Optimization of Ethoxzolamide Analogs with Improved Pharmacokinetic Properties for In Vivo Efficacy against Neisseria gonorrhoeae.

Drug-resistant gonorrhea is caused by the bacterial pathogen Neisseria gonorrhoeae, for which there is no recommended oral treatment. We have demonstrated that the FDA-approved human carbonic anhydrase inhibitor ethoxzolamide potently inhibits N. gonorrhoeae; however, is not effective at reducing N. gonorrhoeae bioburden in a mouse model. Thus, we sought to optimize the pharmacokinetic properties of the ethoxzolamide scaffold. These efforts resulted in analogs with improved activity against N. gonorrhoeae, increased metabolic stability in mouse liver microsomes, and improved Caco-2 permeability compared to ethoxzolamide. Improvement in these properties resulted in increased plasma exposure in vivo after oral dosing. Top compounds were investigated for in vivo efficacy in a vaginal mouse model of gonococcal genital tract infection, and they significantly decreased the gonococcal burden compared to vehicle and ethoxzolamide controls. Altogether, results from this study provide evidence that ethoxzolamide-based compounds have the potential to be effective oral therapeutics against gonococcal infection.

Novel combinatorial approach: Harnessing HIV protease inhibitors to enhance amphotericin B's antifungal efficacy in cryptococcosis.

Cryptococcosis is a fungal infection that is becoming increasingly prevalent worldwide, particularly among individuals with compromised immune systems, such as HIV patients. Amphotericin B (AmB) is the first-line treatment mainly combined with flucytosine. The scarcity and the prohibitive cost of this regimen urge the use of fluconazole as an alternative, leading to increased rates of treatment failure and relapses. Therefore, there is a critical need for efficient and cost-effective therapy to enhance the efficacy of AmB. In this study, we evaluated the efficacy of the HIV protease inhibitors (PIs) to synergize the activity of AmB in the treatment of cryptococcosis. Five PIs (ritonavir, atazanavir, saquinavir, lopinavir, and nelfinavir) were found to synergistically potentiate the killing activity of AmB against Cryptococcus strains with Æ©FICI ranging between 0.09 and 0.5 against 20 clinical isolates. This synergistic activity was further confirmed in a time-kill assay, where different AmB/PIs combinations exhibited fungicidal activity within 24 hrs. Additionally, PIs in combination with AmB exhibited an extended post-antifungal effect on treated cryptococcal cells for approximately 10 hrs compared to 4 hours with AmB alone. This promising activity against cryptococcal cells did not exhibit increased cytotoxicity towards treated kidney cells, ruling out the risk of drug combination-induced nephrotoxicity. Finally, we evaluated the efficacy of AmB/PIs combinations in the Caenorhabditis elegans model of cryptococcosis, where these combinations significantly reduced the fungal burden of the treated nematodes by approximately 2.44 Log10 CFU (92.4%) compared to the untreated worms and 1.40 Log10 ((39.4%) compared to AmB alone. The cost-effectiveness and accessibility of PIs in resource-limited geographical areas compared to other antifungal agents, such as flucytosine, make them an appealing choice for combination therapy.

Effects of Rigidity and Configuration of Charged Moieties within Cationic Amphiphilic Polyproline Helices on Cell Penetration and Antibiotic Activity.

Effective molecular strategies are needed to target pathogenic bacteria that thrive and proliferate within mammalian cells, a sanctuary inaccessible to many therapeutics. Herein, we present a class of cationic amphiphilic polyproline helices (CAPHs) with a rigid placement of the cationic moiety on the polyproline helix and assess the role of configuration of the unnatural proline residues making up the CAPHs. By shortening the distance between the guanidinium side chain and the proline backbone of the agents, a notable increase in cellular uptake and antibacterial activity was observed, whereas changing the configuration of the moieties on the pyrrolidine ring from cis to trans resulted in more modest increases. When the combination of these two activities was evaluated, the more rigid CAPHs were exceptionally effective at eradicating intracellular methicillin-resistant Staphylococcus aureus (MRSA) and Salmonella infections within macrophages, significantly exceeding the clearance with the parent CAPH.

