Rachita Dash

ACS Infect Dis. 2022 Aug 12;8(8):1627-1636. doi: 10.1021/acsinfecdis.2c00229. Epub 2022 Aug 2.

ABSTRACT

The rise of antibiotic-resistant Mycobacterium tuberculosis and non-tuberculous mycobacterial infections has placed ever-increasing importance on discovering new antibiotics to treat these diseases. Recently, a new penem, T405, was discovered to have strong antimicrobial activity against M. tuberculosis and Mycobacteroides abscessus. Here, a penem library of C2 side-chain variants was synthesized, and their antimicrobial activities were evaluated against M. tuberculosis H₃₇Rv and M. abscessus ATCC 19977. Several new penems with antimicrobial activity stronger than the standard-of-care carbapenem antibiotics were identified with some candidates improving on the activity of the lead compound, T405. Moreover, many candidates showed little or no increase in the minimum inhibitory concentration in the presence of serum compared to the highly protein-bound T405. The penems with the strongest activity identified in this study were then biochemically characterized by reaction with the representative l,d-transpeptidase Ldt_Mt2 and the representative penicillin-binding protein d,d-carboxypeptidase DacB2.

PMID:35916356 | PMC:PMC10029149 | DOI:10.1021/acsinfecdis.2c00229

FEBS J. 2023 Jan 25. doi: 10.1111/febs.16734. Online ahead of print.

ABSTRACT

Various d-amino acids have been found in a wide range of organisms, including mammals. Although the physiological functions of various d-amino acids have been reported or suggested, the molecular basis of these biological functions has been elucidated in only a few cases. The identification of a d-amino acid biosynthetic enzyme is a critical step in understanding the mechanism of the physiological functions of d-amino acids. While in vivo functional screening can be a powerful tool for identifying novel metabolic enzymes, none of the existing organisms exhibit growth dependent on d-amino acid other than d-Ala and d-Glu. Here, we report the first organism that exhibits non-canonical d-amino acid auxotrophy. We found that an Escherichia coli strain lacking the major d-Ala and d-Glu biosynthetic enzymes, alr, dadX, and murI, and expressing the mutated d-amino acid transaminase (DAAT) gene from Bacillus sp. YM-1 (MB3000/mdaat⁺ ) grew well when supplemented with certain d-amino acid. A multicopy suppression study with plasmids encoding one of the 51 PLP-dependent enzymes of E. coli showed that MB3000/mdaat⁺ could detect weak and moonlighting racemase activity, such from cystathionine β-lyase (MetC) and a negative regulator of MalT activity/cystathionine β-lyase (MalY)-these exhibit only a few tenths to a few thousandths of the racemization activity of canonical amino acid racemases. We believe that this unique platform will contribute to further research in this field by identifying novel d-amino acid-metabolizing enzymes.

PMID:36695650 | DOI:10.1111/febs.16734

Microbiol Spectr. 2023 Jan 31:e0319722. doi: 10.1128/spectrum.03197-22. Online ahead of print.

ABSTRACT

The majority of preclinical research has shown that Mycobacterium tuberculosis can modify host lipids in various ways. To boost its intramacrophage survival, M. tuberculosis causes host lipids to build up, resulting in the development of lipid-laden foam cells. M. tuberculosis binds to and enters the macrophage via the cell membrane cholesterol. Aggregation of cholesterol in the cell wall of M. tuberculosis and an increase in vascularity at the granuloma site reduce the permeability of rifampicin and isoniazid concentrations. However, very few studies have assessed the effect of statins on drug penetration. Here, we used atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, to observe its effect on the bacterial burden by increasing the drug concentration at the infection site. We looked into how atorvastatin could be used in conjunction with first-line drugs to promote drug permeation. In this study, we detected an accumulation of drugs at the peripheral sites of the lungs and impaired drug distribution to the diseased sites. The efficacy of antituberculosis drugs, with atorvastatin as an adjunct, on the viability of M. tuberculosis cells was demonstrated. A nontoxic statin dosage established phenotypic and normal granuloma vasculature and showed an additive effect with rifampicin and isoniazid. Our data show that statins help to reduce the tuberculosis bacterial burden. Our findings reveal that the bacterial load is connected with impaired drug permeability resulting from lipid accumulation in the bacterial cell wall. Statin therapy combined with antituberculosis medications have the potential to improve treatment in tuberculosis patients. IMPORTANCE Mycobacterium tuberculosis binds to and enters the macrophage via the cell membrane cholesterol. M. tuberculosis limits phagosomal maturation and activation without engaging in phagocytosis. Aggregation of cholesterol in the cell wall of M. tuberculosis and an increase in the vascularity at the granuloma site reduce the permeability of rifampicin and isoniazid concentrations. However, very few studies have assessed the effect of statins on drug penetration, which can be increased through a reduction in cholesterol and vascularity. Herein, we used atorvastatin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, to observe its effect on bacterial burden through increasing the drug concentration at the infection site. Our main research goal is to diminish mycobacterial dissemination and attenuate bacterial growth by increasing drug permeability.

