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Abstract Background The emergence of multidrug-resistant pathogens and the lack of new antimicrobial drugs is a major public health concern that needs urgent and innovative solutions. Endophytic fungi living in unique niches such as in endosymbiosis with plants are increasingly drawing attention as alternative sources of novel and chemically diverse compounds with unique mechanisms of action. Methods In the present study, ten endophytic fungi isolated from the medicinal plant, Sclerocarya birrea were screened for bioactivity against a panel of indicator bacteria. Three bioactive endophytic fungi (strains P02PL2, P02MS1, and P02MS2A) were selected and identified through ITS-rDNA sequencing. The whole broth extracts of the three selected isolates were further screened against contemporary drug-resistant bacterial pathogens. This was followed by partial purification by solid phase extraction and GC–MS analysis of bioactive fractions. Results The bioactive endophytic fungi were identified as Alternaria alternata species (strains P02PL2 and P02MS1) and Nigrospora oryzae (strain P02MS2A). The whole broth extracts from N. oryzae P02MS2A exhibited a MIC of one μg/mL and 16 μg/mL against gram-negative, MDR Pseudomonas 5625574 and gram-positive MRSA 25775 clinical isolates, respectively. After partial purification and GC–MS analysis of whole broth extract from A. alternaria PO2MS1, 2-fluorobenzoic acid heptadecyl was putatively identified as the active compound in fraction C of this extract. This compound was also putatively identified in fraction E of A. alternata P02PL2, fraction B of A. alternata P02MS1 and fraction B of N. oryzae P02MS2A, and interestingly, all these fractions retained activity against the two MDR clinical isolates. Conclusion The putative identification of 2-fluorobenzoic acid heptadecyl compound showing a broad-spectrum of activity, more especially against gram-negative MDR contemporary pathogens is highly encouraging in the initiative at developing novel drugs to combat multi-drug resistance.

Developing new anti-human immunodeficiency virus (HIV) drug candidates that target different sites in HIV-1 replication, with better resistance profiles and lower drug toxicity, is essential to eradicating HIV. This study investigated the potential of fractionated crude extracts of Alternaria alternata as immunomodulatory or anti-HIV drug candidates. Solid-phase extraction (SPE) was used to fractionate A. alternata PO4PR2 using three different columns: MAX (Mixed-mode, strong Anion-eXchange), MCX (Mixed-mode, strong Cation-eXchange), and HLB (Hydrophilic–Lipophilic Balance) with methanol gradient methods (5%, 45%, and 95%). An MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was used to assess the cell viability and cytotoxicity of the fractionated crude extract A. alternata PO4PR2 in the TZM-bl cell lines. This was followed by a luciferase-based antiviral assay to assess the antiviral activity of A. alternata PO4PR2. A time of addition (TOA) assay was performed to ascertain the mechanism of inhibition employed by the fractionated crude extract of A. alternata PO4PR2 in the HIV life cycle. The p24 titer was determined using an ELISA, while a luciferase-based antiviral assay was used to evaluate the HIV percentage inhibition for different HIV-1 replication cycles. The TOA assay was established using antiviral drugs that target different sites in the HIV replication cycle. These included maraviroc, azidothymidine, raltegravir, and amprenavir. The immunomodulatory effect of the fractionated crude extracts on CD4+ T cells was measured by a flow cytometric analysis, for which fluorochrome-labelled monoclonal antibodies were used as markers for activation (CD38 and HLA-DR) and exhaustion (PD-1). The MCX fraction demonstrated a more significant anti-HIV inhibition than that of the fractions generated in other columns, with an IC50 of 0.3619 µg/mL, an HIV inhibition of 77%, 5% HLB (IC50: 0.7232 µg/mL; HIV inhibition of 64%), and 5% MAX (IC50: 5.240 µg/mL; HIV inhibition of 67%). It was evident from the time of addition data that the crude extract and the 5% MCX fraction inhibited viral binding (68%), reverse transcription (75%), integration (98%), and proteolysis (77%). It was shown that A. alternata (the MCX fraction) have a significant inhibitory effect on reverse transcription (75% HIV inhibition) and integration (100% HIV inhibition). The 5% MCX (p = 0.0062), 5% HLB (p = 0.0269), and 5% MAX (p = 0.0117) fractionated A. alternata crude extracts had low levels of CD4+ T cell (CD38 + HLA-DR+) activation compared to those of the AZT treatment, while CD4+ T cell activation was insignificant. The 5% MAX and HLB A. alternata fractions may possess immunomodulatory compounds with less anti-HIV-1 activity. A. alternata could be a key source of innovative anti-HIV drugs with immunomodulatory characteristics.

