Opportunistic Human Pathogen Research Articles

Prevalence and variability of siderophore production in the Achromobacter genus.

Achromobacter spp. are recognized as emerging opportunistic pathogens in humans with various underlying diseases, including cystic fibrosis (CF). Although their pathophysiological traits are increasingly studied, their virulence factors remain incompletely described. Particularly, siderophores that represent important factors of bacterial growth have not yet been studied in this genus. A population-based study was performed to explore the ability of members of the Achromobacter genus to produce siderophores, both overall and in relevant subgroups (Achromobacter species; strain origin, either clinical-from CF or non-CF patients-or environmental). This study provides original data showing that siderophore production is a common trait of Achromobacter strains, particularly observed among clinical strains. The major species, Achromobacter xylosoxidans, encompassed both one of the highest prevalence of siderophore-producing strains and strains producing the largest amounts of siderophores, particularly observed for CF strains. These observations may represent additional advantages accounting for the epidemiological success of this species.

Autolysis of Pseudomonas aeruginosa Quorum-Sensing Mutant Is Suppressed by Staphylococcus aureus through Iron-Dependent Metabolism.

Microorganisms usually coexist as a multifaceted polymicrobial community in the natural habitats and at mucosal sites of the human body. Two opportunistic human pathogens, Pseudomonas aeruginosa and Staphylococcus aureus commonly coexist in the bacterial infections for hospitalized and/or immunocompromised patients. Here, we observed that autolysis of the P. aeruginosa quorum-sensing (QS) mutant (lasRmvfR) was suppressed by the presence of the S. aureus cells in vitro. The QS mutant still displayed killing against S. aureus cells, suggesting the link between the S. aureus-killing activity and the autolysis suppression. Independent screens of the P. aeruginosa transposon mutants defective in the S. aureus-killing and the S. aureus transposon mutants devoid of the autolysis suppression revealed the genetic link between both phenotypes, suggesting that the iron-dependent metabolism involving S. aureus exoproteins might be central to both phenotypes. The autolysis was suppressed by iron treatment as well. These results suggest that the interaction between P. aeruginosa and S. aureus might be governed by mechanisms that necessitate the QS circuitry as well as the metabolism involving the extracellular iron resources during the polymicrobial infections in the human airway.

Redundant essentiality of AsmA-like proteins in Pseudomonas aeruginosa.

Given the importance of the outer membrane (OM) for viability and antibiotic resistance in Gram-negative bacteria, in the last decades, several studies have focused on the characterization of the systems involved in OM biogenesis, which have also been explored as targets for antibacterial drug development. However, the mechanism mediating translocation of glycerophospholipids (GPLs) to the OM remained unknown until recent studies provided evidence that AsmA-like proteins could be responsible for this process. Here, we demonstrate for the first time that AsmA-like proteins are essential and redundant for growth and OM integrity in a Gram-negative bacterium other than the model organism Escherichia coli and demonstrate that the human pathogen Pseudomonas aeruginosa has an additional essential AsmA-like protein that is not present in E. coli, thus expanding the range of AsmA-like proteins that play key functions in Gram-negative bacteria.

Two promising natural lipopeptides from Bacillus subtilis effectively induced membrane permeabilization in Candida glabrata.

Candida glabrata is an important opportunistic human pathogen well known to develop resistance to antifungal drugs. Due to their numerous desirable qualities, antimicrobial lipopeptides have gained significant attention as promising candidates for antifungal drugs. In the present study, two bioactive lipopeptides (AF4 and AF5 m/z 1071.5 and 1085.5, respectively), coproduced and purified from Bacillus subtilis RLID12.1, consist of seven amino acid residues with lipid moieties. In our previous studies, the reversed phased-HPLC purified lipopeptides demonstrated broad-spectrum of antifungal activities against over 110 Candida albicans, Candida non-albicans and mycelial fungi. Two lipopeptides triggered membrane permeabilization of C. glabrata cells, as confirmed by propidium iodide-based flow cytometry, with PI uptake up to 99% demonstrating fungicidal effects. Metabolic inactivation in treated cells was confirmed by FUN-1-based confocal microscopy. Together, the results indicate that these lipopeptides have potentials to be developed into a new set of antifungals for combating fungal infections.

