Inflammasome-mediated glucose limitation induces antibiotic tolerance in Staphylococcus aureus.

Staphylococcus aureus is a leading human pathogen that frequently causes relapsing infections. The failure of antibiotics to eradicate infection contributes to infection relapse. Host-pathogen interactions have a substantial impact on antibiotic susceptibility and the formation of antibiotic tolerant cells. In this study, we interrogate how a major S. aureus virulence factor, α-toxin, interacts with macrophages to alter the microenvironment of the pathogen, thereby influencing its susceptibility to antibiotics. We find α-toxin-mediated activation of the NLRP3 inflammasome induces antibiotic tolerance. Induction of tolerance is driven by increased glycolysis in the host cells, resulting in glucose limitation and ATP depletion in S. aureus. Additionally, inhibition of NLRP3 activation improves antibiotic efficacy in vitro and in vivo, suggesting that this strategy has potential as a host-directed therapeutic to improve outcomes. Our findings identify interactions between S. aureus and the host that result in metabolic crosstalk that can determine the outcome of antimicrobial therapy.

Overcoming biological barriers to improve treatment of a Staphylococcus aureus wound infection.

Chronic wounds frequently become infected with bacterial biofilms which respond poorly to antibiotic therapy. Aminoglycoside antibiotics are ineffective at treating deep-seated wound infections due to poor drug penetration, poor drug uptake into persister cells, and widespread antibiotic resistance. In this study, we combat the two major barriers to successful aminoglycoside treatment against a biofilm-infected wound: limited antibiotic uptake and limited biofilm penetration. To combat the limited antibiotic uptake, we employ palmitoleic acid, a host-produced monounsaturated fatty acid that perturbs the membrane of gram-positive pathogens and induces gentamicin uptake. This novel drug combination overcomes gentamicin tolerance and resistance in multiple gram-positive wound pathogens. To combat biofilm penetration, we examined the ability of sonobactericide, a non-invasive ultrasound-mediated-drug delivery technology to improve antibiotic efficacy using an in vivo biofilm model. This dual approach dramatically improved antibiotic efficacy against a methicillin-resistant Staphylococcus aureus (MRSA) wound infection in diabetic mice.

Antibiotic Tolerance and Treatment Outcomes in Cystic Fibrosis Methicillin-Resistant Staphylococcus aureus Infections.

Methicillin-resistant Staphylococcus aureus (MRSA) is highly prevalent in U.S. cystic fibrosis (CF) patients and is associated with worse clinical outcomes in CF. These infections often become chronic despite repeated antibiotic therapy. Here, we assessed whether bacterial phenotypes, including antibiotic tolerance, can predict the clinical outcomes of MRSA infections. MRSA isolates (n = 90) collected at the incident (i.e., acute) and early infection states from 57 patients were characterized for growth rates, biofilm formation, hemolysis, pigmentation, and vancomycin tolerance. The resistance profiles were consistent with those in prior studies. Isolates from the early stage of infection were found to produce biofilms, and 70% of the isolates exhibited delta-hemolysis, an indicator of agr activity. Strong vancomycin tolerance was present in 24% of the isolates but was not associated with intermediate vancomycin susceptibility. There were no associations between these phenotypic measures, antibiotic tolerance, and MRSA clearance. Our research suggests that additional factors may be relevant for predicting the clearance of MRSA. IMPORTANCE Chronic MRSA infections remain challenging to treat in patients with cystic fibrosis (CF). The ability of the bacterial population to survive high concentrations of bactericidal antibiotics, including vancomycin, despite lacking resistance is considered one of the main reasons for treatment failures. The connection between antibiotic tolerance and treatment outcomes remains unexplored and can be crucial for prognosis and regimen design toward eradication. In this study, we measured the capacity of 90 MRSA isolates from CF patients to form vancomycin-tolerant persister cells and evaluated their correlation with the clinical outcomes. Additionally, various traits that could reflect the metabolism and/or virulence of those MRSA isolates were systematically phenotyped and included for their predictive power. Our research highlights that despite the importance of antibiotic tolerance, additional factors need to be considered for predicting the clearance of MRSA.

Chemoproteomics for Plasmodium Parasite Drug Target Discovery.

