Kadie Holsinger

Clin Microbiol Infect. 2022 Oct 18:S1198-743X(22)00531-6. doi: 10.1016/j.cmi.2022.10.017. Online ahead of print.

ABSTRACT

OBJECTIVES: Rapid detection of bacterial pathogens to species and subspecies level is crucial for appropriate treatment, infection control and public health management. Currently, one of the challenges in clinical microbiology is the discrimination of mycobacterial sub-species within the M. tuberculosis complex (MTBC). Our objective was to evaluate the ability of a biosafe mycobacterial-lipid based approach to identify MTBC cultures and subspecies.

METHODS: A blinded study was performed using 90 mycobacterial clinical isolates strains comprising MTBC strains sub-cultured in Middlebrook 7H11 media supplemented with 10% OADC growth supplement and incubated for up to six weeks at 37°C and using the following 7 reference strains (M. tuberculosis H37Rv, M canettii, M. africanum, M. pinnipedii, M. caprae, M. bovis, M. bovis BCG) grown under the same conditions, in order to set the reference lipid database and test it against the 90 MTBC clinical isolates. Cultured mycobacteria were heat-inactivated and loaded onto the MALDI target followed by addition of the matrix. Acquisition of the data was done using the positive ion mode.

RESULTS: Based on the identification of clear and defined lipid signatures from the 7 reference strains, the method we have developed is fast (<10 mins) and produced interpretable profiles for all but four isolates, caused by poor ionization giving an n = 86 with interpretable spectra. The sensitivity and specificity of the MALDI-ToF, were 94.4 (95% CI 86.4-98.5) and 94.4 (95% CI 72.7-99.9) respectively. .

CONCLUSIONS: Mycobacterial lipid profiling provides for a means of rapid, safe and accurate discrimination of species within the MTBC.

PMID:36270589 | DOI:10.1016/j.cmi.2022.10.017

Plant Mol Biol. 2022 Oct 22. doi: 10.1007/s11103-022-01317-1. Online ahead of print.

ABSTRACT

The C-terminal stretch in SbGPRP1 (Sorghum glycine-rich proline-rich protein) acts as an antimicrobial peptide in the host innate defense mechanism. Cationic antimicrobial proteins or peptides can either bind to the bacterial membrane or target a specific protein on the bacterial membrane thus leading to membrane perturbation. The 197 amino acid polypeptide of SbGPRP1 showed disordered structure at the N-terminal end and ordered conformation at the C-terminal end. In the present study, the expression of N-SbGPRP1, C-SbGPRP1, and ∆SbGPRP1 followed by antimicrobial assays showed potential antimicrobial property of the C-terminal peptide against gram-positive bacteria Bacillus subtilis and phytopathogen Rhodococcus fascians. The SbGPRP1 protein loses its antimicrobial property when the 23 amino acid sequence (GHGGHGVFGGGYGHGGYGHGYGG) from position 136 to 158 is deleted from the protein. Thus, it can be concluded that the 23 amino acid sequence is vital for the said antimicrobial property. NPN assay, SEM analysis, and electrolyte leakage assays showed potent antimicrobial activity for C-SbGPRP1. Overexpression of the C-SbGPRP1 mutant protein in tobacco followed by infection with Rhodococcus fascians inhibited bacterial growth as shown by SEM analysis. To determine if C-SbGPRP1 might target any protein on the bacterial membrane we isolated the bacterial membrane protein from both Bacillus subtilis and Rhodococcus fascians. Bacterial membrane protein that interacted with the column-bound C-SbGPRP1 was eluted and subjected to LC-MS/MS. LC-MS/MS data analysis showed peptide hit with membrane protein YszA from Bacillus subtilis and a membrane protein from Rhodococcus fascians. Isolated bacterial membrane protein from Bacillus subtilis or Rhodococcus fascians was able to reduce the antimicrobial activity of C-SbGPRP1. Furthermore, BiFC experiments showed interactions between C-SbGPRP1 and YszA protein from Bacillus subtilis leading to the conclusion that bacterial membrane protein was targeted in such membrane perturbation leading to antimicrobial activity.

PMID:36271987 | DOI:10.1007/s11103-022-01317-1

Can J Microbiol. 2022 Oct 20. doi: 10.1139/cjm-2022-0135. Online ahead of print.

ABSTRACT

Lactoferrin is an innate glycoprotein with broad antibacterial and antibiofilm properties. Lactoferrin's autonomous antibiofilm activity against Gram-positive bacteria is postulated to involve the cell wall and biofilm components. Thus, the prevention of biomass formation and eradication of preformed biofilms by lactoferrin was investigated using a methicillin-resistant Staphylococcus epidermidis (MRSE) strain. Additionally, the ability of lactoferrin to modulate the expression of the biofilm-associated protein gene (bap) was studied. The bap gene regulates the production of biofilm-associated proteins responsible for bacterial adhesion and aggregation. In the in vitro biofilm assays, lactoferrin prevented biofilm formation and eradicated established biofilms for up to 24 and 72 hours, respectively. Extensive eradication of MRSE biofilm biomass was accompanied by the significant upregulation of bap gene expression. These data suggest the interaction of lactoferrin with the biofilm components and cell wall of MRSE, including the biofilm-associated protein.