Investigation of Cryptococcus Colonization and Mating Genotype in Environmental Samples

Cryptococcus species are fungal pathogens that pose a serious threat to human life and can cause meningoencephalitis in immunocompromised and healthy individuals. It was estimated that approximately 112000 people die every year due to cryptococcal-related infections all over the world, especially in immunocompromised individuals. Cryptococcus species can be found in soil, bat dung, pigeon droppings, and various tree species in addition to humans. Despite the majority of Cryptococcus species being haploid opportunistic human pathogens, it is known that the ability to undergo sexual reproduction plays a significant role in the expansion of species distribution and the increase in virulence. In Cryptococcus species, sexual reproduction is governed by the mating genotype gene region called the MAT locus. Pathogenic Cryptococcus species have two mating types (MATa and MATα), defined by the presence of one of two alternative alleles at a single MAT locus. In this study, various tree species (eucalyptus, olive and carob) in a total of seven regions in Mersin (Gülnar, Göksu, Narlıkuyu, Ayaş, Kızkalesi, and Tarsus) and Hatay provinces were examined to detect Cryptococcus species. The aim of this study was to determine the environmental distribution and sexual genotypes of Cryptococcus species in these regions. In the present study, samples were collected from a total of 750 trees, including olive, eucalyptus, and carob trees. The samples were incubated on Staib agar medium containing 0.1% biphenyl and 0.5% chloramphenicol. Colonies that formed brown pigment were identified as C.neoformans using conventional and molecular methods. The sexual genotypes were determined by comparing the lengths of the STE20 gene from the isolates compared with those of reference C.neoformans strains. Growth was observed in 97 (12.9%) of 750 samples collected from eucalyptus (n= 236), olive (n= 303) and carob (n= 211) trees. All 97 isolates were determined to be C.neoformans var. grubii. The highest positivity was found in Narlıkuyu (78.2%), and from carob (9.4%) and olive (3.5%) trees. Cryptococcus species was not detected in any of the samples derived from eucalyptus trees. Based on the lengths of the STE20 gene, it was determined that all C.neoformans var. grubii isolates were in the MAT Aα genotype. The data obtained regarding the environmental distribution of Cryptococcus species and the distribution of genes involved in sexual reproduction are believed to provide valuable guidance in terms of the potential clinical implications of environmental Cryptococcus hotspots and regional species characteristics in our country.

Thamnostylum piriforme, a novel etiological agent of superficial mycosis

Fungi are opportunistic eukaryotic entities often taking advantage of susceptibilities offered by a host due to its immunocompromised status, changed microbiome, or ruptured physical barriers and eventually cause infections. They either invade the skin superficially or are deep-seated. Superficial mycosis affects the skin, hair, and nails inhabiting the outermost layer, stratum corneum. In the present study, we report a case of superficial mycosis (onychomycosis in particular) in a 45-year-old immunocompetent man who was an ex-defense personnel and presently serving as a security guard at the University of Jammu, District Jammu, Jammu and Kashmir, India. The infection evolved 17 years ago and negatively affected the quality of life of the patient. For the identification of the causal agent, direct microscopy, cultural, micro-morphological, molecular characterization (ITS sequencing), and phylogenetic analysis were taken into account. A mucoralean fungal species, Thamnostylum piriforme, was isolated from the fingernails (left hand) of the investigated patient, which represents a new global report as the causal agent of superficial mycosis. In vitro antifungal susceptibility testing showed T. piriforme sensitivity to itraconazole, amphotericin B and ketoconazole while resistance to fluconazole. Careful selection of optimal therapy for fungal infection based primarily on correct identification and antifungal susceptibility testing could provide effective results during treatment against these opportunistic human fungal pathogens.

Commercial farmed swine harbour a variety of pathogenic bacteria and antimicrobial resistance genes.