Emerging Plasmodium parasite drug resistance is threatening progress towards malaria control and elimination. While recent efforts in cell-based, high-throughput drug screening have produced first-in-class drugs with promising activities against different Plasmodium life cycle stages, most of these antimalarial agents have elusive mechanisms of action. Though challenging to address, target identification can provide valuable information to facilitate lead optimization and preclinical drug prioritization. Recently, proteome-wide methods for direct assessment of drug-protein interactions have emerged as powerful tools in a number of systems, including Plasmodium. In this review, we will discuss current chemoproteomic strategies that have been adapted to antimalarial drug target discovery, including affinity- and activity-based protein profiling and the energetics-based techniques thermal proteome profiling and stability of proteins from rates of oxidation. The successful application of chemoproteomics to the Plasmodium blood stage highlights the potential of these methods to link inhibitors to their molecular targets in more elusive Plasmodium life stages and intracellular pathogens in the future.

Chemoproteomics: Towards Global Drug Target Profiling.

Illuminating drugs' mechanisms of action and their effects on the biomolecules of pathogens and humans is a much-needed next step to facilitate pharmaceutical development. Although studies have linked some drugs to their therapeutic targets by using transcriptomics and genomics, these approaches have intrinsic limitations and cannot directly assess drugs' effects on their protein targets. In this regard, chemoproteomic methods can detect protein-ligand interactions and quantitate the chemical or thermal stability changes of the entire detectible proteomes induced by drugs of interest. These widely applicable techniques have recently been adapted to deconvolute the mechanisms of action of antiparasitic drugs and successfully identified an essential target that was previously not known to be druggable. A continued effort to Integrate chemoproteomics into the drug-development pipeline could greatly improve our understanding of drugs' mechanisms, toxicity and pharmacodynamic properties.

Phosphatidylinositol 3-phosphate and Hsp70 protect Plasmodium falciparum from heat-induced cell death.

Phosphatidylinositol 3-phosphate (PI(3)P) levels in Plasmodium falciparum correlate with tolerance to cellular stresses caused by artemisinin and environmental factors. However, PI(3)P function during the Plasmodium stress response was unknown. Here, we used PI3K inhibitors and antimalarial agents to examine the importance of PI(3)P under thermal conditions recapitulating malarial fever. Live cell microscopy using chemical and genetic reporters revealed that PI(3)P stabilizes the digestive vacuole (DV) under heat stress. We demonstrate that heat-induced DV destabilization in PI(3)P-deficient P. falciparum precedes cell death and is reversible after withdrawal of the stress condition and the PI3K inhibitor. A chemoproteomic approach identified PfHsp70-1 as a PI(3)P-binding protein. An Hsp70 inhibitor and knockdown of PfHsp70-1 phenocopy PI(3)P-deficient parasites under heat shock. Furthermore, PfHsp70-1 downregulation hypersensitizes parasites to heat shock and PI3K inhibitors. Our findings underscore a mechanistic link between PI(3)P and PfHsp70-1 and present a novel PI(3)P function in DV stabilization during heat stress.

A Systematic Analysis of Mosquito-Microbiome Biosynthetic Gene Clusters Reveals Antimalarial Siderophores that Reduce Mosquito Reproduction Capacity.

Advances in infectious disease control strategies through genetic manipulation of insect microbiomes have heightened interest in microbially produced small molecules within mosquitoes. Herein, 33 mosquito-associated bacterial genomes were mined and over 700 putative biosynthetic gene clusters (BGCs) were identified, 135 of which belong to known classes of BGCs. After an in-depth analysis of the 135 BGCs, iron-binding siderophores were chosen for further investigation due to their high abundance and well-characterized bioactivities. Through various metabolomic strategies, eight siderophore scaffolds were identified in six strains of mosquito-associated bacteria. Among these, serratiochelin A and pyochelin were found to reduce female Anopheles gambiae overall fecundity likely by lowering their blood-feeding rate. Serratiochelin A and pyochelin were further found to inhibit the Plasmodium parasite asexual blood and liver stages in vitro. Our work supplies a bioinformatic resource for future mosquito-microbiome studies and highlights an understudied source of bioactive small molecules.

Plasmodium chaperonin TRiC/CCT identified as a target of the antihistamine clemastine using parallel chemoproteomic strategy.

The antihistamine clemastine inhibits multiple stages of the Plasmodium parasite that causes malaria, but the molecular targets responsible for its parasite inhibition were unknown. Here, we applied parallel chemoproteomic platforms to discover the mechanism of action of clemastine and identify that clemastine binds to the Plasmodium falciparum TCP-1 ring complex or chaperonin containing TCP-1 (TRiC/CCT), an essential heterooligomeric complex required for de novo cytoskeletal protein folding. Clemastine destabilized all eight P. falciparum TRiC subunits based on thermal proteome profiling (TPP). Further analysis using stability of proteins from rates of oxidation (SPROX) revealed a clemastine-induced thermodynamic stabilization of the Plasmodium TRiC delta subunit, suggesting an interaction with this protein subunit. We demonstrate that clemastine reduces levels of the major TRiC substrate tubulin in P. falciparum parasites. In addition, clemastine treatment leads to disorientation of Plasmodium mitotic spindles during the asexual reproduction and results in aberrant tubulin morphology suggesting protein aggregation. This clemastine-induced disruption of TRiC function is not observed in human host cells, demonstrating a species selectivity required for targeting an intracellular human pathogen. Our findings encourage larger efforts to apply chemoproteomic methods to assist in target identification of antimalarial drugs and highlight the potential to selectively target Plasmodium TRiC-mediated protein folding for malaria intervention.

