Publications by authors named "Albertus Viljoen"

47 Publications

AFM force-clamp spectroscopy captures the nanomechanics of the Tad pilus retraction.

Nanoscale Horiz 2021 Jun;6(6):489-496

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Croix du Sud, 4-5, bte, L7.07.07, Louvain-la-Neuve B-1348, Belgium.

Motorization of bacterial pili is key to generate traction forces to achieve cellular function. The Tad (or Type IVc) pilus from Caulobacter crescentus is a widespread motorized nanomachine crucial for bacterial survival, evolution and virulence. An unusual bifunctional ATPase motor drives Tad pilus retraction, which helps the bacteria to land on target surfaces. Here, we use a novel platform combining a fluorescence-based screening of piliated bacteria and atomic force microscopy (AFM) force-clamp spectroscopy, to monitor over time (30 s) the nanomechanics and dynamics of the Tad nanofilament retraction under a high constant tension (300 pN). We observe striking transient variations of force and height originating from two phenomena: active pilus retraction and passive hydrophobic interactions between the pilus and the hydrophobic substrate. That the Tad pilus is able to retract under high tensile loading - at a velocity of ∼150 nm s-1 - indicates that this nanomachine is stronger than previously anticipated. Our findings show that pilus retraction and hydrophobic interactions work together to mediate bacterial cell landing and surface adhesion. The motorized pilus retraction actively triggers the cell to approach the substrate. At short distances, passive hydrophobic interactions accelerate the approach phenomenon and promote strong cell-substrate adhesion. This mechanism could provide a strategy to save ATP-based energy by the retraction ATPase. Our force-clamp AFM methodology offers promise to decipher the physics of bacterial nanomotors with high sensitivity and temporal resolution.
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http://dx.doi.org/10.1039/d1nh00158bDOI Listing
June 2021

Staphylococcus aureus vWF-binding protein triggers a strong interaction between clumping factor A and host vWF.

Commun Biol 2021 Apr 12;4(1):453. Epub 2021 Apr 12.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Louvain-la-Neuve, Belgium.

The Staphylococcus aureus cell wall-anchored adhesin ClfA binds to the very large blood circulating protein, von Willebrand factor (vWF) via vWF-binding protein (vWbp), a secreted protein that does not bind the cell wall covalently. Here we perform force spectroscopy studies on living bacteria to unravel the molecular mechanism of this interaction. We discover that the presence of all three binding partners leads to very high binding forces (2000 pN), largely outperforming other known ternary complexes involving adhesins. Strikingly, our experiments indicate that a direct interaction involving features of the dock, lock and latch mechanism must occur between ClfA and vWF to sustain the extreme tensile strength of the ternary complex. Our results support a previously undescribed mechanism whereby vWbp activates a direct, ultra-strong interaction between ClfA and vWF. This intriguing interaction represents a potential target for therapeutic interventions, including synthetic peptides inhibiting the ultra-strong interactions between ClfA and its ligands.
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http://dx.doi.org/10.1038/s42003-021-01986-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8041789PMC
April 2021

AFM Unravels the Unique Adhesion Properties of the Type IVc Pilus Nanomachine.

Nano Lett 2021 04 23;21(7):3075-3082. Epub 2021 Mar 23.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Croix du Sud, 4-5, bte, L7.07.07., B-1348 Louvain-la-Neuve, Belgium.

Bacterial pili are proteinaceous motorized nanomachines that play various functional roles including surface adherence, bacterial motion, and virulence. The surface-contact sensor type IVc (or Tad) pilus is widely distributed in both Gram-positive and Gram-negative bacteria. In , this nanofilament, though crucial for surface colonization, has never been thoroughly investigated at the molecular level. As assembles several surface appendages at specific stages of the cell cycle, we designed a fluorescence-based screen to selectively study single piliated cells and combined it with atomic force microscopy and genetic manipulation to quantify the nanoscale adhesion of the type IVc pilus to hydrophobic substrates. We demonstrate that this nanofilament exhibits high stickiness compared to the canonical type IVa/b pili, resulting mostly from multiple hydrophobic interactions along the fiber length, and that it features nanospring mechanical properties. Our findings may be helpful to better understand the structure-function relationship of bacterial pilus nanomachines.
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http://dx.doi.org/10.1021/acs.nanolett.1c00215DOI Listing
April 2021

AFM in cellular and molecular microbiology.

Cell Microbiol 2021 Mar 12:e13324. Epub 2021 Mar 12.

Louvain Institute of Biomolecular Science and Technology, Université Catholique de Louvain, Louvain-la-Neuve, Belgium.

The unique capabilities of the atomic force microscope (AFM), including super-resolution imaging, piconewton force-sensitivity, nanomanipulation and ability to work under physiological conditions, have offered exciting avenues for cellular and molecular biology research. AFM imaging has helped unravel the fine architectures of microbial cell envelopes at the nanoscale, and how these are altered by antimicrobial treatment. Nanomechanical measurements have shed new light on the elasticity, tensile strength and turgor pressure of single cells. Single-molecule and single-cell force spectroscopy experiments have revealed the forces and dynamics of receptor-ligand interactions, the nanoscale distribution of receptors on the cell surface and the elasticity and adhesiveness of bacterial pili. Importantly, recent force spectroscopy studies have demonstrated that extremely stable bonds are formed between bacterial adhesins and their cognate ligands, originating from a catch bond behaviour allowing the pathogen to reinforce adhesion under shear or tensile stress. Here, we survey how the versatility of AFM has enabled addressing crucial questions in microbiology, with emphasis on bacterial pathogens.
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http://dx.doi.org/10.1111/cmi.13324DOI Listing
March 2021

Seeing and Touching the Mycomembrane at the Nanoscale.