Exploring novel microbial metabolites and drugs for inhibiting Clostridioides difficile.

Clostridioides difficile is an enteric pathogen that can cause a range of illnesses from mild diarrhea to pseudomembranous colitis and even death. This pathogen often takes advantage of microbial dysbiosis provoked by antibiotic use. With the increasing incidence and severity of infections, coupled with high recurrence rates, there is an urgent need to identify innovative therapies that can preserve the healthy state of the gut microbiota. In this study, we screened a microbial metabolite library against C. difficile. From a collection of 527 metabolites, we identified 18 compounds with no previously identified antimicrobial activity and metabolites that exhibited potent activity against C. difficile growth. Of these 18 hits, five drugs and three metabolites displayed the most potent anti-C. difficile activity and were subsequently assessed against 20 clinical isolates of C. difficile. These potent agents included ecteinascidin 770 (minimum inhibitory concentration against 50% of isolates [MIC50] ≤0.06 µg/mL); 8-hydroxyquinoline derivatives, such as broxyquinoline and choloroquinaldol (MIC50 = 0.125 µg/mL); ionomycin calcium salt, carbadox, and robenidine hydrochloride (MIC50 = 1 µg/mL); and dronedarone and milbemycin oxime (MIC50 = 4 µg/mL). Unlike vancomycin and fidaxomicin, which are the standard-of-care anti-C. difficile antibiotics, most of these metabolites showed robust bactericidal activity within 2-8 h with minimal impact on the growth of representative members of the normal gut microbiota. These results suggest that the drugs and microbial metabolite scaffolds may offer alternative avenues to address unmet needs in C. difficile disease prevention and treatment. IMPORTANCE: The most frequent infection associated with hospital settings is Clostridioides difficile, which can cause fatal diarrhea and severe colitis, toxic megacolon, sepsis, and leaky gut. Those who have taken antibiotics for other illnesses that affect the gut's healthy microbiota are more susceptible to C. difficile infection (CDI). Recently, some reports showed higher recurrence rates and resistance to anti-C. difficile, which may compromise the efficacy of CDI treatment. Our study is significant because it is anticipated to discover novel microbial metabolites and drugs with microbial origins that are safe for the intestinal flora, effective against C. difficile, and reduce the risk of recurrence associated with CDI.

Development of Penicillin-Based Carbonic Anhydrase Inhibitors Targeting Multidrug-Resistant Neisseria gonorrhoeae.

The development of antibacterial drugs with new mechanisms of action is crucial in combating the rise of antibiotic-resistant infections. Bacterial carbonic anhydrases (CAs, EC 4.2.1.1) have been validated as promising antibacterial targets against pathogens such as Helicobacter pylori, Neisseria gonorrhoeae, and vancomycin-resistant enterococci. A multitarget strategy is proposed to design penicillin-based CA inhibitor hybrids for tackling resistance by targeting multiple bacterial pathways, thereby resensitizing drug-resistant strains to clinical antibiotics. The sulfonamide derivatives potently inhibited the CAs from N. gonorrhoeae and Escherichia coli with KI values in the range of 7.1-617.2 nM. Computational simulations with the main penicillin-binding protein (PBP) of N. gonorrhoeae indicated that these hybrid derivatives maintained the mechanism of action of the lead ß-lactams. A subset of derivatives showed potent PBP-related antigonococcal effects against multidrug-resistant N. gonorrhoeae strains, with several compounds significantly outperforming both the lead ß-lactam and CA inhibitor drugs (MIC values in the range 0.25 to 0.5 µg/mL).

Laser-assisted surface alloying of titanium with silver to enhance antibacterial and bone-cell mineralization properties of orthopedic implants.