Antiretroviral treatment has significantly reduced human immunodeficiency virus infection and mortality. However, the current treatment regimen is limited by adverse side effects, the emergence of drug resistance, and the inability to eliminate viral reservoirs. Here, fifteen endophytic fungi were isolated from Sclerocarya birrea and Hypoxis plants. Crude extracts of Alternaria alternata (strain ID PO4PR1, PO4PR2, and PO2PL1) of the fifteen isolate’s crude extracts showed anti-HIV-1 activity in TZM-bl cell line at inhibitory concentration (IC50) values ranging from 0.017 to 1.170 μg/ml. The three crude extracts also maintained the virus replication inhibition profile on PBMCs and CD4+ T cells at concentrations ranging from 0.3 to 50.2 ng/ml. Partial purification using the solid phase extraction and analysis with Gas Chromatography-Mass spectrophotometry showed a diverse profile. The bioactive compounds were identified based on peak area, retention time, similarity index. The major compounds from GC-MS analysis of A. Alternata revealed the existence of cyclotrisiloxane octamethyl (22.92%); Propaninitrile (16,67%); Pyrrolol[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methyl propyl) (10.42%); Silane, diethylethoxy(2-ethoxyethyloxy) (4.17%); Coumarin, 3,4-dihydro-4,5,7-trimethyl- 4,5,7-Trimethyl-2-chromanone (13.7%) and 1,2-Cyclobutanedicarbonitrile (2.08%) with previously reported biological activities such as antimicrobial, anti-inflammatory and antioxidant properties. Therefore, these bioactive compounds from A. alternata fungal endophytes could be repurposed as potential anti-HIV agents. This study showed the potential of endophytic fungi, Alternaria alternata from S. birrea, and Hypoxis species as producers of anti-HIV compounds.

The increasing reports of multidrug-resistant Klebsiella pneumoniae have emerged as a public health concern, raising questions about the potential routes for the evolution and dissemination of the pathogenic K. pneumoniae into environmental reservoirs. Potential drivers of the increased incidence of antimicrobial-resistant environmental K. pneumoniae include the eminent global climatic variations as a direct or indirect effect of human activities. The ability of microorganisms to adapt and grow at an exponential rate facilitates the distribution of environmental strains with acquired resistant mutations into water systems, vegetation, and soil which are major intersection points with animals and humans. The bacterial pathogen, K. pneumoniae, is one of the critical-priority pathogens listed by the World Health Organization, mostly associated with hospital-acquired infections. However, the increasing prevalence of pathogenic environmental strains with similar characteristics to clinical-antibiotic-resistant K. pneumoniae isolates is concerning. Considering the eminent impact of global climatic variations in the spread and dissemination of multidrug-resistant bacteria, in this review, we closely assess factors influencing the dissemination of this pathogen resulting in increased interaction with the environment, human beings, and animals. We also look at the recent developments in rapid detection techniques as part of the response measures to improve surveillance and preparedness for potential outbreaks. Furthermore, we discuss alternative treatment strategies that include secondary metabolites such as biosurfactants and plant extracts with high antimicrobial properties.