J Bacteriol 2021 Apr 21;203(10). Epub 2021 Apr 21.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Louvain-la-Neuve, Belgium

Mycobacteria have unique cell envelopes, surface properties, and growth dynamics, which all play a part in the ability of these important pathogens to infect, evade host immunity, disseminate, and resist antibiotic challenges. Recent atomic force microscopy (AFM) studies have brought new insights into the nanometer-scale ultrastructural, adhesive, and mechanical properties of mycobacteria. The molecular forces with which mycobacterial adhesins bind to host factors, like heparin and fibronectin, and the hydrophobic properties of the mycomembrane have been unraveled by AFM force spectroscopy studies. Real-time correlative AFM and fluorescence imaging have delineated a complex interplay between surface ultrastructure, tensile stresses within the cell envelope, and cellular processes leading to division. The unique capabilities of AFM, which include subdiffraction-limit topographic imaging and piconewton force sensitivity, have great potential to resolve important questions that remain unanswered on the molecular interactions, surface properties, and growth dynamics of this important class of pathogens.
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http://dx.doi.org/10.1128/JB.00547-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8088606PMC
April 2021

-Methylation of the Glycopeptidolipid Acyl Chain Defines Surface Hydrophobicity of and Macrophage Invasion.

ACS Infect Dis 2020 10 14;6(10):2756-2770. Epub 2020 Sep 14.

Centre National de la Recherche Scientifique UMR 9004, Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier, 1919 route de Mende, 34293 Montpellier, France.

, an emerging pathogen responsible for severe lung infections in cystic fibrosis patients, displays either smooth (S) or rough (R) morphotypes. The S-to-R transition is associated with reduced levels of glycopeptidolipid (GPL) production and is correlated with increased pathogenicity in animal and human hosts. While the structure of GPL is well established, its biosynthetic pathway is incomplete. In addition, the biological functions of the distinct structural parts of this complex lipid remain elusive. Herein, the gene encoding a putative -methyltransferase was deleted in the S variant. Subsequent biochemical and structural analyses demonstrated that methoxylation of the fatty acyl chain of GPL was abrogated in the mutant, and this defect was rescued upon complementation with a functional gene. In contrast, the introduction of derivatives mutated at residues essential for methyltransferase activity failed to complement GPL defects, indicating that encodes an -methyltransferase. Unexpectedly, phenotypic analyses showed that was more hydrophilic than its parental progenitor, as demonstrated by hexadecane-aqueous buffer partitioning and atomic force microscopy experiments with hydrophobic probes. Importantly, the invasion rate of THP-1 macrophages by was reduced by 50% when compared to the wild-type strain. Together, these results indicate that Fmt -methylates the lipid moiety of GPL and plays a substantial role in conditioning the surface hydrophobicity of as well as in the early steps of the interaction between the bacilli and macrophages.
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http://dx.doi.org/10.1021/acsinfecdis.0c00490DOI Listing
October 2020

Binding Strength of Gram-Positive Bacterial Adhesins.

Front Microbiol 2020 25;11:1457. Epub 2020 Jun 25.

Louvain Institute of Biomolecular Science and Technology, Catholic University of Louvain, Louvain-la-Neuve, Belgium.

Bacterial pathogens are equipped with specialized surface-exposed proteins that bind strongly to ligands on host tissues and biomaterials. These adhesins play critical roles during infection, especially during the early step of adhesion where the cells are exposed to physical stress. Recent single-molecule experiments have shown that staphylococci interact with their ligands through a wide diversity of mechanosensitive molecular mechanisms. Adhesin-ligand interactions are activated by tensile force and can be ten times stronger than classical non-covalent biological bonds. Overall these studies demonstrate that Gram-positive adhesins feature unusual stress-dependent molecular interactions, which play essential roles during bacterial colonization and dissemination. With an increasing prevalence of multidrug resistant infections caused by and , chemotherapeutic targeting of adhesins offers an innovative alternative to antibiotics.
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http://dx.doi.org/10.3389/fmicb.2020.01457DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7330015PMC
June 2020

Synthesis and evaluation of heterocycle structures as potential inhibitors of Mycobacterium tuberculosis UGM.

Bioorg Med Chem 2020 07 2;28(13):115579. Epub 2020 Jun 2.

Laboratoire Synthèse et Isolement de Molécules Bioactives (SIMBA, EA 7502), Université de Tours, Faculté de Pharmacie, Parc de Grandmont, 31 Avenue Monge, 37200 Tours, France. Electronic address:

In this study, we screen three heterocyclic structures as potential inhibitors of UDP-galactopyranose mutase (UGM), an enzyme involved in the biosynthesis of the cell wall of Mycobacterium tuberculosis. In order to understand the binding mode, docking simulations are performed on the best inhibitors. Their activity on Mycobacterium tuberculosis is also evaluated. This study made it possible to highlight an "oxazepino-indole" structure as a new inhibitor of UGM and of M. tuberculosis growth in vitro.
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http://dx.doi.org/10.1016/j.bmc.2020.115579DOI Listing
July 2020

Fast chemical force microscopy demonstrates that glycopeptidolipids define nanodomains of varying hydrophobicity on mycobacteria.

Nanoscale Horiz 2020 06 21;5(6):944-953. Epub 2020 Apr 21.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Croix du Sud, 4-5, bte L7.07.07, B-1348 Louvain-la-Neuve, Belgium.

