The DnaK Chaperone System Buffers the Fitness Cost of Antibiotic Resistance Mutations in Mycobacteria

ABSTRACT Chaperones aid in protein folding and maintenance of protein integrity. In doing so, they have the unique ability to directly stabilize resistance-conferring amino acid substitutions in drug targets and to counter the stress imparted by these substitutions, thus supporting heritable antimicrobial resistance (AMR). We asked whether chaperones support AMR in Mycobacterium smegmatis, a saprophytic model of Mycobacterium tuberculosis, the causative agent of tuberculosis (TB). We show that DnaK associates with many drug targets and that DnaK associates more with AMR-conferring mutant RNA polymerase (RNAP) than with wild-type RNAP. In addition, frequency-of-resistance (FOR) and fitness studies reveal that the DnaK system of chaperones supports AMR in antimicrobial targets in mycobacteria, including RNAP and the ribosome. These findings highlight chaperones as potential targets for drugs to overcome AMR in mycobacteria, including M. tuberculosis, as well as in other pathogens. IMPORTANCE AMR is a global problem, especially for TB. Here, we show that mycobacterial chaperones support AMR in M. smegmatis, a nonpathogenic model of M. tuberculosis, the causative agent of TB. In particular, the mycobacterial DnaK system of chaperones supports AMR in the antimicrobial targets RNA polymerase and the ribosome. This is the first report showing a role for protein chaperones in mediating AMR in mycobacteria. Given the widespread role of protein chaperones in enabling genomic diversity, we anticipate that our findings can be extended to other microbes.

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Mutation ofRv2887, amarR-Like Gene, Confers Mycobacterium tuberculosis Resistance to an Imidazopyridine-Based Agent

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01341-15 ◽

2015 ◽

Vol 59 (11) ◽

pp. 6873-6881 ◽

Cited By ~ 13

Author(s):

Kathryn Winglee ◽

Shichun Lun ◽

Marco Pieroni ◽

Alan Kozikowski ◽

William Bishai

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Targets ◽

Efflux Pump ◽

Transcriptional Regulator ◽

Cross Resistance ◽

Loss Of Function ◽

Strong Activity ◽

Content Type ◽

Novel Drug

ABSTRACTDrug resistance is a major problem inMycobacterium tuberculosiscontrol, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity againstM. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independentM. tuberculosismutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations inRv2887were common to all three MP-III-71-resistant mutants, and we confirmed the role ofRv2887as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified inEscherichia colito negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation ofRv2887abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations ofRv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance ofM. tuberculosisRv2887mutants may involve efflux pump upregulation and also drug methylation.

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Deletion of the rpoZ gene, encoding the ω subunit of RNA polymerase, results in pleiotropic surface-related phenotypes in Mycobacterium smegmatis

Microbiology ◽

10.1099/mic.0.28879-0 ◽

2006 ◽

Vol 152 (6) ◽

pp. 1741-1750 ◽

Cited By ~ 32

Author(s):

Renjith Mathew ◽

Raju Mukherjee ◽

Radhakrishnan Balachandar ◽

Dipankar Chatterji

Keyword(s):

Rna Polymerase ◽

Mycobacterium Smegmatis ◽

Biofilm Formation ◽

Physiological Role ◽

Wild Type ◽

Gene Encoding ◽

Biofilm Growth ◽

Mutant Bacterium ◽

Sliding Motility

The ω subunit, the smallest subunit of bacterial RNA polymerase, is known to be involved in maintaining the conformation of the β′ subunit and aiding its recruitment to the rest of the core enzyme assembly in Escherichia coli. It has recently been shown in Mycobacterium smegmatis, by creating a deletion mutation of the rpoZ gene encoding ω, that the physiological role of the ω subunit also includes providing physical protection to β′. Interestingly, the mutant had altered colony morphology. This paper demonstrates that the mutant mycobacterium has pleiotropic phenotypes including reduced sliding motility and defective biofilm formation. Analysis of the spatial arrangement of biofilms by electron microscopy suggests that the altered phenotype of the mutant arises from a deficiency in generation of extracellular matrix. Complementation of the mutant strain with a copy of the wild-type rpoZ gene integrated in the bacterial chromosome restored both sliding motility and biofilm formation to the wild-type state, unequivocally proving the role of ω in the characteristics observed for the mutant bacterium. Analysis of the cell wall composition demonstrated that the mutant bacterium had an identical glycopeptidolipid profile to the wild-type, but failed to synthesize the short-chain mycolic acids characteristic of biofilm growth in M. smegmatis.

