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<front>
<journal-meta>
<journal-id journal-id-type="publisher-id">Front. Cell. Infect. Microbiol.</journal-id>
<journal-title>Frontiers in Cellular and Infection Microbiology</journal-title>
<abbrev-journal-title abbrev-type="pubmed">Front. Cell. Infect. Microbiol.</abbrev-journal-title>
<issn pub-type="epub">2235-2988</issn>
<publisher>
<publisher-name>Frontiers Media S.A.</publisher-name>
</publisher>
</journal-meta>
<article-meta>
<article-id pub-id-type="doi">10.3389/fcimb.2022.997283</article-id>
<article-categories>
<subj-group subj-group-type="heading">
<subject>Cellular and Infection Microbiology</subject>
<subj-group>
<subject>Review</subject>
</subj-group>
</subj-group>
</article-categories>
<title-group>
<article-title>Unraveling the mechanisms of intrinsic drug resistance in <italic>Mycobacterium tuberculosis</italic>
</article-title>
</title-group>
<contrib-group>
<contrib contrib-type="author">
<name>
<surname>Poulton</surname>
<given-names>Nicholas C.</given-names>
</name>
<uri xlink:href="https://loop.frontiersin.org/people/1759540"/>
</contrib>
<contrib contrib-type="author" corresp="yes">
<name>
<surname>Rock</surname>
<given-names>Jeremy M.</given-names>
</name>
<xref ref-type="author-notes" rid="fn001">
<sup>*</sup>
</xref>
</contrib>
</contrib-group>
<aff id="aff1">
<institution>Laboratory of Host-Pathogen Biology, The Rockefeller University</institution>, <addr-line>New York, NY</addr-line>, <country>United States</country>
</aff>
<author-notes>
<fn fn-type="edited-by">
<p>Edited by: Ben Gold, Weill Cornell Medicine, United States</p>
</fn>
<fn fn-type="edited-by">
<p>Reviewed by: Thomas Dick, Hackensack Meridian Health, United States; Sangeeta Tiwari, The University of Texas at El Paso, United States; Christopher Ealand, University of the Witwatersrand, South Africa</p>
</fn>
<fn fn-type="corresp" id="fn001">
<p>*Correspondence: Jeremy M. Rock, <email xlink:href="mailto:rock@rockefeller.edu">rock@rockefeller.edu</email>
</p>
</fn>
<fn fn-type="other" id="fn002">
<p>This article was submitted to Molecular Bacterial Pathogenesis, a section of the journal Frontiers in Cellular and Infection Microbiology</p>
</fn>
</author-notes>
<pub-date pub-type="epub">
<day>17</day>
<month>10</month>
<year>2022</year>
</pub-date>
<pub-date pub-type="collection">
<year>2022</year>
</pub-date>
<volume>12</volume>
<elocation-id>997283</elocation-id>
<history>
<date date-type="received">
<day>18</day>
<month>07</month>
<year>2022</year>
</date>
<date date-type="accepted">
<day>30</day>
<month>09</month>
<year>2022</year>
</date>
</history>
<permissions>
<copyright-statement>Copyright &#xa9; 2022 Poulton and Rock</copyright-statement>
<copyright-year>2022</copyright-year>
<copyright-holder>Poulton and Rock</copyright-holder>
<license xlink:href="http://creativecommons.org/licenses/by/4.0/">
<p>This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.</p>
</license>
</permissions>
<abstract>
<p>Tuberculosis (TB) is among the most difficult infections to treat, requiring several months of multidrug therapy to produce a durable cure. The reasons necessitating long treatment times are complex and multifactorial. However, one major difficulty of treating TB is the resistance of the infecting bacterium, <italic>Mycobacterium tuberculosis</italic> (Mtb), to many distinct classes of antimicrobials. This review will focus on the major gaps in our understanding of intrinsic drug resistance in Mtb and how functional and chemical-genetics can help close those gaps. A better understanding of intrinsic drug resistance will help lay the foundation for strategies to disarm and circumvent these mechanisms to develop more potent antitubercular therapies.</p>
</abstract>
<kwd-group>
<kwd>tuberculosis</kwd>
<kwd>intrinsic resistance</kwd>
<kwd>chemical genetics</kwd>
<kwd>drug repurposing</kwd>
<kwd>drug discovery</kwd>
</kwd-group>
<contract-num rid="cn001">1DP2AI14485001</contract-num>