Introduction. The northern region of Thailand serves as a crucial area for swine production, contributing to the Thai community food supply. Previous studies have highlighted the presence of foodborne bacterial pathogens originating from swine farms in this region, posing a threat to both human and animal health.Gap statement. Multiple swine bacterial pathogens have been studied at a species level, but the distribution and co-occurrence of bacterial pathogens in agricultural swine has not been well established.Aim. Our study employed the intestinal scraping technique to directly examine the bacterial micro-organisms interacting with the swine host.Methodology. We used shotgun metagenomic sequencing to analyse the bacterial pathogens inhabiting the caecal microbiome of swine from five commercial farms in northern Thailand.Results. A variety of pathogenic and opportunistic bacteria were identified, including Escherichia coli, Clostridium botulinum, Staphylococcus aureus and the Corynebacterium genus. From a One Health perspective, these species are important foodborne and opportunistic pathogens in both humans and agricultural animals, making swine a critical pathogen reservoir that can cause illness in humans, especially farm workers. Additionally, the swine caecal microbiome contains commensal bacteria such as Bifidobacterium, Lactobacillus and Faecalibacterium, which are associated with normal physiology and feed utilization in healthy swine. Antimicrobial resistance genes were also detected in all samples, specifically conferring resistance to tetracycline and aminoglycosides, which have historically been used extensively in swine farming.Conclusion. The findings further support the need for improved sanitation standards in swine farms, and additional monitoring of agricultural animals and farm workers to reduce contamination and improved produce safety for human consumption.

Characterization of haloacid dehalogenase superfamily acid phosphatase from Staphylococcus lugdunensis

The haloacid dehalogenase superfamily implicated in bacterial pathogenesis comprises different enzymes having roles in many metabolic pathways. Staphylococcus lugdunensis, a Gram-positive bacterium, is an opportunistic human pathogen causing infections in the central nervous system, urinary tract, bones, peritoneum, systemic conditions and cutaneous infection. The haloacid dehalogenase superfamily proteins play a significant role in the pathogenicity of certain bacteria, facilitating invasion, survival, and proliferation within host cells. The genome of S. lugdunensis encodes more than ten proteins belonging to this superfamily. However, none of them have been characterized. The present work reports the characterization of one of the haloacid dehalogenase superfamily proteins (SLHAD1) from Staphylococcus lugdunensis. The functional analysis revealed that SLHAD1 is a metal-dependent acid phosphatase, which catalyzes the dephosphorylation of phosphorylated metabolites of cellular pathways, including glycolysis, gluconeogenesis, nucleotides, and thiamine metabolism. Based on the substrate specificity and genomic analysis, the physiological function of SLHAD1 in thiamine metabolism has been tentatively assigned. The crystal structure of SLHAD1, lacking 49 residues at the C-terminal, was determined at 1.7 Å resolution with a homodimer in the asymmetric unit. It was observed that SLHAD1 exhibited time-dependent cleavage at a specific point, occurring through a self-initiated process. A combination of bioinformatics, biochemical, biophysical, and structural studies explored unique features of SLHAD1. Overall, the study revealed a detailed characterization of a critical enzyme of the human pathogen Staphylococcus lugdunensis, associated with several life-threatening infections.

Extracellular Vesicles from a Biofilm of a Clinical Isolate of Candida albicans Negatively Impact on Klebsiella pneumoniae Adherence and Biofilm Formation.

The opportunistic human fungal pathogen Candida albicans produces and releases into the surrounding medium extracellular vesicles (EVs), which are involved in some processes as communication between fungal cells and host-pathogen interactions during infection. Here, we have conducted the isolation of EVs produced by a clinical isolate of C. albicans during biofilm formation and proved their effect towards the ability of the Gram-negative bacterial pathogen Klebsiella pneumoniae to adhere to HaCaT cells and form a biofilm in vitro. The results represent the first evidence of an antagonistic action of fungal EVs against bacteria.

The Designed Pore-Forming Antimicrobial Peptide C14R Combines Excellent Activity against the Major Opportunistic Human Pathogen Pseudomonas aeruginosa with Low Cytotoxicity.