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Characterization of Trypanosoma cruzi Sirtuins as Possible Drug Targets for Chagas Disease

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.04694-14 ◽

2015 ◽

Vol 59 (8) ◽

pp. 4669-4679 ◽

Cited By ~ 23

Author(s):

Nilmar Silvio Moretti ◽

Leonardo da Silva Augusto ◽

Tatiana Mordente Clemente ◽

Raysa Paes Pinto Antunes ◽

Nobuko Yoshida ◽

...

Keyword(s):

Trypanosoma Cruzi ◽

Chagas Disease ◽

Drug Targets ◽

Wild Type ◽

Content Type ◽

Damage Repair ◽

Lysine Deacetylases

ABSTRACTAcetylation of lysine is a major posttranslational modification of proteins and is catalyzed by lysine acetyltransferases, while lysine deacetylases remove acetyl groups. Among the deacetylases, the sirtuins are NAD+-dependent enzymes, which modulate gene silencing, DNA damage repair, and several metabolic processes. As sirtuin-specific inhibitors have been proposed as drugs for inhibiting the proliferation of tumor cells, in this study, we investigated the role of these inhibitors in the growth and differentiation ofTrypanosoma cruzi, the agent of Chagas disease. We found that the use of salermide during parasite infection prevented growth and initial multiplication after mammalian cell invasion byT. cruziat concentrations that did not affect host cell viability. In addition,in vivoinfection was partially controlled upon administration of salermide. There are two sirtuins inT. cruzi, TcSir2rp1 and TcSir2rp3. By using specific antibodies and cell lines overexpressing the tagged versions of these enzymes, we found that TcSir2rp1 is localized in the cytosol and TcSir2rp3 in the mitochondrion. TcSir2rp1 overexpression acts to impair parasite growth and differentiation, whereas the wild-type version of TcSir2rp3 and not an enzyme mutated in the active site improves both. The effects observed with TcSir2rp3 were fully reverted by adding salermide, which inhibited TcSir2rp3 expressed inEscherichia coliwith a 50% inhibitory concentration (IC50) ± standard error of 1 ± 0.5 μM. We concluded that sirtuin inhibitors targeting TcSir2rp3 could be used in Chagas disease chemotherapy.

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Association of ω with the C-Terminal Region of the β′ Subunit Is Essential for Assembly of RNA Polymerase in Mycobacterium tuberculosis

Journal of Bacteriology ◽

10.1128/jb.00159-18 ◽

2018 ◽

Vol 200 (12) ◽

Author(s):

Chunyou Mao ◽

Yan Zhu ◽

Pei Lu ◽

Lipeng Feng ◽

Shiyun Chen ◽

...

Keyword(s):

