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Talaromycosis is a fungal infection caused by an opportunistic dimorphic fungus Talaromyces marneffei. During infection, T. marneffei resides inside phagosomes of human host macrophages where the fungus encounters nutrient scarcities and host-derived oxidative stressors. Previously, we showed that the deletion of acuK, a gene encoding Zn(2)Cys(6) transcription factor, caused a decreased ability for T. marneffei to defend against macrophages, as well as a growth impairment in T. marneffei on both low iron-containing medium and gluconeogenic substrate-containing medium. In this study, a paralogous gene acuM was deleted and characterized. The ΔacuM mutant showed similar defects with the ΔacuK mutant, suggesting their common role in gluconeogenesis and iron homeostasis. Unlike the pathogenic mold Aspergillus fumigatus, the ΔacuK and ΔacuM mutants unexpectedly exhibited normal siderophore production and did not show lower expression levels of genes involved in iron uptake and siderophore synthesis. To identify additional target genes of AcuK and AcuM, RNA-sequencing analysis was performed in the ΔacuK and ΔacuM strains growing in a synthetic dextrose medium with 1% glucose at 25 °C for 36 hours. Downregulated genes in both mutants participated in iron-consuming processes, especially in mitochondrial metabolism and anti-oxidative stress. Importantly, the ΔacuM mutant was sensitive to the oxidative stressors menadione and hydrogen peroxide while the ΔacuK mutant was sensitive to only hydrogen peroxide. The yeast form of both mutants demonstrated a more severe defect in antioxidant properties than the mold form. Moreover, ribosomal and ribosomal biogenesis genes were expressed at significantly lower levels in both mutants, suggesting that AcuK and AcuM could affect the protein translation process in T. marneffei. Our study highlighted the role of AcuK and AcuM as global regulators that control multiple cellular adaptations under various harsh environmental conditions during host infection. These transcription factors could be potentially exploited as therapeutic targets for the treatment of this neglected infectious disease.

The adverse outcomes of fungal infection in mammalian hosts depend on the complex interactions between the host immune system and pathogen virulence-associated traits. The main clinical problems arise when the host response is either too weak to effectively eliminate the pathogen or overly aggressive, resulting in host tissue damage rather than protection. This article will highlight current knowledge regarding the virulence attributions and mechanisms involved in the dual-sided role of the host immune system in the immunopathogenesis of the thermally dimorphic fungus Talaromyces marneffei through the lens of the damage response framework (DRF) of microbial pathogenesis model.

Background and Aim: Antimicrobial resistance is an emerging public health threat. Foodborne illnesses are typically caused by bacteria, such as Escherichia coli, Pseudomonas aeruginosa, Bacillus cereus, and Staphylococcus aureus, which are frequently resistant to common antimicrobial agents. Rice is a staple grain in most parts of the world. Our previous work showed that Phatthalung Sangyod rice seed protein hydrolysates (SYPs), especially SYP4, exhibit antifungal activity against several fungal species that are pathogenic for both humans and animals and are non-cytotoxic to animal red blood cells. In this study, we aimed to determine the effects of the bioactive peptides in SYPs against several pathogenic bacteria in humans and animals. Materials and Methods: After isolating SYP1, it was treated as follows: heated (SYP2), and hydrolyzed using pepsin (SYP3), and proteinase K (SYP4). Then, we used 500 μg of protein to evaluate the antibacterial effects on four pathogenic bacteria, including E. coli, P. aeruginosa, B. cereus, and S. aureus, using agar well diffusion. Using a broth microdilution assay, we determined the minimum inhibitory and bactericidal concentration (MIC and MBC, respectively) values of active SYPs. Using the agar well diffusion and microtube incubation methods, we also assessed the inhibitory effects of SYPs on the bacterial quorum sensing (QS) activity of Chromobacterium violaceum. Sangyod rice seed protein hydrolysates were evaluated for their ability to inhibit the biofilm formation of bacterial cells by a crytal violet assay. Furthermore, using the dropping method, we tested the inhibitory effects of SYPs on the bacterial pigments pyocyanin in P. aeruginosa and staphyloxanthin in S. aureus. Results: Our results showed that the crude protein lysate (SYP1) did not exhibit antibacterial activity against any of the test bacteria. Intriguingly, after boiling (SYP2) and enzymatic hydrolysis (SYP3 and SYP4), the protein hydrolysates were transformed into bioactive peptides and displayed antibacterial properties against all of the test bacteria at a concentration of 500 μg as determined by agar well diffusion. SYP4 demonstrated the highest antibacterial activity as it completely inhibited all test strains, with inhibition zones ranging from 16.88 ± 0.25 to 21.25 ± 0.5 mm, and also yielded the highest MIC/MBC values against P. aeruginosa, B. cereus, and E. coli, at 256 and >256 μg/mL, respectively. We observed that at least 256 μg/mL of SYP4 is required to exhibit optimal antibacterial activity. At 16–128 μg/mL, it exhibited antibiofilm activity against S. aureus. Furthermore, at 256 μg/mL, SYP4 inhibited pyocyanin in P. aeruginosa and staphyloxanthin in S. aureus. Although SYP2 and SYP3 displayed weak antibacterial activity and their MIC values could not be obtained for all bacteria, they showed strong QS inhibition in C. violaceum at 256 μg protein. Moreover, SYP2 and SYP3, at a minimum concentration of 32 μg/mL, significantly reduced violacein production. SYP3 also showed biofilm reduction activity on S. aureus at least 16-512 μg/mL. Conclusion: Sangyod Phatthalung protein hydrolysates exerted excellent inhibitory effects against the growth of bacteria and their virulence factors, such as QS, biofilm formation, and/or pigment production. These factors include zoonotic and foodborne pathogens. Therefore, daily consumption of Sangyod Phatthalung rice might reduce the risk of bacterial pathogenesis and foodborne diseases. In conclusion, functional foods or alternate methods of treating bacterial illnesses may be developed in humans and animals.