The continuous burden of human immunodeficiency virus-1 in Sub-Saharan Africa, coupled with the inability of antiretroviral agents to eradicate HIV-1 from viral reservoirs, the potential risks of drug resistance development, and the development of adverse effects, emphasizes the need to develop a new class of HIV-1 inhibitors. Here, we cultivated four endophytic fungal isolates from a medicinal plant, Albizia adianthifolia with the addition of small epigenetic modifiers, sodium butyrate, and valproic acid, to induce the expression of biosynthetic gene clusters encoding active secondary metabolites with probable anti-HIV activities. We identified a non-toxic crude extract of the endophytic fungus Penicillium chrysogenum treated with sodium butyrate to possess significantly greater anti-HIV activity than the untreated extracts. Penicillium chrysogenum P03MB2 showed anti-HIV activity with an IC50 of 0.6024 µg/mL compared to untreated fungal crude extract (IC50 5.053 µg/mL) when treated with sodium butyrate. The profile of secondary metabolite compounds from the bioactive, partially purified extracts were identified by gas chromatography-mass spectrometry (GC-MS), and more bioactive compounds were detected in treated P. chrysogenum P03MB2 fractions than in untreated fractions. Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro (13.64%), cyclotrisiloxane, hexamethyl (8.18%), cyclotetrasiloxane, octamethyl (7.23%), cyclopentasiloxane, decamethyl (6.36%), quinoline, 1,2-dihydro-2,24-trimethyl (5.45%), propanenitrile (4.55%), deca-6,9-diene (4.55%), dibutyl phthalate (4.55%), and silane[1,1-dimethyl-2-propenyl)oxy]dimethyl (2.73%) were the most abundant compounds. These results indicate that treatment of endophytic fungi with small epigenetic modifiers enhances the secretion of secondary metabolites with stronger anti-HIV-1 properties, acknowledging the feasibility of epigenetic modification as an innovative approach for the discovery of cryptic fungal metabolites which can be developed into therapeutic compounds.

The emergence of drug-resistant Human Immunodeficiency Virus-1 strains against anti-HIV therapies in the clinical pipeline, and the persistence of HIV in cellular reservoirs remains a significant concern. Therefore, there is a continuous need to discover and develop new, safer, and effective drugs targeting novel sites to combat HIV-1. The fungal species are gaining increasing attention as alternative sources of anti-HIV compounds or immunomodulators that can escape the current barriers to cure. Despite the potential of the fungal kingdom as a source for diverse chemistries that can yield novel HIV therapies, there are few comprehensive reports on the progress made thus far in the search for fungal species with the capacity to produce anti-HIV compounds. This review provides insights into the recent research developments on natural products produced by fungal species, particularly fungal endophytes exhibiting immunomodulatory or anti-HIV activities. In this study, we first explore currently existing therapies for various HIV-1 target sites. Then we assess the various activity assays developed for gauging antiviral activity production from microbial sources since they are crucial in the early screening phases for discovering novel anti-HIV compounds. Finally, we explore fungal secondary metabolites compounds that have been characterized at the structural level and demonstrate their potential as inhibitors of various HIV-1 target sites.

The discovery of silent biosynthetic gene clusters (BGCs) in fungi provides unlimited prospects to harness the secondary metabolites encoded by gene clusters for various applications, including pharmaceuticals. Amplifying these prospects is the new interest in exploring fungi living in the extremes, such as those associated with plants (fungal endophytes). Fungal species in endosymbiosis relationship with plants are recognized as the future factories of clinically relevant agents since discovering that they can produce similar metabolites as their plant host. The endophytes produce these compounds in natural environments as a defense mechanism against pathogens that infect the plant host or as a strategy for mitigating competitors. The signaling cascades leading to the expression of silent biosynthetic gene clusters in the natural environment remain unknown. Lack of knowledge on regulatory circuits of biosynthetic gene clusters limits the ability to exploit them in the laboratory. They are often silent and require tailor-designed strategies for activation. Epigenetic modification using small molecular compounds that alter the chromatin network, leading to the changes in secondary metabolites profile, has achieved considerable success. This review aims to comprehensively analyze the secondary metabolite profiles expressed after treatment with various epigenetic modifiers. We first describe the regulatory circuits governing the expression of secondary metabolites in fungi. Following this, we provide a detailed review of the small molecular modifiers, their mechanism(s) of action, and the diverse chemistries resulting from epigenetic modification. We further show that genetic deletion or epigenetic inhibition of histone deacetylases does not always lead to the overexpression or induction of silent secondary metabolites. Instead, the response is more complex and often leads to differential expression of secondary metabolites. Finally, we propose using this strategy as an initial screening tool to dereplicate promising fungal species.