<contract-sponsor id="cn001">National Institutes of Health<named-content content-type="fundref-id">10.13039/100000002</named-content>
</contract-sponsor>
<counts>
<fig-count count="2"/>
<table-count count="1"/>
<equation-count count="0"/>
<ref-count count="173"/>
<page-count count="16"/>
<word-count count="8218"/>
</counts>
</article-meta>
</front>
<body>
<sec id="s1" sec-type="intro">
<title>Introduction</title>
<p>
<italic>Mycobacterium tuberculosis</italic> (Mtb) infection is notoriously difficult to treat. Standard treatment regimens for drug sensitive tuberculosis (TB) typically last for 6 months and involve combination therapy with 2-4 antibiotics, depending on the stage of treatment (<xref ref-type="bibr" rid="B47">Dorman et&#xa0;al., 2021</xref>; <xref ref-type="bibr" rid="B161">WHO, 2021</xref>). Even with 6 months of chemotherapy, 5-10% of patients may experience disease relapse (<xref ref-type="bibr" rid="B81">Lambert et&#xa0;al., 2003</xref>; <xref ref-type="bibr" rid="B104">Merle et&#xa0;al., 2014</xref>; <xref ref-type="bibr" rid="B34">Colangeli et&#xa0;al., 2018</xref>). The difficulty of treating TB can be attributed to a multitude of factors including variable drug penetration into infected lesions (<xref ref-type="bibr" rid="B39">Dartois, 2014</xref>; <xref ref-type="bibr" rid="B88">Lenaerts et&#xa0;al., 2015</xref>) and treatment lapses due to toxic drug side effects (<xref ref-type="bibr" rid="B150">Tostmann et&#xa0;al., 2008</xref>; <xref ref-type="bibr" rid="B136">Seddon et&#xa0;al., 2012</xref>; <xref ref-type="bibr" rid="B138">Si et&#xa0;al., 2018</xref>; <xref ref-type="bibr" rid="B35">Conradie et&#xa0;al., 2020</xref>). However, a major contributor to the difficulty of treating TB is the problem of bacterial drug resistance, which can broadly be classified into two main categories: intrinsic drug resistance and acquired drug resistance (<xref ref-type="bibr" rid="B155">Walker et&#xa0;al., 2015</xref>; <xref ref-type="bibr" rid="B166">Xu et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B13">Batt et&#xa0;al., 2020</xref>). Bacterial drug resistance is phenotypically distinct from drug tolerance and persistence (<xref ref-type="bibr" rid="B21">Brauner et&#xa0;al., 2016</xref>; <xref ref-type="bibr" rid="B10">Balaban et&#xa0;al., 2019</xref>), which will not be reviewed here.</p>
<p>Intrinsic resistance refers to an innate property of a bacterial species that renders an antibacterial, or group of antibacterials, less effective (<xref ref-type="bibr" rid="B15">Blair et&#xa0;al., 2015</xref>; <xref ref-type="bibr" rid="B117">Peterson and Kaur, 2018</xref>). Importantly, intrinsic resistance mechanisms are usually present in all (or almost all) members of a bacterial species. In some cases, genes imparting intrinsic resistance appear to have evolved specifically for protection against antibacterial compounds (<xref ref-type="bibr" rid="B98">Madsen et&#xa0;al., 2005</xref>; <xref ref-type="bibr" rid="B92">Liu et&#xa0;al., 2019</xref>). For example, Mtb encodes <italic>erm(37)</italic>, a 23S rRNA methyltransferase that protects the ribosome from macrolide, lincosamide, and streptogramin binding (<xref ref-type="bibr" rid="B98">Madsen et&#xa0;al., 2005</xref>). <italic>erm(37)</italic> does not have a known role in bacterial growth, virulence, or stress tolerance and likely evolved to protect ancestral, soil-dwelling actinobacteria against ribosome-targeting natural products produced by themselves or their neighbors (<xref ref-type="bibr" rid="B106">Morris et&#xa0;al., 2005</xref>). In other cases, genes essential for microbial growth and virulence can contribute to intrinsic drug resistance (<xref ref-type="bibr" rid="B145">Tan et&#xa0;al., 2012</xref>; <xref ref-type="bibr" rid="B13">Batt et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B51">Dulberger et&#xa0;al., 2020</xref>). For example, many essential genes in Mtb are involved in cell envelope biosynthesis and regulation. The Mtb cell envelope protects Mtb from host immune pressure and serves as a selective barrier to antibiotic penetration (<xref ref-type="bibr" rid="B70">Johnson et&#xa0;al., 2019</xref>; <xref ref-type="bibr" rid="B116">Peterson et&#xa0;al., 2021</xref>).</p>