PMID:36265186 | DOI:10.1139/cjm-2022-0135

Front Immunol. 2022 Oct 7;13:971883. doi: 10.3389/fimmu.2022.971883. eCollection 2022.

ABSTRACT

The Apextrin C-terminal (ApeC) domain is a new protein domain largely specific to aquatic invertebrates. In amphioxus, a short-form ApeC-containing protein (ACP) family is capable of binding peptidoglycan (PGN) and agglutinating bacteria via its ApeC domain. However, the functions of ApeC in other phyla remain unknown. Here we examined 130 ACPs from gastropods and bivalves, the first and second biggest mollusk classes. They were classified into nine groups based on their phylogenetics and architectures, including three groups of short-form ACPs, one group of apextrins and two groups of ACPs of complex architectures. No groups have orthologs in other phyla and only four groups have members in both gastropods and bivalves, suggesting that mollusk ACPs are highly diversified. We selected one bivalve ACP (CgACP1; from the oyster Crossostrea gigas) and one gastropod ACP (BgACP1; from the snail Biomphalaria glabrata) for functional experiments. Both are highly-expressed, secreted short-form ACPs and hence comparable to the amphioxus ACPs previously reported. We found that recombinant CgACP1 and BgACP1 bound with yeasts and several bacteria with different affinities. They also agglutinated these microbes, but showed no inhibiting or killing effects. Further analyses show that both ACPs had high affinities to the Lys-type PGN from S. aureus but weak or no affinities to the DAP-type PGN from Bacillus subtilis. Both recombinant ACPs displayed weak or no affinities to other microbial cell wall components, including lipopolysaccharide (LPS), lipoteichoic acid (LTA), zymosan A, chitin, chitosan and cellulose, as well as to several PGN moieties, including muramyl dipeptide (MDP), N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). Besides, CgACP1 had the highest expression in the gill and could be greatly up-regulated quickly after bacterial challenge. This is reminiscent of the amphioxus ACP1/2 which serve as essential mucus lectins in the gill. Taken together, the current findings from mollusk and amphioxus ACPs suggest several basic common traits for the ApeC domains, including the high affinity to Lys-type PGN, the bacterial binding and agglutinating capacity, and the role as mucus proteins to protect the mucosal surface.

PMID:36275759 | PMC:PMC9585378 | DOI:10.3389/fimmu.2022.971883

J Med Chem. 2022 Oct 21. doi: 10.1021/acs.jmedchem.2c01275. Online ahead of print.

ABSTRACT

To develop novel antibiotics, targeting the early steps of cell wall peptidoglycan biosynthesis seems to be a promising strategy that is still underutilized. MurA, the first enzyme in this pathway, is targeted by the clinically used irreversible inhibitor fosfomycin. However, mutations in its binding site can cause bacterial resistance. We herein report a series of novel reversible pyrrolidinedione-based MurA inhibitors that equally inhibit wild type (WT) MurA and the fosfomycin-resistant MurA C115D mutant, showing an additive effect with fosfomycin for the inhibition of WT MurA. For the most potent inhibitor 46 (IC₅₀ = 4.5 μM), the mode of inhibition was analyzed using native mass spectrometry and protein NMR spectroscopy. The compound class was nontoxic against human cells and highly stable in human S9 fraction, human plasma, and bacterial cell lysate. Taken together, this novel compound class might be further developed toward antibiotic drug candidates that inhibit cell wall synthesis.

PMID:36269107 | DOI:10.1021/acs.jmedchem.2c01275

ACS Infect Dis. 2022 Nov 11;8(11):2374-2388. doi: 10.1021/acsinfecdis.2c00460. Epub 2022 Oct 20.

ABSTRACT

A key mechanism driving antimicrobial resistance (AMR) stems from the ability of bacteria to up-regulate efflux pumps upon exposure to drugs. The resistance gained by this up-regulation is pliable because of the tight regulation of efflux pump levels. This leads to temporary enhancement in survivability of bacteria due to higher efflux pump levels in the presence of antibiotics, which can be reversed when the cells are no longer exposed to the drug. Knowledge of the extent of resistance thus gained would inform intervention strategies aimed at mitigating AMR. Here, we combine mathematical modeling and experiments to quantify the maximum extent of resistance that efflux pump up-regulation can confer via phenotypic induction in the presence of drugs and genotypic abrogation of regulation. Our model describes the dynamics of drug transport in and out of cells coupled with the associated regulation of efflux pump levels and predicts the increase in the minimum inhibitory concentration (MIC) of drugs due to such regulation. To test the model, we measured the uptake and efflux as well as the MIC of the compound ethidium bromide (EtBr), a substrate of the efflux pump LfrA, in wild-type Mycobacterium smegmatis mc²155, as well as in two laboratory-generated strains. Our model captured the observed EtBr levels and MIC fold-changes quantitatively. Further, the model identified key parameters associated with the resulting resistance, variations in which could underlie the extent to which such resistance arises across different drug-bacteria combinations, potentially offering tunable handles to optimize interventions aimed at minimizing AMR.

