Cryptococcus neoformans is a common opportunistic pathogen, exhibiting pronounced neurotropism that often results in cryptococcal meningitis. Its invasive ability is closely associated with multiple factors, including capsular polysaccharides, melanin, hydrolases, and adaptability to the host environment. Conventional diagnostic methods such as fungal culture and India ink staining, though still in use, have notable limitations, whereas emerging techniques like molecular diagnostics, imaging technologies, and biochips have significantly enhanced the diagnostic accuracy and sensitivity. In clinical treatment, amphotericin B and fluconazole are widely used as first-line antifungals, while the resistance to azoles is a growing problem and results in an elevated rate of clinical treatment failure. This is mainly attributed to target alterations, upregulation of efflux pump expression and genomic ploidy changes. Recent studies on virulence factors and resistance mechanisms have driven the development of novel antifungal therapies, including drug repurposing, novel drug development, and innovative drug delivery strategies. This article reviews the latest research in the virulence factors, diagnostic techniques, antifungal resistance mechanisms, and therapeutic development of C. neoformans, providing insights into the clinical management of cryptococcosis.

[Objective] Phosphate-solubilizing bacteria (PSB) can increase available phosphorus by promoting the transformation of different phosphorus forms in soil. However, the phosphate-solubilizing ability of PSB is influenced by soil texture. Therefore, we studied the changes of phosphorus fractions in red soil with different textures and treated with PSB, with the aims of improving the efficient utilization of soil phosphorus. [Methods] We collected the rhizosphere soil samples of four main Camellia oleifera varieties (‘Huashuo’ ‘Huaxin’ ‘Huajin’ and ‘Changlin No. 40’) in Liuyang City, Hunan Province. PSB strains were isolated, purified, screened, and identified by plate coating, transparent circle method, molybdenum-antimony anti-spectrophotometric method, and 16S rRNA gene sequencing, and a batch of highly efficient PSB strains were obtained. The phosphate-solubilizing abilities of the obtained PSB strains were investigated with different phosphorus sources (iron phosphate, aluminum phosphate, calcium phosphate, and calcium phytate). PSB were inoculated into the red soil samples added with 0, 20% and 40% perlite (0%PR, 20%PR and 40PR%) to clarify the changes in phosphorus fractions in the red soil samples with different textures. [Results] A total of 57 strains of PSB were isolated from the rhizosphere of C. oleifera, of which strains CL37, HS5, and CL36 exhibited stronger phosphate-solubilizing abilities. The three strains were identified by 16S rRNA gene sequencing and named Pantoea sp. CL37, Burkholderia sp. HS5, and Burkholderia sp. CL36, respectively. The three strains showed significant differences in their ability to solubilize different phosphorus sources, with the highest solubilizing ability for calcium phytate and calcium phosphate. Compared with CK, the inoculation of PSB increased the available phosphorus (AP) in soil by 8.90%-54.60% and 1.90%-56.00% in 20%PR and 40%PR, respectively. The inoculation with PSB increased Fe-P, Resin-P, NaHCO₃-Pi, and NaOH-Pi, which showed a tendency of first increasing and then decreasing along with the increase in the addition of perlite in red soil. Meanwhile, PSB decreased the content of HCl-Pi and Residual-P, and the decrease in HCl-Pi was more pronounced in 20%PR than in 0%PR and 40%PR. Of all treatments, the inoculation with HS5 led to the highest increases in Fe-P, Resin-P, NaHCO₃-Pi, and NaOH-Pi in the soil samples analyzed. Correlation analysis and random forest analysis suggested that AP was mainly affected by Resin-P, Al-P, NaOH-Pi, acid phosphatase, urease, pH, and NaOH-Po. [Conclusion] The phosphate-solubilizing pathways of PSB vary in red soil with different textures. Strain HS5 has a strong phosphorus-transforming ability and is more conducive to phosphorus transformation in the red soil with 20%PR.

