Characterization of a novel endopeptidase Cwl0971 that hydrolyzes peptidoglycan of Clostridioides difficile involved in pleiotropic cellular processes

SummaryClostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe that can cause nosocomial antibiotic-associated intestinal disease. Although the production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, the mechanism of TcdA and TcdB release from intracell remains unclear. In this study, we identified and characterized a new cell wall hydrolase Cwl0971 (endopeptidase, CDR20291_0971) from C. difficile R20291, which is involved in bacterial autolysis. The gene 0971 deletion mutant (R20291Δ0971) generated with CRISPR-AsCpfI exhibited significantly delayed cell autolysis and increased cell viability compared to R20291, and the purified Cwl0971 exhibited hydrolase activity for Bacillus subtilis cell wall. Meanwhile, 0971 gene deletion impaired TcdA and TcdB release due to the decreased cell autolysis in the stationary / late phase of cell growth. Moreover, biofilm formation, germination, and sporulation of the mutant strain decreased significantly compared to the wild type strain. In vivo, the depletion of Cwl097 decreased fitness over the parent strain in a mouse infection model. Collectively, Cwl0971 is involved in cell wall lysis and cell viability, which can affect several phenotypes of R20291. Our data indicate that the endopeptidase Cwl0971 is an attractive target for C. difficile infection therapeutics and prophylactics.

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The zinc transporter ZnuABC is critical for the virulence of Chromobacterium violaceum and contributes to diverse zinc-dependent physiological processes

10.1101/2021.06.03.447022 ◽

2021 ◽

Author(s):

Renato E. R. S. Santos ◽

Waldir P. da Silva Júnior ◽

Simone A. Harrison ◽

Eric P Skaar ◽

Walter J. Chazin ◽

...

Keyword(s):

Mutant Strain ◽

Zinc Uptake ◽

Chromobacterium Violaceum ◽

Host Protein ◽

Infection Model ◽

Uptake System ◽

Environmental Bacterium ◽

Mouse Infection Model ◽

Synthetic Metal

Chromobacterium violaceum is a ubiquitous environmental bacterium that causes sporadic life-threatening infections in humans. How C. violaceum acquires zinc to colonize environmental and host niches is unknown. In this work, we demonstrated that C. violaceum employs the zinc uptake system ZnuABC to overcome zinc limitation in the host, ensuring the zinc supply for several physiological demands. Our data indicated that the C. violaceum ZnuABC transporter is encoded in a zur-CV_RS15045-CV_RS15040-znuCBA operon. This operon was repressed by the zinc uptake regulator Zur and derepressed in the presence of the host protein calprotectin (CP) and the synthetic metal chelator EDTA. A ΔznuCBA mutant strain showed impaired growth under these zinc-chelated conditions. Moreover, the deletion of znuCBA provoked a reduction in violacein production, swimming motility, biofilm formation, and bacterial competition. Remarkably, the ΔznuCBA mutant strain was highly attenuated for virulence in an in vivo mouse infection model and showed a low capacity to colonize the liver, grow in the presence of CP, and resist neutrophil killing. Overall, our findings demonstrate that ZnuABC is essential for C. violaceum virulence, contributing to subvert the zinc-based host nutritional immunity.

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Methionyl-tRNA synthetase inhibitor has potent in vivo activity in a novel Giardia lamblia luciferase murine infection model

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/dkz567 ◽

2020 ◽

Vol 75 (5) ◽

pp. 1218-1227

Author(s):

Samantha A Michaels ◽

Han-Wei Shih ◽

Bailin Zhang ◽

Edelmar D Navaluna ◽

Zhongsheng Zhang ◽

...