Escherichia Coli ◽

Mycobacterium Tuberculosis ◽

Rna Polymerase ◽

Essential Role ◽

Β Subunit ◽

Content Type ◽

E Coli ◽

Core Enzyme ◽

Loop Region

ABSTRACT The ω subunit is the smallest subunit of bacterial RNA polymerase (RNAP). Although homologs of ω are essential in both eukaryotes and archaea, this subunit has been known to be dispensable for RNAP in Escherichia coli and in other bacteria. In this study, we characterized an indispensable role of the ω subunit in Mycobacterium tuberculosis . Unlike the well-studied E. coli RNAP, the M. tuberculosis RNAP core enzyme cannot be functionally assembled in the absence of the ω subunit. Importantly, substitution of M. tuberculosis ω with ω subunits from E. coli or Thermus thermophilus cannot restore the assembly of M. tuberculosis RNAP. Furthermore, by replacing different regions in M. tuberculosis ω with the corresponding regions from E. coli ω, we found a nonconserved loop region in M. tuberculosis ω essential for its function in RNAP assembly. From RNAP structures, we noticed that the location of the C-terminal region of the β′ subunit (β′CTD) in M. tuberculosis RNAP but not in E. coli or T. thermophilus RNAP is close to the ω loop region. Deletion of this β′CTD in M. tuberculosis RNAP destabilized the binding of M. tuberculosis ω on RNAP and compromised M. tuberculosis core assembly, suggesting that these two regions may function together to play a role in ω-dependent RNAP assembly in M. tuberculosis . Sequence alignment of the ω loop and the β′CTD regions suggests that the essential role of ω is probably restricted to mycobacteria. Together, our study characterized an essential role of M. tuberculosis ω and highlighted the importance of the ω loop region in M. tuberculosis RNAP assembly. IMPORTANCE DNA-dependent RNA polymerase (RNAP), which consists of a multisubunit core enzyme (α 2 ββ′ω) and a dissociable σ subunit, is the only enzyme in charge of transcription in bacteria. As the smallest subunit, the roles of ω remain the least well studied. In Escherichia coli and some other bacteria, the ω subunit is known to be nonessential for RNAP. In this study, we revealed an essential role of the ω subunit for RNAP assembly in the human pathogen Mycobacterium tuberculosis , and a mycobacterium-specific ω loop that plays a role in this function was also characterized. Our study provides fresh insights for further characterizing the roles of bacterial ω subunit.

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The Role of Replication Clamp-Loader Protein HolC of Escherichia coli in Overcoming Replication/Transcription Conflicts

mBio ◽

10.1128/mbio.00184-21 ◽

2021 ◽

Vol 12 (2) ◽

Author(s):

Deani L. Cooper ◽

Taku Harada ◽

Samia Tamazi ◽

Alexander E. Ferrazzoli ◽

Susan T. Lovett

Keyword(s):

Escherichia Coli ◽

Dna Replication ◽

Rna Polymerase ◽

Transcription Elongation ◽

Clamp Loader ◽

Content Type ◽

Growth Defects ◽

Transcriptional Termination ◽

Transcription Complexes

ABSTRACT In Escherichia coli, DNA replication is catalyzed by an assembly of proteins, the DNA polymerase III holoenzyme. This complex includes the polymerase and proofreading subunits, the processivity clamp, and clamp loader complex. The holC gene encodes an accessory protein (known as χ) to the core clamp loader complex and is the only protein of the holoenzyme that binds to single-strand DNA binding protein, SSB. HolC is not essential for viability, although mutants show growth impairment, genetic instability, and sensitivity to DNA damaging agents. In this study, we isolate spontaneous suppressor mutants in a ΔholC strain and identify these by whole-genome sequencing. Some suppressors are alleles of RNA polymerase, suggesting that transcription is problematic for holC mutant strains, or alleles of sspA, encoding stringent starvation protein. Using a conditional holC plasmid, we examine factors affecting transcription elongation and termination for synergistic or suppressive effects on holC mutant phenotypes. Alleles of RpoA (α), RpoB (β), and RpoC (β′) RNA polymerase holoenzyme can partially suppress loss of HolC. In contrast, mutations in transcription factors DksA and NusA enhanced the inviability of holC mutants. HolC mutants showed enhanced sensitivity to bicyclomycin, a specific inhibitor of Rho-dependent termination. Bicyclomycin also reverses suppression of holC by rpoA, rpoC, and sspA. An inversion of the highly expressed rrnA operon exacerbates the growth defects of holC mutants. We propose that transcription complexes block replication in holC mutants and that Rho-dependent transcriptional termination and DksA function are particularly important to sustain viability and chromosome integrity. IMPORTANCE Transcription elongation complexes present an impediment to DNA replication. We provide evidence that one component of the replication clamp loader complex, HolC, of Escherichia coli is required to overcome these blocks. This genetic study of transcription factor effects on holC growth defects implicates Rho-dependent transcriptional termination and DksA function as critical. It also implicates, for the first time, a role of SspA, stringent starvation protein, in avoidance or tolerance of replication/replication conflicts. We speculate that HolC helps avoid or resolve collisions between replication and transcription complexes, which become toxic in HolC’s absence.

