Combatting Antibiotic-Resistant Staphylococcus aureus: Discovery of TST1N-224, a Potent Inhibitor Targeting Response Regulator VraRC, through Pharmacophore-Based Screening and Molecular Characterizations.

Staphylococcus aureus (S. aureus) is a major global health concern, causing various infections and presenting challenges due to antibiotic resistance. In particular, methicillin-resistant S. aureus, vancomycin-intermediate S. aureus (VISA), and vancomycin-resistant S. aureus pose significant obstacles in treating S. aureus infections. Therefore, the critical need for novel drugs to counter these resistant forms is pressing. Two-component systems (TCSs), integral to bacterial regulation, offer promising targets for disruption. In this study, a comprehensive approach, involving pharmacophore-based inhibitor screening, along with biochemical and biophysical analyses were conducted to identify, characterize, and validate potential inhibitors targeting the response regulator VraRC of S. aureus. The constructed pharmacophore model, Phar-VRPR-N3, demonstrated effectiveness in identifying a potent inhibitor, TST1N-224 (IC50 = 60.2 ± 4.0 µM), against the formation of the VraRC-DNA complex. Notably, TST1N-224 exhibited strong binding to VraRC (KD = 23.4 ± 1.2 µM) using a fast-on-fast-off binding mechanism. Additionally, NMR-based molecular modeling revealed that TST1N-224 predominantly interacts with the α9- and α10-helixes of the DNA-binding domain of VraR, where the interactive and functionally essential residues (N165, K180, S184, and R195) act as hotspots for structure-based inhibitor optimization. Furthermore, TST1N-224 evidently enhanced the susceptibility of VISA to both vancomycin and methicillin. Importantly, TST1N-224 distinguished by 1,2,5,6-tetrathiocane with the 3 and 8 positions modified with ethanesulfonates holds significant potential as a lead compound for the development of new antimicrobial agents.

Deubiquitination module is critical for oxidative stress response and biofilm formation in Candida glabrata.

Candidiasis is one of the most important fungal diseases and generally refers to diseases of the skin or mucosal tissues caused by Candida species. Candida glabrata is an opportunistic human fungal pathogen. Infection with C. glabrata has significantly increased due to innate antifungal drug tolerance and the ability to adhere to mucocutaneous surfaces. Spt-Ada-Gcn5 acetyltransferase complex contains two different post-translational modifications, histone acetylation (HAT) module and deubiquitination (DUB) module, which are decisive in gene regulation and highly conserved in eukaryotes. Previous research in our laboratory found that the HAT module ADA2 could regulate C. glabrata oxidative stress tolerance, drug tolerance, cell wall integrity, and virulence. However, the roles of the DUB module that is comprised of UBP8, SGF11, SGF73, and SUS1 genes in those phenotypes are not yet understood. In this study, we found that DUB module genes UBP8, SGF11, and SUS1, but not SGF73 positively regulate histone H2B DUB. Furthermore, ubp8, sgf11, and sus1 mutants exhibited decreased biofilm formation and sensitivity to cell wall-perturbing agent sodium dodecyl sulfate and antifungal drug amphotericin B. In addition, the sgf73 mutant showed increased biofilm formation but was susceptible to oxidative stresses, antifungal drugs, and cell wall perturbing agents. The ubp8, sgf11, and sus1 mutants showed marginal hypovirulence, whereas the sgf73 mutant exhibited virulence similar to the wild type in a murine systemic infection model. In conclusion, the C. glabrata DUB module plays distinct roles in H2B ubiquitination, oxidative stress response, biofilm formation, cell wall integrity, and drug tolerance, but exhibits minor roles in virulence.

In this study, we found that the deubiquitination (DUB) module of the Spt-Ada-Gcn5 acetyltransferase complex is involved in H2B DUB, oxidative stress response, biofilm formation, cell wall integrity, and drug tolerance in the human fungal pathogen Candida glabrata. The multiple functions controlled by the DUB module exhibit conserved and divergent functions between Saccharomyces cerevisiae, C. albicans, and C. glabrata.