Mycobacterium abscessus is an emerging multidrug-resistant bacterial pathogen causing severe lung infections in cystic fibrosis patients. A remarkable trait of this mycobacterial species is its ability to form morphologically smooth (S) and rough (R) colonies. The S-to-R transition is caused by the loss of glycopeptidolipids (GPLs) in the outer layer of the cell envelope and correlates with an increase in cording and virulence. Despite the physiological and medical importance of this morphological transition, whether it involves changes in cell surface properties remains unknown. Herein, we combine recently developed quantitative imaging (QI) atomic force microscopy (AFM) with hydrophobic tips to quantitatively map the surface structure and hydrophobicity of M. abscessus at high spatiotemporal resolution, and to assess how these properties are modulated by the S-to-R transition and by treatment with an inhibitor of the mycolic acid transporter MmpL3. We discover that loss of GPLs leads to major modifications in surface hydrophobicity, without any apparent change in cell surface ultrastructure. While R bacilli are homogeneously hydrophobic, S bacilli feature unusual variations of nanoscale hydrophobic properties. These previously undescribed cell surface nanodomains are likely to play critical roles in bacterial adhesion, aggregation, phenotypic heterogeneity and transmission, and in turn in virulence and pathogenicity. Our study also suggests that MmpL3 inhibitors show promise in nanomedicine as chemotherapeutic agents to interfere with the highly hydrophobic nature of the mycobacterial cell wall. The advantages of QI-AFM with hydrophobic tips are the ability to map chemical and structural properties simultaneously and at high resolution, applicable to a wide range of biosystems.
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http://dx.doi.org/10.1039/c9nh00736aDOI Listing
June 2020

How Microbes Use Force To Control Adhesion.

J Bacteriol 2020 05 27;202(12). Epub 2020 May 27.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Louvain-la-Neuve, Belgium

Microbial adhesion and biofilm formation are usually studied using molecular and cellular biology assays, optical and electron microscopy, or laminar flow chamber experiments. Today, atomic force microscopy (AFM) represents a valuable addition to these approaches, enabling the measurement of forces involved in microbial adhesion at the single-molecule level. In this minireview, we discuss recent discoveries made applying state-of-the-art AFM techniques to microbial specimens in order to understand the strength and dynamics of adhesive interactions. These studies shed new light on the molecular mechanisms of adhesion and demonstrate an intimate relationship between force and function in microbial adhesins.
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http://dx.doi.org/10.1128/JB.00125-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253613PMC
May 2020

Mechanical Forces between Mycobacterial Antigen 85 Complex and Fibronectin.

Cells 2020 03 14;9(3). Epub 2020 Mar 14.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Croix du Sud, 4-5, bte L7.07.07, B-1348 Louvain-la-Neuve, Belgium.

Adhesion to extracellular matrix proteins is an important first step in host invasion, employed by many bacterial pathogens. In mycobacteria, the secreted Ag85 complex proteins, involved in the synthesis of the cell envelope, are known to bind to fibronectin (Fn) through molecular forces that are currently unknown. In this study, single-molecule force spectroscopy is used to study the strength, kinetics and thermodynamics of the Ag85-Fn interaction, focusing on the multidrug-resistant species. Single Ag85 proteins bind Fn with a strength of ~75 pN under moderate tensile loading, which compares well with the forces reported for other Fn-binding proteins. The binding specificity is demonstrated by using free Ag85 and Fn peptides with active binding sequences. The Ag85-Fn rupture force increases with mechanical stress (i.e., loading rate) according to the Friddle-Noy-de Yoreo theory. From this model, we extract thermodynamic parameters that are in good agreement with previous affinity determinations by surface plasmon resonance. Strong bonds (up to ~500 pN) are observed under high tensile loading, which may favor strong mycobacterial attachment in the lung where cells are exposed to high shear stress or during hematogenous spread which leads to a disseminated infection. Our results provide new insight into the pleiotropic functions of an important mycobacterial virulence factor that acts as a stress-sensitive adhesin.
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http://dx.doi.org/10.3390/cells9030716DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7140604PMC
March 2020

The endogenous galactofuranosidase GlfH1 hydrolyzes mycobacterial arabinogalactan.

J Biol Chem 2020 04 27;295(15):5110-5123. Epub 2020 Feb 27.

Univ. Lille, CNRS, UMR8576 - UGSF - Unit[c33c]zpi;● de Glycobiologie Structurale et Fonctionnelle, F-59000 Lille, France

Despite impressive progress made over the past 20 years in our understanding of mycolylarabinogalactan-peptidoglycan (mAGP) biogenesis, the mechanisms by which the tubercle bacillus adapts its cell wall structure and composition to various environmental conditions, especially during infection, remain poorly understood. Being the central portion of the mAGP complex, arabinogalactan (AG) is believed to be the constituent of the mycobacterial cell envelope that undergoes the least structural changes, but no reports exist supporting this assumption. Herein, using recombinantly expressed mycobacterial protein, bioinformatics analyses, and kinetic and biochemical assays, we demonstrate that the AG can be remodeled by a mycobacterial endogenous enzyme. In particular, we found that the mycobacterial GlfH1 (Rv3096) protein exhibits exo-β-d-galactofuranose hydrolase activity and is capable of hydrolyzing the galactan chain of AG by recurrent cleavage of the terminal β-(1,5) and β-(1,6)-Galf linkages. The characterization of this galactosidase represents a first step toward understanding the remodeling of mycobacterial AG.
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http://dx.doi.org/10.1074/jbc.RA119.011817DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7152746PMC
April 2020

Scratching the Surface: Bacterial Cell Envelopes at the Nanoscale.

mBio 2020 02 25;11(1). Epub 2020 Feb 25.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Louvain-la-Neuve, Belgium