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Potential Role of Endonuclease Inhibition and Other Targets in the Treatment of Influenza

Current Drug Targets ◽

10.2174/1389450120666190801115130 ◽

2020 ◽

Vol 21 (2) ◽

pp. 202-211

Author(s):

Doreen Szollosi ◽

Ashley Bill

Keyword(s):

Rna Polymerase ◽

Drug Targets ◽

Rna Virus ◽

Neuraminidase Inhibitor ◽

Viral Fusion ◽

Dependent Endonuclease ◽

Resistant Strains ◽

Polymerase Basic 1 ◽

Novel Drug

Background: Influenza is a single-stranded RNA virus that is highly contagious and infects millions of people in the U.S. annually. Due to complications, approximately 959,000 people were hospitalized and another 79,400 people died during the 2017-2018 flu season. While the best methods of prevention continue to be vaccination and hygiene, antiviral treatments may help reduce symptoms for those who are infected. Until recently, the only antiviral drugs in use have been the neuraminidase inhibitors: oseltamivir, zanamivir, and peramivir. Objective: We reviewed novel drug targets that can be used in the treatment of influenza, particularly in the case of neuraminidase inhibitor-resistant strains that may emerge. Results: More recently, a drug with a new mechanism of action has been approved. Baloxavir marboxil inhibits the influenza cap-dependent endonuclease that is needed for the virus to initiate replication within the host cell. This endonuclease target is within the polymerase acid (PA) subunit of RNA polymerase. Since the RNA-dependent RNA polymerase consists of two other subunits, polymerase basic 1 and 2, RNA polymerase has several targets that prevent viral replication. Other targets still under investigation include viral kinases, endocytosis, and viral fusion. Conclusion: Due to the possibility of viral mutations and resistance, it is important to have antivirals with different mechanisms available, especially in the case of a new pandemic strain. Several novel antivirals are within various stages of development and may represent new classes of treatments that can reduce symptoms and complications in those patients who may be at higher risk.

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Ergothioneine Is a Secreted Antioxidant in Mycobacterium smegmatis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02572-12 ◽

2013 ◽

Vol 57 (7) ◽

pp. 3202-3207 ◽

Cited By ~ 47

Author(s):

Carine Sao Emani ◽

Monique J. Williams ◽

Ian J. Wiid ◽

Nicholas F. Hiten ◽

Albertus J. Viljoen ◽

...

Keyword(s):

Oxidative Stress ◽

Mycobacterium Smegmatis ◽

Double Mutant ◽

Culture Media ◽

Growth Cycle ◽

Low Molecular Weight ◽

Deficient Mutant ◽

Wild Type ◽

Content Type

ABSTRACTErgothioneine (ERG) and mycothiol (MSH) are two low-molecular-weight thiols synthesized by mycobacteria. The role of MSH has been extensively investigated in mycobacteria; however, little is known about the role of ERG in mycobacterial physiology. In this study, quantification of ERG at various points in the growth cycle ofMycobacterium smegmatisrevealed that a significant portion of ERG is found in the culture media, suggesting that it is actively secreted. A mutant ofM. smegmatislackingegtD(MSMEG_6247) was unable to synthesize ERG, confirming its role in ERG biosynthesis. Deletion ofegtDfrom wild-typeM. smegmatisand an MSH-deficient mutant did not affect their susceptibility to antibiotics tested in this study. The ERG- and MSH-deficient double mutant was significantly more sensitive to peroxide than either of the single mutants lacking either ERG or MSH, suggesting that both thiols play a role in protectingM. smegmatisagainst oxidative stress and that ERG is able to partly compensate for the loss of MSH.