Orthopedic device-related infection (ODRI) poses a significant threat to patients with titanium-based implants. The challenge lies in developing antibacterial surfaces that preserve the bulk mechanical properties of titanium implants while exhibiting characteristics similar to bone tissue. In response, we present a two-step approach: silver nanoparticle (AgNP) coating followed by selective laser-assisted surface alloying on commonly used titanium alumina vanadium (TiAl6V4) implant surfaces. This process imparts antibacterial properties without compromising the bulk mechanical characteristics of the titanium alloy. Systematic optimization of laser beam power (8-40 W) resulted in an optimized surface (32 W) with uniform TiAg alloy formation. This surface displayed a distinctive hierarchical mesoporous textured surface, featuring cauliflower-like nanostructures measuring between 5-10 nm uniformly covering spatial line periods of 25 µm while demonstrating homogenous elemental distribution of silver throughout the laser processed surface. The optimized laser processed surface exhibited prolonged superhydrophilicity (40 days) and antibacterial efficacy (12 days) against Staphylococcus aureus and Escherichia coli. Additionally, there was a significant twofold increase in bone mineralization compared to the pristine Ti6Al4V surface (p < 0.05). Rockwell hardness tests confirmed minimal (<1%) change in bulk mechanical properties compared to the pristine surface. This innovative laser-assisted approach, with its precisely tailored surface morphology, holds promise for providing enduring antibacterial and osteointegration properties, rendering it an optimal choice for modifying load-bearing implant devices without altering material bulk characteristics.

Lansoprazole interferes with fungal respiration and acts synergistically with amphotericin B against multidrug-resistant Candida auris.

Candida auris has emerged as a problematic fungal pathogen associated with high morbidity and mortality. Amphotericin B (AmB) is the most effective antifungal used to treat invasive fungal candidiasis, with resistance rarely observed among clinical isolates. However, C. auris possesses extraordinary resistant profiles against all available antifungal drugs, including AmB. In our pursuit of potential solutions, we screened a panel of 727 FDA-approved drugs. We identified the proton pump inhibitor lansoprazole (LNP) as a potent enhancer of AmB's activity against C. auris. LNP also potentiates the antifungal activity of AmB against other medically important species of Candida and Cryptococcus. Our investigations into the mechanism of action unveiled that LNP metabolite(s) interact with a crucial target in the mitochondrial respiratory chain (complex III, known as cytochrome bc1). This interaction increases oxidative stress within fungal cells. Our results demonstrated the critical role of an active respiratory function in the antifungal activity of LNP. Most importantly, LNP restored the efficacy of AmB in an immunocompromised mouse model, resulting in a 1.7-log (∼98%) CFU reduction in the burden of C. auris in the kidneys. Our findings strongly advocate for a comprehensive evaluation of LNP as a cytochrome bc1 inhibitor for combating drug-resistant C. auris infections.

Enhanced antifungal activity of posaconazole against Candida auris by HIV protease inhibitors, atazanavir and saquinavir.

The increasing incidence and dissemination of multidrug-resistant Candida auris represents a serious global threat. The emergence of pan-resistant C. auris exhibiting resistance to all three classes of antifungals magnifies the need for novel therapeutic interventions. We identified that two HIV protease inhibitors, atazanavir and saquinavir, in combination with posaconazole exhibited potent activity against C. auris in vitro and in vivo. Both atazanavir and saquinavir exhibited a remarkable synergistic activity with posaconazole against all tested C. auris isolates and other medically important Candida species. In a time-kill assay, both drugs restored the fungistatic activity of posaconazole, resulting in reduction of 5 and 5.6 log10, respectively. Furthermore, in contrast to the individual drugs, the two combinations effectively inhibited the biofilm formation of C. auris by 66.2 and 81.2%, respectively. Finally, the efficacy of the two combinations were tested in a mouse model of C. auris infection. The atazanavir/posaconazole and saquinavir/posaconazole combinations significantly reduced the C. auris burden in mice kidneys by 2.04- (99.1%) and 1.44-log10 (96.4%) colony forming unit, respectively. Altogether, these results suggest that the combination of posaconazole with the HIV protease inhibitors warrants further investigation as a new therapeutic regimen for the treatment of C. auris infections.