[Objective] To express the recombinant nonstructural protein NSP1 of the porcine reproductive and respiratory syndrome virus (PRRSV) strain NADC 30, evaluate its immune effect in mice, and explore the potential value of the nonstructural proteins of PRRSV as vaccine antigens. [Methods] The prokaryotic expression system was used to express NSP1 of NADC30. After purification, the expression and antibody reactivity of NSP1 in vitro were identified by Western blotting. After mice were immunized with NSP1, the levels of cellular and humoral immunity induced by NSP1 were measured. The level of neutralizing antibody induced by NSP1 was evaluated by the virus neutralization assay. [Results] The target gene of NSP1 was connected to the Escherichia coli pET-28a vector for prokaryotic expression. Western blotting identified that the recombinant protein NSP1 was correctly expressed and had antibody reactivity. After mice were immunized with the confirmed recombinant protein NSP1, the levels of interferon (IFN)-γ and tumor necrosis factor (TNF)-α in the spleen lymphocytes of mice were elevated, and cellular immunity was stimulated. At the same time, the recombinant protein NSP1 significantly improved the proliferation of spleen lymphocytes in mice. ELISA results suggested that the level of specific antibodies in the serum rose after immunization. Further analysis of the specific antibody subtypes (IgG2a and IgG1) produced showed that the type of immunity stimulated by recombinant NSP1 was biased to Th2 humoral immunity. In addition, the virus neutralization assay showed that the recombinant protein NSP1 had a good virus neutralization ability, with the neutralization titer of 1:37 on day 28 and 1:31 on day 42, which were significantly higher than those of the PBS control group and had no difference from those of the commercial vaccine group. [Conclusion] The recombinant nonstructural protein NSP1 of PRRSV can stimulate cellular and humoral immunity in mice and has a good virus neutralization ability, which provides a new idea for the development of next-generation PRRSV vaccines.

Aeromonas hydrophila is a pathogen that can infect both fish and mammals, including humans. [Objective] To construct the tolA-deleted strain of A. hydrophila ATCC 7966 and use this strain to explore the biological functions of tolA. [Methods] We constructed the tolA-deleted strain AhΔtolA by homologous recombination and characterized the physiological phenotype of AhΔtolA. Transcriptome sequencing was performed to compare the gene expression between the wild type (WT) and AhΔtolA. [Results] The cell morphology of AhΔtolA was changed. The deletion of tolA significantly enhanced the sensitivity to sodium deoxycholate and oxidative stress, while significantly reducing the biofilm formation and the expression levels of several virulence genes. The yield of outer membrane vesicles was significantly increased in AhΔtolA. Transcriptomic analysis data showed that a total of 300 differentially expressed genes (DEGs) were screened between WT and AhΔtolA, including171 genes with up-regulated expression and 129 genes with down-regulated expression. GO enrichment analysis showed that the DEGs were mainly enriched in the oxidation-reduction process, metabolic process, outer membrane, and oxidoreductase activity. KEGG pathway enrichment analysis showed that the DEGs were mainly enriched in the biosynthesis of secondary metabolites, microbial metabolism in diverse environments, biosynthesis of cofactors, and biosynthesis of amino acids. [Conclusion] This study gives an insight into the roles of tolA in A. hydrophila and provides information about the metabolic pathways involving tolA. These results provide a theoretical reference for the prevention and control of A. hydrophila.