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Mutations in theembC-embAIntergenic Region Contribute to Mycobacterium tuberculosis Resistance to Ethambutol

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03285-14 ◽

2014 ◽

Vol 58 (11) ◽

pp. 6837-6843 ◽

Cited By ~ 23

Author(s):

Zhenling Cui ◽

Yuanyuan Li ◽

Song Cheng ◽

Hua Yang ◽

Junmei Lu ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Mycobacterium Smegmatis ◽

Transcriptional Activity ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Mobility Shift ◽

Content Type ◽

Clinical Strains ◽

Gel Mobility Shift

ABSTRACTThe rapid increase inMycobacterium tuberculosisresistance to ethambutol (EMB) threatens the diagnosis and treatment of tuberculosis (TB). We investigated the role of mutations in theembC-embAintergenic region (IGR) in EMB-resistant clinical strains from east China. A total of 767M. tuberculosisclinical strains were collected and analyzed for their drug susceptibility to EMB using the MGIT 960 system and MIC assay, and theembC-embAIGRs of these strains were sequenced. The transcriptional activity of theembC-embAIGR mutations was examined by reporter gene assays in recombinantMycobacterium smegmatisstrains, and the effect of IGR mutations on its binding to EmbR, a transcription regulator ofembAB, was analyzed by gel mobility shift assays. Correlation coefficient analysis showed that theembC-embAIGR mutation is associated with EMB resistance. The clinical strains carrying IGR mutations had a much higher level ofembAandembBmRNA as well as higher MICs to EMB. IGR mutations had higher transcriptional activity when transformed intoM. smegmatisstrains. Mutated IGRs bound to EmbR with much higher affinity than wild-type fragments. The sensitivity of molecular drug susceptibility testing (DST) with IGR mutations as an additional marker increased from 65.5% to 73.5%. Mutations of theembC-embAIGR enhance the binding of EmbR to the promoter region ofembABand increase the expression ofembAB, thus contributing to EMB resistance. Therefore, identification of IGR mutations as markers of EMB resistance could increase the sensitivity of molecular DST.

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Phenotypic Suppression of Streptomycin Resistance by Mutations in Multiple Components of the Translation Apparatus

Journal of Bacteriology ◽

10.1128/jb.00219-15 ◽

2015 ◽

Vol 197 (18) ◽

pp. 2981-2988 ◽

Cited By ~ 3

Author(s):

Jennifer F. Carr ◽

Hannah J. Lee ◽

Joshua B. Jaspers ◽

Albert E. Dahlberg ◽

Gerwald Jogl ◽

...

Keyword(s):

Antibiotic Resistance ◽

Long Range ◽

Experimental Evolution ◽

Thermus Thermophilus ◽

Thermophilic Bacterium ◽

Streptomycin Resistance ◽

Resistance Mutations ◽

Content Type ◽

Translation Apparatus ◽

Antibiotic Resistance Mutations

ABSTRACTThe bacterial ribosome and its associated translation factors are frequent targets of antibiotics, and antibiotic resistance mutations have been found in a number of these components. Such mutations can potentially interact with one another in unpredictable ways, including the phenotypic suppression of one mutation by another. These phenotypic interactions can provide evidence of long-range functional interactions throughout the ribosome and its functional complexes and potentially give insights into antibiotic resistance mechanisms. In this study, we used genetics and experimental evolution of the thermophilic bacteriumThermus thermophilusto examine the ability of mutations in various components of the protein synthesis apparatus to suppress the streptomycin resistance phenotypes of mutations in ribosomal protein S12, specifically those located distant from the streptomycin binding site. With genetic selections and strain constructions, we identified suppressor mutations in EF-Tu or in ribosomal protein L11. Using experimental evolution, we identified amino acid substitutions in EF-Tu or in ribosomal proteins S4, S5, L14, or L19, some of which were found to also relieve streptomycin resistance. The wide dispersal of these mutations is consistent with long-range functional interactions among components of the translational machinery and indicates that streptomycin resistance can result from the modulation of long-range conformational signals.IMPORTANCEThe thermophilic bacteriumThermus thermophilushas become a model system for high-resolution structural studies of macromolecular complexes, such as the ribosome, while its natural competence for transformation facilitates genetic approaches. Genetic studies ofT. thermophilusribosomes can take advantage of existing high-resolution crystallographic information to allow a structural interpretation of phenotypic interactions among mutations. Using a combination of genetic selections, strain constructions, and experimental evolution, we find that certain mutations in the translation apparatus can suppress the phenotype of certain antibiotic resistance mutations. Suppression of resistance can occur by mutations located distant in the ribosome or in a translation factor. These observations suggest the existence of long-range conformational signals in the translating ribosome, particularly during the decoding of mRNA.