PMID:36264222 | DOI:10.1021/acsinfecdis.2c00460

Comput Intell Neurosci. 2022 Oct 10;2022:2271788. doi: 10.1155/2022/2271788. eCollection 2022.

ABSTRACT

The nucleotide oligomerization domain (NOD)-like receptors (NLRs) are a group of intracellular proteins that are essential for controlling the host's innate immune response. The cytosolic nucleotide binding oligomerization domains 1 and 2 receptors (NOD1 and NOD2) are the most widely investigated NLRs. As pattern recognition receptors (PRRs), NOD1 and NOD2 may recognize and bind endogenous damage associated molecular patterns (DAMPs) and external pathogenic associated molecular patterns (PAMPs), directing the activation of inflammatory caspases through engaging the adaptor protein RIP2, which further activates the NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways, thereby mediating host innate immunity and regulating the adaptive immunity. Previous research has identified NOD1 and NOD2 as key players in inflammatory disease and host-microbial defense. Despite numerous studies claiming that NOD1 and NOD2 are linked to tumorigenesis and tumor development, it is still unclear whether NOD1 and NOD2 act as cancer's friends or foes. In this review, we focus on concluding the current research progress on the role of NOD1 and NOD2 in a variety of cancers and discussing the potential reasons for the contradicting role of NOD1 and NOD2 in cancers. This review may help better understand the role of NOD1 and NOD2 in cancer and shed light on NOD1 and NOD2 as potential therapeutic targets for tumor immunotherapy.

PMID:36262606 | PMC:PMC9576356 | DOI:10.1155/2022/2271788

J Med Chem. 2022 Oct 27;65(20):14144-14179. doi: 10.1021/acs.jmedchem.2c01349. Epub 2022 Oct 18.

ABSTRACT

The clinical success of linezolid for treating Gram-positive infections paired with the high conservation of bacterial ribosomes predicts that if oxazolidinones were engineered to accumulate in Gram-negative bacteria, then this pharmacological class would find broad utility in eradicating infections. Here, we report an investigative study of a strategically designed library of oxazolidinones to determine the effects of molecular structure on accumulation and biological activity. Escherichia coli, Acinetobacter baumannii, and Pseudomonas aeruginosa strains with varying degrees of compromise (in efflux and outer membrane) were used to identify motifs that hinder permeation across the outer membrane and/or enhance efflux susceptibility broadly and specifically between species. The results illustrate that small changes in molecular structure are enough to overcome the efflux and/or permeation issues of this scaffold. Three oxazolidinone analogues (3e, 8d, and 8o) were identified that exhibit activity against all three pathogens assessed, a biological profile not observed for linezolid.

PMID:36257060 | DOI:10.1021/acs.jmedchem.2c01349

J Med Chem. 2022 Oct 18. doi: 10.1021/acs.jmedchem.2c01437. Online ahead of print.

ABSTRACT

Due to the emergence of antimicrobial resistance and the lack of new antibacterial agents, it has become urgent to discover and develop new antibacterial agents against multidrug-resistant pathogens. Antimicrobial peptides (AMPs) serve as the first line of defense for the host. In this work, we have designed, synthesized, and biologically evaluated a series of phenyl sulfide derivatives by biomimicking the structural features and biological functions of AMPs. Among these derivatives, the most promising compound 17 exhibited potent antibacterial activity against Gram-positive bacteria (minimum inhibitory concentrations = 0.39-1.56 μg/mL), low hemolytic activity (HC₅₀ > 200 μg/mL), and high membrane selectivity. In addition, 17 can rapidly kill Gram-positive bacteria within 0.5 h through membrane-targeting action and avoid antibiotic resistance. More importantly, 17 showed high in vivo efficacy against Staphylococcus aureus in a murine corneal infection model. Therefore, 17 has great potential as a lead compound for the treatment of Gram-positive bacterial infections.

PMID:36256884 | DOI:10.1021/acs.jmedchem.2c01437

Chemistry. 2022 Oct 18. doi: 10.1002/chem.202202991. Online ahead of print.

