The Antituberculosis Drug Ethambutol Selectively Blocks Apical Growth in CMN Group Bacteria

ABSTRACT Members of the genus Mycobacterium are the most prevalent cause of infectious diseases. Mycobacteria have a complex cell envelope containing a peptidoglycan layer and an additional arabinogalactan polymer to which a mycolic acid bilayer is linked; this complex, multilayered cell wall composition (mAGP) is conserved among all CMN group bacteria. The arabinogalactan and mycolic acid synthesis pathways constitute effective drug targets for tuberculosis treatment. Ethambutol (EMB), a classical antituberculosis drug, inhibits the synthesis of the arabinose polymer. Although EMB acts bacteriostatically, its underlying molecular mechanism remains unclear. Here, we used Corynebacterium glutamicum and Mycobacterium phlei as model organisms to study the effects of EMB at the single-cell level. Our results demonstrate that EMB specifically blocks apical cell wall synthesis, but not cell division, explaining the bacteriostatic effect of EMB. Furthermore, the data suggest that members of the family Corynebacterineae have two dedicated machineries for cell elongation (elongasome) and cytokinesis (divisome). IMPORTANCE Antibiotic treatment of bacterial pathogens has contributed enormously to the increase in human health. Despite the apparent importance of antibiotic treatment of bacterial infections, surprisingly little is known about the molecular functions of antibiotic actions in the bacterial cell. Here, we analyzed the molecular effects of ethambutol, a first-line antibiotic against infections caused by members of the genus Mycobacterium. We find that this drug selectively blocks apical cell growth but still allows for effective cytokinesis. As a consequence, cells survive ethambutol treatment and adopt a pneumococcal cell growth mode with cell wall synthesis only at the site of cell division. However, combined treatment of ethambutol and beta-lactam antibiotics acts synergistically and effectively stops cell proliferation. IMPORTANCE Antibiotic treatment of bacterial pathogens has contributed enormously to the increase in human health. Despite the apparent importance of antibiotic treatment of bacterial infections, surprisingly little is known about the molecular functions of antibiotic actions in the bacterial cell. Here, we analyzed the molecular effects of ethambutol, a first-line antibiotic against infections caused by members of the genus Mycobacterium. We find that this drug selectively blocks apical cell growth but still allows for effective cytokinesis. As a consequence, cells survive ethambutol treatment and adopt a pneumococcal cell growth mode with cell wall synthesis only at the site of cell division. However, combined treatment of ethambutol and beta-lactam antibiotics acts synergistically and effectively stops cell proliferation.

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A Staphylococcus aureus clpX Mutant Used as a Unique Screening Tool to Identify Cell Wall Synthesis Inhibitors that Reverse β-Lactam Resistance in MRSA

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.691569 ◽

2021 ◽

Vol 8 ◽

Author(s):

Kristoffer T. Bæk ◽

Camilla Jensen ◽

Maya A. Farha ◽

Tobias K. Nielsen ◽

Ervin Paknejadi ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

High Throughput Screening ◽

Bacterial Infections ◽

Screening Tool ◽

Acquired Resistance ◽

Teichoic Acid ◽

Acid Synthesis ◽

Biologically Active ◽

Cell Wall Synthesis

Staphylococcus aureus is a leading cause of bacterial infections world-wide. Staphylococcal infections are preferentially treated with β-lactam antibiotics, however, methicillin-resistant S. aureus (MRSA) strains have acquired resistance to this superior class of antibiotics. We have developed a growth-based, high-throughput screening approach that directly identifies cell wall synthesis inhibitors capable of reversing β-lactam resistance in MRSA. The screen is based on the finding that S. aureus mutants lacking the ClpX chaperone grow very poorly at 30°C unless specific steps in teichoic acid synthesis or penicillin binding protein (PBP) activity are inhibited. This property allowed us to exploit the S. aureus clpX mutant as a unique screening tool to rapidly identify biologically active compounds that target cell wall synthesis. We tested a library of ∼50,000 small chemical compounds and searched for compounds that inhibited growth of the wild type while stimulating growth of the clpX mutant. Fifty-eight compounds met these screening criteria, and preliminary tests of 10 compounds identified seven compounds that reverse β-lactam resistance of MRSA as expected for inhibitors of teichoic acid synthesis. The hit compounds are therefore promising candidates for further development as novel combination agents to restore β-lactam efficacy against MRSA.