The diminishing portfolio of mankind's available antibiotics urges science to develop novel potent drugs. Here, we present a peptide fitting the typical blueprint of amphipathic and membrane-active antimicrobial peptides, denominated C14R. This 2 kDa peptide consists of 16 amino acid residues, with seven being either hydrophobic, aromatic, or non-polar, and nine being polar or positively charged, strictly separated on opposite sides of the predicted α-helix. The affinity of the peptide C14R to P. aeruginosa membranes and its intrinsic tendency to productively insert into membranes of such composition were analyzed by dynamic simulations. Its biological impact on the viability of two different P. aeruginosa reference strains was demonstrated by determining the minimal inhibitory concentrations (MICs), which were found to be in the range of 10-15 µg/mL. C14R's pore-forming capability was verified in a permeabilization assay based on the peptide-triggered uptake of fluorescent dyes into the bacterial cells. Finally, the peptide was used in radial diffusion assays, which are commonly used for susceptibility testing of antimicrobial peptides in clinical microbiology. In comparison to reference strains, six clinical P. aeruginosa isolates were clearly affected, thereby paving the way for further in-depth analyses of C14R as a promising new AMP drug in the future.

Housefly gut microbiomes as a reservoir and facilitator for the spread of antibiotic resistance.

Arthropods, such as houseflies, play a significant role in the dissemination of antimicrobial resistance (AMR); however, their impact has often been overlooked in comparison to other AMR vectors. Understanding the contribution of arthropods to the spread of AMR is critical for implementing robust policies to mitigate the spread of AMR across One Health sectors, affecting animals and environmental habitats as well as humans. In this study, we investigated the in situ transfer of a gfp-labelled AMR plasmid (IncA/C carrying an mcr-8 gene, pA/C_MCR-8) in the gut microbiota of houseflies (Musca domestica) by applying single-cell sorting, 16S rRNA gene amplicon sequencing and whole-genome sequencing. Our findings demonstrate that the pA/C_MCR-8-positive Escherichia coli donor strain is capable of colonizing the gut microbiome of houseflies and persists in the housefly intestine for 5 days; however, no transfer was detectable above the detection threshold of 10-5 per cell. The conjugative plasmid pA/C_MCR-8 demonstrated a high transfer frequency ranging from 4.1 × 10-3 to 5.0 × 10-3 per cell in vitro and exhibited transfer across various bacterial phyla, primarily encompassing Pseudomonadota and Bacillota. Phylogenic analysis has revealed that Providencia stuartii, a human opportunistic pathogen, is a notable recipient of pA/C_MCR-8. The conjugation assays further revealed that newly formed P. stuartii transconjugants readily transfer pA/C_MCR-8 to other clinically relevant pathogens (e.g. Klebsiella pneumoniae). Our findings indicate the potential transfer of AMR plasmids from houseflies to human opportunistic pathogens and further support the adoption of a One Health approach in developing infection control policies that address AMR across clinical settings.

Genomic analysis of multidrug-resistant Delftia tsuruhatensis isolated from raw bovine milk.

Delftia tsuruhatensis is a gram-negative, aerobic bacterium mostly known as an organic pollutant degrading and growth-promoting microorganism. However, it recently emerged as an opportunistic human pathogen. To date, the source of D. tsuruhatensis infection is not clear. The majority of studies of D. tsuruhatensis have focused on environmental or clinical strains, while investigations of D. tsuruhatensis strains isolated from food sources are limited. In the present study, we report the case of D. tsuruhatensis isolation from raw bovine milk. Classical bacteriology approaches, as well as next-generation sequencing and comparative genomics, were used to characterize the features of the D. tsuruhatensis MR-6/3H strain. The MR-6/3H strain was resistant to 19 antimicrobials among 23 tested, including all aminoglycosides, phenicol, trimethoprim-sulfamethoxazole, and almost all β-lactams. Phylogenetically, the MR-6/3H was close to clinical origin strains, including those previously isolated in Russia. Comparative genomics revealed the presence of putative antimicrobial resistance genes in the MR-6/3H isolate, mostly associated with efflux systems. Notably, genus-specific OXA-926-like β-lactamase was also detected. In all, 27 putative virulence factors were predicted, the majority of which were associated with motility, adherence, stress survival, siderophore synthesis, and immunomodulation. In the MR-6/3H genome, the five prophage regions were identified, including two with intact levels. Integrons and CRISPR-Cas systems were not detected in the MR-6/3H isolate. Thus, our findings suggest that raw milk can be the potential source of and transmission route for the dissemination of multidrug-resistant D. tsuruhatensis.