Novel phenylthiazoles with a tert-butyl moiety: promising antimicrobial activity against multidrug-resistant pathogens with enhanced ADME properties.

The structure-activity relationship of a new tert-butylphenylthiazole series, with a pyrimidine linker, was investigated. We wished to expand knowledge of this novel class of antibiotics by generating 21 new derivatives bearing ≥2 heteroatoms in their side chains. Their activity was examined against isolates of methicillin-resistant Staphylococcus aureus (MRSA), Clostridium difficile, Escherichia coli, Neisseria gonorrhoeae, and Candida albicans. Two compounds with 1,2-diaminocyclohexane as a nitrogenous side chain showed promising activity against the highly infectious MRSA USA300 strain, with a minimum inhibitory concentration (MIC) of 4 µg mL-1. One of these two compounds demonstrated potent activity against C. difficile, with a MIC of 4 µg mL-1. Moderate activities against a C. difficile strain with a MIC of 8 µg mL-1 were noted. Some new compounds possessed antifungal activity against a wild fluconazole-resistant C. albicans strain, with MIC values of 4-16 µg mL-1. ADME and metabolism-simulation studies were performed for the most promising compound and compared with lead compounds. Our results revealed that one compound possessed greater penetration of bacterial membranes and metabolic resistance, which aided a longer duration of action against MRSA.

Inhibition of pathogenic bacterial carbonic anhydrases by monothiocarbamates.

Carbonic anhydrases (CAs) from the pathogenic bacteria Nesseria gonorrhoeae and vancomycin-resistant enterococci (VRE) have recently been validated as antibacterial drug targets. Here we explored the inhibition of the α-CA from N. gonorrhoeae (α-NgCA), of α- and γ-class enzymes from Enterococcus faecium (α-EfCA and γ-EfCA) with a panel of aliphatic, heterocyclic and aryl-alkyl primary/secondary monothiocarbamates (MTCs). α-NgCA was inhibited in vitro with KIs ranging from 0.367 to 0.919 µM. The compounds inhibited the α-EfCA and γ-EfCA with KI ranges of 0.195-0.959 µM and of 0.149-1.90 µM, respectively. Some MTCs were also investigated for their inhibitory effects on the growth of clinically-relevant N. gonorrhoeae and VRE strains. No inhibitory effects on the growth of VRE were noted for all MTCs, whereas one compound (13) inhibited the growth N. gonorrhoeae strains at concentrations ranging from 16 to 64 µg/mL. This suggests that compound 13 may be a potential antibacterial agent against N. gonorrhoeae.

Naphthylthiazoles: a class of broad-spectrum antifungals.

Cryptococcal infections remain a major cause of mortality worldwide due to the ability of Cryptococci to pass through the blood-brain barrier (BBB) causing lethal meningitis. The limited number of available therapeutics, which exhibit limited availability, severe toxicity and low tolerability, necessitates the development of new therapeutics. Investigating the antifungal activity of a novel series of naphthylthiazoles provided trans-diaminocyclohexyl derivative 18 with many advantageous attributes as a potential therapeutic for cryptococcal meningitis. Briefly, the antimycotic activity of 18 against cryptococcal strains was highly comparable to that of amphotericin-B and fluconazole with MIC values as low as 1 µg mL-1. Moreover, compound 18 possessed additional advantages over fluconazole; it significantly reduced the intracellular burden of Cryptococci and markedly inhibited cryptococcal biofilm formation. Initial PK assessment of 18 indicated its ability to reach the CNS after oral administration with high permeability, and it maintained therapeutic plasma concentrations for 18 h. Its antifungal activity extended to other clinically relevant strains, such as fluconazole-resistant C. auris.

Colonization efficiency of multidrug-resistant Neisseria gonorrhoeae in a female mouse model.