PMID:36719189 | DOI:10.1128/spectrum.03197-22

Front Cell Infect Microbiol. 2023 Jan 12;12:1064148. doi: 10.3389/fcimb.2022.1064148. eCollection 2022.

ABSTRACT

INTRODUCTION: Routine efficacy assessments of new tuberculosis (TB) treatments include quantitative solid culture or routine liquid culture, which likely miss quantification of drug tolerant bacteria. To improve these assessments, comparative analyses using additional measures such as quantification of differentially culturable tubercle bacteria (DCTB) are required. Essential for enabling this is a comparative measure of TB treatment responses using routine solid and liquid culture with liquid limiting dilutions (LLDs) that detect DCTB in sputum.

METHODS: We recruited treatment-naïve TB patients, with and without HIV-infection, and serially quantified their sputum for DCTB over the course of treatment.

RESULTS: Serial sputum sampling in 73 individuals during their first 14 days of treatment demonstrated that clearance of DCTB was slower compared to routine solid culture. Treatment response appeared to be characterized by four patterns: (1) Classic bi-phasic bacterial clearance; (2) early non-responders with slower clearance; (3) paradoxical worsening with an increase in bacterial count upon treatment initiation; and (4) non-responders with no change in bacterial load. During treatment, LLDs displayed greater bacterial yield when compared with quantitative solid culture. Upon treatment completion, 74% [46/62] of specimens displayed residual DCTB and within this group, two recurrences were diagnosed. Residual DCTB upon treatment completion was associated with a higher proportion of MGIT culture, GeneXpert, and smear positivity at two months post treatment. No recurrences occurred in the group without residual DCTB.

DISCUSSION: These data indicate that DCTB assays detect distinct subpopulations of organisms in sputum that are missed by routine solid and liquid culture, and offer important alternatives for efficacy assessments of new TB treatments. The residual DCTB observed upon treatment completion suggests that TB treatment does not always eliminate all bacterial populations, a finding that should be investigated in larger cohorts.

PMID:36710965 | PMC:PMC9877613 | DOI:10.3389/fcimb.2022.1064148

Sci Adv. 2023 Jan 27;9(4):eabg6808. doi: 10.1126/sciadv.abg6808. Epub 2023 Jan 27.

ABSTRACT

Real-time localization and microbial activity information of indigenous gut microbiota over an extended period of time remains a challenge with existing visualizing methods. Here, we report a metabolic fluorine labeling (MEFLA)-based strategy for monitoring the dynamic gut microbiota via ¹⁹F magnetic resonance imaging (¹⁹F MRI). In situ labeling of different microbiota subgroups is achieved by using a panel of peptidoglycan-targeting MEFLA probes containing ¹⁹F atoms of different chemical shifts, and subsequent real-time in vivo imaging is accomplished by multiplexed hotspot ¹⁹F MRI with high sensitivity and unlimited penetration. Using this method, we realize extended visualization (>24 hours) of native gut microbes located at different intestinal sections and semiquantitative analysis of their metabolic dynamics modulated by various conditions, such as the host death and different β-lactam antibiotics. Our strategy holds great potential for noninvasive and real-time assessing of the metabolic activities and locations of the highly dynamic gut microbiota.