Keyword(s):

Giardia Lamblia ◽

Trna Synthetase ◽

Luciferase Reporter ◽

Infection Model ◽

Cell Assay ◽

Mouse Infection Model ◽

Mouse Infection ◽

Methionyl Trna Synthetase

Abstract Background Methionyl-tRNA synthetase (MetRS) inhibitors are under investigation for the treatment of intestinal infections caused by Giardia lamblia. Objectives To properly analyse the therapeutic potential of the MetRS inhibitor 1717, experimental tools including a robust cell-based assay and a murine model of infection were developed based on novel strains of G. lamblia that employ luciferase reporter systems to quantify viable parasites. Methods Systematic screening of Giardia-specific promoters and luciferase variants led to the development of a strain expressing the click beetle green luciferase. Further modifying this strain to express NanoLuc created a dual reporter strain capable of quantifying parasites in both the trophozoite and cyst stages. These strains were used to develop a high-throughput cell assay and a mouse infection model. A library of MetRS inhibitors was screened in the cell assay and Compound-1717 was tested for efficacy in the mouse infection model. Results Cell viability in in vitro compound screens was quantified via bioluminescence readouts while infection loads in mice were monitored with non-invasive whole-animal imaging and faecal analysis. Compound-1717 was effective in clearing mice of Giardia infection in 3 days at varying doses, which was supported by data from enzymatic and phenotypic cell assays. Conclusions The new in vitro and in vivo assays based on luciferase expression by engineered G. lamblia strains are useful for the discovery and development of new therapeutics for giardiasis. MetRS inhibitors, as validated by Compound-1717, have promising anti-giardiasis properties that merit further study as alternative therapeutics.

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Putative Role of β-1,3 Glucans in Candida albicans Biofilm Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01056-06 ◽

2006 ◽

Vol 51 (2) ◽

pp. 510-520 ◽

Cited By ~ 250

Author(s):

Jeniel Nett ◽

Leslie Lincoln ◽

Karen Marchillo ◽

Randall Massey ◽

Kathleen Holoyda ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Cell Viability ◽

Amphotericin B ◽

Cell Walls ◽

Positive Impact ◽

Indirect Evidence ◽

Phenotypic Properties

ABSTRACT Biofilms are microbial communities, embedded in a polymeric matrix, growing attached to a surface. Nearly all device-associated infections involve growth in the biofilm life style. Biofilm communities have characteristic architecture and distinct phenotypic properties. The most clinically important phenotype involves extraordinary resistance to antimicrobial therapy, making biofilm infections very difficulty to cure without device removal. The current studies examine drug resistance in Candida albicans biofilms. Similar to previous reports, we observed marked fluconazole and amphotericin B resistance in a C. albicans biofilm both in vitro and in vivo. We identified biofilm-associated cell wall architectural changes and increased β-1,3 glucan content in C. albicans cell walls from a biofilm compared to planktonic organisms. Elevated β-1,3 glucan levels were also found in the surrounding biofilm milieu and as part of the matrix both from in vitro and in vivo biofilm models. We thus investigated the possible contribution of β-glucans to antimicrobial resistance in Candida albicans biofilms. Initial studies examined the ability of cell wall and cell supernatant from biofilm and planktonic C. albicans to bind fluconazole. The cell walls from both environmental conditions bound fluconazole; however, four- to fivefold more compound was bound to the biofilm cell walls. Culture supernatant from the biofilm, but not planktonic cells, bound a measurable amount of this antifungal agent. We next investigated the effect of enzymatic modification of β-1,3 glucans on biofilm cell viability and the susceptibility of biofilm cells to fluconazole and amphotericin B. We observed a dose-dependent killing of in vitro biofilm cells in the presence of three different β-glucanase preparations. These same concentrations had no impact on planktonic cell viability. β-1,3 Glucanase markedly enhanced the activity of both fluconazole and amphotericin B. These observations were corroborated with an in vivo biofilm model. Exogenous biofilm matrix and commercial β-1,3 glucan reduced the activity of fluconazole against planktonic C. albicans in vitro. In sum, the current investigation identified glucan changes associated with C. albicans biofilm cells, demonstrated preferential binding of these biofilm cell components to antifungals, and showed a positive impact of the modification of biofilm β-1,3 glucans on drug susceptibility. These results provide indirect evidence suggesting a role for glucans in biofilm resistance and present a strong rationale for further molecular dissection of this resistance mechanism to identify new drug targets to treat biofilm infections.

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Simultaneous Administration of 2-Aminoethyl Diphenylborinate and Chloroquine Reverses Chloroquine Resistance in Malaria Parasites

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04805-14 ◽

2015 ◽

Vol 59 (5) ◽

pp. 2890-2892 ◽

Cited By ~ 1

Author(s):

Ehab Mossaad ◽

Wakako Furuyama ◽

Masahiro Enomoto ◽

Satoru Kawai ◽

Katsuhiko Mikoshiba ◽

...