ABSTRACT

Soluble fragments of peptidoglycan called muropeptides are released from the cell wall of bacteria as part of their metabolism or as a result of biological stresses. These compounds trigger immune responses in mammals and plants. In bacteria, they play a major role in the induction of antibiotic resistance. The development of efficient methods to produce muropeptides is therefore desirable both to address their mechanism of action and to design new antibacterial and immunostimulant agents. Here, we engineered the peptidoglycan recycling pathway of Escherichia coli to produce N-acetyl-β-D-glucosaminyl-(1→4)-1,6-anhydro-N-acetyl-β-D-muramic acid (GlcNAc-anhMurNAc), a common precursor of Gram-negative and Gram-positive muropeptides. Inactivation of the hexosaminidase nagZ gene allowed the efficient production of this key disaccharide, providing access to Gram-positive muropeptides through subsequent chemical peptide conjugation. E. coli strains deficient in both NagZ hexosaminidase and amidase activities further enabled the in vivo production of Gram-negative muropeptides containing meso-diaminopimelic acid, a rarely available amino acid.

PMID:36256497 | DOI:10.1002/chem.202202991

ACS Infect Dis. 2022 Oct 14;8(10):2149-2160. doi: 10.1021/acsinfecdis.2c00263. Epub 2022 Sep 7.

ABSTRACT

Optimization of compound permeation into Gram-negative bacteria is one of the most challenging tasks in the development of antibacterial agents. Two permeability barriers─the passive diffusion barrier of the outer membrane (OM) and active drug efflux─act synergistically to protect cells from the antibacterial action of compounds. In Escherichia coli (E. coli) and relatives, these two barriers sieve compounds based on different physicochemical properties that are defined by their interactions with OM porins and efflux pumps, respectively. In this study, we critically tested the hypothesis that the best substrates and inhibitors of efflux pumps are compounds that can effectively permeate the OM and are available at relatively high concentrations in the periplasm. For this purpose, we filtered a large subset of the ZINC15 database of commercially available compounds for compounds containing a primary amine, a chemical feature known to facilitate the uptake through E. coli general porins. The assembled library was screened by ensemble docking to AcrA, the periplasmic component of the AcrAB-TolC efflux pump, followed by experimental testing of the top predicted binders for antibacterial activities, efflux recognition, and inhibition. We found that the filtered primary amine library is a rich source of compounds with efflux-inhibiting activities and identified efflux pump inhibitors with novel chemical scaffolds effective against E. coli AcrAB-TolC and efflux pumps of multidrug-resistant clinical isolates of Acinetobacter baumannii. However, primary amines are not required for the recognition of compounds by efflux pumps and their efflux-inhibitory activities.

PMID:36070489 | DOI:10.1021/acsinfecdis.2c00263

J Chem Inf Model. 2022 Oct 24;62(20):5023-5033. doi: 10.1021/acs.jcim.2c00767. Epub 2022 Oct 10.

ABSTRACT

Passive diffusion across biomembranes is an important mechanism of permeation for multiple drugs, including antibiotics. However, this process is frequently neglected while studying drug uptake and, in our view, warrants further investigation. Here, we apply molecular dynamics simulations to investigate the impact of changes in molecular hydrophobicity on the permeability of a series of inhibitors of the quorum sensing of <i>Pseudomonas aeruginosa</i>, previously discovered by us, across a membrane model. Overall, we show that permeation across this membrane model does not correlate with the molecule's hydrophobicity. We demonstrate that using a simple model for permeation, based on the difference between the maximum and minimum of the free energy profile, outperforms the inhomogeneous solubility-diffusion model, yielding a permeability ranking that better agrees with the experimental results, especially for hydrophobic permeants. The calculated differences in permeability could not explain differences in <i>in bacterio</i> activity. Nevertheless, substantial differences in molecular orientation along the permeation pathway correlate with the <i>in bacterio</i> activity, emphasizing the importance of analyzing, at an atomistic level, the permeation pathway of these solutes.

PMID:36214845 | DOI:10.1021/acs.jcim.2c00767

Microbiol Spectr. 2022 Oct 6:e0307522. doi: 10.1128/spectrum.03075-22. Online ahead of print.

ABSTRACT

An estimated one-third of the world's population is infected with Mycobacterium tuberculosis, with the majority being vaccinated with Mycobacterium bovis BCG. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a threat, and we must understand how SARS-CoV-2 can modulate both BCG immunity and tuberculosis pathogenesis. Interestingly, neither BCG vaccination nor tuberculosis infection resulted in differences in clinical outcomes associated with SARS-CoV-2 in transgenic mice. Surprisingly, earlier M. tuberculosis infection resulted in lower SARS-CoV-2 viral loads, mediated by the heightened immune microenvironment of the murine lungs, unlike vaccination with BCG, which had no impact. In contrast, M. tuberculosis-infected tissues had increased bacterial loads and decreased histiocytic inflammation in the lungs following SARS-CoV-2 superinfection. SARS-CoV-2 modulated BCG-induced type 17 responses while decreasing type 1 and increasing type 2 cytokines in M. tuberculosis-infected mice. These findings challenge initial findings of BCG's positive impact on SARS-CoV-2 infection and suggest potential ramifications for M. tuberculosis reactivation upon SARS-CoV-2 superinfection. IMPORTANCE Prior to SARS-CoV-2, M. tuberculosis was the leading infectious disease killer, with an estimated one-third of the world's population infected and 1.7 million deaths a year. Here, we show that SARS-CoV-2 superinfection caused increased bacterial dissemination in M. tuberculosis-infected mice along with immune and pathological changes. SARS-CoV-2 also impacted the immunity of BCG-vaccinated mice, resulting in decreased interleukin-17 (IL-17) levels, while offering no protective effect against SARS-CoV-2. These results demonstrate that SARS-CoV-2 may have a deleterious effect on the ongoing M. tuberculosis pandemic and potentially limit BCG's efficacy.