Rifabutin: a repurposed antibiotic with high potential against planktonic and biofilm staphylococcal clinical isolates.

Staphylococcus aureus poses a significant threat as an opportunistic pathogen in humans, and animal medicine, particularly in the context of hospital-acquired infections (HAIs). Effective treatment is a significant challenge, contributing substantially to the global health burden. While antibiotic therapy remains the primary approach for staphylococcal infections, its efficacy is often compromised by the emergence of resistant strains and biofilm formation. The anticipated solution is the discovery and development of new antibacterial agents. However, this is a time consuming and expensive process with limited success rates. One potential alternative for addressing this challenge is the repurposing of existing antibiotics. This study investigated the potential of rifabutin (RFB) as a repurposed antibiotic for treating S. aureus infections. The minimum inhibitory concentration (MIC) of rifabutin was assessed by the broth microdilution method, in parallel to vancomycin, against 114 clinical isolates in planktonic form. The minimum biofilm inhibitory concentration (MBIC50) was determined by an adaptation of the broth microdilution method, followed by MTT assay, against a subset of selected 40 clinical isolates organized in biofilms. The study demonstrated that RFB MIC ranged from 0.002 to 6.250 μg/mL with a MIC50 of 0.013 μg/mL. RFB also demonstrated high anti-biofilm activity in the subset of 40 clinical isolates, with confirmed biofilm formation, with no significant MBIC50 differences observed between the MSSA and MRSA strains, in contrast to that observed for the VAN. These results highlight the promising efficacy of RFB against staphylococcal clinical isolates with different resistance patterns, whether in planktonic and biofilm forms.

Electrophysiology of fluoride channels in the yeasts Saccharomyces cerevisiae and Candida albicans.

Tight regulation of molecules moving through the cell membrane is particularly important for free-living microorganisms because of their small cell volumes and frequent changes in the chemical composition of the extracellular environment. This is true for nutrients, but even more so for toxic molecules. Traditionally, the transport of these diverse molecules in microorganisms has been studied on cell populations rather than on single cells, mainly because of technical difficulties. The goal of this chapter is to make available a detailed method to prepare yeast spheroplasts to study the movement of fluoride ions across the plasma membrane of single cells by the patch-clamp technique. In this procedure, three steps are critical to achieve high resistance (GΩ) seals between the membrane and the glass electrode: (1) appropriate removal of the cell wall by enzymatic treatment; (2) balance between the osmotic strength of sealing solutions and cell membrane turgor; and (3) meticulous morphological inspection of spheroplasts suitable for gigaseal formation. We show now that this method, originally developed for Saccharomyces cerevisiae, can also be applied to Candida albicans, an opportunistic human pathogen.

Development and design of CRISPR-based diagnostic for Acinetobacter baumannii by employing off-target gene editing of sgRNA

Acinetobacter baumannii is widely recognized as a human opportunistic pathogen in nosocomial infections. The proliferation of multidrug-resistant strains of A. baumannii has presented an array of difficulties for clinical anti-infective therapies and diagnostic procedures, owing to the existence of numerous variations. The development of therapy utilizing CRISPR/Cas9 for treatment and diagnosis necessitates an in-depth study of potential off-target consequences. The objective of this work is to assess potential off-target effects associated with a single guide RNA (sgRNA) designed to identify several variants present in A. baumannii. The current investigation involved the identification of Cas12 nuclease-specific protospacer adjacent motif (PAM) and downstream target sequences. This was achieved by utilizing computational tools and software to analyze conserved sections of the A. baumannii siderophore protein gene. Further, the in-silico expression vector was created with the SnapGene software. A total of 24 potential off-target sequences were identified in these sequences with 100% query identity with 96 different A. baumannii strains. In addition, a target-specific oligonucleotide single-guide RNA (sgRNA) template was synthesized by appending an additional nucleotide 'G' to the 5′ end. This research uses A. baumannii as an example of a problem that affects all treatments and diagnosis procedures to illustrate the significance of screening off-targets in different variants of a pathogen. Our findings may impact the safety and effectiveness of CRISPR/Cas9, which may have wider implications for additional targets that are currently being used therapeutically.