Keyword(s):

Chloroquine Resistance ◽

Infection Model ◽

Simultaneous Administration ◽

Plasmodium Chabaudi ◽

Content Type ◽

Mouse Infection Model ◽

Complete Reversal ◽

The Mean

ABSTRACTA nearly complete reversal of chloroquine (CQ) resistance in the CQ-resistantPlasmodium falciparumK-1 strain, with a significant decrease in the mean ± standard deviation (SD) 50% inhibitory concentration (IC50) from 1,050 ± 95 nM to 14 ± 2 nM, was achievedin vitroby the simultaneous administration of 2-aminoethyl diphenylborinate (2-APB). The CQ resistance-reversing activity of 2-APB, which showed the same efficacy as verapamil, was also observed in anin vivomouse infection model with the CQ-resistantPlasmodium chabaudiAS(30CQ) strain.

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Mutants of Mycobacterium tuberculosis Lacking Three of the Five rpf-Like Genes Are Defective for Growth In Vivo and for Resuscitation In Vitro

Infection and Immunity ◽

10.1128/iai.73.5.3038-3043.2005 ◽

2005 ◽

Vol 73 (5) ◽

pp. 3038-3043 ◽

Cited By ~ 117

Author(s):

Katrina J. Downing ◽

Vladimir V. Mischenko ◽

Margarita O. Shleeva ◽

Danielle I. Young ◽

Michael Young ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Micrococcus Luteus ◽

Most Probable Number ◽

Infection Model ◽

Term Survival ◽

Probable Number ◽

Mouse Infection Model ◽

Nonculturable State

ABSTRACT Mycobacterium tuberculosis contains five genes, rpfA through rpfE, that bear significant homology to the resuscitation-promoting factor (rpf) gene of Micrococcus luteus, whose product is required to resuscitate the growth of dormant cultures of M. luteus and is essential for the growth of this organism. Previous studies have shown that deletion of any one of the five rpf-like genes did not affect the growth or survival of M. tuberculosis in vitro. In conjunction with the results of whole-genome expression profiling, this finding was indicative of their functional redundancy. In this study, we demonstrate that the single deletion mutants are phenotypically similar to wild-type M. tuberculosis H37Rv in vivo. The deletion of individual rpf-like genes had no discernible effect on the growth or long-term survival of M. tuberculosis in liquid culture, and the ability to resuscitate spontaneously from a nonculturable state in a most probable number assay was also unaffected for the three strains tested (the ΔrpfB, ΔrpfD, and ΔrpfE strains). In contrast, two multiple strains, KDT8 (ΔrpfA-mutation ΔrpfC ΔrpfB) and KDT9 (ΔrpfA ΔrpfC ΔrpfD), which lack three of the five rpf-like genes, were significantly yet differentially attenuated in a mouse infection model. These mutants were also unable to resuscitate spontaneously in vitro, demonstrating the importance of the Rpf-like proteins of M. tuberculosis in resuscitation from the nonculturable state. These results strongly suggest that the biological functions of the five rpf-like genes of M. tuberculosis are not wholly redundant and underscore the potential utility of these proteins as targets for therapeutic intervention.

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Monoclonal antibodies targeting surface exposed epitopes of Candida albicans cell wall proteins confer in vivo protection in an infection model

10.1101/2021.09.29.462385 ◽

2021 ◽

Author(s):

Soumya Palliyil ◽

Mark Mawer ◽

Sami Alwafi ◽

Lily Fogg ◽

Giuseppe Buda De Cesare ◽

...

Keyword(s):

Cell Wall ◽

Candida Albicans ◽

Fungal Infections ◽

Yeast Cells ◽

Infection Model ◽

Cell Wall Proteins ◽

Peptide Antigens ◽

Log Reduction ◽

Pre Treatment

MAb based immunotherapies targeting systemic and deep-seated fungal infections are still in their early stages of development with currently no licensed antifungal mAbs available. The cell wall glycoproteins of Candida albicans are potential targets for therapeutic antibody generation due to their extracellular location and key involvement in fungal pathogenesis. We describe phage display based generation of recombinant human antibodies specifically targeting two key cell wall proteins (CWPs) in C. albicans - Utr2 and Pga31, using peptide antigens representing the surface exposed regions of CWPs at elevated levels during in vivo infection. Reformatted mAbs preferentially recognised C. albicans hyphal forms compared to yeast cells and an increased binding in cells pre-treated with caspofungin. In macrophage interaction assays, mAb pre-treatment resulted in a faster engulfment of C. albicans cells suggesting opsonophagocytosis. Finally, in a series of clinically predictive, mouse models of systemic candidiasis, our lead mAb achieved an improved survival (83%) and several log reduction of fungal burden in the kidneys, similar to levels achieved for the fungicidal drug caspofungin, and superior to any anti-Candida mAb.