PMID:36200898 | DOI:10.1128/spectrum.03075-22

Front Cell Infect Microbiol. 2022 Sep 20;12:916247. doi: 10.3389/fcimb.2022.916247. eCollection 2022.

ABSTRACT

The genome of Mycobacterium tuberculosis (Mtb) harbors the genetic machinery for assembly of the Fimbrial low-molecular-weight protein (Flp) type IV pilus. Presumably, the Flp pilus is essential for pathogenesis. However, it remains unclear whether the pili genes are transcribed in culture or during infection of host cells. This study aimed to shed light on the expression of the Flp pili-assembly genes (tadZ, tadA, tadB, tadC, flp, tadE, and tadF) in Mtb growing under different growth conditions (exponential phase, stationary phase, and dormancy NRP1 and NRP2 phases induced by hypoxia), during biofilm formation, and in contact with macrophages and alveolar epithelial cells. We found that expression of tad/flp genes was significantly higher in the stationary phase than in exponential or NRP1 or NRP2 phases suggesting that the bacteria do not require type IV pili during dormancy. Elevated gene expression levels were recorded when the bacilli were in contact for 4 h with macrophages or epithelial cells, compared to mycobacteria propagated alone in the cultured medium. An antibody raised against a 12-mer peptide derived from the Flp pilin subunit detected the presence of Flp pili on intra- and extracellular bacteria infecting eukaryotic cells. Altogether, these are compelling data showing that the Flp pili genes are expressed during the interaction of Mtb with host cells and highlight a role for Flp pili in colonization and invasion of the host, subsequently promoting bacterial survival during dormancy.

PMID:36204636 | PMC:PMC9531140 | DOI:10.3389/fcimb.2022.916247

ACS Omega. 2022 Sep 23;7(39):34997-35013. doi: 10.1021/acsomega.2c03711. eCollection 2022 Oct 4.

ABSTRACT

Metabolic labeling paired with click chemistry is a powerful approach for selectively imaging the surfaces of diverse bacteria. Herein, we explored the feasibility of labeling the lipopolysaccharide (LPS) of Myxococcus xanthus-a Gram-negative predatory social bacterium known to display complex outer membrane (OM) dynamics-via growth in the presence of distinct azido (-N₃) analogues of 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo). Determination of the LPS carbohydrate structure from strain DZ2 revealed the presence of one Kdo sugar in the core oligosaccharide, modified with phosphoethanolamine. The production of 8-azido-8-deoxy-Kdo (8-N₃-Kdo) was then greatly improved over previous reports via optimization of the synthesis of its 5-azido-5-deoxy-d-arabinose precursor to yield gram amounts. The novel analogue 7-azido-7-deoxy-Kdo (7-N₃-Kdo) was also synthesized, with both analogues capable of undergoing in vitro strain-promoted azide-alkyne cycloaddition (SPAAC) "click" chemistry reactions. Slower and faster growth of M. xanthus was displayed in the presence of 8-N₃-Kdo and 7-N₃-Kdo (respectively) compared to untreated cells, with differences also seen for single-cell gliding motility and type IV pilus-dependent swarm community expansion. While the surfaces of 8-N₃-Kdo-grown cells were fluorescently labeled following treatment with dibenzocyclooctyne-linked fluorophores, the surfaces of 7-N₃-Kdo-grown cells could not undergo fluorescent tagging. Activity analysis of the KdsB enzyme required to activate Kdo prior to its integration into nascent LPS molecules revealed that while 8-N₃-Kdo is indeed a substrate of the enzyme, 7-N₃-Kdo is not. Though a lack of M. xanthus cell aggregation was shown to expedite growth in liquid culture, 7-N₃-Kdo-grown cells did not manifest differences in intrinsic clumping relative to untreated cells, suggesting that 7-N₃-Kdo may instead be catabolized by the cells. Ultimately, these data provide important insights into the synthesis and cellular processing of valuable metabolic labels and establish a basis for the elucidation of fundamental principles of OM dynamism in live bacterial cells.

PMID:36211050 | PMC:PMC9535733 | DOI:10.1021/acsomega.2c03711

ACS Omega. 2022 Sep 20;7(39):34921-34928. doi: 10.1021/acsomega.2c03481. eCollection 2022 Oct 4.