Background and Aim: Immune cells require toll-like receptor 4 (TLR4) to respond to lipopolysaccharides (LPS) by releasing pro-inflammatory cytokines. Peripheral blood mononuclear cells (PBMCs) are used to assess changes in cytokines released in response to diseases or pathogens. This study aimed to assess TLR4 gene expression in PBMCs from Leghorn chicken and the release of related cytokines. Materials and Methods: Peripheral blood mononuclear cells were isolated from blood samples obtained from Leghorn chicks. The PBMC cultures were stimulated with various concentrations of LPS (0.01-1 µg/ml). Polymerase chain reaction was used to detect TLR4 expression. The production of tumor necrosis factor-alpha (TNF-α) and interleukins (IL-1β and IL-6) was quantified using an enzyme-linked immunosorbent assay. Results: We found that TLR4 was expressed in both non-stimulated and stimulated Leghorn chicken PBMCs. In addition, the release of TNF-α, IL-1β, and IL-6 levels in Leghorn chicken PBMCs increased significantly with an increase in LPS concentration (0.01–1 µg/mL) (p < 0.05). Conclusion: Although TLR4 was expressed in both non-stimulated and stimulated Leghorn chicken PBMCs, its expression was significantly higher in LPS-stimulated PBMCs Therefore, the chicken’s endotoxin response can be assessed by evaluating the pro-inflammatory cytokine production from PBMCs.

Background and Aim: Probiotics are beneficial microorganisms for humans and animals. In this study, we developed a microencapsulated probiotic with antibacterial activity against avian pathogenic Escherichia coli (APEC). Materials and Methods: Alignment of the 16S rRNA sequences of the isolate WU222001 with those deposited in GenBank revealed that the isolate was Pediococcus acidilactici with 99.6% homology. This bacterium was characterized as a probiotic based on its tolerance toward in vitro gastrointestinal tract (GIT) conditions, hydrophobicity, and auto-aggregation. The antibacterial activity of the probiotic’s culture supernatant against APEC was investigated using a broth microdilution assay. Pediococcus acidilactici was microencapsulated using sodium alginate and agar with diameters ranging from 47 to 61 µm. Then, physicochemical characteristics and stability of the microcapsules were determined. Results: The isolate was characterized as a probiotic based on its resistance to low pH, bile salts, and pancreatin, with relative values of 79.2%, 70.95%, and 90.64%, respectively. Furthermore, the bacterium exhibited 79.56% auto-aggregation and 55.25% hydrophobicity at 24 h. The probiotic’s culture supernatant exhibited strong antibacterial activity against clinical APEC isolates with minimum inhibitory concentration and minimum bactericidal concentration of 12.5% and 25% v/v, respectively. Microencapsulation-enhanced bacterial viability in GIT compared to free cells. Moreover, 89.65% of the encapsulated cells were released into the simulated intestinal fluid within 4 h. The viable count in microcapsules was 63.19% after 3 months of storage at 4°C. Conclusion: The results indicated that the culture supernatant of P. acidilactici inhibited the growth of APEC. In addition, microencapsulation extends the viability of P. acidilactici under harsh conditions, indicating its potential application in the feed production.

