Kadie Holsinger

Antimicrob Agents Chemother. 2023 Jan 30:e0137722. doi: 10.1128/aac.01377-22. Online ahead of print.

ABSTRACT

Gram-negative bacteria are notoriously more resistant to antibiotics than Gram-positive bacteria, primarily due to the presence of the outer membrane and a plethora of active efflux pumps. However, the potency of antibiotics also varies dramatically between different Gram-negative pathogens, suggesting major mechanistic differences in how antibiotics penetrate permeability barriers. Two approaches are used broadly to analyze how permeability barriers affect intracellular accumulation of antibiotics. One compares the antibacterial activities of compounds, while the other measures the total intracellular concentrations of compounds in nongrowing cells, with both approaches using strains harboring wild-type or genetically modified efflux systems and permeability barriers. Whether the two assays provide similar mechanistic insights remains unclear. In this study, we analyzed the intracellular accumulation and antibacterial activities of antibiotics representative of major clinical classes in three Gram-negative pathogens of high clinical importance, Pseudomonas aeruginosa, Escherichia coli, and Acinetobacter baumannii. We found that both assays are informative about properties of permeability barriers, but there is no quantitative agreement between the assays. Our results show that the three pathogens differ dramatically in their permeability barriers, with the outer membrane playing the dominant role in E. coli and P. aeruginosa but efflux dominating in A. baumannii. However, even compounds of the same chemotype may use different permeation pathways depending on small chemical modifications. Accordingly, a classification analysis revealed limited conservation of molecular properties that define compound penetration into the three bacteria.

PMID:36715507 | DOI:10.1128/aac.01377-22

Anal Chem. 2023 Jan 24. doi: 10.1021/acs.analchem.2c05140. Online ahead of print.

ABSTRACT

Effective identification of multiple pathogenic bacteria in unknown samples is important for disease prevention and control but remains a challenge yet. A single-mode array-based sensing approach is simple and sensitive, but it usually relies on the use of multiple cross-reactive receptors to construct sensor arrays, which is cumbersome and insufficiently accurate. Here, we developed a sensor array with colorimetric and photothermal dual mode of differentiating multiple pathogenic bacteria. The sensor array was based on boronic acid-functionalized Au-Fe₃O₄ nanoparticles (BA-GMNPs), which not only possess localized surface plasmon resonance properties, showing a burgundy color similar to that of AuNPs, but also exhibit mild superparamagnetism, allowing for the differentiation of bacteria before and after binding to the nanoparticles. Immobilization of BA-GMNPs on the bacterial cell surface by covalent bonding would diminish NaCl-induced assembly of BA-GMNPs. Different BA-GMNPs@bacterial complexes differed in their ability to resist assembly and produced different colorimetric and photothermal response signals. A unique molecular fingerprint of each bacterium was obtained by linear discriminant analysis of the response patterns, demonstrating an effective differentiation among the six species studied. Compared with single-mode sensing arrays based on multiple receptors, this method only requires the preparation of a single nanomaterial, which produces two signal outputs for the identification of multiple bacteria with better differentiation. It can distinguish not only multiple pathogenic bacteria but also Gram-negative and Gram-positive bacteria, and, more importantly, it can perform preliminary discrimination of unknown samples.

PMID:36693785 | DOI:10.1021/acs.analchem.2c05140

Microb Biotechnol. 2023 Jan 27. doi: 10.1111/1751-7915.14213. Online ahead of print.