ABSTRACT

Strain-promoted azide-alkyne cycloaddition (SPAAC) reactions like click chemistry have the potential to be highly scalable, robust, and cost-effective methods for generating small- and large-molecule conjugates for a variety of applications. However, despite method improvements, the rates of copper-based click chemistry reactions continue to be much faster than the rates of copper-free click chemistry reactions, which makes broader deployment of click chemistry challenging from a safety and compatibility standpoint. In this study, we used a zwitterionic detergent, namely, lauryldimethylamine N-oxide (LDAO), in a copper-free click chemistry reaction to investigate its impact on the generation of conjugate vaccines (CVs). For this, we utilized an Xpress cell-free protein synthesis (CFPS) platform to generate a proprietary variant of CRM197 (eCRM) containing non-native amino acids (nnAA) with azide-containing side chains as a carrier protein for conjugation to several clinically relevant dibenzocyclooctyne (DBCO)-derivatized S. pneumoniae serotypes (types 3, 5, 18C, and 19A). For conjugation, we performed copper-free click chemistry in the presence and absence of LDAO. Our results show that the addition of LDAO significantly enhanced the reaction kinetics to generate larger conjugates, which were similarly immunogenic and equally stable to conjugates generated without LDAO. Most importantly, the addition of LDAO substantially improved the efficiency of the conjugation process. Thus, our results for the first time show that the addition of a zwitterionic surfactant to a copper-free click chemistry reaction can significantly accelerate the reaction kinetics along with improving the efficiency of the conjugation process.

PMID:36211053 | PMC:PMC9535640 | DOI:10.1021/acsomega.2c03481

Microbiol Spectr. 2022 Sep 29:e0256722. doi: 10.1128/spectrum.02567-22. Online ahead of print.

ABSTRACT

Resistance to antibiotics is an increasing problem and necessitates novel antibacterial therapies. The polyketide antibiotics cervimycin A to D are natural products of Streptomyces tendae HKI 0179 with promising activity against multidrug-resistant staphylococci and vancomycin-resistant enterococci. To initiate mode of action studies, we selected cervimycin C- and D-resistant (CmR) Staphylococcus aureus strains. Genome sequencing of CmR mutants revealed amino acid exchanges in the essential histidine kinase WalK, the Clp protease proteolytic subunit ClpP or the Clp ATPase ClpC, and the heat shock protein DnaK. Interestingly, all characterized CmR mutants harbored a combination of mutations in walK and clpP or clpC. In vitro and in vivo analyses showed that the mutations in the Clp proteins abolished ClpP or ClpC activity, and the deletion of clpP rendered S. aureus but not all Bacillus subtilis strains cervimycin-resistant. The essential gene walK was the second mutational hotspot in the CmR S. aureus strains, which decreased WalK activity in vitro and generated a vancomycin-intermediate resistant phenotype, with a thickened cell wall, a lower growth rate, and reduced cell lysis. Transcriptomic and proteomic analyses revealed massive alterations in the CmR strains compared to the parent strain S. aureus SG511, with major shifts in the heat shock regulon, the metal ion homeostasis, and the carbohydrate metabolism. Taken together, mutations in the heat shock genes clpP, clpC, and dnaK, and the walK kinase gene in CmR mutants induced a vancomycin-intermediate resistant phenotype in S. aureus, suggesting cell wall metabolism or the Clp protease system as primary target of cervimycin. IMPORTANCE Staphylococcus aureus is a frequent cause of infections in both the community and hospital setting. Resistance development of S. aureus to various antibiotics is a severe problem for the treatment of this pathogen worldwide. New powerful antimicrobial agents against Gram-positives are needed, since antibiotics like vancomycin fail to cure vancomycin-intermediate resistant S. aureus (VISA) and vancomycin-resistant enterococci (VRE) infections. One candidate substance with promising activity against these organisms is cervimycin, which is an antibiotic complex with a yet unknown mode of action. In our study, we provide first insights into the mode of action of cervimycins. By characterizing cervimycin-resistant S. aureus strains, we revealed the Clp system and the essential kinase WalK as mutational hotspots for cervimycin resistance in S. aureus. It further emerged that cervimycin-resistant S. aureus strains show a VISA phenotype, indicating a role of cervimycin in perturbing the bacterial cell envelope.

PMID:36173303 | DOI:10.1128/spectrum.02567-22

J Biomol Struct Dyn. 2022 Oct;40(17):7645-7655. doi: 10.1080/07391102.2021.1899052. Epub 2021 Mar 15.