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Cell Wall, Cell Division, and Cell Growth

Plant Growth and Development ◽

10.1016/b978-012660570-9/50142-8 ◽

2002 ◽

pp. 23-74 ◽

Cited By ~ 1

Author(s):

Lalit M. Srivastava

Keyword(s):

Cell Wall ◽

Cell Division ◽

Cell Growth ◽

Wall Cell

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Bacteriophage SP01 Gene Product 56 Inhibits Bacillus subtilis Cell Division by Interacting with FtsL and Disrupting Pbp2B and FtsW Recruitment

Journal of Bacteriology ◽

10.1128/jb.00463-20 ◽

2020 ◽

Vol 203 (2) ◽

pp. e00463-20

Author(s):

Amit Bhambhani ◽

Isabella Iadicicco ◽

Jules Lee ◽

Syed Ahmed ◽

Max Belfatto ◽

...

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Cell Division ◽

Gene Product ◽

Fluorescent Protein ◽

Lytic Bacteriophage ◽

Cell Wall Synthesis ◽

Content Type ◽

Ftsz Ring ◽

Green Fluorescent

ABSTRACTPrevious work identified gene product 56 (gp56), encoded by the lytic bacteriophage SP01, as being responsible for inhibition of Bacillus subtilis cell division during its infection. Assembly of the essential tubulin-like protein FtsZ into a ring-shaped structure at the nascent site of cytokinesis determines the timing and position of division in most bacteria. This FtsZ ring serves as a scaffold for recruitment of other proteins into a mature division-competent structure permitting membrane constriction and septal cell wall synthesis. Here, we show that expression of the predicted 9.3-kDa gp56 of SP01 inhibits later stages of B. subtilis cell division without altering FtsZ ring assembly. Green fluorescent protein-tagged gp56 localizes to the membrane at the site of division. While its localization does not interfere with recruitment of early division proteins, gp56 interferes with the recruitment of late division proteins, including Pbp2b and FtsW. Imaging of cells with specific division components deleted or depleted and two-hybrid analyses suggest that gp56 localization and activity depend on its interaction with FtsL. Together, these data support a model in which gp56 interacts with a central part of the division machinery to disrupt late recruitment of the division proteins involved in septal cell wall synthesis.IMPORTANCE Studies over the past decades have identified bacteriophage-encoded factors that interfere with host cell shape or cytokinesis during viral infection. The phage factors causing cell filamentation that have been investigated to date all act by targeting FtsZ, the conserved prokaryotic tubulin homolog that composes the cytokinetic ring in most bacteria and some groups of archaea. However, the mechanisms of several phage factors that inhibit cytokinesis, including gp56 of bacteriophage SP01 of Bacillus subtilis, remain unexplored. Here, we show that, unlike other published examples of phage inhibition of cytokinesis, gp56 blocks B. subtilis cell division without targeting FtsZ. Rather, it utilizes the assembled FtsZ cytokinetic ring to localize to the division machinery and to block recruitment of proteins needed for septal cell wall synthesis.