Developing new anti-human immunodeficiency virus (HIV) drug candidates that target different sites in HIV-1 replication, with better resistance profiles and lower drug toxicity, is essential to eradicating HIV. This study investigated the potential of fractionated crude extracts of Alternaria alternata as immunomodulatory or anti-HIV drug candidates. Solid-phase extraction (SPE) was used to fractionate A. alternata PO4PR2 using three different columns: MAX (Mixed-mode, strong Anion-eXchange), MCX (Mixed-mode, strong Cation-eXchange), and HLB (Hydrophilic–Lipophilic Balance) with methanol gradient methods (5%, 45%, and 95%). An MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) assay was used to assess the cell viability and cytotoxicity of the fractionated crude extract A. alternata PO4PR2 in the TZM-bl cell lines. This was followed by a luciferase-based antiviral assay to assess the antiviral activity of A. alternata PO4PR2. A time of addition (TOA) assay was performed to ascertain the mechanism of inhibition employed by the fractionated crude extract of A. alternata PO4PR2 in the HIV life cycle. The p24 titer was determined using an ELISA, while a luciferase-based antiviral assay was used to evaluate the HIV percentage inhibition for different HIV-1 replication cycles. The TOA assay was established using antiviral drugs that target different sites in the HIV replication cycle. These included maraviroc, azidothymidine, raltegravir, and amprenavir. The immunomodulatory effect of the fractionated crude extracts on CD4+ T cells was measured by a flow cytometric analysis, for which fluorochrome-labelled monoclonal antibodies were used as markers for activation (CD38 and HLA-DR) and exhaustion (PD-1). The MCX fraction demonstrated a more significant anti-HIV inhibition than that of the fractions generated in other columns, with an IC50 of 0.3619 µg/mL, an HIV inhibition of 77%, 5% HLB (IC50: 0.7232 µg/mL; HIV inhibition of 64%), and 5% MAX (IC50: 5.240 µg/mL; HIV inhibition of 67%). It was evident from the time of addition data that the crude extract and the 5% MCX fraction inhibited viral binding (68%), reverse transcription (75%), integration (98%), and proteolysis (77%). It was shown that A. alternata (the MCX fraction) have a significant inhibitory effect on reverse transcription (75% HIV inhibition) and integration (100% HIV inhibition). The 5% MCX (p = 0.0062), 5% HLB (p = 0.0269), and 5% MAX (p = 0.0117) fractionated A. alternata crude extracts had low levels of CD4+ T cell (CD38 + HLA-DR+) activation compared to those of the AZT treatment, while CD4+ T cell activation was insignificant. The 5% MAX and HLB A. alternata fractions may possess immunomodulatory compounds with less anti-HIV-1 activity. A. alternata could be a key source of innovative anti-HIV drugs with immunomodulatory characteristics. [ABSTRACT FROM AUTHOR]