Tafenoquine: A Step toward Malaria Elimination.

There is a pressing need for compounds with broad-spectrum activity against malaria parasites at various life cycle stages to achieve malaria elimination. However, this goal cannot be accomplished without targeting the tenacious dormant liver-stage hypnozoite that causes multiple relapses after the first episode of illness. In the search for the magic bullet to radically cure Plasmodium vivax malaria, tafenoquine outperformed other candidate drugs and was approved by the U.S. Food and Drug Administration in 2018. Tafenoquine is an 8-aminoquinoline that inhibits multiple life stages of various Plasmodium species. Additionally, its much longer half-life allows for single-dose treatment, which will improve the compliance rate. Despite its approval and the long-time use of other 8-aminoquinolines, the mechanisms behind tafenoquine's activity and adverse effects are still largely unknown. In this Perspective, we discuss the plausible underlying mechanisms of tafenoquine's antiparasitic activity and highlight its role as a cellular stressor. We also discuss potential drug combinations and the development of next-generation 8-aminoquinolines to further improve the therapeutic index of tafenoquine for malaria treatment and prevention.

Discovery of Druggable Host Factors Critical to Plasmodium Liver-Stage Infection.

Plasmodium parasites undergo an obligatory and asymptomatic developmental stage within the liver before infecting red blood cells to cause malaria. The hijacked host pathways critical to parasite infection during this hepatic phase remain poorly understood. Here, we implemented a forward genetic screen to identify over 100 host factors within the human druggable genome that are critical to P. berghei infection in hepatoma cells. Notably, we found knockdown of genes involved in protein trafficking pathways to be detrimental to parasite infection. The disruption of protein trafficking modulators, including COPB2 and GGA1, decreases P. berghei parasite size, and an immunofluorescence study suggests that these proteins are recruited to the Plasmodium parasitophorous vacuole in infected hepatocytes. These findings reveal that various host intracellular protein trafficking pathways are subverted by Plasmodium parasites during the liver stage and provide new insights into their manipulation for growth and development.

Close the ring to break the cycle: tandem quinolone-alkyne-cyclisation gives access to tricyclic pyrrolo[1,2-a]quinolin-5-ones with potent anti-protozoal activity.

Expanding the chemical space of quinolones led to a tandem quinolone-alkyne-cyclisation reaction allowing chemoselective control of the synthesis of tricyclic pyrrolo[1,2-a]quinolin-5-ones. Importantly, we discovered anti-protozoal activity against Plasmodium and Toxoplasma with specific potency of one of the compounds against the liver stage of the malaria parasite in the nanomolar range.

Plasmodium PK9 Inhibitors Promote Growth of Liver-Stage Parasites.

There is a scarcity of pharmacological tools to interrogate protein kinase function in Plasmodium parasites, the causative agent of malaria. Among Plasmodium's protein kinases, those characterized as atypical represent attractive drug targets as they lack sequence similarity to human proteins. Here, we describe takinib as a small molecule to bind the atypical P. falciparum protein kinase 9 (PfPK9). PfPK9 phosphorylates the Plasmodium E2 ubiquitin-conjugating enzyme PfUBC13, which mediates K63-linkage-specific polyubiquitination. Takinib is a potent human TAK1 inhibitor, thus we developed the Plasmodium-selective takinib analog HS220. We demonstrate that takinib and HS220 decrease K63-linked ubiquitination in P. falciparum, suggesting PfPK9 inhibition in cells. Takinib and HS220 induce a unique phenotype where parasite size in hepatocytes increases, yet high compound concentrations decrease the number of parasites. Our studies highlight the role of PK9 in regulating parasite development and the potential of targeting Plasmodium kinases for malaria control.

Identification of Hsp90 Inhibitors with Anti-Plasmodium Activity.