ABSTRACT

Antimicrobial peptides play a crucial role in innate immunity, whose components are mainly peptide-based molecules with antibacterial properties. Indeed, the exploration of the immune system over the past 40 years has revealed a number of natural peptides playing a pivotal role in the defence mechanisms of vertebrates and invertebrates, including amphibians, insects, and mammalians. This review provides a discussion regarding the antibacterial mechanisms of peptide-based agents and their structure-activity relationships (SARs) with the aim of describing a topic that is not yet fully explored. Some growing evidence suggests that innate immunity should be strongly considered for the development of novel antibiotic peptide-based libraries. Also, due to the constantly rising concern of antibiotic resistance, the development of new antibiotic drugs is becoming a priority of global importance. Hence, the study and the understanding of defence phenomena occurring in the immune system may inspire the development of novel antibiotic compound libraries and set the stage to overcome drug-resistant pathogens. Here, we provide an overview of the importance of peptide-based antibacterial sources, focusing on accurately selected molecular structures, their SARs including recently introduced modifications, their latest biotechnology applications, and their potential against multi-drug resistant pathogens. Last, we provide cues to describe how antibacterial peptides show a better scope of action selectivity than several anti-infective agents, which are characterized by non-selective activities and non-targeted actions toward pathogens.

PMID:36705032 | DOI:10.1111/1751-7915.14213

Med Res Rev. 2023 Jan 29. doi: 10.1002/med.21936. Online ahead of print.

ABSTRACT

Modified and synthetic α-amino acids are known to show diverse applications. Histidine, which possesses numerous applications when subjected to synthetic modifications, is one such amino acid. The utility of modified histidines varies widely from remarkable biological activities to catalysis, and from nanotechnology to polymer chemistry. This renders histidine residue an important place in scientific research. Histidine is a well-studied scaffold and constitutes the active site of various enzymes catalyzing important reactions in the biological systems. A rational modification in histidine structure with a distinctly developed protocol extensively changes its physical and chemical properties. The utilization of modified histidines in search of potent, target selective and proteostable scaffolds is vital in the development of bioactive peptides with enhanced drug-likeliness. This review is a compilation and analysis of reported side-chain ring modifications at histidine followed by applications of ring-modified histidines in the synthesis of various categories of bioactive peptides and peptidomimetics.

PMID:36710510 | DOI:10.1002/med.21936

Chembiochem. 2023 Jan 30. doi: 10.1002/cbic.202200693. Online ahead of print.

ABSTRACT

Serving as an exoskeletal scaffold, peptidoglycan is a polymeric macromolecule that is essential and conserved across all bacteria, yet is absent in mammalian cells, which renders bacterial peptidoglycan a well-established excellent antibiotic target. In addition, soluble peptidoglycan fragments derived from bacteria are increasingly recognized as key signalling molecules in mediating diverse intra- and inter-species communications in nature, including in gut microbiota-host crosstalk. Each bacteria species encodes multiple redundant enzymes for key enzymatic activities involved in peptidoglycan assembly and breakdown. In this review, we discuss recent findings on the biochemical activities of major peptidoglycan enzymes, including peptidoglycan glycosyltransferases (PGT) and transpeptidases (TPs) in the final stage of peptidoglycan assembly, as well as peptidoglycan glycosidases, lytic transglucosylases (LTs), amidases, endopeptidases (EPs) and carboxypeptidases (CPs) in peptidoglycan turnover and metabolism. Biochemical characterisations of these enzymes provide valuable insights on their substrate specificity, regulation mechanisms and potential modes of inhibition.

PMID:36715567 | DOI:10.1002/cbic.202200693

Virulence. 2023 Jan 24:2171641. doi: 10.1080/21505594.2023.2171641. Online ahead of print.