Talaromyces amestolkiae Infection in an AIDS Patient with Cryptococcal Meningitis.

Concurrent infections caused by multiple fungal pathogens in immunocompromised patients can pose diagnostic and treatment challenges. Here, we presented the first reported case in Taiwan of an AIDS patient who had concurrent infection with Cryptococcus neoformans meningitis and Talaromyces amestolkiae lymphadenopathy. The patient presented with an enlarged inguinal lymph node and was diagnosed with T. amestolkiae lymphadenitis. The species T. amestolkiae was identified using DNA sequencing, which had the capability of differentiating it from other Talaromyces species. The patient was discharged from the hospital following treatment with amphotericin B and subsequent administration of voriconazole. This case highlights the importance of maintaining a suspicion of co-infections and utilizing appropriate diagnostic tools, such as DNA sequencing, to identify possible pathogens. Further studies are needed to determine the optimal treatment for T. amestolkiae and other co-infecting fungal pathogens.

The Gcn5-Ada2-Ada3 histone acetyltransferase module has divergent roles in pathogenesis of Candida glabrata.

Candida glabrata is an opportunistic fungal pathogen and the second most prevalent species isolated from candidiasis patients. C. glabrata has intrinsic tolerance to antifungal drugs and oxidative stresses and the ability to adhere to mucocutaneous surfaces. However, knowledge about the regulation of its virulence traits is limited. The Spt-Ada-Gcn5 acetyltransferase (SAGA) complex modulates gene transcription by histone acetylation through the histone acetyltransferase (HAT) module comprised of Gcn5-Ada2-Ada3. Previously, we showed that the ada2 mutant was hypervirulent but displayed decreased tolerance to antifungal drugs and cell wall perturbing agents. In this study, we further characterized the functions of Ada3 and Gcn5 in C. glabrata. We found that single, double, or triple deletions of the HAT module, as expected, resulted in a decreased level of acetylation on histone H3 lysine 9 (H3K9) and defective growth. These mutants were more susceptible to antifungal drugs, oxidative stresses, and cell wall perturbing agents compared with the wild-type. In addition, HAT module mutants exhibited enhanced agar invasion and upregulation of adhesin and proteases encoding genes, whereas the biofilm formation of those mutants was impaired. Interestingly, HAT module mutants exhibited enhanced induction of catalases (CTA1) expression upon treatment with H2O2 compared with the wild-type. Lastly, although ada3 and gcn5 exhibited marginal hypervirulence, the HAT double and triple mutants were hypervirulent in a murine model of candidiasis. In conclusion, the HAT module of the SAGA complex plays unique roles in H3K9 acetylation, drug tolerance, oxidative stress response, adherence, and virulence in C. glabrata.

The present study characterizes the functions of the conserved histone acetyltransferase module in the pathogenesis of the pathogenic yeast Candida glabrata. The results indicated that this module has divergent roles in the pathogenesis of C. glabrata.

Deciphering Structure-Function Relationship Unveils Salt-Resistant Mode of Action of a Potent MRSA-Inhibiting Antimicrobial Peptide, RR14.