ABSTRACT

Mycobacterium tuberculosis cell wall is intricate and impermeable to many agents. A D, D-carboxypeptidase (DacB1) is one of the enzymes involved in the biosynthesis of cell wall peptidoglycan and catalyzes the terminal D-alanine cleavage from pentapeptide precursors. Catalytic activity and mechanism by which DacB1 functions is poorly understood. Herein, we investigated the acylation mechanism of DacB1 by β-lactams using a 6-membered ring transition state model that involves a catalytic water molecule in the reaction pathway. The full transition states (TS) optimization plus frequency were achieved using the ONIOM (B3LYP/6-31 + G(d): AMBER) method. Subsequently, the activation free energies were computed via single-point calculations on fully optimized structures using B3LYP/6-311++(d,p): AMBER and M06-2X/6-311++(d,p): AMBER with an electronic embedding scheme. The 6-membered ring transition state is an effective model to examine the inactivation of DacB1 via acylation by β-lactams antibiotics (imipenem, meropenem, and faropenem) in the presence of the catalytic water. The ΔG^# values obtained suggest that the nucleophilic attack on the carbonyl carbon is the rate-limiting step with 13.62, 19.60 and 30.29 kcal mol^-1 for Imi-DacB1, Mero-DacB1 and Faro-DacB1, respectively. The electrostatic potential (ESP) and natural bond orbital (NBO) analysis provided significant electronic details of the electron-rich region and charge delocalization, respectively, based on the concerted 6-membered ring transition state. The stabilization energies of charge transfer within the catalytic reaction pathway concurred with the obtained activation free energies. The outcomes of this study provide important molecular insight into the inactivation of D, D-carboxypeptidase by β-lactams.Communicated by Ramaswamy H. Sarma.

PMID:33719919 | DOI:10.1080/07391102.2021.1899052

Subcell Biochem. 2022;99:379-391. doi: 10.1007/978-3-031-00793-4_12.

ABSTRACT

The mycobacteria genus is responsible for numerous infectious diseases that have afflicted the human race since antiquity-tuberculosis and leprosy in particular. An important contributor to their evolutionary success is their unique cell envelope, which constitutes a quasi-impermeable barrier, protecting the microorganism from external threats, antibiotics included. The arabinofuranosyltransferases are a family of enzymes, unique to the Actinobacteria family that mycobacteria genus belongs to, that are critical to building of this cell envelope. In this chapter, we will analyze available structures of members of the mycobacterial arabinofuranosyltransferase, clarify their function, as well as explore the common themes present amongst this family of enzymes, as revealed by recent research.

PMID:36151383 | DOI:10.1007/978-3-031-00793-4_12

Antimicrob Agents Chemother. 2022 Sep 26:e0209121. doi: 10.1128/aac.02091-21. Online ahead of print.

ABSTRACT

Multidrug-resistant (MDR) tuberculosis (TB) is defined by the resistance of Mycobacterium tuberculosis, the causative organism, to the first-line antibiotics rifampicin and isoniazid. Mitigating or reversing resistance to these drugs offers a means of preserving and extending their use in TB treatment. R-loops are RNA/DNA hybrids that are formed in the genome during transcription, and they can be lethal to the cell if not resolved. RNase HI is an enzyme that removes R-loops, and this activity is essential in M. tuberculosis: knockouts of rnhC, the gene encoding RNase HI, are nonviable. This essentiality makes it a candidate target for the development of new antibiotics. In the model organism Mycolicibacterium smegmatis, RNase HI activity is provided by two enzymes, RnhA and RnhC. We show that the partial depletion of RNase HI activity in M. smegmatis, by knocking out either of the genes encoding RnhA or RnhC, led to the accumulation of R-loops. The sensitivity of the knockout strains to the antibiotics moxifloxacin, streptomycin, and rifampicin was increased, the latter by a striking near 100-fold. We also show that R-loop accumulation accompanies partial transcriptional inhibition, suggesting a mechanistic basis for the synergy between RNase HI depletion and rifampicin. A model of how transcriptional inhibition can potentiate R-loop accumulation is presented. Finally, we identified four small molecules that inhibit recombinant RnhC activity and that also potentiated rifampicin activity in whole-cell assays against M. tuberculosis, supporting an on-target mode of action and providing the first step in developing a new class of antimycobacterial drug.

PMID:36154174 | DOI:10.1128/aac.02091-21

Biophys J. 2022 Sep 20;121(18):3486-3498. doi: 10.1016/j.bpj.2022.08.007. Epub 2022 Aug 13.