ABSTRACT

In many Gram-positive bacteria, the transpeptidase enzyme sortase A (SrtA) anchors surface proteins to cell wall and plays a critical role in the bacterial pathogenesis. Here, we show that in Staphylococcus aureus, an important human pathogen, the SrtA is phosphorylated by serine/threonine protein kinase Stk1. S. aureus SrtA can also be phosphorylated by small-molecule phosphodonor acetyl phosphate (AcP) in vitro. We determined that various amino acid residues of S. aureus SrtA are subject to phosphorylation, primarily on its catalytic site residue cysteine-184 in the context of a bacterial cell lysate. Both Stk1 and AcP-mediated phosphorylation inhibited the enzyme activity of SrtA in vitro. Consequently, deletion of gene (i.e. stp1) encoding serine/threonine phosphatase Stp1, the corresponding phosphatase of Stk1, caused an increase in the phosphorylation level of SrtA. The stp1 deletion mutant mimicked the phenotypic traits of srtA deletion mutant (i.e. attenuated growth where either hemoglobin or heme as a sole iron source and reduced liver infections in a mouse model of systemic infection). Importantly, the phenotypic defects of the stp1 deletion mutant can be alleviated by overexpressing srtA. Taken together, our finding suggests that phosphorylation plays an important role in modulating the activity of SrtA in S. aureus.

PMID:36694285 | DOI:10.1080/21505594.2023.2171641

Infect Immun. 2023 Jan 30:e0050022. doi: 10.1128/iai.00500-22. Online ahead of print.

ABSTRACT

The peptidoglycan of Staphylococcus aureus is a critical cell envelope constituent and virulence factor that subverts host immune defenses and provides protection against environmental stressors. Peptidoglycan chains of the S. aureus cell wall are processed to characteristically short lengths by the glucosaminidase SagB. It is well established that peptidoglycan is an important pathogen-associated molecular pattern (PAMP) that is recognized by the host innate immune system and promotes production of proinflammatory cytokines, including interleukin-1β (IL-1β). However, how bacterial processing of peptidoglycan drives IL-1β production is comparatively unexplored. Here, we tested the involvement of staphylococcal glucosaminidases in shaping innate immune responses and identified SagB as a mediator of IL-1β production. A ΔsagB mutant fails to promote IL-1β production by macrophages and dendritic cells, and processing of peptidoglycan by SagB is essential for this response. SagB-dependent IL-1β production by macrophages is independent of canonical pattern recognition receptor engagement and NLRP3 inflammasome-mediated caspase activity. Instead, treatment of macrophages with heat-killed cells from a ΔsagB mutant leads to reduced caspase-independent cleavage of pro-IL-1β, resulting in accumulation of the pro form in the macrophage cytosol. Furthermore, SagB is required for virulence in systemic infection and promotes IL-1β production in a skin and soft tissue infection model. Taken together, our results suggest that the length of S. aureus cell wall glycan chains can drive IL-1β production by innate immune cells through a previously undescribed mechanism related to IL-1β maturation.

PMID:36715551 | DOI:10.1128/iai.00500-22

Mol Pharm. 2023 Jan 25. doi: 10.1021/acs.molpharmaceut.2c00821. Online ahead of print.

ABSTRACT

Small-molecule drugs have been employed for years as therapeutics in the pharmaceutical industry. However, small-molecule drugs typically have short in vivo half-lives which is one of the largest impediments to the success of many potentially valuable pharmacologically active small molecules. The undesirable pharmacokinetics and pharmacology associated with some small molecules have led to the development of a new class of bioconjugates known as chemically programmed antibodies (cPAbs). cPAbs are bioconjugates in which antibodies are used to augment small molecules with effector functions and prolonged pharmacokinetic profiles, where the pharmacophore of the small molecule is harnessed for target binding and therefore biological targeting. Many different small molecules can be conjugated to large proteins such as full monoclonal antibodies (IgG), fragment crystallizable regions (Fc), or fragment antigen binding regions (Fab). In order to successfully and site-specifically conjugate small molecules to any class of antibodies (IgG, Fc, or Fab), the molecules must be derivatized with a functional group for ease of conjugation without altering the pharmacology of the small molecules. In this Review, we summarize the different synthetic or biological methods that have been employed to produce cPAbs. These unique chemistries have potential to be applied to other fields of antibody modification such as antibody drug conjugates, radioimmunoconjugates, and fluorophore-tagged antibodies.

PMID:36696533 | DOI:10.1021/acs.molpharmaceut.2c00821

Int J Mol Sci. 2023 Jan 8;24(2):1242. doi: 10.3390/ijms24021242.