Antimicrobial peptide AMP-17 induces protection against systemic candidiasis and interacts synergistically with fluconazole against Candida albicans biofilm.

Candida albicans, a common commensal and opportunistic fungal pathogen in humans, can occasionally progress to disseminated candidiasis which is a serious condition with a high morbidity and fatality rate. The emergence of drug-resistant fungal strains compels us to look for an efficient treatment solution. Our earlier studies have demonstrated that the unique antimicrobial peptide AMP-17 from Musca domestica has a strong antifungal impact on C. albicans in vitro. Here, we verified the therapeutic effects of AMP-17 on systemic candidiasis in vivo and the peptide interacts with fluconazole, a common antifungal medication, to treat systemic candidiasis. In the disseminated candidiasis model of Galleria mellonella and mice challenged with C. albicans, AMP-17 increased the survival rates of infected larvae and mice to 66.7 and 75%, respectively. Furthermore, the peptide lowered the load of C. albicans in the infected larvae and the kidneys of the mice by nearly 90%. Additional histological examination and measurements of plasma cytokines showed that the injection of AMP-17 markedly reduced the inflammatory response and balanced cytokine expression. Furthermore, checkerboard micro dilution experiments demonstrated that AMP-17 and fluconazole worked in synergy to inhibit C. albicans in the biofilm mode. According to morphological studies, AMP-17 and fluconazole together decreased the production of hyphae throughout the C. albicans biofilm formation process, loosening the mature biofilms' structure and lowering the amount of carbohydrates in the extracellular matrix (ECM) of the biofilms. Taken together, these results showed that AMP-17 would be a viable treatment for systemic candidiasis and might be a different approach to combating Candida biofilm, either by itself or in conjunction with fluconazole.

Measuring Stress Phenotypes in Cryptococcus neoformans.

Cryptococcus neoformans is an opportunistic human fungal pathogen capable of surviving in a wide range of environments and hosts. It has been developed as a model organism to study fungal pathogenesis due to its fully sequenced haploid genome and optimized gene deletion and mutagenesis protocols. These methods have greatly aided indetermining the relationship between Cryptococcus genotype and phenotype. Furthermore, the presence of congenic mata and matα strains associated with a defined sexual cycle has helped further understand cryptococcal biology. Several in vitro stress conditions have been optimized to closely mimic the stress that yeast encounter in the environment or within the infected host. These conditions have proven to be extremely useful in elucidating the role of several genes in allowing yeast to adapt and survive in hostile external environments. This chapter describes various in vitro stress conditions that could be used to test the sensitivity of different mutant strains, as well as the protocol for preparing them. We have also included a list of mutants that could be used as a positive control strain when testing the sensitivity of the desired strain to a specific stress.

Discovery of a lagriamide polyketide by integrated genome mining, isotopic labeling, and untargeted metabolomics.