The rapid occurrence of gonococcal resistance to all classes of antibiotics could lead to untreatable gonorrhea. Thus, development of novel anti-Neisseria gonorrhoeae drugs is urgently needed. Neisseria gonorrhoeae FA1090 is the most used in gonococcal infection mouse models because of its natural resistance to streptomycin. Streptomycin inhibits the urogenital commensal flora that permits gonococcal colonization. However, this strain is drug-susceptible and cannot be used to investigate the efficacy of novel agents against multidrug-resistant N. gonorrhoeae. Hence, to test the in vivo efficacy of new therapeutics against N. gonorrhoeae resistant to the frontline antibiotics, azithromycin, or ceftriaxone, we constructed streptomycin-resistant mutants of N. gonorrhoeae CDC-181 (azithromycin-resistant) and WHO-X (ceftriaxone-resistant). We identified the inoculum size needed to successfully colonize mice. Both mutants, CDC-181-rpsLA128G and WHO-X-rpsLA128G, colonized the genital tract of mice for 14 days with 100% colonization observed for at least 7 days. CDC-181-rpsLA128G demonstrated better colonization of the murine genital tract compared to WHO-X-rpsLA128G. Lower inoculum of WHO-X-rpsLA128G (105 and 106 CFU) colonized mice better than higher inoculum. Overall, our results indicate that CDC-181-rpsLA128G and WHO-X-rpsLA128G can colonize the lower genital tract of mice and are suitable to be used in mouse models to investigate the efficacy of antigonococcal agents.

Antisense inhibition of RNA polymerase α subunit of Clostridioides difficile.

Clostridioides difficile, the causative agent of antibiotic-associated diarrhea and pseudomembranous colitis, has emerged as a major enteric pathogen in recent years. Antibiotic treatment perturbs the gut microbiome homeostasis, which facilitates the colonization and proliferation of the pathogen in the host intestine. Paradoxically, the clinical repertoire for C. difficile infection includes the antibiotics vancomycin and/or fidaxomicin. The current therapies do not address the perturbed gut microbiome, which supports the recurrence of infection after cessation of antibiotic therapy. Peptide nucleic acids (PNAs) are novel alternatives to traditional antimicrobial therapy capable of forming strong and stable complexes with RNA and DNA, thus permitting targeted inhibition of specific genes. Here, we report a novel PNA that can target the RNA polymerase α subunit (rpoA) in C. difficile. The designed anti-rpoA construct inhibited clinical isolates of C. difficile (minimum inhibitory concentration values ranged between 4 and 8 µM) and exhibited bactericidal activity. Furthermore, silencing of the rpoA gene suppressed the expression of genes that encode virulence factors [toxin A (tcdA), toxin B (tcdB)] in C. difficile, and the gene that encodes the transcription factor stage 0 sporulation protein (spoOA). Interestingly, the efficacy of the designed PNA conjugate remained unaffected even when tested at different pH levels and against a high inoculum of the pathogen. The rpoA-TAT conjugate was very specific against C. difficile and did not inhibit members of the beneficial gut microflora. Taken altogether, our study confirms that the rpoA gene can be a promising narrow-spectrum therapeutic target to curb C. difficile infection. IMPORTANCE The widespread use of antibiotics can destroy beneficial intestinal microflora, opening the door for spores of Clostridioides difficile to run rampant in the digestive system, causing life-threatening diarrhea. Alternative approaches to target this deadly pathogen are urgently needed. We utilized targeted therapeutics called peptide nucleic acids (PNAs) to inhibit gene expression in C. difficile. Inhibition of the RNA polymerase α subunit gene (rpoA) by PNA was found to be lethal for C. difficile and could also disarm its virulence factors. Additionally, antisense inhibition of the C. difficile rpoA gene did not impact healthy microflora. We also propose a novel approach to manipulate gene expression in C. difficile without the need for established genetic tools.

Substituted salicylic acid analogs offer improved potency against multidrug-resistant Neisseria gonorrhoeae and good selectivity against commensal vaginal bacteria.