<p>Acquired drug resistance refers to antibiotic resistance that evolves through specific chromosomal mutations or horizontal gene transfer (<xref ref-type="bibr" rid="B117">Peterson and Kaur, 2018</xref>; <xref ref-type="bibr" rid="B52">Evans et&#xa0;al., 2020</xref>). In Mtb all acquired drug resistance arises as a result of mutation since there is no evidence for recent horizontal gene transfer in Mtb (<xref ref-type="bibr" rid="B16">Boritsch et&#xa0;al., 2016</xref>). Many of the mutations that confer high-level acquired drug resistance in Mtb have been well studied and characterized (<xref ref-type="bibr" rid="B155">Walker et&#xa0;al., 2015</xref>; <xref ref-type="bibr" rid="B36">CRyPTIC Consortium, 2018</xref>; <xref ref-type="bibr" rid="B65">Hunt et&#xa0;al., 2019</xref>). For example, partial loss-of-function mutations in the isoniazid (INH)-activating enzyme <italic>katG</italic> are the primary mechanism by which INH resistance emerges (<xref ref-type="bibr" rid="B170">Zhang et&#xa0;al., 1992</xref>). Rifampicin resistance emerges primarily through point mutations in the rifampicin resistance determining region on the beta subunit of RNA polymerase (<italic>rpoB</italic>) (<xref ref-type="bibr" rid="B167">Yamada et&#xa0;al., 1985</xref>; <xref ref-type="bibr" rid="B147">Telenti et&#xa0;al., 1993</xref>). Although many resistance-conferring mutations have been identified over the years, there is a growing appreciation for drug resistance mutations that fall outside the drug activator or target and which typically confer low-to-intermediate resistance (<xref ref-type="bibr" rid="B163">Wong et&#xa0;al., 2011</xref>; <xref ref-type="bibr" rid="B34">Colangeli et&#xa0;al., 2018</xref>; <xref ref-type="bibr" rid="B61">Hicks et&#xa0;al., 2020</xref>). Such low-to-intermediate resistance is clinically relevant (<xref ref-type="bibr" rid="B34">Colangeli et&#xa0;al., 2018</xref>) but much more poorly understood. While there remains much to be explored regarding acquired drug resistance in Mtb, this is a topic covered extensively in other reviews (including reviews in this series) and will not be a major focus here.</p>
<p>This review will first briefly outline existing methods used to define intrinsic drug resistance mechanisms in Mtb. We will then review our current understanding and knowledge gaps of intrinsic drug resistance in Mtb and highlight how functional and chemical-genetics (<xref ref-type="bibr" rid="B133">Sassetti et&#xa0;al., 2001</xref>; <xref ref-type="bibr" rid="B75">Kim et&#xa0;al., 2013</xref>; <xref ref-type="bibr" rid="B18">Bosch et&#xa0;al., 2021</xref>) can help close those gaps. We end with a brief discussion on how existing genetic approaches could be expanded to further intrinsic drug resistance research.</p>
</sec>
<sec id="s2">
<title>Chemical-genetic approaches to define intrinsic drug resistance mechanisms in Mtb</title>
<p>Chemical-genetic studies have been a pillar of biology for decades. This vast body of literature covers studies of species from all three domains of life and serves as a rich resource for understanding basic biology as well as informing drug discovery efforts (<xref ref-type="bibr" rid="B112">Parsons et&#xa0;al., 2006</xref>; <xref ref-type="bibr" rid="B87">Leh&#xe1;r et&#xa0;al., 2008</xref>; <xref ref-type="bibr" rid="B107">Nichols et&#xa0;al., 2011</xref>; <xref ref-type="bibr" rid="B23">Brown et&#xa0;al., 2014</xref>; <xref ref-type="bibr" rid="B25">Cacace et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B8">Antonova-Koch et&#xa0;al., 2018</xref>). Broadly speaking, chemical-genetics is the study of how genetic alterations influence the activity of a chemical compound. The simplest form of chemical-genetics relies on spontaneous mutagenesis to study the relationships between genes and drugs. In a given population of bacteria, drug resistance mutations can arise spontaneously at a low frequency and can be isolated by plating on a selective antibiotic concentration (<xref ref-type="bibr" rid="B95">Luria and Delbr&#xfc;ck, 1943</xref>; <xref ref-type="bibr" rid="B69">Jin and Gross, 1988</xref>). Genomes of drug-resistant clones can then be sequenced to determine the mutations causing drug resistance. This simple yet elegant approach has been used for decades to identify some of the most common mechanisms of antibiotic resistance.</p>
