were morphologically characterized and phenotyped by MHC class II markers and CD68. The increased production of TNF-α, IL-1β and iNOS supported the infiltration of these cells during the neuroinflammatory process. In the proteomic analysis of A. hydrophila isolated from brain tissue, we found chemotactic and transport proteins, proteolytic enzymes and enzymes associated with the dismutation of nitric oxide (NO), as well as motor proteins and those responsible for cell division. After characterizing the most abundant proteins during the course of infection, we investigated the druggability index of these proteins and identified promising peptide sequences as molecular targets that are similar among bacteria. Thus, these findings deepened the understanding of the pathophysiology of meningitis caused by A. hydrophila. Moreover, through the proteomics analysis, important mechanisms and pathways used by the pathogen to subvert the host response were revealed, providing insights for the development of novel antibiotics and vaccines.

Characterization of a new bifunctional endo-1,4-ß-xylanase/esterase found in the rumen metagenome.

Metagenomic data mining of the Nellore cattle rumen microbiota identified a new bifunctional enzyme, endo-1,4-ß-xylanase/esterase, which was subsequently overexpressed in E. coli BL21 (DE3). This enzyme was stable at pH intervals of 5 to 6.5 and temperatures between 30 and 45 °C, and under the test conditions, it had a Vmax of 30.959 ± 2.334 µmol/min/mg, Km of 3.6 ± 0.6 mM and kcat of 2.323 ± 175 s-1. Additionally, the results showed that the enzyme is tolerant to NaCl and organic solvents and therefore is suitable for industrial environments. Xylanases are widely applicable, and the synergistic activity of endo-1,4-ß-xylanase/esterase in a single molecule will improve the degradation efficiency of heteroxylans via the creation of xylanase binding sites. Therefore, this new molecule has the potential for use in lignocellulosic biomass processing and as an animal feed food additive and could improve xylooligosaccharide production efficiency.

Proteomic analysis capsule synthesis and redox mechanisms in the intracellular survival of group B Streptococcus in fish microglia

Group B Streptococcus (GBS) causes meningitis in neonates and Nile tilapia (Oreochromis niloticus). The molecular mechanisms regulating the intracellular survival of this pathogen in the host cell are complex and crucial for the progression of infection. Thus, we propose the use of GBS-infected Nile tilapia microglia as an in vitro model system simulating infection caused by homologous bacteria in humans. We used this model to evaluate the phagocytic activity, as well as the functional aspects of the capsular proteins A, B, C, and D and the major redox enzymes, and the synergistic role of mechanisms/proteins involved in blocking phagocytic process. We observed that in the intracellular phase, GBS showed enhanced synthesis of the polysaccharide capsule and used superoxide dismutase, thioredoxin, NADH oxidase, and alkyl hydroperoxide reductase to scavenge reactive oxygen species and reactive nitrogen species produced by the host cell. Furthermore, although these virulence mechanisms were effective during the initial hours of infection, they were not able to subvert microglial responses, which partially neutralized the infection. Altogether, our findings provided important information regarding the intracellular survival mechanisms of GBS and perspectives for the production of new drugs and vaccines, through the druggability analysis of specific proteins. In conclusion, tilapia microglia serve as a potent in vitro experimental model for the study of meningitis.

Meningitis Caused by Streptococcus agalactiae in Nile Tilapia (Oreochromis niloticus): Infection and Inflammatory Response.

Streptococcus agalactiae (Sta) of Lancefield group B is the primary etiological agent of bacterial meningitis in Nile tilapia and newborn humans. Thus, the study of this disease is of fundamental importance for aquaculture and human medicine. Additionally, elucidation of the mechanisms involved in the host-pathogenic response is important for the success of new therapies. In the present study, we elucidated important aspects of the innate immune response in the brain tissue of Nile tilapia (Oreochromis niloticus) infected by Sta. The neuroinflammatory process in the meninges started with the migration of MHC class II and CD68 + cells, production of TNF-alpha, and the effective immune response to Sta was mediated by the increased iNOs+. In conclusion, the present study brings a partial understanding of the pathophysiological and neuroinflammatory mechanisms in meningitis in Sta infected tilapia, enabling important advances in the therapy of this disease as well as the possibility of using this biological model to understand human meningitis.

Meningitis caused by Streptococcus agalactiae in nile tilapia (Oreochromis niloticus): infection and inflammatory response

Streptococcus agalactiae (Sta) of Lancefield group B is the primary etiological agent of bacterial meningitis in Nile tilapia and newborn humans. Thus, the study of this disease is of fundamental importance for aquaculture and human medicine. Additionally, elucidation of the mechanisms involved in the host–pathogenic response is important for the success of new therapies. In the present study, we elucidated important aspects of the innate immune response in the brain tissue of Nile tilapia (Oreochromis niloticus) infected by Sta. The neuroinflammatory process in the meninges started with the migration of MHC class II and CD68 + cells, production of TNF-alpha, and the effective immune response to Sta was mediated by the increased iNOs+. In conclusion, the present study brings a partial understanding of the pathophysiological and neuroinflammatory mechanisms in meningitis in Sta infected tilapia, enabling important advances in the therapy of this disease as well as the possibility of using this biological model to understand human meningitis.

Proteoliposomes harboring alkaline phosphatase and nucleotide pyrophosphatase as matrix vesicle biomimetics.

We have established a proteoliposome system as an osteoblast-derived matrix vesicle (MV) biomimetic to facilitate the study of the interplay of tissue-nonspecific alkaline phosphatase (TNAP) and NPP1 (nucleotide pyrophosphatase/phosphodiesterase-1) during catalysis of biomineralization substrates. First, we studied the incorporation of TNAP into liposomes of various lipid compositions (i.e. in pure dipalmitoyl phosphatidylcholine (DPPC), DPPC/dipalmitoyl phosphatidylserine (9:1 and 8:2), and DPPC/dioctadecyl-dimethylammonium bromide (9:1 and 8:2) mixtures. TNAP reconstitution proved virtually complete in DPPC liposomes. Next, proteoliposomes containing either recombinant TNAP, recombinant NPP1, or both together were reconstituted in DPPC, and the hydrolysis of ATP, ADP, AMP, pyridoxal-5'-phosphate (PLP), p-nitrophenyl phosphate, p-nitrophenylthymidine 5'-monophosphate, and PP(i) by these proteoliposomes was studied at physiological pH. p-Nitrophenylthymidine 5'-monophosphate and PLP were exclusively hydrolyzed by NPP1-containing and TNAP-containing proteoliposomes, respectively. In contrast, ATP, ADP, AMP, PLP, p-nitrophenyl phosphate, and PP(i) were hydrolyzed by TNAP-, NPP1-, and TNAP plus NPP1-containing proteoliposomes. NPP1 plus TNAP additively hydrolyzed ATP, but TNAP appeared more active in AMP formation than NPP1. Hydrolysis of PP(i) by TNAP-, and TNAP plus NPP1-containing proteoliposomes occurred with catalytic efficiencies and mild cooperativity, effects comparable with those manifested by murine osteoblast-derived MVs. The reconstitution of TNAP and NPP1 into proteoliposome membranes generates a phospholipid microenvironment that allows the kinetic study of phosphosubstrate catabolism in a manner that recapitulates the native MV microenvironment.