The bacterial cell envelope is essential for viability, the environmental gatekeeper and first line of defense against external stresses. For most bacteria, the envelope biosynthesis is also the site of action of some of the most important groups of antibiotics. It is a complex, often multicomponent structure, able to withstand the internally generated turgor pressure. Thus, elucidating the architecture and dynamics of the cell envelope is important, to unravel not only the complexities of cell morphology and maintenance of integrity but also how interventions such as antibiotics lead to death. To address these questions requires the capacity to visualize the cell envelope via high-spatial resolution approaches. In recent years, atomic force microscopy (AFM) has brought novel molecular insights into the assembly, dynamics, and functions of bacterial cell envelopes. The ultrafine resolution and physical sensitivity of the technique have revealed a wealth of ultrastructural features that are invisible to traditional optical microscopy techniques or imperceptible in their true physiological state by electron microscopy. Here, we discuss recent progress in our use of AFM imaging for understanding the architecture and dynamics of the bacterial envelope. We survey recent studies that demonstrate the power of the technique to observe isolated membranes and live cells at (sub)nanometer resolution and under physiological conditions and to track structural dynamics in response to growth or to drugs.
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http://dx.doi.org/10.1128/mBio.03020-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7042696PMC
February 2020

What makes bacterial pathogens so sticky?

Mol Microbiol 2020 04 16;113(4):683-690. Epub 2020 Jan 16.

Louvain Institute of Biomolecular Science and Technology, UCLouvain, Louvain-la-Neuve, Belgium.

Pathogenic bacteria use a variety of cell surface adhesins to promote binding to host tissues and protein-coated biomaterials, as well as cell-cell aggregation. These cellular interactions represent the first essential step that leads to host colonization and infection. Atomic force microscopy (AFM) has greatly contributed to increase our understanding of the specific interactions at play during microbial adhesion, down to the single-molecule level. A key asset of AFM is that adhesive interactions are studied under mechanical force, which is highly relevant as surface-attached pathogens are often exposed to physical stresses in the human body. These studies have identified sophisticated binding mechanisms in adhesins, which represent promising new targets for antiadhesion therapy.
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http://dx.doi.org/10.1111/mmi.14448DOI Listing
April 2020

The TetR Family Transcription Factor MAB_2299c Regulates the Expression of Two Distinct MmpS-MmpL Efflux Pumps Involved in Cross-Resistance to Clofazimine and Bedaquiline in Mycobacterium abscessus.

Antimicrob Agents Chemother 2019 10 23;63(10). Epub 2019 Sep 23.

Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier, CNRS UMR 9004, Montpellier, France

is a human pathogen responsible for severe respiratory infections, particularly in patients with underlying lung disorders. Notorious for being highly resistant to most antimicrobials, new therapeutic approaches are needed to successfully treat -infected patients. Clofazimine (CFZ) and bedaquiline (BDQ) are two antibiotics used for the treatment of multidrug-resistant tuberculosis and are considered alternatives for the treatment of pulmonary disease. To get insights into their mechanisms of resistance in , we previously characterized the TetR transcriptional regulator MAB_2299c, which controls expression of the genes, encoding an MmpS-MmpL efflux pump. Here, studies identified a second () target of MAB_2299c. A palindromic DNA sequence upstream of , sharing strong homology with the one located upstream of , was found to form a complex with the MAB_2299c regulator in electrophoretic mobility shift assays. Deletion of in a wild-type strain led to increased susceptibility to both CFZ and BDQ. In addition, deletion of these genes in a CFZ/BDQ-susceptible mutant lacking as well as further exacerbated the sensitivity of this strain to both drugs and inside macrophages. Overall, these results indicate that encodes a new MmpS-MmpL efflux pump system involved in the intrinsic resistance to CFZ and BDQ. They also support the view that MAB_2299c controls the expression of two separate MmpS-MmpL efflux pumps, substantiating the importance of as a marker of resistance to be considered when assessing drug susceptibility in clinical isolates.
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http://dx.doi.org/10.1128/AAC.01000-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6761555PMC
October 2019

1-Benzo[]Imidazole Derivatives Affect MmpL3 in Mycobacterium tuberculosis.

Antimicrob Agents Chemother 2019 10 23;63(10). Epub 2019 Sep 23.

Institute of Medical Biology, Polish Academy of Sciences, Lodz, Poland

1-benzo[]imidazole derivatives exhibit antitubercular activity at a nanomolar range of concentrations and are not toxic to human cells, but their mode of action remains unknown. Here, we showed that these compounds are active against intracellular To identify their target, we selected drug-resistant mutants and then used whole-genome sequencing to unravel mutations in the essential gene, which encodes the integral membrane protein that catalyzes the export of trehalose monomycolate, a precursor of the mycobacterial outer membrane component trehalose dimycolate (TDM), as well as mycolic acids bound to arabinogalactan. The drug-resistant phenotype was also observed in the parental strain overexpressing the alleles carrying the mutations identified in the resistors. However, no cross-resistance was observed between 1-benzo[]imidazole derivatives and SQ109, another MmpL3 inhibitor, or other first-line antitubercular drugs. Metabolic labeling and quantitative thin-layer chromatography (TLC) analysis of radiolabeled lipids from cultures treated with the benzoimidazoles indicated an inhibition of trehalose dimycolate (TDM) synthesis, as well as reduced levels of mycolylated arabinogalactan, in agreement with the inhibition of MmpL3 activity. Overall, this study emphasizes the pronounced activity of 1-benzo[]imidazole derivatives in interfering with mycolic acid metabolism and their potential for therapeutic application in the fight against tuberculosis.
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http://dx.doi.org/10.1128/AAC.00441-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6761528PMC
October 2019

Nitrogen deprivation induces triacylglycerol accumulation, drug tolerance and hypervirulence in mycobacteria.

Sci Rep 2019 06 17;9(1):8667. Epub 2019 Jun 17.

Aix-Marseille Univ, CNRS, LISM, IMM FR3479, Marseille, France.