<p>Other applications of chemical-genetics in Mtb rely on active disruption of target genes (<xref ref-type="fig" rid="f1">
<bold>Figure&#xa0;1</bold>
</xref>). One such technique, transposon mutagenesis, involves phage-mediated transduction and integration of a mariner transposon at random TA dinucleotide sequences in the Mtb genome (<xref ref-type="bibr" rid="B133">Sassetti et&#xa0;al., 2001</xref>; <xref ref-type="bibr" rid="B41">Dejesus et&#xa0;al., 2017</xref>). As typically used in Mtb, this approach results in the irreversible inactivation of target genes and is thus restricted to the analysis of <italic>in vitro</italic> non-essential genes as mutants for <italic>in vitro</italic> essential genes are lost during library construction. Despite the strong GC bias in the Mtb genome, the overwhelming majority of Mtb genes are sufficiently susceptible to transposition for this technique to work efficiently at genome scale (<xref ref-type="bibr" rid="B41">Dejesus et&#xa0;al., 2017</xref>). Transposon sequencing (TnSeq) has been used to study chemical-genetic interactions in axenic culture (<xref ref-type="bibr" rid="B166">Xu et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B54">Furi&#xf3; et&#xa0;al., 2021</xref>; <xref ref-type="bibr" rid="B148">Thiede et&#xa0;al., 2022</xref>) as well as in macrophage and mouse models of infection (<xref ref-type="bibr" rid="B14">Bellerose et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B78">Kreutzfeldt et&#xa0;al., 2022</xref>).</p>
<fig id="f1" position="float">
<label>Figure&#xa0;1</label>
<caption>
<p>Techniques for large-scale genetic studies in Mtb: Illustration depicting the principles of <bold>(A)</bold> TnSeq, <bold>(B)</bold> regulated proteolysis, and <bold>(C)</bold> CRISPR interference as implemented in mycobacteria. <bold>(A)</bold> Transposon sequencing (TnSeq) involves transduction of Mtb with a phage encoding a mariner transposon, which will randomly insert at TA dinucleotides in the Mtb genome (<xref ref-type="bibr" rid="B133">Sassetti et&#xa0;al., 2001</xref>; <xref ref-type="bibr" rid="B41">Dejesus et&#xa0;al., 2017</xref>). The absence of transposon insertion events, as determined by next generation sequencing, indicates the essentiality of that gene under the conditions assayed. <bold>(B)</bold> Regulated proteolysis involves the addition of a C-terminal &#x201c;DAS&#x201d; tag to an endogenous Mtb gene (<xref ref-type="bibr" rid="B75">Kim et&#xa0;al., 2013</xref>; <xref ref-type="bibr" rid="B70">Johnson et&#xa0;al., 2019</xref>). Addition or removal of tetracycline, depending on the variant of TetR being used, will induce expression of the proteolytic adaptor, SspB, and facilitate ClpXP-mediated degradation of the DAS-tagged protein. The level of target knockdown can be modulated by using different Tet-regulated promoters to drive <italic>sspB</italic> expression. <bold>(C)</bold> CRISPR interference (CRISPRi) utilizes a single guide RNA (sgRNA) to localize a nuclease-dead Cas9 enzyme to a specific target sequence in the Mtb genome, sterically blocking transcription initiation or elongation. Modulation of target knockdown level can be achieved by designing an sgRNA targeting a divergent protospacer adjacent motif (PAM) sequence, which is directly adjacent to the sgRNA target sequence (<xref ref-type="bibr" rid="B128">Rock et&#xa0;al., 2017</xref>), or modulating the extent of complementarity between the sgRNA and the DNA target (<xref ref-type="bibr" rid="B123">Qi et&#xa0;al., 2013</xref>; <xref ref-type="bibr" rid="B153">Vigouroux et&#xa0;al., 2018</xref>; <xref ref-type="bibr" rid="B59">Hawkins et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B18">Bosch et&#xa0;al., 2021</xref>; <xref ref-type="bibr" rid="B100">Mathis et&#xa0;al., 2021</xref>). Figure graphics were generated using Biorender software.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-12-997283-g001.tif"/>