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A High Throughput Screen for Inhibitors of Fungal Cell Wall Synthesis

CrossRef Listing of Deleted DOIs ◽

10.1177/108705710200700408 ◽

2002 ◽

Vol 7 (4) ◽

pp. 359-366 ◽

Cited By ~ 8

Author(s):

Jonathan M. Evans ◽

Phillip G. Zaworski ◽

Christian N. Parker

Keyword(s):

Cell Wall ◽

Cell Growth ◽

High Throughput ◽

High Throughput Screening ◽

Osmotic Shock ◽

Fungal Cell Wall ◽

Cell Wall Synthesis ◽

Fungal Cell ◽

Unique Nature ◽

Pharmacologically Active

Fungal cell wall synthesis is essential for viability, requiring the activity of genes involved in environmental sensing, precursor synthesis, transport, secretion, and assembly. This multitude of potential targets, the availability of known agents targeting this pathway, and the unique nature of fungal cell wall synthesis make this pathway an appealing target for drug discovery. Here we describe the adaptation of an assay monitoring cell wall synthesis for high-throughput screening. The assay requires fungal cell growth, in the presence of the test compound, for 3 h before the cells are subjected to osmotic shock in the presence of a dye that stains DNA. Miniaturization of the assay to a 384-well plate format and removing a mechanical transfer led to subtle changes in the assay characteristics. Validation of the assay with a library of known pharmacologically active agents has identified a number of different classes of compounds that are active in this assay, causing aberrant cell wall morphology and in many cases the inhibition of fungal cell growth.

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Beta-Lactam Antibiotics Induce a Lethal Malfunctioning of the Bacterial Cell Wall Synthesis Machinery

Cell ◽

10.1016/j.cell.2014.11.017 ◽

2014 ◽

Vol 159 (6) ◽

pp. 1300-1311 ◽

Cited By ~ 246

Author(s):

Hongbaek Cho ◽

Tsuyoshi Uehara ◽

Thomas G. Bernhardt

Keyword(s):

Cell Wall ◽

Bacterial Cell ◽

Bacterial Cell Wall ◽

Cell Wall Synthesis ◽

Beta Lactam ◽

Beta Lactam Antibiotics

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Bactericidal action of peptide antibiotic AS-48 against Escherichia coli K-12

Canadian Journal of Microbiology ◽

10.1139/m89-048 ◽

1989 ◽

Vol 35 (2) ◽

pp. 318-321 ◽

Cited By ~ 20

Author(s):

A. Gálvez ◽

E. Valdivia ◽

M. Martínez ◽

M. Maqueda

Keyword(s):

Escherichia Coli ◽

Cell Wall ◽

Cell Growth ◽

Mode Of Action ◽

Cytoplasmic Membrane ◽

Peptide Antibiotic ◽

Biosynthetic Pathways ◽

Bactericidal Action ◽

Cell Wall Synthesis ◽

K 12

Peptide antibiotic AS-48 exerts a bactericidal mode of action on exponential cultures of Escherichia coli K-12 through a multi-hit kinetics interaction. AS-48 causes a parallel and gradual cessation of all biosynthetic pathways monitored (protein, RNA, DNA, and cell wall synthesis), the rate of incorporation of labeled precursors, the rate of O2 consumption, and cell growth. These effects have been attributed to alterations of cytoplasmic membrane functions.Key words: Escherichia coli, peptide antibiotic, bactericide.

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A cell wall damage response mediated by a sensor kinase/response regulator pair enables beta-lactam tolerance

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1520333113 ◽

2015 ◽

Vol 113 (2) ◽

pp. 404-409 ◽

Cited By ~ 34

Author(s):

Tobias Dörr ◽

Laura Alvarez ◽

Fernanda Delgado ◽

Brigid M. Davis ◽

Felipe Cava ◽

...