PMID:36706178 | DOI:10.1126/sciadv.abg6808

Angew Chem Int Ed Engl. 2023 Jan 26. doi: 10.1002/anie.202217777. Online ahead of print.

ABSTRACT

The general lack of permeability of small molecules observed for Mycobacterium tuberculosis (Mtb) is most ascribed to its unique cell envelope. More specifically, the outer mycomembrane is hypothesized to be the principal determinant for access of antibiotics to their molecular targets. We describe a novel assay that combines metabolic tagging of the peptidoglycan, which sits directly beneath the mycomembrane, click chemistry of test molecules, and a fluorescent labeling chase step, to measure the permeation of small molecules. We showed that the assay workflow was robust and compatible with high-throughput analysis in mycobacteria by testing a small panel of azide-tagged molecules. The general trend is similar across the two types of mycobacteria with some notable exceptions. We anticipate that this assay platform will lay the foundation for medicinal chemistry efforts to understand and improve uptake of both existing drugs and newly-discovered compounds into mycobacteria.

PMID:36700874 | DOI:10.1002/anie.202217777

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

Appl Microbiol Biotechnol. 2023 Feb;107(2-3):867-879. doi: 10.1007/s00253-022-12350-x. Epub 2022 Dec 31.

ABSTRACT

Biofilm-forming Staphylococcus aureus can easily accumulate on various food contact surfaces which induce cross-contamination and are difficult to eliminate in the food industry. This study aimed to evaluate the anti-biofilm effects of natural product biochanin A against S. aureus. Results showed that biochanin A effectively eradicated established S. aureus biofilms on different food-contact materials. Fluorescence microscopic analyses suggested that biochanin A disintegrated the established biofilms by dissociate extracellular polymeric substance (EPS) in matrix. In addition, biochanin A at the sub-MIC concentration also effectively inhibited the biofilm formation by regulating the expression of biofilm-related genes (icaA, srtA, eno) and suppressing the release of EPS in biofilm matrix. Molecular docking also demonstrated that biochanin A conducted strong interactions with biofilm-related proteins (Ica A, Sortase A, and Enolase). These findings demonstrated that biochanin A has the potential to be developed as a potent agent against S. aureus biofilm in food industries. KEY POINTS: • Anti-biofilm effect of biochanin A against S. aureus was revealed for the first time. • Biofilm of S. aureus on various food-contact surfaces were efficiently eradicated. • Biochanin A prevented S. aureus biofilm formation via reducing EPS production.

PMID:36585511 | DOI:10.1007/s00253-022-12350-x

Virulence. 2023 Dec;14(1):2171641. doi: 10.1080/21505594.2023.2171641.

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 haemoglobin or haem 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 | PMC:PMC9928477 | DOI:10.1080/21505594.2023.2171641

ACS Omega. 2022 Dec 7;7(50):46693-46701. doi: 10.1021/acsomega.2c05652. eCollection 2022 Dec 20.

ABSTRACT

Post-translational modifications (PTMs) of proteins increase the functional diversity of the proteome and play crucial regulatory roles in cellular processes. Ubiquitination is a highly regulated and reversible PTM accomplished by a complex multistep process with the sequential action of several specific ubiquitinating (E1-E3) and deubiquitinating enzymes. The different types of ubiquitination (mono-, poly-mono-, and poly-) and the presence of several target sites in a single substrate add to its complexity, which makes the in vitro reconstitution of this ubiquitin (Ub) machinery a quite cumbersome process. Defects in components of the ubiquitination process also contribute to disease pathogenesis, especially cancer and neurodegeneration. This makes them of interest as potential therapeutic targets. Therefore, the development of efficient and reliable methods that will generate a highly homogeneous ubiquitinated peptide and protein conjugate is a topical subject area of research. In this report, we describe the development of a simple and efficient in vitro sortase-mediated chemoenzymatic strategy for semisynthesis of defined and homogeneous ubiquitin conjugates with more than 90% yield. This was achieved by engineering a sortase recognition motif in the dynamic C-terminus of ubiquitin and its conjugation to an isopeptide-linked di-Gly appended peptide LMFK(ε-GG)TEG corresponding to the ubiquitination site residues ₃₈₃LMFKTEG₃₈₉ of p53. The defined and homogeneous ubiquitin conjugates were also weighed for their recognition propensity by deubiquitinating enzymes. This facile semisynthesis of ubiquitin conjugates establishes a simple one-step sortase-mediated chemoenzymatic route for the synthesis of homogeneous and defined isopeptide-linked polypeptides and will help in understanding the complexity of the ubiquitination machinery as well as designing isopeptide drugs and therapeutics.