Microorganisms from the order Burkholderiales have been the source of a number of important classes of natural products in recent years. For example, study of the beetle-associated symbiont Burkholderia gladioli led to the discovery of the antifungal polyketide lagriamide; an important molecule from the perspectives of both biotechnology and chemical ecology. As part of a wider project to sequence Burkholderiales genomes from our in-house Burkholderiales library we identified a strain containing a biosynthetic gene cluster (BGC) similar to the original lagriamide BGC. Structure prediction failed to identify any candidate masses for the products of this BGC from untargeted metabolomics mass spectrometry data. However, genome mining from publicly available databases identified fragments of this BGC from a culture collection strain of Paraburkholderia. Whole genome sequencing of this strain revealed the presence of a homologue of this BGC with very high sequence identity. Stable isotope feeding of the two strains in parallel using our newly developed IsoAnalyst platform identified the product of this lagriamide-like BGC directly from the crude fermentation extracts, affording a culturable supply of this interesting compound class. Using a combination of bioinformatic, computational and spectroscopic methods we defined the absolute configurations for all 11 chiral centers in this new metabolite, which we named lagriamide B. Biological testing of lagriamide B against a panel of 21 bacterial and fungal pathogens revealed antifungal activity against the opportunistic human pathogen Aspergillus niger, while image-based Cell Painting analysis indicated that lagriamide B also causes actin filament disruption in U2-OS osteosarcoma cells.

Hexadecanoic acid analogs as potential CviR-mediated quorum sensing inhibitors in Chromobacterium violaceum: an in silico study

Chromobacterium violaceum is a Gram-negative, rod-shaped and opportunistic human pathogen. C. violaceum is resistant to various antibiotics due to the production of quorum sensing (QS)-controlled virulence factor and biofilm formation. Hence, we need to find alternative strategies to overcome the antimicrobial resistance and biofilm formation in Gram-negative bacteria. QS is a mechanism in which bacteria’s ability to regulate the virulence factors and biofilm formations leads to disease progression. Previously, hexadecanoic acid was identified as a CviR-mediated quorum-sensing inhibitor. In this study, we aimed to discover potential analogs of hexadecanoic acid as a CviR-mediated quorum-sensing inhibitor against C. violaceum by using ADME/T prediction, density functional theory, molecular docking, molecular dynamics and free energy binding calculations. ADME/T properties predicted for analogs were acceptable for human oral absorption and feasibility. The highest occupied molecular orbitals and lowest unoccupied molecular orbitals gap energies predicted and found oleic acid with –0.3748 energies. Docosatrienoic acid exhibited the highest binding affinity −8.15 Kcal/mol and strong and stable interactions with the amino acid residues on the active site of the CviR protein. These compounds on MD simulations for 100 ns show strong hydrogen-bonding interactions with the protein and remain stable inside the active site. Our results suggest hexadecanoic acid analogs could serve as anti-QS and anti-biofilm molecules for treating C. violaceum infections. However, further validation and investigation of these inhibitors against CviR are needed to claim their candidacy for clinical trials. Communicated by Ramaswamy H. Sarma

Systematic identification and characterization of five transcription factors mediating the oxidative stress response in Candida albicans

Candida albicans is an opportunistic human fungal pathogen that causes superficial and systemic infections, particularly in immunocompromised individuals. In response to C. albicans infection, innate immune cells of the host produce and accumulate reactive oxygen species (ROS), which can lead to irreversible damage and apoptosis of fungal cells. Several transcription factors involved in this oxidative stress response have been identified; however, a systematic study to identify the transcription factors that mediate the oxidative stress response has not yet been conducted. Here, we screened a comprehensive transcription factor mutant library consisting of 211 transcription factor deletion mutant strains in the presence and absence of hydrogen peroxide (H2O2), a potent ROS inducer, and identified five transcription factors (Skn7, Dpb4, Cap1, Dal81, and Stp2) that are sensitive to H2O2. Genome-wide transcriptional profiling revealed that H2O2 induces a discrete set of differentially regulated genes among the five identified transcription factor mutant strains. Functional enrichment analysis identified KEGG pathways pertaining to glycolysis/gluconeogenesis, amino sugar and nucleotide sugar metabolism, and ribosome synthesis as the most enriched pathways. GO term analysis of the top common differentially expressed genes among the transcription factor mutant strains identified hexose catabolism and iron transport as the most enriched GO terms upon exposure to H2O2.This study is the first to systematically identify and characterise the transcription factors involved in the response to H2O2. Based on our transcriptional profiling results, we found that exposure to H2O2 modulates several downstream genes involved in fungal virulence. Overall, this study sheds new light on the metabolism, physiological functions, and cellular processes involved in the H2O2-induced oxidative stress response in C. albicans.