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TgATAT-Mediated α-Tubulin Acetylation Is Required for Division of the Protozoan Parasite Toxoplasma gondii

mSphere ◽

10.1128/msphere.00088-15 ◽

2016 ◽

Vol 1 (1) ◽

Cited By ~ 4

Author(s):

Joseph M. Varberg ◽

Leah R. Padgett ◽

Gustavo Arrizabalaga ◽

William J. Sullivan

Keyword(s):

Toxoplasma Gondii ◽

Drug Target ◽

Drug Targets ◽

Protozoan Parasite ◽

World Population ◽

Content Type ◽

Tubulin Acetylation ◽

Parasite Replication ◽

New Treatments

ABSTRACT Toxoplasma gondii is an opportunistic parasite that infects at least one-third of the world population. New treatments for the disease (toxoplasmosis) are needed since current drugs are toxic to patients. Microtubules are essential cellular structures built from tubulin that show promise as antimicrobial drug targets. Microtubules can be regulated by chemical modification, such as acetylation on lysine 40 (K40). To determine the role of K40 acetylation in Toxoplasma and whether it is a liability to the parasite, we performed mutational analyses of the α-tubulin gene. Our results indicate that parasites cannot survive without K40 acetylation unless microtubules are stabilized with a secondary mutation. Additionally, we identified the parasite enzyme that acetylates α-tubulin (TgATAT). Genetic disruption of TgATAT caused severe defects in parasite replication, further highlighting the importance of α-tubulin K40 acetylation in Toxoplasma and its promise as a potential new drug target. Toxoplasma gondii is a widespread protozoan parasite that causes potentially life-threatening opportunistic disease. New inhibitors of parasite replication are urgently needed, as the current antifolate treatment is also toxic to patients. Microtubules are essential cytoskeletal components that have been selectively targeted in microbial pathogens; further study of tubulin in Toxoplasma may reveal novel therapeutic opportunities. It has been noted that α-tubulin acetylation at lysine 40 (K40) is enriched during daughter parasite formation, but the impact of this modification on Toxoplasma division and the enzyme mediating its delivery have not been identified. We performed mutational analyses to provide evidence that K40 acetylation stabilizes Toxoplasma microtubules and is required for parasite replication. We also show that an unusual Toxoplasma homologue of α-tubulin acetyltransferase (TgATAT) is expressed in a cell cycle-regulated manner and that its expression peaks during division. Disruption of TgATAT with CRISPR/Cas9 ablates K40 acetylation and induces replication defects; parasites appear to initiate mitosis yet exhibit incomplete or improper nuclear division. Together, these findings establish the importance of tubulin acetylation, exposing a new vulnerability in Toxoplasma that could be pharmacologically targeted. IMPORTANCE Toxoplasma gondii is an opportunistic parasite that infects at least one-third of the world population. New treatments for the disease (toxoplasmosis) are needed since current drugs are toxic to patients. Microtubules are essential cellular structures built from tubulin that show promise as antimicrobial drug targets. Microtubules can be regulated by chemical modification, such as acetylation on lysine 40 (K40). To determine the role of K40 acetylation in Toxoplasma and whether it is a liability to the parasite, we performed mutational analyses of the α-tubulin gene. Our results indicate that parasites cannot survive without K40 acetylation unless microtubules are stabilized with a secondary mutation. Additionally, we identified the parasite enzyme that acetylates α-tubulin (TgATAT). Genetic disruption of TgATAT caused severe defects in parasite replication, further highlighting the importance of α-tubulin K40 acetylation in Toxoplasma and its promise as a potential new drug target.

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