Multidrug-resistant (MDR) bacteria lead to considerable morbidity and mortality, threatening public health worldwide. In particular, infections of methicillin-resistant Staphylococcus aureus (MRSA) in hospital and community settings are becoming a serious health problem. Antimicrobial peptides (AMPs) are considered novel therapeutic targets against MDR bacteria. However, salt sensitivity reduces the bactericidal potency of AMPs, posing a major obstacle for their development as antibiotics. Thus, the design and development of salt-insensitive peptides with potent antibacterial activity is imperative. Here, we employed biochemical and biophysical examinations coupled with molecular modeling to systematically investigate the structure-function relationship of a novel salt-insensitive AMP, RR14. The secondary structure of RR14 was characterized as an apparent α-helix, a structure that confers strong membrane-permeabilizing ability targeting bacterial-mimetic membranes. Additionally, the bioactive structure of RR14 was determined in complex with dodecylphosphocholine (DPC) micelles, where it possesses a central α-helical segment comprising residues R4 to K13 (R4-K13). RR14 was observed to orient itself into the DPC micelle with its N terminus and the α-helical segment (I5-R10) buried inside the micelles, which is essential for membrane permeabilization and bactericidal activity. Moreover, the specific and featured arrangement of positively charged residues of RR14 on its amphipathic helical conformation has great potential to render its strong salt resistance ability. Our study explored the structure-function relationship of RR14, explaining its possible mode of action against MRSA and other microbes. The insights obtained are of great applicability for the development of new antibacterial agents. IMPORTANCE Many antimicrobial peptides have been observed to become inactive in the presence of high salt concentrations. To further develop new and novel AMPs with potent bactericidal activity and salt insensitivity, understanding the structural basis for salt resistance is important. Here, we employed biochemical and biophysical examinations to systematically investigate the structure-function relationship of a novel salt-insensitive AMP, RR14. RR14 was observed to orient itself into DPC micelles with the N terminus and the α-helical segment (I5-R10) buried inside the micelles, which is essential for membrane permeabilization and bactericidal activity. Moreover, the specific and featured arrangement of cationic residues of RR14 on its amphipathic helical conformation renders its strong salt resistance ability. The insights obtained are of great applicability for developing new antibacterial agents.

Unraveling the Pathobiological Role of the Fungal KEOPS Complex in Cryptococcus neoformans.

The KEOPS (kinase, putative endopeptidase, and other proteins of small size) complex has critical functions in eukaryotes; however, its role in fungal pathogens remains elusive. Herein, we comprehensively analyzed the pathobiological functions of the fungal KEOPS complex in Cryptococcus neoformans (Cn), which causes fatal meningoencephalitis in humans. We identified four CnKEOPS components: Pcc1, Kae1, Bud32, and Cgi121. Deletion of PCC1, KAE1, or BUD32 caused severe defects in vegetative growth, cell cycle control, sexual development, general stress responses, and virulence factor production, whereas deletion of CGI121 led to similar but less severe defects. This suggests that Pcc1, Kae1, and Bud32 are the core KEOPS components, and Cgi121 may play auxiliary roles. Nevertheless, all KEOPS components were essential for C. neoformans pathogenicity. Although the CnKEOPS complex appeared to have a conserved linear arrangement of Pcc1-Kae1-Bud32-Cgi121, as supported by physical interaction between Pcc1-Kae1 and Kae1-Bud32, CnBud32 was found to have a unique extended loop region that was critical for the KEOPS functions. Interestingly, CnBud32 exhibited both kinase activity-dependent and -independent functions. Supporting its pleiotropic roles, the CnKEOPS complex not only played conserved roles in t6A modification of ANN codon-recognizing tRNAs but also acted as a major transcriptional regulator, thus controlling hundreds of genes involved in various cellular processes, particularly ergosterol biosynthesis. In conclusion, the KEOPS complex plays both evolutionarily conserved and divergent roles in controlling the pathobiological features of C. neoformans and could be an anticryptococcal drug target. IMPORTANCE The cellular function and structural configuration of the KEOPS complex have been elucidated in some eukaryotes and archaea but have never been fully characterized in fungal pathogens. Here, we comprehensively analyzed the pathobiological roles of the KEOPS complex in the globally prevalent fungal meningitis-causing pathogen C. neoformans. The CnKEOPS complex, composed of a linear arrangement of Pcc1-Kae1-Bud32-Cgi121, not only played evolutionarily conserved roles in growth, sexual development, stress responses, and tRNA modification but also had unique roles in controlling virulence factor production and pathogenicity. Notably, a unique extended loop structure in CnBud32 is critical for the KEOPS complex in C. neoformans. Supporting its pleiotropic roles, transcriptome analysis revealed that the CnKEOPS complex governs several hundreds of genes involved in carbon and amino acid metabolism, pheromone response, and ergosterol biosynthesis. Therefore, this study provides novel insights into the fungal KEOPS complex that could be exploited as a potential antifungal drug target.