ABSTRACT

Tuberculosis (TB) of the central nervous system (CNS) presents high mortality due to brain damage and inflammation events. The formation and deposition of immune complexes (ICs) in the brain microvasculature during Mycobacterium tuberculosis (Mtb) infection are crucial for its pathobiology. The relevance of ICs to Mtb antigens in the pathogenesis of CNS-TB has been poorly explored. Here, we aimed to establish a murine experimental model of ICs-mediated brain vasculitis induced by cell wall antigens of Mtb. We administered a cell wall extract of the prototype pathogenic Mtb strain H37Rv to male BALB/c mice by subcutaneous and intravenous routes. Serum concentration and deposition of ICs onto blood vessels were determined by polyethylene glycol precipitation, ELISA, and immunofluorescence. Histopathological changes in the brain, lung, spleen, liver, and kidney were evaluated by hematoxylin and eosin staining. Our results evidenced that vasculitis developed in the studied tissues. High serum levels of ICs and vascular deposition were evident in the brain, lung, and kidneys early after the last cell wall antigen administration. Cell wall Mtb antigens induce strong type III hypersensitivity reactions and the development of systemic vasculitis with brain vascular changes and meningitis, supporting a role for ICs in the pathogenesis of TB.

PMID:36674759 | PMC:PMC9866931 | DOI:10.3390/ijms24021242

ACS Omega. 2022 Dec 30;8(2):2485-2490. doi: 10.1021/acsomega.2c06964. eCollection 2023 Jan 17.

ABSTRACT

Microbicides with distinct antibacterial mechanisms show potential to combat multi-drug resistance bacteria. We herein report peptidoglycan-directed chemical ligation (PGCL) between alkyne-bearing vancomycin and an azide-tagged cationic polymer. The former binds peptidoglycan and inhibits peptidoglycan crosslinking, while the latter interferes the integrity of the bacterial membrane. PGCL results in enhanced bactericidal activity against Gram-positive Staphylococcus aureus (S. aureus) over Gram-negative Escherichia coli (E. coli). These data indicate the potential of PGCL to selectively and synergistically inhibit Gram-positive pathogens via dual modality antibacterial mechanisms of peptidoglycan-inhibiting antibiotics and bacterial membrane-disrupting polycations.

PMID:36687063 | PMC:PMC9850734 | DOI:10.1021/acsomega.2c06964

Biochem Soc Trans. 2023 Jan 20:BST20220611. doi: 10.1042/BST20220611. Online ahead of print.

ABSTRACT

The bacterial genus Mycobacterium comprises numerous pathogenic species including M. tuberculosis, the causative agent of the disease tuberculosis. Mycobacteria are obligate aerobes that generate cellular energy through oxidative phosphorylation, the combined activities of the electron transport chain (ETC) and adenosine triphosphate (ATP) synthase. This reliance on oxidative phosphorylation makes the process an attractive target for development of drugs to treat mycobacterial infections. However, targeting the ETC is complicated by the highly branched nature of the chain in mycobacteria and the ability of mycobacteria to alter the expression of ETC constituents in different growth conditions. Here, we review recent characterization of the branched and flexible ETC in mycobacteria, with an emphasis on the structural characterization of mycobacterial ETC complexes by electron cryomicroscopy.

PMID:36661265 | DOI:10.1042/BST20220611

Pharmaceuticals (Basel). 2022 Dec 23;16(1):23. doi: 10.3390/ph16010023.