Mycobacteria share with other actinomycetes the ability to produce large quantities of triacylglycerol (TAG), which accumulate as intracytoplasmic lipid inclusions (ILI) also known as lipid droplets (LD). Mycobacterium tuberculosis (M. tb), the etiologic agent of tuberculosis, acquires fatty acids from the human host which are utilized to synthesize TAG, subsequently stored in the form of ILI to meet the carbon and nutrient requirements of the bacterium during long periods of persistence. However, environmental factors governing mycobacterial ILI formation and degradation remain poorly understood. Herein, we demonstrated that in the absence of host cells, carbon excess and nitrogen starvation promote TAG accumulation in the form of ILI in M. smegmatis and M. abscessus, used as surrogate species of M. tb. Based on these findings, we developed a simple and reversible in vitro model to regulate ILI biosynthesis and hydrolysis in mycobacteria. We also showed that TAG formation is tgs1 dependent and that lipolytic enzymes mediate TAG breakdown. Moreover, we confirmed that the nitrogen-deprived and ILI-rich phenotype was associated with an increased tolerance towards several drugs used for treating mycobacterial infections. Importantly, we showed that the presence of ILI substantially enhanced the bacterial burden and granuloma abundance in zebrafish embryos infected with lipid-rich M. abscessus as compared to embryos infected with lipid-poor M. abscessus, suggesting that ILI are actively contributing to mycobacterial virulence and pathogenesis.
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http://dx.doi.org/10.1038/s41598-019-45164-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6572852PMC
June 2019

Verapamil Improves the Activity of Bedaquiline against Mycobacterium abscessus In Vitro and in Macrophages.

Antimicrob Agents Chemother 2019 09 23;63(9). Epub 2019 Aug 23.

Institut de Recherche en Infectiologie de Montpellier, Université de Montpellier, CNRS UMR 9004, Montpellier, France

Due to intrinsic multidrug resistance, pulmonary infections with are extremely difficult to treat. Previously, we demonstrated that bedaquiline is highly effective against both and Here, we report that verapamil improves the efficacy of bedaquiline activity against clinical isolates and low-level resistant strains, both and in macrophages. Verapamil may have clinical potential as adjunctive therapy provided that sufficiently high doses can be safely achieved.
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http://dx.doi.org/10.1128/AAC.00705-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6709475PMC
September 2019

Lsr2 Is an Important Determinant of Intracellular Growth and Virulence in .

Front Microbiol 2019 30;10:905. Epub 2019 Apr 30.

2I, UVSQ, INSERM, Université Paris-Saclay, Versailles, France.

, a pathogen responsible for severe lung infections in cystic fibrosis patients, exhibits either smooth (S) or rough (R) morphotypes. The S-to-R transition correlates with inhibition of the synthesis and/or transport of glycopeptidolipids (GPLs) and is associated with an increase of pathogenicity in animal and human hosts. Lsr2 is a small nucleoid-associated protein highly conserved in mycobacteria, including , and is a functional homolog of the heat-stable nucleoid-structuring protein (H-NS). It is essential in but not in the non-pathogenic model organism . It acts as a master transcriptional regulator of multiple genes involved in virulence and immunogenicity through binding to AT-rich genomic regions. Previous transcriptomic studies, confirmed here by quantitative PCR, showed increased expression of () in R morphotypes when compared to their S counterparts, suggesting a possible role of this protein in the virulence of the R form. This was addressed by generating knock-out mutants in both S (Δ-S) and R (Δ-R) variants, demonstrating that this gene is dispensable for growth. We show that the wild-type S variant, Δ-S and Δ-R strains were more sensitive to HO as compared to the wild-type R variant of . Importantly, virulence of the Lsr2 mutants was considerably diminished in cellular models (macrophage and amoeba) as well as in infected animals (mouse and zebrafish). Collectively, these results emphasize the importance of Lsr2 in virulence.
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http://dx.doi.org/10.3389/fmicb.2019.00905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6503116PMC
April 2019

Mutations in the MAB_2299c TetR Regulator Confer Cross-Resistance to Clofazimine and Bedaquiline in .

Antimicrob Agents Chemother 2019 01 21;63(1). Epub 2018 Dec 21.

Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier, CNRS UMR 9004, Montpellier, France

New therapeutic approaches are needed against , a respiratory mycobacterial pathogen that evades efforts to successfully treat infected patients. Clofazimine and bedaquiline, two drugs used for the treatment of multidrug-resistant tuberculosis, are being considered as alternatives for the treatment of lung diseases caused by With the aim to understand the mechanism of action of these agents in , we sought herein to determine the means by which can develop resistance. Spontaneous resistant strains selected on clofazimine, followed by whole-genome sequencing, identified mutations in , encoding a putative TetR transcriptional regulator. Unexpectedly, mutants with these mutations were also cross-resistant to bedaquiline. MAB_2299c was found to bind to its target DNA, located upstream of the divergently oriented gene cluster, encoding MmpS/MmpL membrane proteins. Point mutations or deletion of was associated with the concomitant upregulation of the and transcripts and accounted for this cross-resistance. Strikingly, deletion of and in the mutant strain restored susceptibility to bedaquiline and clofazimine. Overall, these results expand our knowledge with respect to the regulatory mechanisms of the MmpL family of proteins and a novel mechanism of drug resistance in this difficult-to-treat respiratory mycobacterial pathogen. Therefore, may represent an important marker of resistance to be considered in the treatment of diseases with clofazimine and bedaquiline in clinical settings.
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http://dx.doi.org/10.1128/AAC.01316-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6325171PMC
January 2019

MmpL8 controls virulence and production of a previously unknown glycolipid family.

Proc Natl Acad Sci U S A 2018 10 9;115(43):E10147-E10156. Epub 2018 Oct 9.