Keyword(s):

Cell Wall ◽

Response Regulator ◽

Cell Envelope ◽

Normal Growth ◽

Cell Diameter ◽

Cell Wall Synthesis ◽

Beta Lactam ◽

Cell Wall Damage ◽

A Cell

The bacterial cell wall is critical for maintenance of cell shape and survival. Following exposure to antibiotics that target enzymes required for cell wall synthesis, bacteria typically lyse. Although several cell envelope stress response systems have been well described, there is little knowledge of systems that modulate cell wall synthesis in response to cell wall damage, particularly in Gram-negative bacteria. Here we describe WigK/WigR, a histidine kinase/response regulator pair that enablesVibrio cholerae, the cholera pathogen, to survive exposure to antibiotics targeting cell wall synthesis in vitro and during infection. Unlike wild-typeV. cholerae, mutants lackingwigRfail to recover following exposure to cell-wall–acting antibiotics, and they exhibit a drastically increased cell diameter in the absence of such antibiotics. Conversely, overexpression ofwigRleads to cell slimming. Overexpression of activated WigR also results in increased expression of the full set of cell wall synthesis genes and to elevated cell wall content. WigKR-dependent expression of cell wall synthesis genes is induced by various cell-wall–acting antibiotics as well as by overexpression of an endogenous cell wall hydrolase. Thus, WigKR appears to monitor cell wall integrity and to enhance the capacity for increased cell wall production in response to damage. Taken together, these findings implicate WigKR as a regulator of cell wall synthesis that controls cell wall homeostasis in response to antibiotics and likely during normal growth as well.

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Tol-Pal System and Rgs Proteins Interact to Promote Unipolar Growth and Cell Division in Sinorhizobium meliloti

mBio ◽

10.1128/mbio.00306-20 ◽

2020 ◽

Vol 11 (3) ◽

Cited By ~ 1

Author(s):

Elizaveta Krol ◽

Hamish C. L. Yau ◽

Marcus Lechner ◽

Simon Schäper ◽

Gert Bange ◽

...

Keyword(s):

Cell Wall ◽

Cell Division ◽

Cell Growth ◽

Cell Elongation ◽

Sinorhizobium Meliloti ◽

Rgs Proteins ◽

Polar Growth ◽

Content Type ◽

Cell Pole ◽

Growing Cell

ABSTRACT Sinorhizobium meliloti is an alphaproteobacterium belonging to the Rhizobiales. Bacteria from this order elongate their cell wall at the new cell pole, generated by cell division. Screening for protein interaction partners of the previously characterized polar growth factors RgsP and RgsM, we identified the inner membrane components of the Tol-Pal system (TolQ and TolR) and novel Rgs (rhizobial growth and septation) proteins with unknown functions. TolQ, Pal, and all Rgs proteins, except for RgsE, were indispensable for S. meliloti cell growth. Six of the Rgs proteins, TolQ, and Pal localized to the growing cell pole in the cell elongation phase and to the septum in predivisional cells, and three Rgs proteins localized to the growing cell pole only. The putative FtsN-like protein RgsS contains a conserved SPOR domain and is indispensable at the early stages of cell division. The components of the Tol-Pal system were required at the late stages of cell division. RgsE, a homolog of the Agrobacterium tumefaciens growth pole ring protein GPR, has an important role in maintaining the normal growth rate and rod cell shape. RgsD is a periplasmic protein with the ability to bind peptidoglycan. Analysis of the phylogenetic distribution of the Rgs proteins showed that they are conserved in Rhizobiales and mostly absent from other alphaproteobacterial orders, suggesting a conserved role of these proteins in polar growth. IMPORTANCE Bacterial cell proliferation involves cell growth and septum formation followed by cell division. For cell growth, bacteria have evolved different complex mechanisms. The most prevalent growth mode of rod-shaped bacteria is cell elongation by incorporating new peptidoglycans in a dispersed manner along the sidewall. A small share of rod-shaped bacteria, including the alphaproteobacterial Rhizobiales, grow unipolarly. Here, we identified and initially characterized a set of Rgs (rhizobial growth and septation) proteins, which are involved in cell division and unipolar growth of Sinorhizobium meliloti and highly conserved in Rhizobiales. Our data expand the knowledge of components of the polarly localized machinery driving cell wall growth and suggest a complex of Rgs proteins with components of the divisome, differing in composition between the polar cell elongation zone and the septum.

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