ABSTRACT

The Toll-like receptor 4 (TLR4) signaling pathway plays a central role in the prompt defense against infectious challenge and provides immediate response to Gram-negative bacterial infection. The TLR4/MD-2 complex can sense and respond to various pathogen-associated molecular patterns (PAMPs) with bacterial lipopolysaccharide (LPS) being the most potent and the most frequently occurring activator of the TLR4-mediated inflammation. TLR4 is believed to be both a friend and foe since improperly regulated TLR4 signaling can result in the overactivation of immune responses leading to sepsis, acute lung injury, or pathologic chronic inflammation involved in cancer and autoimmune disease. TLR4 is also considered a legitimate target for vaccine adjuvant development since its activation can boost the adaptive immune responses. The dual action of the TLR4 complex justifies the efforts in the development of both TLR4 antagonists as antisepsis drug candidates or remedies for chronic inflammatory diseases and TLR4 agonists as vaccine adjuvants or immunotherapeutics. In this review, we provide a brief overview of the biochemical evidences for possible pharmacologic applications of TLR4 ligands as therapeutics and report our systematic studies on the design, synthesis, and immunobiological evaluation of carbohydrate-based TLR4 antagonists with nanomolar affinity for MD-2 as well as disaccharide-based TLR4 agonists with picomolar affinity for the TLR4/MD-2 complex.

PMID:36678520 | DOI:10.3390/ph16010023

Sensors (Basel). 2023 Jan 4;23(2):561. doi: 10.3390/s23020561.

ABSTRACT

The biosensing of bacterial pathogens is of a high priority. Electrochemical biosensors are an important future tool for rapid bacteria detection. A monolayer of bacterial-binding peptides can serve as a recognition layer in such detection devices. Here, we explore the potential of random peptide mixtures (RPMs) composed of phenylalanine and lysine in random sequences and of controlled length, to form a monolayer that can be utilized for sensing. RPMs were found to assemble in a thin and diluted layer that attracts various bacteria. Faradaic electrochemical impedance spectroscopy was used with modified gold electrodes to measure the charge-transfer resistance (R_CT) caused due to the binding of bacteria to RPMs. Pseudomonas aeruginosa was found to cause the most prominent increase in R_CT compared to other model bacteria. We show that the combination of highly accessible antimicrobial RPMs and electrochemical analysis can be used to generate a new promising line of bacterial biosensors.

PMID:36679359 | PMC:PMC9866871 | DOI:10.3390/s23020561

Int J Mol Sci. 2023 Jan 10;24(2):1332. doi: 10.3390/ijms24021332.

ABSTRACT

To date, a number of lantibiotics have been shown to use lipid II-a highly conserved peptidoglycan precursor in the cytoplasmic membrane of bacteria-as their molecular target. The α-component (Lchα) of the two-component lantibiotic lichenicidin, previously isolated from the Bacillus licheniformis VK21 strain, seems to contain two putative lipid II binding sites in its N-terminal and C-terminal domains. Using NMR spectroscopy in DPC micelles, we obtained convincing evidence that the C-terminal mersacidin-like site is involved in the interaction with lipid II. These data were confirmed by the MD simulations. The contact area of lipid II includes pyrophosphate and disaccharide residues along with the first isoprene units of bactoprenol. MD also showed the potential for the formation of a stable N-terminal nisin-like complex; however, the conditions necessary for its implementation in vitro remain unknown. Overall, our results clarify the picture of two component lantibiotics mechanism of antimicrobial action.

PMID:36674846 | PMC:PMC9863751 | DOI:10.3390/ijms24021332

J Org Chem. 2023 Feb 3;88(3):1319-1326. doi: 10.1021/acs.joc.2c01342. Epub 2023 Jan 19.

ABSTRACT

Previously, we developed a method for the detection of unprotected amino groups based on their reversible reaction with N-hydroxyphthalimide (NHPI) to form intensely colored products, which can be useful when conducting solid-phase peptide synthesis. Here, we describe a structure-activity relationship study of NHPI derivatives to identify the derivative best suited for this method using a spectrophotometer toward the estimation of chemical yields. We found that the products resulting from the reaction of the derivative with an unprotected amino group were only intensely colored if the structure of the derivative incorporated an NHPI framework. We also prepared five peptides, including those containing N-methyl and D-amino acid, and Pro residues, using our reversible detection method to detect unprotected amino groups. The mechanism of the detection reaction was also studied by the structural analysis of the NHPI (1) and diisopropylamine complex and concluded to entail salt formation between the N-hydroxy group and amine.