Université de Versailles Saint Quentin en Yvelines, INSERM UMR1173, 78000 Versailles, France;

is a peculiar rapid-growing (RGM) capable of surviving within eukaryotic cells thanks to an arsenal of virulence genes also found in slow-growing mycobacteria (SGM), such as A screen based on the intracellular survival in amoebae and macrophages (MΦ) of an transposon mutant library revealed the important role of MAB_0855, a yet uncharacterized Mycobacterial membrane protein Large (MmpL). Large-scale comparisons with SGM and RGM genomes uncovered MmpL12 proteins as putative orthologs of MAB_0855 and a locus-scale synteny between the and loci. A KO mutant of the gene, designated herein as , had impaired adhesion to MΦ and displayed a decreased intracellular viability. Despite retaining the ability to block phagosomal acidification, like the WT strain, the mutant was delayed in damaging the phagosomal membrane and in making contact with the cytosol. Virulence attenuation of the mutant was confirmed in vivo by impaired zebrafish killing and a diminished propensity to induce granuloma formation. The previously shown role of MmpL in lipid transport prompted us to investigate the potential lipid substrates of MmpL8 Systematic lipid analysis revealed that MmpL8 was required for the proper expression of a glycolipid entity, a glycosyl diacylated nonadecyl diol (GDND) alcohol comprising different combinations of oleic and stearic acids. This study shows the importance of MmpL8 in modifying interactions between the bacteria and phagocytic cells and in the production of a previously unknown glycolipid family.
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http://dx.doi.org/10.1073/pnas.1812984115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6205491PMC
October 2018

Delineating the Physiological Roles of the PE and Catalytic Domains of LipY in Lipid Consumption in Mycobacterium-Infected Foamy Macrophages.

Infect Immun 2018 09 22;86(9). Epub 2018 Aug 22.

Aix-Marseille Univ, CNRS, LISM, IMM FR3479, Marseille, France

Within tuberculous granulomas, a subpopulation of resides inside foamy macrophages (FM) that contain abundant cytoplasmic lipid bodies (LB) filled with triacylglycerol (TAG). Upon fusion of LB with -containing phagosomes, TAG is hydrolyzed and reprocessed by the bacteria into their own lipids, which accumulate as intracytosolic lipid inclusions (ILI). This phenomenon is driven by many mycobacterial lipases, among which LipY participates in the hydrolysis of host and bacterial TAG. However, the functional contribution of LipY's PE domain to TAG hydrolysis remains unclear. Here, enzymatic studies were performed to compare the lipolytic activities of recombinant LipY and its truncated variant lacking the N-terminal PE domain, LipY(ΔPE). Complementarily, an FM model was used where bone marrow-derived mouse macrophages were infected with BCG strains either overexpressing LipY or LipY(ΔPE) or carrying a deletion mutation prior to being exposed to TAG-rich very-low-density lipoprotein (VLDL). Results indicate that truncation of the PE domain correlates with increased TAG hydrolase activity. Quantitative electron microscopy analyses showed that (i) in the presence of lipase inhibitors, large ILI (ILI) were not formed because of an absence of LB due to inhibition of VLDL-TAG hydrolysis or inhibition of LB-neutral lipid hydrolysis by mycobacterial lipases, (ii) ILI profiles in the strain overexpressing LipY(ΔPE) were reduced, and (iii) the number of ILI profiles in the Δ mutant was reduced by 50%. Overall, these results delineate the role of LipY and its PE domain in host and mycobacterial lipid consumption and show that additional mycobacterial lipases take part in these processes.
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http://dx.doi.org/10.1128/IAI.00394-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6105901PMC
September 2018

Glycopeptidolipids, a Double-Edged Sword of the Complex.

Front Microbiol 2018 5;9:1145. Epub 2018 Jun 5.

Centre National de la Recherche Scientifique, Institut de Recherche en Infectiologie de Montpellier, UMR 9004, Université de Montpellier, Montpellier, France.

is a rapidly-growing species causing a diverse panel of clinical manifestations, ranging from cutaneous infections to severe respiratory disease. Its unique cell wall, contributing largely to drug resistance and to pathogenicity, comprises a vast panoply of complex lipids, among which the glycopeptidolipids (GPLs) have been the focus of intense research. These lipids fulfill various important functions, from sliding motility or biofilm formation to interaction with host cells and intramacrophage trafficking. Being highly immunogenic, the induction of a strong humoral response is likely to select for rough low-GPL producers. These, in contrast to the smooth high-GPL producers, display aggregative properties, which strongly impacts upon intracellular survival. A propensity to grow as extracellular cords allows these low-GPL producing bacilli to escape the innate immune defenses. Transitioning from high-GPL to low-GPL producers implicates mutations within genes involved in biosynthesis or transport of GPL. This leads to induction of an intense pro-inflammatory response and robust and lethal infections in animal models, explaining the presence of rough isolates in patients with decreased pulmonary functions. Herein, we will discuss how, thanks to the generation of defined GPL mutants and the development of appropriate cellular and animal models to study pathogenesis, GPL contribute to biology and physiopathology.
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http://dx.doi.org/10.3389/fmicb.2018.01145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5996870PMC
June 2018

Structural rearrangements occurring upon cofactor binding in the Mycobacterium smegmatis β-ketoacyl-acyl carrier protein reductase MabA.

Acta Crystallogr D Struct Biol 2018 05 24;74(Pt 5):383-393. Epub 2018 Apr 24.

Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier, CNRS UMR 9004, 34293 Montpellier, France.