Natural alkaloid tryptanthrin exhibits novel anticryptococcal activity.

Cryptococcal meningitis is a prevalent invasive fungal infection that causes around 180 000 deaths annually. Currently, treatment for cryptococcal meningitis is limited and new therapeutic options are needed. Historically, medicinal plants are used to treat infectious and inflammatory skin infections. Tryptanthrin is a natural product commonly found in these plants. In this study, we demonstrated that tryptanthrin had antifungal activity with minimum inhibitory concentration (MIC) of 2 µg/ml against Cryptococcus species and of 8 µg/ml against Trichophyton rubrum. Further analysis demonstrated that tryptanthrin exerted fungistatic and potent antifungal activity at elevated temperature. In addition, tryptanthrin exhibited a synergistic effect with the calcineurin inhibitors FK506 and cyclosporine A against Cryptococcus neoformans. Furthermore, our data showed that tryptanthrin induced cell cycle arrest at the G1/S phase by regulating the expression of genes encoding cyclins and the SBF/MBF complex (CLN1, MBS1, PCL1, and WHI5) in C. neoformans. Screening of a C. neoformans mutant library further revealed that tryptanthrin was associated with various transporters and signaling pathways such as the calcium transporter (Pmc1) and protein kinase A signaling pathway. In conclusion, tryptanthrin exerted novel antifungal activity against Cryptococcus species through a mechanism that interferes with the cell cycle and signaling pathways. LAY SUMMARY: The natural product tryptanthrin had antifungal activity against Cryptococcus species by interfering cell cycle and exerted synergistic effects with immunosuppressants FK506 and cyclosporine A. Our findings suggest that tryptanthrin may be a potential drug or adjuvant for the treatment of cryptococcosis.

The histone acetyltransferase GcnE regulates conidiation and biofilm formation in Aspergillus fumigatus.

Histone modifications play a crucial role in eukaryotic gene regulation. The Spt-Ada-Gcn5-acetyltransferase (SAGA) complex controls histone acetylation, with Gcn5 (GcnE) acting as the acetyltransferase. In the Aspergillus species, GcnE has been shown to regulate asexual development and secondary metabolism. Apart from this, GcnE is required for pathogenicity in plant fungal pathogen A. flavus; however, the role of GcnE in the pathogenicity of human pathogenic fungus A. fumigatus is unknown. In this study, we uncovered the key roles of GcnE in A. fumigatus conidiation, stress responses, and biofilm formation. We observed that deletion of gcnE resulted in aberrant conidiation in which conidiophores displayed abnormal phialide formation. In addition, the ΔgcnE mutant grew slightly faster under limited nitrogen sources (1 mM of ammonium or nitrate) compared to the wild type. The ΔgcnE mutant exhibited increased susceptibility to cell wall-perturbing agents, H2O2 and menadione but enhanced tolerance to LiCl. Furthermore, we showed that GcnE is involved in biofilm formation, and overexpression of adherence-related genes such as somA or uge3 partially rescued biofilm formation defects in the ΔgcnE mutant background. Interestingly, GcnE was not required for virulence in a neutropenic murine model of invasive aspergillosis. These results suggest that GcnE is critical for conidiation and biofilm formation but not virulence in A. fumigatus.

Conserved and Divergent Functions of the cAMP/PKA Signaling Pathway in Candida albicans and Candida tropicalis.