PMID:36655852 | DOI:10.1021/acs.joc.2c01342

Front Cell Infect Microbiol. 2022 Dec 30;12:1124187. doi: 10.3389/fcimb.2022.1124187. eCollection 2022.

NO ABSTRACT

PMID:36644772 | PMC:PMC9837101 | DOI:10.3389/fcimb.2022.1124187

J Biol Chem. 2023 Jan 12:102910. doi: 10.1016/j.jbc.2023.102910. Online ahead of print.

ABSTRACT

Lipids are important nutrients for Mycobacterium tuberculosis (Mtb) to support bacterial survival in mammalian tissues and host cells. Fatty acids and cholesterol are imported across the Mtb cell wall via the dedicated Mce1 and Mce4 transporters, respectively. It is thought that the Mce1 and Mce4 transporters are comprised of subunits that confer substrate specificity and proteins that couple lipid transport to ATP hydrolysis, similar to other bacterial ABC transporters. However, unlike canonical bacterial ABC transporters, Mce1 and Mce4 appear to share a single ATPase, MceG. Previously, it was established that Mce1 and Mce4 are destabilized when key transporter subunits are rendered nonfunctional; therefore, we investigated here the role of MceG in Mce1 and Mce4 protein stability. We determined that key residues in the Walker B domain of MceG are required for the Mce1- and Mce4-mediated transport of fatty acids and cholesterol. Previously it has been established that Mce1 and Mce4 are destabilized and/or degraded when key transporter subunits are rendered nonfunctional, thus we investigated a role for MceG in stabilizing Mce1 and Mce4. Using an unbiased quantitative proteomic approach, we demonstrate that Mce1 and Mce4 proteins are specifically degraded in mutants lacking MceG. Furthermore, bacteria expressing Walker B mutant variants of MceG failed to stabilize Mce1 and Mce4, and we show that deleting MceG impacts the fitness of Mtb in the lungs of mice. Thus, we conclude that MceG represents an enzymatic weakness that can be potentially leveraged to disable and destabilize both the Mce1 and Mce4 transporters in Mtb.

PMID:36642182 | DOI:10.1016/j.jbc.2023.102910

Exp Eye Res. 2023 Jan 9:109383. doi: 10.1016/j.exer.2023.109383. Online ahead of print.

ABSTRACT

Noninfectious exudative conjunctivitis can be experimentally produced in rabbits by application of the apoptogenic bacterial cell wall peptidoglycan, muramyl dipeptide (MDP) to the ocular surface. The purpose of this study was to investigate the acute conjunctival cytopathology induced by unilateral ocular surface exposure to MDP. Hematoxylin and eosin staining assessed bilateral tear cytopathology and conjunctival histopathology. The caspases levels in conjunctival tissue and tears were measured in standard assays utilizing p-nitroanaline tagged caspase-specific substrates. Immunofluorescent antibody identified intracellular caspase-3, nuclear factor-κβ (NF-κβ), and oxidative DNA damage (8-OHdG; 8-oxo-2'-deoxyguanosine) in tear and conjunctiva cells. DNA extracted from conjunctival tissues and pooled tear fluids were visualized by ethydium bromide agarose gel electrophoresis. Onset of ipsilateral conjunctivitis was due to an epitheliopathy characterized by loss of conjunctival epithelial cell adherence, exuviation of conjunctival epithelial cells, and neutrophil infiltration. Caspase-3 levels were significantly higher in exuviated cells in ipsilateral than contralateral tear (p's ≤ 0.001) collected at 3-5 h post MDP. Significantly higher caspase-2, -3, -6, -8 and -9 (p's ≤ 0.03) levels were detected in ipsilateral than contralateral conjunctival tissue at 5 h. Polymeric DNA was detected in ipsilateral but not contralateral conjunctival tissue and tears. Caspase-3, NF-κβ, and 8-OHdG positive neutrophils were detected in bilateral conjunctiva and tear. The caspase-3/NF-κβ epithelial cells and polymeric DNA in conjunctival tissue and shedding of caspase positive cells and polymeric DNA into ipsilateral tears support MDP induction of acute programmed cell death in vivo. The results suggest that ipsilateral exudative conjunctivitis is due to acute caspase-mediated conjunctival epitheliopathy induced following topical exposure to the bacterial peptidoglycan MDP.