Malaria remains a global health burden partly due to Plasmodium parasite resistance to first-line therapeutics. The molecular chaperone heat shock protein 90 (Hsp90) has emerged as an essential protein for blood-stage Plasmodium parasites, but details about its function during malaria's elusive liver stage are unclear. We used target-based screens to identify compounds that bind to Plasmodium falciparum and human Hsp90, which revealed insights into chemotypes with species-selective binding. Using cell-based malaria assays, we demonstrate that all identified Hsp90-binding compounds are liver- and blood-stage Plasmodium inhibitors. Additionally, the Hsp90 inhibitor SNX-0723 in combination with the phosphatidylinositol 3-kinase inhibitor PIK-75 synergistically reduces the liver-stage parasite load. Time course inhibition studies with the Hsp90 inhibitors and expression analysis support a role for Plasmodium Hsp90 in late-liver-stage parasite development. Our results suggest that Plasmodium Hsp90 is essential to liver- and blood-stage parasite infections and highlight an attractive route for development of species-selective PfHsp90 inhibitors that may act synergistically in combination therapies to prevent and treat malaria.

Diversity and enterotype in gut bacterial community of adults in Taiwan.

BACKGROUND: Gastrointestinal microbiota, particularly gut microbiota, is associated with human health. The biodiversity of gut microbiota is affected by ethnicities and environmental factors such as dietary habits or medicine intake, and three enterotypes of the human gut microbiome were announced in 2011. These enterotypes are not significantly correlated with gender, age, or body weight but are influenced by long-term dietary habits. However, to date, only two enterotypes (predominantly consisting of Bacteroides and Prevotella) have shown these characteristics in previous research; the third enterotype remains ambiguous. Understanding the enterotypes can improve the knowledge of the relationship between microbiota and human health. RESULTS: We obtained 181 human fecal samples from adults in Taiwan. Microbiota compositions were analyzed using next-generation sequencing (NGS) technology, which is a culture-independent method of constructing microbial community profiles by sequencing 16S ribosomal DNA (rDNA). In these samples, 17,675,898 sequencing reads were sequenced, and on average, 215 operational taxonomic units (OTUs) were identified for each sample. In this study, the major bacteria in the enterotypes identified from the fecal samples were Bacteroides, Prevotella, and Enterobacteriaceae, and their correlation with dietary habits was confirmed. A microbial interaction network in the gut was observed on the basis of the amount of short-chain fatty acids, pH value of the intestine, and composition of the bacterial community (enterotypes). Finally, a decision tree was derived to provide a predictive model for the three enterotypes. The accuracies of this model in training and independent testing sets were 97.2 and 84.0%, respectively. CONCLUSIONS: We used NGS technology to characterize the microbiota and constructed a predictive model. The most significant finding was that Enterobacteriaceae, the predominant subtype, could be a new subtype of enterotypes in the Asian population.

Characterization of Lipid-Protein Interactions Using Nonquenched Fluorescent Liposomal Nanovesicles and Yeast Proteome Microarrays.

Studying lipid-protein interactions is central to understanding lipid signaling, a key regulatory system in cells. To better identify lipid-binding proteins, we developed a nonquenched fluorescent (NQF) liposome that is able to carry both fluorescent molecules and a lipid of interest. By combining the strength of NQF liposomes with protein microarray technology, the method presented here facilitates high-throughput screening of lipid-protein interactions. This protocol describes how to prepare NQF liposomes and apply the fabricated liposomes to yeast proteome microarrays.

Protein Microarrays: Flexible Tools for Scientific Innovation.

Protein microarrays have emerged as a powerful tool for the scientific community, and their greatest advantage lies in the fact that thousands of reactions can be performed in a parallel and unbiased manner. The first high-density protein microarray, dubbed the "yeast proteome array," consisted of approximately 5800 full-length yeast proteins and was initially used to identify protein-lipid interactions. Further assays were subsequently developed to allow measurement of protein-DNA, protein-RNA, and protein-protein interactions, as well as four well-known posttranslational modifications: phosphorylation, acetylation, ubiquitylation, and SUMOylation. In this introduction, we describe the advent of high-density protein microarrays, as well as current methods for assessing a wide variety of protein interactions and posttranslational modifications.

Heterogeneous Ribonucleoprotein K (hnRNP K) Binds miR-122, a Mature Liver-Specific MicroRNA Required for Hepatitis C Virus Replication.

Heterogeneous ribonucleoprotein K (hnRNP K) binds to the 5' untranslated region of the hepatitis C virus (HCV) and is required for HCV RNA replication. The hnRNP K binding site on HCV RNA overlaps with the sequence recognized by the liver-specific microRNA, miR-122. A proteome chip containing ∼17,000 unique human proteins probed with miR-122 identified hnRNP K as one of the strong binding proteins. In vitro kinetic study showed hnRNP K binds miR-122 with a nanomolar dissociation constant, in which the short pyrimidine-rich residues in the central and 3' portion of the miR-122 were required for hnRNP K binding. In liver hepatocytes, miR-122 formed a coprecipitable complex with hnRNP K. High throughput Illumina DNA sequencing of the RNAs precipitated with hnRNP K was enriched for mature miR-122. SiRNA knockdown of hnRNP K in human hepatocytes reduced the levels of miR-122. These results show that hnRNP K is a cellular protein that binds and affects the accumulation of miR-122. Its ability to also bind HCV RNA near the miR-122 binding site suggests a role for miR-122 recognition of HCV RNA.