[Objective] To analyze the metabolic substrates required for the sclerotial formation of Rhizoctonia solani and understand the influence of nutritional elements and environmental factors on this process. [Methods] Biolog phenotypic microarray was used to study the effects of 663 nutritional substances, 96 osmotic environments, and 96 pH environments on the sclerotial formation of R. solani. [Results] Among the tested nutritional substances and environmental conditions, 19/95 carbon sources, 21/95 nitrogen sources, 16/94 phosphorus and sulfur sources, 69/94 nutritional supplements, 61/282 peptide nitrogen sources, 28/96 osmotic environments, and 40/96 pH environments induced the sclerotial formation of R. solani. Notably, N-acetyl-d-glucosamine, uridine 3′-monophosphate, phosphoryl choline, and five dipeptides (Arg-Trp, Met-Arg, Pro-Phe, Val-Tyr, and Val-Met), as well as three environmental conditions of 10 mmol/L and 20 mmol/L ammonium sulfate at pH 8.0, and pH 4.5+l-proline, significantly induced the sclerotial formation of R. solani. R. solani formed sclerotia in the environments with a broad range of pH 4.0-10.0. The KEGG analysis indicated that the substances inducing sclerotial formation were primarily involved in metabolic pathways, ABC transporters, secondary metabolite biosynthesis, and d-amino acid metabolism. [Conclusion] Nutrient limitation and environmental stress are key factors inducing the sclerotial formation of R. solani. Under nutrient-restricted conditions, the suitable substances for inducing sclerotial formation include five carbon sources (d-sorbitol, d-xylose, N-acetyl-d-galactosamine, d-arabinose, and d-melezitose), three nitrogen sources (N-acetyl-d-glucosamine, adenosine, and thymidine), two phosphorus sources (uridine 3′-monophosphate and phosphoryl choline), one nutritional supplement (Tween-80), and five peptide nitrogen sources (Arg-Trp, Met-Arg, Pro-Phe, Val-Tyr, and Val-Met). The suitable osmotic environments were 10 mmol/L and 20 mmol/L ammonium sulfate at pH 8.0, and the suitable pH environments were pH 4.0-4.5 and pH 9.5-10.0. These findings provide a foundation for understanding the sclerotial formation mechanism of R. solani.

[Objective] To explore the technical feasibility of using transgenic microalgae to control Aedes that transmit diseases such as dengue fever. [Methods] Taking the neurotransmitter transporter gamma-aminobutyric acid receptor gene (gat) of Aedes as a target, we constructed a shRNA-expressing vector and then transferred the vector into Chlamydomonas reinhardtiii CC124 and Chlorella vulgaris HOC5 through electroporation. The recombinant microalgae were used to feed the larvae and adults of Aedes albopictus. [Results] The transgenic algae delayed the larval development and had obvious lethal effects on the larvae and adults of A. albopictus, causing the mortality rates of 75.56% and 58.67%, respectively. The expression level of gat in the larvae was significantly down-regulated. [Conclusion] Deploying gat-shRNA transgenic microalgae in enclosed water environments to suppress mosquito populations in the vicinity is technically feasible. This strategy provides a new perspective on using biological methods to control mosquitoes and block the transmission of severe infectious diseases such as dengue fever and Zika virus disease.

Tuberculosis is an ancient zoonotic disease that poses a serious threat to the health of humans and animals worldwide. Mycobacteriumtuberculosis (Mtb) is the primary causative agent of tuberculosis. As a bridge between innate and adaptive immunity, dendritic cells (DCs) play a pivotal role in controlling Mtb infection by utilizing their potent antigen-presenting capacity to activate the adaptive immune response of the host and thus resist further infection. In recent years, more and more studies have shown that Mtb can evade host immune defenses by regulating DC differentiation and maturation, interfering with phagocytosis and autophagy, and inhibiting the expression of antigen presentation-related molecules, thus causing persistent infection. This review summarizes the current research on the molecular mechanisms of Mtb in regulating DC antigen presentation, aiming to provide insights for further study of Mtb-DC interaction mechanism and development of prevention and control strategies for tuberculosis.

Bacillus subtilis is a generally recognized as safe (GRAS) probiotic and an excellent industrial chassis strain. It possesses advantages such as strong heterologous protein secretion capability, robust growth with low-quality carbon sources, and negligible codon bias. Since 2016, clustered regularly interspaced short palindromic repeats (CRISPR)-based gene editing has been successfully applied to B. subtilis, enabling precise genetic modifications, including point mutations, gene knockout, foreign gene insertion, gene expression regulation, and base editing. These advancements have significantly promoted the development of B. subtilis as an efficient microbial cell factory and have shown broad application potential in agriculture, pharmaceuticals, food production, and synthetic biology. This paper systematically review the development of the CRISPR system in B. subtilis and summarize its application in the efficient production of various products. The aim is to provide insights into the targeted optimization of metabolic pathways in B. subtilisvia the CRISPR system to achieve efficient and stable industrial production of target products, as well as to offer references for the further development and application of novel gene editing systems.