Background: The emergence of multidrug-resistant pathogens and the lack of new antimicrobial drugs is a major public health concern that needs urgent and innovative solutions. Endophytic fungi living in unique niches such as in endosymbiosis with plants are increasingly drawing attention as alternative sources of novel and chemically diverse compounds with unique mechanisms of action. Methods: In the present study, ten endophytic fungi isolated from the medicinal plant, Sclerocarya birrea were screened for bioactivity against a panel of indicator bacteria. Three bioactive endophytic fungi (strains P02PL2, P02MS1, and P02MS2A) were selected and identified through ITS-rDNA sequencing. The whole broth extracts of the three selected isolates were further screened against contemporary drug-resistant bacterial pathogens. This was followed by partial purification by solid phase extraction and GC–MS analysis of bioactive fractions. Results: The bioactive endophytic fungi were identified as Alternaria alternata species (strains P02PL2 and P02MS1) and Nigrospora oryzae (strain P02MS2A). The whole broth extracts from N. oryzae P02MS2A exhibited a MIC of one μg/mL and 16 μg/mL against gram-negative, MDR Pseudomonas 5625574 and gram-positive MRSA 25775 clinical isolates, respectively. After partial purification and GC–MS analysis of whole broth extract from A. alternaria PO2MS1, 2-fluorobenzoic acid heptadecyl was putatively identified as the active compound in fraction C of this extract. This compound was also putatively identified in fraction E of A. alternata P02PL2, fraction B of A. alternata P02MS1 and fraction B of N. oryzae P02MS2A, and interestingly, all these fractions retained activity against the two MDR clinical isolates. Conclusion: The putative identification of 2-fluorobenzoic acid heptadecyl compound showing a broad-spectrum of activity, more especially against gram-negative MDR contemporary pathogens is highly encouraging in the initiative at developing novel drugs to combat multi-drug resistance. [ABSTRACT FROM AUTHOR]

The continuous burden of human immunodeficiency virus-1 in Sub-Saharan Africa, coupled with the inability of antiretroviral agents to eradicate HIV-1 from viral reservoirs, the potential risks of drug resistance development, and the development of adverse effects, emphasizes the need to develop a new class of HIV-1 inhibitors. Here, we cultivated four endophytic fungal isolates from a medicinal plant, Albizia adianthifolia with the addition of small epigenetic modifiers, sodium butyrate, and valproic acid, to induce the expression of biosynthetic gene clusters encoding active secondary metabolites with probable anti-HIV activities. We identified a non-toxic crude extract of the endophytic fungus Penicillium chrysogenum treated with sodium butyrate to possess significantly greater anti-HIV activity than the untreated extracts. Penicillium chrysogenum P03MB2 showed anti-HIV activity with an IC50 of 0.6024 µg/mL compared to untreated fungal crude extract (IC50 5.053 µg/mL) when treated with sodium butyrate. The profile of secondary metabolite compounds from the bioactive, partially purified extracts were identified by gas chromatography-mass spectrometry (GC-MS), and more bioactive compounds were detected in treated P. chrysogenum P03MB2 fractions than in untreated fractions. Pyrrolo[1,2-a]pyrazine-1,4-dione, hexahydro (13.64%), cyclotrisiloxane, hexamethyl (8.18%), cyclotetrasiloxane, octamethyl (7.23%), cyclopentasiloxane, decamethyl (6.36%), quinoline, 1,2-dihydro-2,24-trimethyl (5.45%), propanenitrile (4.55%), deca-6,9-diene (4.55%), dibutyl phthalate (4.55%), and silane[1,1-dimethyl-2-propenyl)oxy]dimethyl (2.73%) were the most abundant compounds. These results indicate that treatment of endophytic fungi with small epigenetic modifiers enhances the secretion of secondary metabolites with stronger anti-HIV-1 properties, acknowledging the feasibility of epigenetic modification as an innovative approach for the discovery of cryptic fungal metabolites which can be developed into therapeutic compounds. [ABSTRACT FROM AUTHOR]

The emergence of drug-resistant Human Immunodeficiency Virus-1 strains against anti-HIV therapies in the clinical pipeline, and the persistence of HIV in cellular reservoirs remains a significant concern. Therefore, there is a continuous need to discover and develop new, safer, and effective drugs targeting novel sites to combat HIV-1. The fungal species are gaining increasing attention as alternative sources of anti-HIV compounds or immunomodulators that can escape the current barriers to cure. Despite the potential of the fungal kingdom as a source for diverse chemistries that can yield novel HIV therapies, there are few comprehensive reports on the progress made thus far in the search for fungal species with the capacity to produce anti-HIV compounds. This review provides insights into the recent research developments on natural products produced by fungal species, particularly fungal endophytes exhibiting immunomodulatory or anti-HIV activities. In this study, we first explore currently existing therapies for various HIV-1 target sites. Then we assess the various activity assays developed for gauging antiviral activity production from microbial sources since they are crucial in the early screening phases for discovering novel anti-HIV compounds. Finally, we explore fungal secondary metabolites compounds that have been characterized at the structural level and demonstrate their potential as inhibitors of various HIV-1 target sites. [ABSTRACT FROM AUTHOR]