PMID:36634837 | DOI:10.1016/j.exer.2023.109383

Nat Biomed Eng. 2023 Jan 12. doi: 10.1038/s41551-022-00991-2. Online ahead of print.

ABSTRACT

Systematically identifying functional peptides is difficult owing to the vast combinatorial space of peptide sequences. Here we report a machine-learning pipeline that mines the hundreds of billions of sequences in the entire virtual library of peptides made of 6-9 amino acids to identify potent antimicrobial peptides. The pipeline consists of trainable machine-learning modules (for performing empirical selection, classification, ranking and regression tasks) assembled sequentially following a coarse-to-fine design principle to gradually narrow down the search space. The leading three antimicrobial hexapeptides identified by the pipeline showed strong activities against a wide range of clinical isolates of multidrug-resistant pathogens. In mice with bacterial pneumonia, aerosolized formulations of the identified peptides showed therapeutic efficacy comparable to penicillin, negligible toxicity and a low propensity to induce drug resistance. The machine-learning pipeline may accelerate the discovery of new functional peptides.

PMID:36635418 | DOI:10.1038/s41551-022-00991-2

Se Pu. 2023 Jan;41(1):1-13. doi: 10.3724/SP.J.1123.2022.11015.

ABSTRACT

Since Nobel Laureate K. B. Sharpless first introduced the concept of click chemistry in 2001, such reactions have become a powerful modular synthesis tool. Click chemistry reactions have rapidly expanded into many scientific fields, such as materials and life science, owing to their distinct advantages, which include mild conditions, fast reaction rates, high yields, low by-product generation, and simple separation and purification procedures. Nowadays, click chemistry reactions have become an essential means of designing and preparing separation materials; thus, interest in this synthetic technique has quickly grown. Here, the development of click chemistry and its unique advantages are briefly described firstly. The reports on click chemistry-based chromatographic separation materials published in the past five years are then systematically reviewed, focusing on two major separation fields: column chromatography and membrane chromatography. Meanwhile, recent advances in the separation materials obtained from three common types of click reactions, namely, azido-alkyne, thiol-alkene, and thiol-alkyne, are summarized. Finally, an outlook on the future of click chemistry is provided in developing efficient chromatographic separation materials.

PMID:36633072 | PMC:PMC9837675 | DOI:10.3724/SP.J.1123.2022.11015

mBio. 2023 Jan 11:e0307322. doi: 10.1128/mbio.03073-22. Online ahead of print.