[Objective] To explore the physiochemical response mechanism of a model algal strain Chlamydomonas reinhardtii that hyper-accumulates oils to exposure of naphthenic acids (NAs). [Methods] The impacts of a typical NA, cyclohexanecarboxylic acid (CHCA), on the physiochemical parameters, including growth, photosynthetic activity, pH value of the culture, uptake of nitrogen and phosphorus, and biochemical constituents (lipids, carbohydrates, proteins, and pigments), of C. reinhardtii with high initial cell density under high light-nitrogen repletion (HL+N) and high light-nitrogen deprivation (HL-N) conditions were studied. [Results] The exposure to CHCA prominently promoted the uptake of phosphorus by C. reinhardtii under HL+N, while significantly inceasing the relative abundance of saturated C16:0 and decreasing the relative abundance of polyunsaturated C18:3n3. In contrast, CHCA treatment significantly inhibited the photosynthetic activity and phosphorus uptake but did not affect the fatty acid profile of C. reinhardtii under HL-N stress. In addition, the growth, pH value of the culture, and content of lipids, carbohydrates, proteins, and pigments, in C. reinhardtii under both HL+N and HL-N conditions all remained relatively constant when subjected to CHCA exposure. [Conclusion] The tolerance of microalgae to environmental stress can be visualized by growth curves, photosythetic activity, uptake of nitrogen and phosphorus, and key biochemical constituents. HL+N improves the tolerance of C. reinhardtii to NAs by promoting the uptake of phosphorus and altering the fatty acid profile, while the case was contrary under HL-N. These findings are beneficial for establishing strategies on effective cultivation of microalgae that highly tolerate NAs.

[Objective] This study characterized a novel esterase EstE from Streptomyces griseus by heterologous expression in Escherichia coli and systematically evaluates its thermostability, alkaline stability, and the effects of various additives (metal ions, detergents, and organic solvents) on its enzymatic activity to explore its potential for industrial applications. [Methods] We synthesized the gene estE' encoding the same amino acid sequence as the native gene by optimizing the original sequence of estE from S. griseus. We then constructed the recombinant plasmid carrying the optimized gene by ligating the gene into the pET-28b(+) vector. The esterase EstE was then expressed under the induction of IPTG and purified via Co²⁺ affinity chromatography. Furthermore, the enzymatic properties of the purified EstE were determined by the p-nitrophenol method, and bioinformatics analysis was performed for this enzyme. [Results] EstE consisted of 289 amino acid residues, with a molecular weight of 31.6 kDa. It belonged to the GDS(L) family, with Ser¹⁶, Asp¹⁹⁴, and His²²⁴ forming its catalytic triad. The enzyme showed the optimal activity at 40 ℃ and pH 8.5, with the highest catalytic efficiency (specific activity of 61.03 U/mg) observed in the case of p-nitrophenyl acetate as a substrate. EstE demonstrated robust thermostability, with the relative activity of 50% after 156.11 h of incubation at 40 ℃ and 2.67 h of incubation at 100 ℃. Moreover, it showed excellent alkaline stability, with the relative activity exceeding 80% after incubation at pH 8.5 for 100 h. In addition, this enzyme exhibited excellent tolerance to organic solvents, maintaining stable activity in the presence of 30% DMSO. [Conclusion] A novel esterase EstE from S. griseus is successfully obtained through heterologous expression, demonstrating excellent catalytic properties, thermostability, alkaline stability, and organic solvent tolerance, positioning it as a promising candidate for industrial applications.