Fungal species undergo many morphological transitions to adapt to changing environments, an important quality especially in fungal pathogens. For decades, Candida albicans has been one of the most prevalent human fungal pathogens, and recently, the prevalence of Candida tropicalis as a causative agent of candidiasis has increased. In C. albicans, the ability to switch between yeast and hyphal forms is thought to be a key virulence factor and is regulated by multiple signaling cascades—including the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA), calcineurin, high-osmolarity glycerol (HOG), and mitogen-activated protein kinases (MAPK) signaling pathways—upon receiving environmental cues. The cAMP/PKA signaling pathway also triggers white-opaque switching in C. albicans. However, studies on C. tropicalis morphogenesis are limited. In this minireview, we discuss the regulation of the yeast-hypha transition, virulence, and white-opaque switching through the cAMP/PKA pathway in the closely related species C. albicans and C. tropicalis.

Protein kinase A governs growth and virulence in Candida tropicalis.

Candida tropicalis is one of the most important human fungal pathogens causing superficial infections in locations such as the oral mucosa and genital tract, as well as systemic infections with high mortality. In its sister species Candida albicans, the cyclic AMP/protein kinase A (cAMP/PKA) pathway regulates fungal adhesion and dimorphism, both of which correlate closely with virulence. CaTpk1 and CaTpk2, the catalytic subunits of PKA, not only share redundant functions in hyphal growth, adhesion, and biofilm formation, but also have distinct roles in stress responses and pathogenesis, respectively. However, studies on PKA in the emerging fungal pathogen C. tropicalis are limited. Our results suggest that Tpk1 is involved in cell wall integrity and drug tolerance. The tpk2/tpk2 mutants, which have no protein kinase A activity, have reduced hyphal growth and adhesion. In addition, the tpk1/tpk1 tpk2/tpk2 double deletion mutant demonstrated delayed growth and impaired hyphal formation. In a murine model of systemic infection, both TPK1 and TPK2 were required for full virulence. We further found that EFG1 and HWP1 expression is regulated by PKA, while BCR1, FLO8, GAL4, and RIM101 are upregulated in the tpk1/tpk1 tpk2/tpk2 mutant. This study demonstrates that Tpk1 is involved in drug tolerance and cell wall integrity, while Tpk2 serves as a key regulator in dimorphism and adhesion. Both Tpk1 and Tpk2 are required for growth and full virulence in C. tropicalis.

Transcription Factor SomA Is Required for Adhesion, Development and Virulence of the Human Pathogen Aspergillus fumigatus.

The transcription factor Flo8/Som1 controls filamentous growth in Saccharomyces cerevisiae and virulence in the plant pathogen Magnaporthe oryzae. Flo8/Som1 includes a characteristic N-terminal LUG/LUH-Flo8-single-stranded DNA binding (LUFS) domain and is activated by the cAMP dependent protein kinase A signaling pathway. Heterologous SomA from Aspergillus fumigatus rescued in yeast flo8 mutant strains several phenotypes including adhesion or flocculation in haploids and pseudohyphal growth in diploids, respectively. A. fumigatus SomA acts similarly to yeast Flo8 on the promoter of FLO11 fused with reporter gene (LacZ) in S. cerevisiae. FLO11 expression in yeast requires an activator complex including Flo8 and Mfg1. Furthermore, SomA physically interacts with PtaB, which is related to yeast Mfg1. Loss of the somA gene in A. fumigatus resulted in a slow growth phenotype and a block in asexual development. Only aerial hyphae without further differentiation could be formed. The deletion phenotype was verified by a conditional expression of somA using the inducible Tet-on system. A adherence assay with the conditional somA expression strain indicated that SomA is required for biofilm formation. A ptaB deletion strain showed a similar phenotype supporting that the SomA/PtaB complex controls A. fumigatus biofilm formation. Transcriptional analysis showed that SomA regulates expression of genes for several transcription factors which control conidiation or adhesion of A. fumigatus. Infection assays with fertilized chicken eggs as well as with mice revealed that SomA is required for pathogenicity. These data corroborate a complex control function of SomA acting as a central factor of the transcriptional network, which connects adhesion, spore formation and virulence in the opportunistic human pathogen A. fumigatus.