Background and Aim: Fungal zoonoses are an economic and public health concern because they can cause various degrees of morbidity and mortality in animals and humans. To combat this issue, alternative natural antifungals, such as products derived from rice protein hydrolysates or rice antifungal protein/peptide are being considered because they are highly bioactive and exhibit various functional properties. Thailand is a leading rice producer and exporter. Among the various cultivated rice varieties, Sangyod rice (Oryza sativa L.) is exclusively indigenous to Thailand’s Phatthalung province; it has a Thai geographical indication tag. Here, we investigated whether the Phatthalung Sangyod rice seeds have bioactive antifungal peptides. Materials and Methods: Antifungal activity in four Sangyod rice seed extracts (SYPs) – namely, (1) the crude lysate, SYP1; (2) the heat-treated lysate, SYP2; (3) the heat- and pepsin digested lysate, SYP3; and (4) the heat- and proteinase K-digested lysate, SYP4 – was analyzed. Protein concentrations in these SYPs were determined using the Bradford assay. The total phenolic compound content was determined using the modified Folin–Ciocalteu method in a 96-well microplate. Then, the SYP protein pattern was determined using the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Subsequently, using the agar well diffusion method, the antifungal properties of these SYPs were tested against ten medically important pathogenic fungi. The minimal inhibitory concentration (MIC) and minimal fungicidal concentration values were determined for the active SYPs – SYP2-4. Finally, the clinical safety of SYP4 was determined using a hemolytic assay (using canine red blood cells [RBCs]). Results: The crude lysate SYP1 did not show antifungal activity against any of the ten tested pathogenic fungi. Surprisingly, hydrolysates SYP2, SYP3, and SYP4 displayed antifungal properties against the ten tested pathogenic fungi. Thus, heat and enzymatic hydrolysis seem to transform the bioactivity of the crude protein extract – SYP1. Further, SYP4 shows the most effective antifungal activity. It completely inhibited Cryptococcus neoformans, Talaromyces marneffei yeast phase, Trichophyton mentagrophytes, and Trichophyton rubrum. A partial inhibitory action on Candida albicans and Microsporum gypseum was possessed while showing the least activity to C. neoformans. SYP4 was nontoxic to canine RBCs. Hemolysis of canine RBCs was undetectable at 1 × MIC and 2 × MIC concentrations; therefore, it can be safely used in further applications. Conclusion: These results indicate that heat and proteinase K hydrolyzed SYP is a very potent antifungal preparation against animal and human fungal pathogens and it can be used in future pharmaceuticals and functional foods.

Background and Aim: Bacillus cereus and Staphylococcus aureus cause foodborne intoxication in humans and animals. Pathogens can produce biofilms controlled by the quorum sensing system. The study aimed to investigate the antibacterial, antibiofilm, and anti-quorum sensing activities of Coffea canephora P. ex Fr. (Robusta coffee) extracts against B. cereus and S. aureus. Materials and Methods: Ethanol extracts of fruit peels and seeds of Robusta coffee were tested for antibacterial activity against B. cereus and S. aureus using a broth microdilution assay. Reduction of the biofilm formation and elimination of the viability of mature biofilm-grown cells of B. cereus and S. aureus were determined. Inhibition of quorum sensing activity in Chromobacterium violaceum by the extracts was investigated using the disk diffusion method and flask incubation assay. Results: Fresh fruit peel extract showed the strongest antibacterial activity against B. cereus and S. aureus with minimum inhibitory concentration (MIC) values of 2 and 4 mg/mL, respectively. However, the extracts did not inhibit Escherichia coli, avian pathogenic E. coli, and Pseudomonas aeruginosa at 8 mg/mL. Significant inhibition of biofilm formation at 1/2 × MIC of the fresh peel extract was detected in B. cereus (56.37%) and S. aureus (39.69 %), respectively. At 8 × MIC of the fresh peel extract, a significant elimination of the mature biofilm viability was detected in B. cereus (92.48%) and S. aureus (74.49%), respectively. The results showed that fresh and dried peel fruit extracts at 1/2 × MIC significantly reduced violacein production with the highest percentage inhibition ranging from 44.53 to 47.48% at 24 h (p ≤ 0.05). Conclusion: The results of the present study suggest the potential therapeutic benefits of Robusta coffee extracts in inhibiting the growth, biofilm, and quorum sensing of both B. cereus and S. aureus. The results put forward an alternative strategy to control the foodborne intoxications caused by both pathogens.

The aim of the experiment was to determine the effects of mangosteen wood vinegar as a potential additive on nutrient digestibility in growing pigs. A 4x4 Latin square design was used in this study. Four crossbred (Duroc x Landrace x Large White) barrows averaging 19.41±0.81 kg in body weight were allotted 4 diets, mixed with mangosteen wood vinegar at levels of 0, 0.2, 0.4, and 0.8%, respectively. The pigs were raised in individual metabolism crates. Feces and urine samples were collected 4 times a day for 5 days for data collection. The results showed that the apparent nutrient digestibility of dry matter, ether extract, crude fiber, ash, nitrogen-free extract, apparent biological value, and digestible energy were not significantly different (P>0.05) in pigs fed with different diets. However, pigs fed with the diets mixed with mangosteen wood vinegar at levels of 0.2, 0.4, and 0.8%, respectively had significantly (P