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In Vitro and In Vivo Activity of AS101 against Carbapenem-Resistant Acinetobacter baumannii

Pharmaceuticals ◽

10.3390/ph14080823 ◽

2021 ◽

Vol 14 (8) ◽

pp. 823

Author(s):

Tsung-Ying Yang ◽

Sung-Pin Tseng ◽

Heather Nokulunga Dlamini ◽

Po-Liang Lu ◽

Lin Lin ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Antibacterial Activities ◽

Treated Group ◽

Infection Model ◽

High Dose ◽

Mouse Infection Model ◽

Carbapenem Resistant ◽

Time Kill

The increasing trend of carbapenem-resistant Acinetobacter baumannii (CRAB) worldwide has become a concern, limiting therapeutic alternatives and increasing morbidity and mortality rates. The immunomodulation agent ammonium trichloro (dioxoethylene-O,O′-) tellurate (AS101) was repurposed as an antimicrobial agent against CRAB. Between 2016 and 2018, 27 CRAB clinical isolates were collected in Taiwan. The in vitro antibacterial activities of AS101 were evaluated using broth microdilution, time-kill assay, reactive oxygen species (ROS) detection and electron microscopy. In vivo effectiveness was assessed using a sepsis mouse infection model. The MIC range of AS101 for 27 CRAB isolates was from 0.5 to 32 µg/mL, which is below its 50% cytotoxicity (approximately 150 µg/mL). Bactericidal activity was confirmed using a time-kill assay. The antibacterial mechanism of AS101 was the accumulation of the ROS and the disruption of the cell membrane, which, in turn, results in cell death. The carbapenemase-producing A. baumannii mouse sepsis model showed that AS101 was a better therapeutic effect than colistin. The mice survival rate after 120 h was 33% (4/12) in the colistin-treated group and 58% (7/12) in the high-dose AS101 (3.33 mg/kg/day) group. Furthermore, high-dose AS101 significantly decreased bacterial population in the liver, kidney and spleen (all p < 0.001). These findings support the concept that AS101 is an ideal candidate for further testing in future studies.

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Two Acinetobacter baumannii Isolates Obtained From a Fatal Necrotizing Fasciitis Infection Display Distinct Genomic and Phenotypic Characteristics in Comparison to Type Strains

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.635673 ◽

2021 ◽

Vol 11 ◽

Author(s):

Jennifer T. Grier ◽

Brock A. Arivett ◽

Maria S. Ramírez ◽

Renee J. Chosed ◽

Jessica A. Bigner ◽

...

Keyword(s):