In mycobacteria, the ketoacyl-acyl carrier protein (ACP) reductase MabA (designated FabG in other bacteria) catalyzes the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products. This first reductive step in the fatty-acid biosynthesis elongation cycle is essential for bacteria, which makes MabA/FabG an interesting drug target. To date, however, very few molecules targeting FabG have been discovered and MabA remains the only enzyme of the mycobacterial type II fatty-acid synthase that lacks specific inhibitors. Despite the existence of several MabA/FabG crystal structures, the structural rearrangement that occurs upon cofactor binding is still not fully understood. Therefore, unlocking this knowledge gap could help in the design of new inhibitors. Here, high-resolution crystal structures of MabA from Mycobacterium smegmatis in its apo, NADP-bound and NADPH-bound forms are reported. Comparison of these crystal structures reveals the structural reorganization of the lid region covering the active site of the enzyme. The crystal structure of the apo form revealed numerous residues that trigger steric hindrance to the binding of NADPH and substrate. Upon NADPH binding, these residues are pushed away from the active site, allowing the enzyme to adopt an open conformation. The transition from an NADPH-bound to an NADP-bound form is likely to facilitate release of the product. These results may be useful for subsequent rational drug design and/or for in silico drug-screening approaches targeting MabA/FabG.
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http://dx.doi.org/10.1107/S2059798318002917DOI Listing
May 2018

Mechanistic and Structural Insights Into the Unique TetR-Dependent Regulation of a Drug Efflux Pump in .

Front Microbiol 2018 5;9:649. Epub 2018 Apr 5.

CNRS UMR 9004, Institut de Recherche en Infectiologie de Montpellier, Université de Montpellier, Montpellier, France.

is an emerging human pathogen causing severe pulmonary infections and is refractory to standard antibiotherapy, yet few drug resistance mechanisms have been reported in this organism. Recently, mutations in leading to up-regulation of the MmpS5/MmpL5 efflux pump were linked to increased resistance to thiacetazone derivatives. Herein, the DNA-binding activity of MAB_4384 was investigated by electrophoretic mobility shift assays using the palindromic sequence IR located upstream of . Introduction of point mutations within IR identified the sequence requirements for optimal binding of the regulator. Moreover, formation of the protein/IR complex was severely impaired for MAB_4384 harboring D14N or F57L substitutions. IR reporter fusions in demonstrated increased β-galactosidase activity either in strains lacking a functional MAB_4384 or in cultures treated with the TAC analogs. In addition, X-ray crystallography confirmed a typical TetR homodimeric structure of MAB_4384 and unraveled a putative ligand binding site in which the analogs could be docked. Overall, these results support drug recognition of the MAB_4384 TetR regulator, alleviating its binding to IR and steering up-regulation of MmpS5/MmpL5. This study provides new mechanistic and structural details of TetR-dependent regulatory mechanisms of efflux pumps and drug resistance in mycobacteria.
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http://dx.doi.org/10.3389/fmicb.2018.00649DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5895659PMC
April 2018

A Simple and Rapid Gene Disruption Strategy in : On the Design and Application of Glycopeptidolipid Mutants.

Front Cell Infect Microbiol 2018 14;8:69. Epub 2018 Mar 14.

Centre National de la Recherche Scientifique UMR 9004, Institut de Recherche en Infectiologie de Montpellier, Université de Montpellier, Montpellier, France.

Little is known about the disease-causing genetic determinants that are used by , increasingly acknowledged as an important emerging pathogen, notably in cystic fibrosis. The presence or absence of surface exposed glycopeptidolipids (GPL) conditions the smooth (S) or rough (R) subsp. () variants, respectively, which are characterized by distinct infective programs. However, only a handful of successful gene knock-out and conditional mutants have been reported in , testifying that genetic manipulation of this mycobacterium is difficult. To facilitate gene disruption and generation of conditional mutants in , we have designed a one-step single cross-over system that allows the rapid and simple generation of such mutants. Cloning of as small as 300 bp of the target gene allows for efficient homologous recombination to occur without additional exogenous recombination-promoting factors. The presence of tdTomato on the plasmids allows easily sifting out the large background of mutants spontaneously resistant to antibiotics. Using this strategy in the S genetic background and the target gene , necessary for GPL synthesis and transport, nearly 100% of red fluorescent clones exhibited a rough morphotype and lost GPL on the surface, suggesting that most red fluorescent colonies obtained after transformation incorporated the plasmid through homologous recombination into the chromosome. This system was further exploited to generate another strain with reduced GPL levels to explore how the presence of these cell wall-associated glycolipids influences hydrophobicity as well as virulence in the zebrafish model of infection. This mutant exhibited a more pronounced killing phenotype in zebrafish embryos compared to its S progenitor and this effect correlated with the production of abscesses in the central nervous system. Overall, these results suggest that the near-complete absence of GPL on the bacterial surface is a necessary condition for optimal pathogenesis of this mycobacterium. They also suggest that GPL content affects hydrophobicity of , potentially altering the aerosol transmission, which is of particular importance from an epidemiological and clinical perspective.
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http://dx.doi.org/10.3389/fcimb.2018.00069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5861769PMC
March 2019

Alkylated/aminated nitroimidazoles and nitroimidazole-7-chloroquinoline conjugates: Synthesis and anti-mycobacterial evaluation.

Bioorg Med Chem Lett 2018 05 8;28(8):1309-1312. Epub 2018 Mar 8.

Department of Chemistry, Guru Nanak Dev University, Amritsar 143005, India. Electronic address:

The success in exploring anti-tubercular potency of nitroimidazole and quinoline, the core moieties of recently approved anti-tubercular drugs instigated us to synthesize a series of alkylated/aminated 2-methyl-5-nitroimidazoles and nitroimidazole-7-chloroquinoline conjugates and to evaluate them for their activities against Mycobacterium tuberculosis as well as for their cytotoxicity towards the J774 murine macrophage cell line. Although the synthesized compounds did not surpass the activity of the standard drug Isoniazid, they have appreciable activities with minimal cytotoxicity. The synthesized nitroimidazole-7-chloroquinoline conjugate, 11c, having butyl chain as linker, proved to be the most potent among the series with an MIC value of 2.2 μg/mL.
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http://dx.doi.org/10.1016/j.bmcl.2018.03.021DOI Listing
May 2018

Identification of genes required for growth in vivo with a prominent role of the ESX-4 locus.