Drug-resistant Neisseria gonorrhoeae represents a major threat to public health; without new effective antibiotics, untreatable gonococcal infections loom as a real possibility. In a previous drug-repurposing study, we reported that salicylic acid had good potency against azithromycin-resistant N. gonorrhoeae. We now report that the anti-gonococcal activity in this scaffold is easily lost by inopportune substitution, but that select substituted naphthyl analogs (3b, 3o and 3p) have superior activity to salicylic acid itself. Furthermore, these compounds retained potency against multiple ceftriaxone- and azithromycin-resistant strains, exhibited rapid bactericidal activity against N. gonorrhoeae, and showed high tolerability to mammalian cells (CC50 > 128 µg/mL). Promisingly, these compounds also show very weak growth inhibition of commensal vaginal bacteria.

Saquinavir potentiates itraconazole's antifungal activity against multidrug-resistant Candida auris in vitro andin vivo.

Candida species are highly opportunistic yeasts that are responsible for serious invasive fungal infections among immunocompromised patients worldwide. Due to the increase in drug resistance and incidence of infections, there is an urgent need to develop new antifungals and to identify co-drugs that can sensitize drug-resistant Candida to antifungals. The objective of this study was to assess the effect of saquinavir on the activity of azole antifungals against C. auris. The in vitro interaction of saquinavir and three azole antifungals (itraconazole, voriconazole, and fluconazole) was evaluated against a panel of C. auris isolates. The itraconazole/saquinavir combination exhibited a synergistic (SYN) relationship against all C. auris isolates tested with the fractional inhibitory concentration index ranging from 0.03 to 0.27. Moreover, a time-kill kinetics assay revealed that saquinavir restored the itraconazole's fungistatic activity against C. auris. Furthermore, saquinavir restored itraconazole's antifungal activity against other clinically important Candida species. The mechanistic investigation indicated that saquinavir significantly inhibited efflux pumps, glucose utilization, and ATP synthesis in Candida. Finally, a murine model of C. auris infection was used to evaluate the efficacy of the itraconazole/saquinavir combination in the presence of ritonavir (as a pharmacokinetic enhancer). The combination significantly reduced the fungal burden in the kidneys by 0.93-log10 colony-forming units (88%) compared to itraconazole alone. This study identified that saquinavir exhibits a potent SYN relationship in combination with itraconazole against Candida species, which warrants further consideration.

Candida auris is a multi-drug resistant fungal pathogen with limited treatment options. In this study, we identified that the antiviral drug, saquinavir, is capable of synergizing and restoring the activity of antifungals against C. auris.

Lopinavir and ritonavir act synergistically with azoles against Candida auris in vitro and in a mouse model of disseminated candidiasis.

INTRODUCTION AND OBJECTIVES: The emergence of Candida auris has created a global health challenge. Azole antifungals are the most affected antifungal class because of the extraordinary capability of C. auris to develop resistance against these drugs. Here, we used a combinatorial therapeutic approach to sensitize C. auris to azole antifungals. METHODS AND RESULTS: We have demonstrated the capability of the HIV protease inhibitors lopinavir and ritonavir, at clinically relevant concentrations, to be used with azole antifungals to treat C. auris infections both in vitro and in vivo. Both lopinavir and ritonavir exhibited potent synergistic interactions with the azole antifungals, particularly with itraconazole against 24/24 (100%) and 31/34 (91%) of tested C. auris isolates, respectively. Furthermore, ritonavir significantly interfered with the fungal efflux pump, resulting in a significant increase in Nile red fluorescence by 44%. In a mouse model of C. auris systemic infection, ritonavir boosted the activity of lopinavir to work synergistically with fluconazole and itraconazole and significantly reduced the kidney fungal burden by a 1.2 log (∼94%) and 1.6 log (∼97%) CFU, respectively. CONCLUSION: Our results urge further comprehensive assessment of azoles and HIV protease inhibitors as a novel drug regimen for the treatment of serious invasive C. auris infections.