Molecular epidemiology of ciprofloxacin-resistant extended-spectrum ß-lactamase-producing Klebsiella pneumoniae in Taiwan.

Fluoroquinolone resistance in extended-spectrum ß-lactamases (ESBL)-producing isolates results in very few antimicrobial treatment options. In Taiwan's Surveillance of Antimicrobial Resistance (TSAR) III program, 124 (52.8%) cases of ESBL-producing Klebsiella pneumoniae (ESBL-KP) were resistant to ciprofloxacin. The prevalence of plasmid-mediated quinolone resistance (PMQR) determinants and chromosomal quinolone resistance-determining regions (QRDR) of gyrA and parC genes among ESBL-KP isolates was assessed via PCR sequencing. Chromosomal QRDR mutations were present in most of the 123 (96.8%) cases of ciprofloxacin-resistant ESBL-KP isolates. Sixty-six (53.2%) isolates had at least one PMQR gene. qnrB2, qnrB4, and qnrS1 were detected in 26, 19, and 13 isolates, respectively, whereas qnrA, qnrC, and qnrD were not detected. ESBL genes were transferable via conjugation with either aac(6')Ib-cr or qnrB in 63.6% of the isolates carrying PMQR genes. QnrB was associated with either CTX-M-15 or SHV-12, and aac(6')Ib-cr was linked to CTX-M-3 or CTX-M-14 in plasmids. qnrS did not co-transfer with ESBL genes. Clonal spread of PMQR genes harboring ESBL-KP isolates was observed in three hospitals. QnrA, which is common in Asia, was unexpectedly absent in ESBL-KP in Taiwan. Aside from transmission via clonal spread for ciprofloxacin-resistant ESBL-KP, concomitant transference of PMQR genes with either bla(CTX-M) or bla(SHV) via plasmid was common.

Widespread dissemination of aminoglycoside resistance genes armA and rmtB in Klebsiella pneumoniae isolates in Taiwan producing CTX-M-type extended-spectrum beta-lactamases.

Among 235 extended-spectrum beta-lactamase-producing Klebsiella pneumoniae (ESBL) isolates collected from a nationwide surveillance performed in Taiwan, 102 (43.4%) were resistant to amikacin. Ninety-two of these 102 (90.2%) isolates were carrying CTX-M-type beta-lactamases individually or concomitantly with SHV-type or CMY-2 beta-lactamases. The armA and rmtB alleles were individually detected in 44 and 37 of these 92 isolates, respectively. One isolate contained both armA and rmtB. The coexistence of the aac(6')-Il and rmtB genes was detected in three isolates. CTX-M-type beta-lactamase genes belonging to either group 1 (CTX-M-3 and CTX-M-15) or group 9 (CTX-M-14) were found in all armA- or rmtB-bearing ESBL-producing K. pneumoniae isolates, and all were conjugatively transferable. All except one of the isolates bearing armA produced CTX-M enzymes of group 1, and the remaining isolate bearing armA produced a group 9 CTX-M-type beta-lactamase. On the contrary, in the majority of rmtB carriers, the CTX-M-type beta-lactamase belonged to group 9 (62.2%). Molecular typing revealed that the amikacin-resistant ESBL-producing K. pneumoniae isolates were epidemiologically unrelated, indicating that the acquisition of resistance was not through the spread of a resistant clone or a resistance plasmid. A tandem repeat or multiple copies of bla(CTX-M-3) were found in some armA-bearing isolates. An ISEcp1 insert was found in all CTX-M ESBL-producing K. pneumoniae isolates carrying armA or rmtB. In conclusion, the concomitant presence of a 16S rRNA methylase gene (armA or rmtB) and bla(CTX-M) among amikacin-resistant ESBL-producing K. pneumoniae isolates is widespread in Taiwan.