Talaromyces marneffei is a dimorphic pathogenic fungus causing opportunistic infection in immunocompromised patients. It is a facultative intracellular pathogen and is usually found inside the host macrophages during infection. Alternative carbons and iron are the important nutrients associated with intracellular survival and pathogenesis of T. marneffei. This study reported the importance of the transcription factor AcuK in control of gluconeogenesis and iron acquisition in T. marneffei. Deletion of acuK gene in T. marneffei resulted in retardation of growth and germination in both mold and yeast phases. Microscopically, ΔacuK showed double nuclei hyphae. However, the yeast cells showed normal morphology. The ΔacuK failed to grow in iron-limiting conditions. Additionally, it could not grow in a medium containing gluconeogenic carbon sources. Moreover, ΔacuK showed higher susceptibility to macrophage killing than the wild type. These results demonstrated that AcuK controlled both iron acquisition and gluconeogenesis, and it could contribute to the pathogenicity of this fungus.

Antifungal proteins (AFPs) are able to inhibit a wide spectrum of fungi without significant toxicity to the hosts. This study examined the antifungal activity of AFPs isolated from a Thai medicinal plant, Rhinacanthus nasutus, against the human pathogenic fungus Talaromycesmarneffei. This dimorphic fungus causes systemic infections in immunocompromised individuals and is endemic in Southeast Asian countries. The R. nasutus crude protein extract inhibited the growth of T. marneffei. The anti-T. marneffei activity was completely lost when treated with proteinase K and pepsin, indicating that the antifungal activity was dependent on a protein component. The total protein extract from R. nasutus was partially purified by size fractionation to ≤10, 10–30, and ≥30 kDa fractions and tested for the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC). All fractions showed anti-T. marneffei activity with the MIC and MFC values of 32 to 128 μg/mL and >128 μg/mL, respectively. In order to determine the mechanism of inhibition, all fractions were tested with T. marneffei mutant strains affected in G-protein signaling and cell wall integrity pathways. The anti-T. marneffei activity of the 10–30 kDa fraction was abrogated by deletion of gasA and gasC, the genes encoding alpha subunits of heterotrimeric G-proteins, indicating that the inhibitory effect is related to intracellular signaling through G-proteins. The work demonstrates that antifungal proteins isolated from R. nasutus represent sources for novel drug development.

Talaromyces marneffei is an opportunistic, dimorphic fungal pathogen that causes a disseminated infection in people with a weakened immunological status. The ability of this fungus to acquire nutrients inside the harsh environment of the macrophage phagosome is presumed to contribute to its pathogenicity. The transcription factors AcuM and AcuK are known to regulate gluconeogenesis and iron acquisition in Aspergillus fumigatus. This study demonstrated that they are also involved in both of these processes in the dimorphic fungus T. marneffei. Expression of acuM and acuK genes was determined by real time-polymerase chain reaction (RT-PCR) on the cells grown in media containing gluconeogenic substrates and various iron concentrations. We found that the acuM and acuK transcript levels were sequentially reduced when growing the fungus in increasing amounts of iron. The acuM transcript was upregulated in the gluconeogenic condition, while the acuK transcript showed upregulation only in the acetate medium in the yeast phase. These results suggest the involvement of acuM and acuK in gluconeogenesis and iron homeostasis in T. marneffei.

Antifungal proteins or peptides (AFPs) are the natural products produced by several life forms including plants as the first-line defenses to infections. The AFPs from Thai plants can be used as new therapeutic agents in the world with growing resistance to conventional antifungals. This study determined the antifungal activities of protein extracts from Thai medicinal plants against important human opportunistic fungi, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, and Talaromyces marneffei. Total crude protein supernatants and their precipitated proteins from 10 Thai medicinal plants with the historical usage for treatment of fungal infection were prepared. Most of the protein extracts showed antifungal activities to the tested fungi. The most effective reactivity found in the extracts from Rhinacanthus nasutus, Andrographis paniculata, and Psidium guajava by showing highest activity to T. marneffei ATCC200051 (yeast phase), followed by C. neoformans ATCC90112, C. albicans ATCC90028, T. marneffei ATCC200051 (mold phase), and A. fumigatus NCPF7367. The precipitated proteins from R. nasutus and A. paniculata containing antifungal properties were selected for partial purification by size cut-off membrane centrifugation and tested for antifungal activities. A colorimetric broth microdilution method was used to determine the minimal inhibitory concentration (MIC) and minimal fungicidal concentration (MFC) to anti-T. marneffei. The partially purified fractions from A. paniculata, and R. nasutus showed anti-T. marneffei activity with the MIC and MFC values ranged from 2 to 128 µg/mL and 16 to >128 µg/mL, respectively. Therefore, A. paniculata and R. nasutus can be further subjected to the study of the therapeutic antifungals. [ABSTRACT FROM AUTHOR]