Exploring novel aryl/heteroaryl-isosteres of phenylthiazole against multidrug-resistant bacteria.

Antimicrobial resistance has become a concern as a worldwide threat. A novel scaffold of phenylthiazoles was recently evaluated against multidrug-resistant Staphylococci to control the emergence and spread of antimicrobial resistance, showing good results. Several structural modifications are needed based on the structure-activity relationships (SARs) of this new antibiotic class. Previous studies revealed the existence of two key structural features essential for the antibacterial activity, the guanidine head and lipophilic tail. In this study, a new series of twenty-three phenylthiazole derivatives were synthesized utilizing the Suzuki coupling reaction to explore the lipophilic part. The in vitro antibacterial activity was evaluated against a range of clinical isolates. The three most promising compounds, 7d, 15d and 17d, with potent MIC values against MRSA USA300 were selected for further antimicrobial evaluation. The tested compounds exhibited potent results against the tested MSSA, MRSA, and VRSA strains (concentration: 0.5 to 4 µg mL-1). Compound 15d inhibited MRSA USA400 at a concentration of 0.5 µg mL-1 (one-fold more potent than vancomycin) and showed low MIC values against ten clinical isolates, including linezolid-resistant strain MRSA NRS119 and three vancomycin-resistant isolates VRSA 9/10/12. Moreover, compound 15d retained its potent antibacterial activity using the in vivo model by the burden reduction of MRSA USA300 in skin-infected mice. The tested compounds also showed good toxicity profiles and were found to be highly tolerable to Caco-2 cells at concentrations of up to 16 µg mL-1, with 100% of the cells remaining viable.

Fingerprint Stimulated Raman Scattering Imaging Unveils Ergosteryl Ester as a Metabolic Signature of Azole-Resistant Candida albicans.

Candida albicans (C. albicans), a major fungal pathogen, causes life-threatening infections in immunocompromised individuals. Fluconazole (FLC) is recommended as first-line therapy for treatment of invasive fungal infections. However, the widespread use of FLC has resulted in increased antifungal resistance among different strains of Candida, especially C. albicans, which is a leading source of hospital-acquired infections. Here, by hyperspectral stimulated Raman scattering imaging of single fungal cells in the fingerprint window and pixel-wise spectral unmixing, we report aberrant ergosteryl ester accumulation in azole-resistant C. albicans compared to azole-susceptible species. This accumulation was a consequence of de novo lipogenesis. Lipid profiling by mass spectroscopy identified ergosterol oleate to be the major species stored in azole-resistant C. albicans. Blocking ergosterol esterification by oleate and suppressing sterol synthesis by FLC synergistically suppressed the viability of C. albicans in vitro and limited the growth of biofilm on mouse skin in vivo. Our findings highlight a metabolic marker and a new therapeutic strategy for targeting azole-resistant C. albicans by interrupting the esterified ergosterol biosynthetic pathway.

Cell-surface anchoring of Listeria adhesion protein on L. monocytogenes is fastened by internalin B for pathogenesis.

Listeria adhesion protein (LAP) is a secreted acetaldehyde alcohol dehydrogenase (AdhE) that anchors to an unknown molecule on the Listeria monocytogenes (Lm) surface, which is critical for its intestinal epithelium crossing. In the present work, immunoprecipitation and mass spectrometry identify internalin B (InlB) as the primary ligand of LAP (KD ∼ 42 nM). InlB-deleted and naturally InlB-deficient Lm strains show reduced LAP-InlB interaction and LAP-mediated pathology in the murine intestine and brain invasion. InlB-overexpressing non-pathogenic Listeria innocua also displays LAP-InlB interplay. In silico predictions reveal that a pocket region in the C-terminal domain of tetrameric LAP is the binding site for InlB. LAP variants containing mutations in negatively charged (E523S, E621S) amino acids in the C terminus confirm altered binding conformations and weaker affinity for InlB. InlB transforms the housekeeping enzyme, AdhE (LAP), into a moonlighting pathogenic factor by fastening on the cell surface.