A human proteome microarray identifies that the heterogeneous nuclear ribonucleoprotein K (hnRNP K) recognizes the 5' terminal sequence of the hepatitis C virus RNA.

Stem-loop I (SL1) located in the 5' untranslated region of the hepatitis C virus (HCV) genome initiates binding to miR-122, a microRNA required for hepatitis HCV replication. However, proteins that bind SL1 remain elusive. In this study, we employed a human proteome microarray, comprised of ∼17,000 individually purified human proteins in full-length, and identified 313 proteins that recognize HCV SL1. Eighty-three of the identified proteins were annotated as liver-expressing proteins, and twelve of which were known to be associated with hepatitis virus. siRNA-induced silencing of eight out of 12 candidate genes led to at least 25% decrease in HCV replication efficiency. In particular, knockdown of heterogeneous nuclear ribonucleoprotein K (hnRNP K) reduced HCV replication in a concentration-dependent manner. Ultra-violet-crosslinking assay also showed that hnRNP K, which functions in pre-mRNA processing and transport, showed the strongest binding to the HCV SL1. We observed that hnRNP K, a nuclear protein, is relocated in the cytoplasm in HCV-expressing cells. Immunoprecipitation of the hnRNP K from Huh7.5 cells stably expressing HCV replicon resulted in the co-immunoprecipitation of SL1. This work identifies a cellular protein that could have an important role in the regulation of HCV RNA gene expression and metabolism.

Profiling lipid-protein interactions using nonquenched fluorescent liposomal nanovesicles and proteome microarrays.

Fluorescent liposomal nanovesicles (liposomes) are commonly used for lipid research and/or signal enhancement. However, the problem of self-quenching with conventional fluorescent liposomes limits their applications because these liposomes must be lysed to detect the fluorescent signals. Here, we developed a nonquenched fluorescent (NQF)1 liposome by optimizing the proportion of sulforhodamine B (SRB) encapsulant and lissamine rhodamine B-dipalmitoyl phosphatidylethanol (LRB-DPPE) on a liposomal surface for signal amplification. Our study showed that 0.3% of LRB-DPPE with 200 µm of SRB provided the maximal fluorescent signal without the need to lyse the liposomes. We also observed that the NQF liposomes largely eliminated self-quenching effects and produced greatly enhanced signals than SRB-only liposomes by 5.3-fold. To show their application in proteomics research, we constructed NQF liposomes that contained phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2) and profiled its protein interactome using a yeast proteome microarray. Our profiling led to the identification of 162 PI(3,5)P2-specific binding proteins (PI(3,5)P2-BPs). We not only recovered many proteins that possessed known PI(3,5)P2-binding domains, but we also found two unknown Pfam domains (Pfam-B_8509 and Pfam-B_10446) that were enriched in our dataset. The validation of many newly discovered PI(3,5)P2-BPs was performed using a bead-based affinity assay. Further bioinformatics analyses revealed that the functional roles of 22 PI(3,5)P2-BPs were similar to those associated with PI(3,5)P2, including vesicle-mediated transport, GTPase, cytoskeleton, and kinase. Among the 162 PI(3,5)P2-BPs, we found a novel motif, HRDIKP[ES]NJLL that showed statistical significance. A docking simulation showed that PI(3,5)P2 interacted primarily with lysine or arginine side chains of the newly identified PI(3,5)P2-binding kinases. Our study showed that this new tool would greatly benefit profiling lipid-protein interactions in high-throughput studies.

Broadband micro-Michelson interferometer with multi-optical-path beating using a sphered-end hollow fiber.

We demonstrate a high-sensitivity broadband (1250-1650 nm) fiber micro-Michelson interferometer using a single-mode fiber end-spliced with a sphered-end hollow-core fiber. The hollow core is slightly smaller than the solid core of a single-mode fiber, so the fractional power of the core mode is converted into cladding modes. The excited cladding modes propagate at distinct optical paths along the hollow-core fiber and have individual foci outside the spherical lens. The reflected core mode, generated at the solid core-air interface, and the reflected cladding modes, generated at external material, interfere with each other to produce beating in the interference signals.