Antiretroviral treatment has significantly reduced human immunodeficiency virus infection and mortality. However, the current treatment regimen is limited by adverse side effects, the emergence of drug resistance, and the inability to eliminate viral reservoirs. Here, fifteen endophytic fungiwere isolated fromSclerocarya birrea and Hypoxis plants. Crude extracts of Alternaria alternata (strain ID PO4PR1, PO4PR2, and PO2PL1) of the fifteen isolate’s crude extracts showed anti-HIV- 1 activity in TZM-bl cell line at inhibitory concentration (IC50) values ranging from 0.017 to 1.170 μg/ml. The three crude extracts also maintained the virus replication inhibition profile on PBMCs and CD4+ T cells at concentrations ranging from 0.3 to 50.2 ng/ml. Partial purification using the solid phase extraction and analysis with Gas Chromatography-Mass spectrophotometry showed a diverse profile. The bioactive compounds were identified based on peak area, retention time, similarity index. The major compounds from GC-MS analysis of A. Alternata revealed the existence of cyclotrisiloxane octamethyl (22.92%); Propaninitrile (16,67%); Pyrrolol[1,2-a]pyrazine-1,4-dione, hexahydro-3-(2-methyl propyl) (10.42%); Silane, diethylethoxy(2-ethoxyethyloxy) (4.17%); Coumarin, 3,4- dihydro-4,5,7-trimethyl- 4,5,7-Trimethyl-2-chromanone (13.7%) and 1,2- Cyclobutanedicarbonitrile (2.08%) with previously reported biological activities such as antimicrobial, anti-inflammatory and antioxidant properties. Therefore, these bioactive compounds from A. alternata fungal endophytes could be repurposed as potential anti-HIV agents. This study showed the potential of endophytic fungi, Alternaria alternata from S. birrea, and Hypoxis species as producers of anti-HIV compounds. [ABSTRACT FROM AUTHOR]

The discovery of silent biosynthetic gene clusters (BGCs) in fungi provides unlimited prospects to harness the secondary metabolites encoded by gene clusters for various applications, including pharmaceuticals. Amplifying these prospects is the new interest in exploring fungi living in the extremes, such as those associated with plants (fungal endophytes). Fungal species in endosymbiosis relationship with plants are recognized as the future factories of clinically relevant agents since discovering that they can produce similar metabolites as their plant host. The endophytes produce these compounds in natural environments as a defense mechanism against pathogens that infect the plant host or as a strategy for mitigating competitors. The signaling cascades leading to the expression of silent biosynthetic gene clusters in the natural environment remain unknown. Lack of knowledge on regulatory circuits of biosynthetic gene clusters limits the ability to exploit them in the laboratory. They are often silent and require tailor-designed strategies for activation. Epigenetic modification using small molecular compounds that alter the chromatin network, leading to the changes in secondary metabolites profile, has achieved considerable success. This review aims to comprehensively analyze the secondary metabolite profiles expressed after treatment with various epigenetic modifiers. We first describe the regulatory circuits governing the expression of secondary metabolites in fungi. Following this, we provide a detailed review of the small molecular modifiers, their mechanism(s) of action, and the diverse chemistries resulting from epigenetic modification. We further show that genetic deletion or epigenetic inhibition of histone deacetylases does not always lead to the overexpression or induction of silent secondary metabolites. Instead, the response is more complex and often leads to differential expression of secondary metabolites. Finally, we propose using this strategy as an initial screening tool to dereplicate promising fungal species. [ABSTRACT FROM AUTHOR]