ABSTRACT

The bacterial cell membrane is an interface for cell envelope synthesis, protein secretion, virulence factor assembly, and a target for host cationic antimicrobial peptides (CAMPs). To resist CAMP killing, several Gram-positive pathogens encode the multiple peptide resistance factor (MprF) enzyme that covalently attaches cationic amino acids to anionic phospholipids in the cell membrane. While E. faecalis encodes two mprF paralogs, MprF2 plays a dominant role in conferring resistance to killing by the CAMP human β-defensin 2 (hBD-2) in E. faecalis strain OG1RF. The goal of the current study is to understand the broader lipidomic and functional roles of E. faecalis mprF. We analyzed the lipid profiles of parental wild-type and mprF mutant strains and show that while ΔmprF2 and ΔmprF1 ΔmprF2 mutants completely lacked cationic lysyl-phosphatidylglycerol (L-PG), the ΔmprF1 mutant synthesized ~70% of L-PG compared to the parent. Unexpectedly, we also observed a significant reduction of PG in ΔmprF2 and ΔmprF1 ΔmprF2. In the mprF mutants, particularly ΔmprF1 ΔmprF2, the decrease in L-PG and phosphatidylglycerol (PG) is compensated by an increase in a phosphorus-containing lipid, glycerophospho-diglucosyl-diacylglycerol (GPDGDAG), and D-ala-GPDGDAG. These changes were accompanied by a downregulation of de novo fatty acid biosynthesis and an accumulation of long-chain acyl-acyl carrier proteins (long-chain acyl-ACPs), suggesting that the suppression of fatty acid biosynthesis was mediated by the transcriptional repressor FabT. Growth in chemically defined media lacking fatty acids revealed severe growth defects in the ΔmprF1 ΔmprF2 mutant strain, but not the single mutants, which was partially rescued through supplementation with palmitic and stearic acids. Changes in lipid homeostasis correlated with lower membrane fluidity, impaired protein secretion, and increased biofilm formation in both ΔmprF2 and ΔmprF1 ΔmprF2, compared to the wild type and ΔmprF1. Collectively, our findings reveal a previously unappreciated role for mprF in global lipid regulation and cellular physiology, which could facilitate the development of novel therapeutics targeting MprF. IMPORTANCE The cell membrane plays a pivotal role in protecting bacteria against external threats, such as antibiotics. Cationic phospholipids such as lysyl-phosphatidyglycerol (L-PG) resist the action of cationic antimicrobial peptides through electrostatic repulsion. Here we demonstrate that L-PG depletion has several unexpected consequences in Enterococcus faecalis, including a reduction of phosphatidylglycerol (PG), enrichment of a phosphorus-containing lipid, reduced fatty acid synthesis accompanied by an accumulation of long-chain acyl-acyl carrier proteins (long chain acyl-ACPs), lower membrane fluidity, and impaired secretion. These changes are not deleterious to the organism as long as exogenous fatty acids are available for uptake from the culture medium. Our findings suggest an adaptive mechanism involving compensatory changes across the entire lipidome upon removal of a single phospholipid modification. Such adaptations must be considered when devising antimicrobial strategies that target membrane lipids.

PMID:36629455 | DOI:10.1128/mbio.03073-22

Curr Biol. 2023 Jan 9;33(1):R33-R36. doi: 10.1016/j.cub.2022.11.025.

ABSTRACT

A new study reveals how Mycobacterium tuberculosis evades anti-bacterial immunity by modifying the plasma membrane phospholipid composition of infected macrophages, thereby blocking the host's pyroptosis response and supporting chronic infection.

PMID:36626862 | DOI:10.1016/j.cub.2022.11.025

Front Microbiol. 2022 Dec 21;13:1056608. doi: 10.3389/fmicb.2022.1056608. eCollection 2022.

ABSTRACT

Mycobacterium tuberculosis possesses a dynamic cell envelope, which consists of a peptidoglycan layer, a mycolic acid layer, and an arabinogalactan polysaccharide. This envelope possesses a highly complex and unique structure representing a barrier that protects and assists the growth of M. tuberculosis and allows its adaptation to the host. It regulates the immune response of the host cells, causing their damage. Therefore, the cell envelope of M. tuberculosis is an attractive target for vaccine and drug development. The emergence of multidrug-resistant as well as extensively drug resistant tuberculosis and co-infection with HIV prevented an effective control of this disease. Thus, the discovery and development of new drugs is a major keystone for TB treatment and control. This review mainly summarizes the development of drug enzymes involved in the biosynthesis of the cell wall in M. tuberculosis, and other potential drug targets in this pathway, to provide more effective strategies for the development of new drugs.

PMID:36620019 | PMC:PMC9810820 | DOI:10.3389/fmicb.2022.1056608