Proc Natl Acad Sci U S A 2018 01 17;115(5):E1002-E1011. Epub 2018 Jan 17.

Université de Versailles Saint Quentin en Yvelines, INSERM UMR1173, 78000 Versailles, France;

, a rapidly growing mycobacterium (RGM) and an opportunistic human pathogen, is responsible for a wide spectrum of clinical manifestations ranging from pulmonary to skin and soft tissue infections. This intracellular organism can resist the bactericidal defense mechanisms of amoebae and macrophages, an ability that has not been observed in other RGM. can up-regulate several virulence factors during transient infection of amoebae, thereby becoming more virulent in subsequent respiratory infections in mice. Here, we sought to identify the genes required for replication within amoebae. To this end, we constructed and screened a transposon () insertion library of an subspcies clinical isolate for attenuated clones. This approach identified five genes within the ESX-4 locus, which in encodes an ESX-4 type VII secretion system that exceptionally also includes the ESX conserved EccE component. To confirm the screening results and to get further insight into the contribution of ESX-4 to growth and survival in amoebae and macrophages, we generated a deletion mutant of that encodes a core structural element of ESX-4. This mutant was less efficient at blocking phagosomal acidification than its parental strain. Importantly, and in contrast to the wild-type strain, it also failed to damage phagosomes and showed reduced signs of phagosome-to-cytosol contact, as demonstrated by a combination of cellular and immunological assays. This study attributes an unexpected and genuine biological role to the underexplored mycobacterial ESX-4 system and its substrates.
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http://dx.doi.org/10.1073/pnas.1713195115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5798338PMC
January 2018

Cyclipostins and cyclophostin analogs inhibit the antigen 85C from both and .

J Biol Chem 2018 02 4;293(8):2755-2769. Epub 2018 Jan 4.

Institut de Recherche en Infectiologie de Montpellier (IRIM), Université de Montpellier, CNRS UMR9004, 34293 Montpellier, France; INSERM, IRIM, 34293 Montpellier, France. Electronic address:

An increasing prevalence of cases of drug-resistant tuberculosis requires the development of more efficacious chemotherapies. We previously reported the discovery of a new class of cyclipostins and cyclophostin (CyC) analogs exhibiting potent activity against both and in infected macrophages. Competitive labeling/enrichment assays combined with MS have identified several serine or cysteine enzymes in lipid and cell wall metabolism as putative targets of these CyC compounds. These targets included members of the antigen 85 (Ag85) complex ( Ag85A, Ag85B, and Ag85C), responsible for biosynthesis of trehalose dimycolate and mycolylation of arabinogalactan. Herein, we used biochemical and structural approaches to validate the Ag85 complex as a pharmacological target of the CyC analogs. We found that CyC, CyC, and CyC bind covalently to the catalytic Ser residue in Ag85C; inhibit mycolyltransferase activity ( the transfer of a fatty acid molecule onto trehalose); and reduce triacylglycerol synthase activity, a property previously attributed to Ag85A. Supporting these results, an X-ray structure of Ag85C in complex with CyC disclosed that this inhibitor occupies Ag85C's substrate-binding pocket. Importantly, metabolic labeling of cultures revealed that the CyC compounds impair both trehalose dimycolate synthesis and mycolylation of arabinogalactan. Overall, our study provides compelling evidence that CyC analogs can inhibit the activity of the Ag85 complex and in mycobacteria, opening the door to a new strategy for inhibiting Ag85. The high-resolution crystal structure obtained will further guide the rational optimization of new CyC scaffolds with greater specificity and potency against .
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http://dx.doi.org/10.1074/jbc.RA117.000760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827452PMC
February 2018

Controlling Extra- and Intramacrophagic by Targeting Mycolic Acid Transport.

Front Cell Infect Microbiol 2017 1;7:388. Epub 2017 Sep 1.

Centre National de la Recherche Scientifique UMR9004, Institut de Recherche en Infectiologie de Montpellier, Université de MontpellierMontpellier, France.

is a rapidly growing mycobacterium (RGM) causing serious infections especially among cystic fibrosis patients. Extremely limited therapeutic options against and a rise in infections with this mycobacterium require novel chemotherapies and a better understanding of how the bacterium causes infection. Different from most RGM, can survive inside macrophages and persist for long durations in infected tissues. We recently delineated differences in the infective programs followed by smooth (S) and rough (R) variants of . Unexpectedly, we found that the S variant behaves like pathogenic slow growing mycobacteria, through maintaining a block on the phagosome maturation process and by inducing phagosome-cytosol communications. On the other hand, R variant infection triggers autophagy and apoptosis, reminiscent of the way that macrophages control RGM. However, the R variant has an exquisite capacity to form extracellular cords, allowing these bacteria to rapidly divide and evade phagocytosis. Therefore, new chemotherapeutic interventions against need to efficiently deal with both the reservoir of intracellular bacilli and the extracellular cords. In this context, we recently identified two chemical entities that were very effective against both populations. Although being structurally unrelated these two chemotypes inhibit the activity of the essential mycolic acid transporter, MmpL3. In this Perspective, we aimed to highlight recent insights into how interacts with phagocytic cells and how the inhibition of mycolic acid transport in this pathogenic RGM could be an efficient means to control both intracellular and extracellular populations of the bacterium.
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http://dx.doi.org/10.3389/fcimb.2017.00388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5585149PMC
July 2018