PMID:36570257 | PMC:PMC9773336 | DOI:10.1021/acsomega.2c05652

Philos Trans R Soc Lond B Biol Sci. 2023 Feb 27;378(1871):20220038. doi: 10.1098/rstb.2022.0038. Epub 2023 Jan 11.

ABSTRACT

Ribosomal incorporation of d-α-amino acids (dAA) and N-methyl-l-α-amino acids (^MeAA) with negatively charged sidechains, such as d-Asp, d-Glu, ^MeAsp and ^MeGlu, into nascent peptides is far more inefficient compared to those with neutral or positively charged ones. This is because of low binding affinity of their aminoacyl-transfer RNA (tRNA) to elongation factor-thermo unstable (EF-Tu), a translation factor responsible for accommodation of aminoacyl-tRNA onto ribosome. It is well known that EF-Tu binds to two parts of aminoacyl-tRNA, the amino acid moiety and the T-stem; however, the amino acid binding pocket of EF-Tu bearing Glu and Asp causes electric repulsion against the negatively charged amino acid charged on tRNA. To circumvent this issue, here we adopted two strategies: (i) use of an EF-Tu variant, called EF-Sep, in which the Glu216 and Asp217 residues in EF-Tu are substituted with Asn216 and Gly217, respectively; and (ii) reinforcement of the T-stem affinity using an artificially developed chimeric tRNA, tRNA^Pro1E2, whose T-stem is derived from Escherichia coli tRNA^Glu that has high affinity to EF-Tu. Consequently, we could successfully enhance the incorporation efficiencies of d-Asp, d-Glu, ^MeAsp and ^MeGlu and demonstrated for the first time, to our knowledge, ribosomal synthesis of macrocyclic peptides containing multiple d-Asp or ^MeAsp. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

PMID:36633283 | PMC:PMC9835608 | DOI:10.1098/rstb.2022.0038

Antibiotics (Basel). 2022 Dec 8;11(12):1772. doi: 10.3390/antibiotics11121772.

ABSTRACT

Cell-penetrating peptides (CPPs) are natural or engineered peptide sequences with the intrinsic ability to internalize into a diversity of cell types and simultaneously transport hydrophilic molecules and nanomaterials, of which the cellular uptake is often limited. In addition to this primordial activity of cell penetration without membrane disruption, multivalent antimicrobial activity accompanies some CPPs. Antimicrobial peptides (AMPs) with cell-penetrability exert their effect intracellularly, and they are of great interest. CPPs with antimicrobial activity (CPAPs) comprise a particular class of bioactive peptides that arise as promising agents against difficult-to-treat intracellular infections. This short review aims to present the antibacterial, antiparasitic, and antiviral effects of various cell-penetrating antimicrobial peptides currently documented. Examples include the antimicrobial effects of different CPAPs against bacteria that can propagate intracellularly, like Staphylococcus sp., Streptococcus sp., Chlamydia trachomatis, Escherichia coli, Mycobacterium sp., Listeria sp., Salmonella sp. among others. CPAPs with antiviral effects that interfere with the intracellular replication of HIV, hepatitis B, HPV, and herpes virus. Additionally, CPAPs with activity against protozoa of the genera Leishmania, Trypanosoma, and Plasmodium, the etiological agents of Leishmaniasis, Chagas' Disease, and Malaria, respectively. The information provided in this review emphasizes the potential of multivalent CPAPs, with anti-infective properties for application against various intracellular infections. So far, CPAPs bear a promise of druggability for the translational medical use of CPPs alone or in combination with chemotherapeutics. Moreover, CPAPs could be an exciting alternative for pharmaceutical design and treating intracellular infectious diseases.

PMID:36551429 | PMC:PMC9774436 | DOI:10.3390/antibiotics11121772