ABSTRACT

Polymyxins are increasingly used as the last-line therapeutic option for the treatment of infections caused by multidrug-resistant Gram-negative bacteria. However, efforts to address the resistance in superbugs are compromised by a poor understanding of the bactericidal modes because high-resolution detection of the cell structure is still lacking. By performing molecular dynamics simulations at a coarse-grained level, here we show that polymyxin B (PmB) disrupts Gram-negative bacterial membranes by altering lipid homeostasis and asymmetry. We found that the binding of PmBs onto the asymmetric outer membrane (OM) loosens the packing of lipopolysaccharides (LPS) and induces unbalanced bending torque between the inner and outer leaflets, which in turn triggers phospholipids to flip from the inner leaflet to the outer leaflet to compensate for the stress deformation. Meanwhile, some LPSs may be detained on the inner membrane (IM). Then, the lipid-scrambled OM undergoes phase separation. Defects are created at the boundaries between LPS-rich domains and phospholipid-rich domains, which consequently facilitate the uptake of PmB across the OM. Finally, PmBs target LPSs detained on the IM and similarly perturb the IM. This lipid Scramble, membrane phase Separation, and peptide Translocation model depicts a novel mechanism by which polymyxins kill bacteria and sheds light on developing a new generation of polymyxins or antibiotic adjuvants with improved killing activities and higher therapeutic indices.

PMID:35964158 | PMC:PMC9515121 | DOI:10.1016/j.bpj.2022.08.007

Front Microbiol. 2022 Sep 6;13:985871. doi: 10.3389/fmicb.2022.985871. eCollection 2022.

ABSTRACT

Beta-lactams have been excluded from tuberculosis therapy due to the intrinsic resistance of Mycobacterium tuberculosis (Mtb) to this antibiotic class, usually attributed to a potent beta-lactamase, BlaC, and to an unusually complex cell wall. In this pathogen, the peptidoglycan is cross-linked by penicillin-binding proteins (PBPs) and L,D-transpeptidases, the latter resistant to inhibition by most beta-lactams. However, recent studies have shown encouraging results of beta-lactam/beta-lactamase inhibitor combinations in clinical strains. Additional research on the mechanisms of action and resistance to these antibiotics and other inhibitors of peptidoglycan synthesis, such as the glycopeptides, is crucial to ascertain their place in alternative regimens against drug-resistant strains. Within this scope, we applied selective pressure to generate mutants resistant to amoxicillin, meropenem or vancomycin in Mtb H37Rv or Mycolicibacterium smegmatis (Msm) mc²-155. These were phenotypically characterized, and whole-genome sequencing was performed. Mutations in promising targets or orthologue genes were inspected in Mtb clinical strains to establish potential associations between altered susceptibility to beta-lactams and the presence of key genomic signatures. The obtained isolates had substantial increases in the minimum inhibitory concentration of the selection antibiotic, and beta-lactam cross-resistance was detected in Mtb. Mutations in L,D-transpeptidases and major PBPs, canonical targets, or BlaC were not found. The transcriptional regulator PhoP (Rv0757) emerged as a common denominator for Mtb resistance to both amoxicillin and meropenem, while Rv2864c, a lipoprotein with PBP activity, appears to be specifically involved in decreased susceptibility to the carbapenem. Nonetheless, the mutational pattern detected in meropenem-resistant mutants was different from the yielded by amoxicillin-or vancomycin-selected isolates, suggesting that distinct pathways may participate in increased resistance to peptidoglycan inhibitors, including at the level of beta-lactam subclasses. Cross-resistance between beta-lactams and antimycobacterials was mostly unnoticed, and Msm meropenem-resistant mutants from parental strains with previous resistance to isoniazid or ethambutol were isolated at a lower frequency. Although cell-associated nitrocefin hydrolysis was increased in some of the isolates, our findings suggest that traditional assumptions of Mtb resistance relying largely in beta-lactamase activity and impaired access of hydrophilic molecules through lipid-rich outer layers should be challenged. Moreover, the therapeutical potential of the identified Mtb targets should be explored.

PMID:36147841 | PMC:PMC9485614 | DOI:10.3389/fmicb.2022.985871

Membranes (Basel). 2022 Sep 16;12(9):891. doi: 10.3390/membranes12090891.

ABSTRACT

Antimicrobial peptides (AMPs) can be directed to specific membranes based on differences in lipid composition. In this study, we performed atomistic and coarse-grained simulations of different numbers of the designed AMP adepantin-1 with a eukaryotic membrane, cytoplasmic Gram-positive and Gram-negative membranes, and an outer Gram-negative membrane. At the core of adepantin-1's behavior is its amphipathic α-helical structure, which was implemented in its design. The amphipathic structure promotes rapid self-association of peptide in water or upon binding to bacterial membranes. Aggregates initially make contact with the membrane via positively charged residues, but with insertion, the hydrophobic residues are exposed to the membrane's hydrophobic core. This adaptation alters the aggregate's stability, causing the peptides to diffuse in the polar region of the membrane, mostly remaining as a single peptide or pairing up to form an antiparallel dimer. Thus, the aggregate's proposed role is to aid in positioning the peptide into a favorable conformation for insertion. Simulations revealed the molecular basics of adepantin-1 binding to various membranes, and highlighted peptide aggregation as an important factor. These findings contribute to the development of novel anti-infective agents to combat the rapidly growing problem of bacterial resistance to antibiotics.

PMID:36135911 | DOI:10.3390/membranes12090891