Cell Viability ◽

Necrotizing Fasciitis ◽

Acinetobacter Baumannii ◽

Galleria Mellonella ◽

Alveolar Epithelial Cell ◽

Fatal Case ◽

Comparative Genomic ◽

Infection Model ◽

Tissue Samples

Acinetobacter baumannii has been recognized as a critical pathogen that causes severe infections worldwide not only because of the emergence of extensively drug-resistant (XDR) derivatives, but also because of its ability to persist in medical environments and colonize compromised patients. While there are numerous reports describing the mechanisms by which this pathogen acquires resistance genes, little is known regarding A. baumannii’s virulence functions associated with rare manifestations of infection such as necrotizing fasciitis, making the determination and implementation of alternative therapeutic targets problematic. To address this knowledge gap, this report describes the analysis of the NFAb-1 and NFAb-2 XDR isolates, which were obtained at two time points during a fatal case of necrotizing fasciitis, at the genomic and functional levels. The comparative genomic analysis of these isolates with the ATCC 19606T and ATCC 17978 strains showed that the NFAb-1 and NFAb-2 isolates are genetically different from each other as well as different from the ATCC 19606T and ATCC 17978 clinical isolates. These genomic differences could be reflected in phenotypic differences observed in these NFAb isolates. Biofilm, cell viability and flow cytometry assays indicate that all tested strains caused significant decreases in A549 human alveolar epithelial cell viability with ATCC 17978, NFAb-1 and NFAb-2 producing significantly less biofilm and significantly more hemolysis and capacity for intracellular invasion than ATCC 19606T. NFAb-1 and NFAb-2 also demonstrated negligible surface motility but significant twitching motility compared to ATCC 19606T and ATCC 17978, likely due to the presence of pili exceeding 2 µm in length, which are significantly longer and different from those previously described in the ATCC 19606T and ATCC 17978 strains. Interestingly, infection with cells of the NFAb-1 isolate, which were obtained from a premortem blood sample, lead to significantly higher mortality rates than NFAb-2 bacteria, which were obtained from postmortem tissue samples, when tested using the Galleria mellonella in vivo infection model. These observations suggest potential changes in the virulence phenotype of the A. baumannii necrotizing fasciitis isolates over the course of infection by mechanisms and cell processes that remain to be identified.

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Discovery of (3-Benzyl-5-hydroxyphenyl)carbamates as New Antitubercular Agents with Potent In Vitro and In Vivo Efficacy

Molecules ◽

10.3390/molecules24102021 ◽

2019 ◽

Vol 24 (10) ◽

pp. 2021 ◽

Cited By ~ 2

Author(s):

Ya-Juan Cheng ◽

Zhi-Yong Liu ◽

Hua-Ju Liang ◽

Cui-Ting Fang ◽

Niu-Niu Zhang ◽

...

Keyword(s):

Oral Administration ◽

Inhibitory Activity ◽

Multidrug Resistant ◽

Infection Model ◽

Antitubercular Agents ◽

In Vivo Efficacy ◽

Mouse Infection Model ◽

Good Potential

A series of 3-amino-5-benzylphenol derivatives were designed and synthesized. Among them, (3-benzyl-5-hydroxyphenyl)carbamates were found to exert good inhibitory activity against M. tuberculosis H37Ra, H37Rv and clinically isolated multidrug-resistant M. tuberculosis strains (MIC = 0.625–6.25 μg/mL). The privileged compounds 3i and 3l showed moderate cytotoxicity against cell line A549. Compound 3l also exhibited potent in vivo inhibitory activity on a mouse infection model via the oral administration. The results demonstrated 3-hydroxyphenylcarbamates as a class of new antitubercular agents with good potential.

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An essential bifunctional enzyme in Mycobacterium tuberculosis for itaconate dissimilation and leucine catabolism

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1906606116 ◽

2019 ◽

Vol 116 (32) ◽

pp. 15907-15913 ◽

Cited By ~ 11

Author(s):

Hua Wang ◽

Alexander A. Fedorov ◽

Elena V. Fedorov ◽

Debbie M. Hunt ◽

Angela Rodgers ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Resistance Mechanism ◽

Infection Model ◽

Enzymatic Assays ◽

X Ray Crystallography ◽

Mouse Infection Model ◽

Leucine Catabolism ◽

Global Population

Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis. One-fourth of the global population is estimated to be infected with Mtb, accounting for ∼1.3 million deaths in 2017. As part of the immune response to Mtb infection, macrophages produce metabolites with the purpose of inhibiting or killing the bacterial cell. Itaconate is an abundant host metabolite thought to be both an antimicrobial agent and a modulator of the host inflammatory response. However, the exact mode of action of itaconate remains unclear. Here, we show that Mtb has an itaconate dissimilation pathway and that the last enzyme in this pathway, Rv2498c, also participates in l-leucine catabolism. Our results from phylogenetic analysis, in vitro enzymatic assays, X-ray crystallography, and in vivo Mtb experiments, identified Mtb Rv2498c as a bifunctional β-hydroxyacyl-CoA lyase and that deletion of the rv2498c gene from the Mtb genome resulted in attenuation in a mouse infection model. Altogether, this report describes an itaconate resistance mechanism in Mtb and an l-leucine catabolic pathway that proceeds via an unprecedented (R)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) stereospecific route in nature.

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