</fig>
<p>More recently, genetic techniques have been developed that are more applicable to the study of <italic>in vitro</italic> essential Mtb genes. One such technique relies on a regulated proteolysis system, wherein the protein of interest is tagged with a C-terminal &#x201c;degron&#x201d; that is recognized by a tetracycline-regulated proteolytic adapter. Upon addition or removal of tetracycline (depending on the variant of the TetR used in the study), the <italic>sspB</italic> adapter is expressed and the corresponding protein is degraded (<xref ref-type="bibr" rid="B75">Kim et&#xa0;al., 2013</xref>). In a tour de force, Johnson et&#xa0;al. used a barcoded library of Mtb degron mutants coupled with next generation sequencing to profile over 50,000 compounds to identify target-compound chemical-genetic interactions. The authors identify the putative molecular target for over 40 of these compounds, some of which are active against novel therapeutic targets such as the essential efflux pump <italic>efpA</italic> (<xref ref-type="bibr" rid="B70">Johnson et&#xa0;al., 2019</xref>). Throughout the review we will use the term &#x201c;degron libraries&#x201d; to refer to this regulated proteolysis technique.</p>
<p>Blending some of the attractive capabilities of both TnSeq and the degron approach, CRISPR interference (CRISPRi) has been used by several labs, including our own, to perform targeted transcriptional inhibition of essential and non-essential genes (<xref ref-type="bibr" rid="B30">Choudhary et&#xa0;al., 2015</xref>; <xref ref-type="bibr" rid="B44">de Wet et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B18">Bosch et&#xa0;al., 2021</xref>; <xref ref-type="bibr" rid="B102">McNeil et&#xa0;al., 2021</xref>). This technique leverages the targeting specificity of CRISPR-Cas systems to localize a catalytically dead Cas9 protein to a gene of interest, serving as a steric block to transcription (<xref ref-type="bibr" rid="B123">Qi et&#xa0;al., 2013</xref>; <xref ref-type="bibr" rid="B115">Peters et&#xa0;al., 2016</xref>). Recently, we have used this system at genome-scale to profile a select group of antitubercular drugs (<xref ref-type="bibr" rid="B90">Li et&#xa0;al., 2022</xref>).</p>
</sec>
<sec id="s3">
<title>The mycobacterial envelope as a first line of intrinsic antibiotic resistance</title>
<p>It has long been appreciated that mycobacteria have a high level of intrinsic resistance to a diverse set of antibiotics (<xref ref-type="bibr" rid="B67">Jarlier and Nikaido, 1994</xref>; <xref ref-type="bibr" rid="B57">Gygli et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B166">Xu et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B13">Batt et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B51">Dulberger et&#xa0;al., 2020</xref>). This phenotype has generally been attributed to the relative impermeability of the mycobacterial envelope, which is distinct from those of classic Gram-negative or Gram-positive bacterial species. The Mtb envelope is a complex network composed primarily of peptidoglycan, arabinogalactan, and mycolic acids, which we will refer to as the mAGP complex. At the innermost layer closest to the plasma membrane is the peptidoglycan (PG), which is itself covalently linked to a network of arabinogalactan (AG) polymers (<xref ref-type="bibr" rid="B51">Dulberger et&#xa0;al., 2020</xref>). Connected to the AG by esterification is a thick layer of long-chain fatty acids called mycolic acids which form a pseudo-outer membrane bilayer known as the mycobacterial outer membrane (MOM) or mycomembrane (<xref ref-type="bibr" rid="B66">Jankute et&#xa0;al., 2015</xref>). Interspersed in the mycolic acids are a select group of proteins including porin-like proteins (<xref ref-type="bibr" rid="B139">Siroy et&#xa0;al., 2008</xref>; <xref ref-type="bibr" rid="B156">Wang et&#xa0;al., 2020</xref>) and secretion systems (<xref ref-type="bibr" rid="B9">Ates et&#xa0;al., 2015</xref>; <xref ref-type="bibr" rid="B149">Tiwari et&#xa0;al., 2020</xref>), as well as virulence associated glycolipids such as phthiocerol dimycocerosates (PDIMs) (<xref ref-type="bibr" rid="B127">Rens et&#xa0;al., 2021</xref>). Outside of the MOM is a mycobacterial capsule which is composed primarily of complex carbohydrates such as &#x3b1;-D-glucan and D-arabino-D-mannan, but also a select group of lipids and proteins (<xref ref-type="bibr" rid="B142">Stokes et&#xa0;al., 2004</xref>; <xref ref-type="bibr" rid="B72">Kalscheuer et&#xa0;al., 2019</xref>). Due to the typical growth conditions used in mycobacterial media, the capsule is generally stripped from the Mtb cell surface and is not studied during axenic growth (<xref ref-type="bibr" rid="B142">Stokes et&#xa0;al., 2004</xref>). The implications of this fact will be discussed later in this review.</p>
<p>The intrinsic resistance of Mtb to many different classes of antibiotics is often attributed to the impermeability of the MOM (<xref ref-type="bibr" rid="B67">Jarlier and Nikaido, 1994</xref>; <xref ref-type="bibr" rid="B13">Batt et&#xa0;al., 2020</xref>). Hydrophilic solutes are unable to traverse the mycolic acids and are thought to rely on protein-mediated translocation <italic>via</italic> porin-like proteins (<xref ref-type="bibr" rid="B67">Jarlier and Nikaido, 1994</xref>; <xref ref-type="bibr" rid="B9">Ates et&#xa0;al., 2015</xref>; <xref ref-type="bibr" rid="B13">Batt et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B156">Wang et&#xa0;al., 2020</xref>). Hydrophobic compounds are thought to get stuck in a poorly fluid mycolic acid sink and fail to efficiently traverse the MOM. There are substantial data to support this model using both chemical and genetic disruption of the mycolic acid network to potentiate antibiotic uptake and activity (<xref ref-type="bibr" rid="B91">Liu and Nikaido, 1999</xref>; <xref ref-type="bibr" rid="B83">Larrouy-Maumus et&#xa0;al., 2016</xref>; <xref ref-type="bibr" rid="B166">Xu et&#xa0;al., 2017</xref>). The clinical implications of this phenomenon can be seen by the synergistic interaction between rifampicin, which inhibits RNA polymerase (<xref ref-type="bibr" rid="B26">Campbell et&#xa0;al., 2001</xref>), and ethambutol, which inhibits arabinogalactan and lipoarabinomannan (LAM) biosynthesis (<xref ref-type="bibr" rid="B55">Goude et&#xa0;al., 2009</xref>; <xref ref-type="bibr" rid="B101">McNeil et&#xa0;al., 2019</xref>; <xref ref-type="bibr" rid="B171">Zhang et&#xa0;al., 2020</xref>). Because arabinogalactan serves as an anchor for the mycolic acid layer, AG inhibitors like ethambutol also perturb the MOM (<xref ref-type="bibr" rid="B74">Kilburn and Takayama, 1981</xref>; <xref ref-type="bibr" rid="B105">Mikusov&#xe1; et&#xa0;al., 1995</xref>; <xref ref-type="bibr" rid="B51">Dulberger et&#xa0;al., 2020</xref>). Despite its relatively modest <italic>in vitro</italic> and <italic>in vivo</italic> activity, ethambutol is included as part of the first-line RIPE (<bold>
<underline>r</underline>
</bold>ifampicin, <bold>
<underline>i</underline>
</bold>soniazid, <bold>
<underline>p</underline>
</bold>yrazinamide, <bold>
<underline>e</underline>
</bold>thambutol) regimen for drug sensitive TB. It has previously been suggested that ethambutol mainly serves as a &#x201c;safety net&#x201d; to prevent the emergence of rifampicin and isoniazid resistant TB (<xref ref-type="bibr" rid="B50">Dub&#xe9; et&#xa0;al., 1997</xref>). More recently, the clinical importance of ethambutol has been attributed to the efficient distribution of this drug throughout TB lung lesions (<xref ref-type="bibr" rid="B173">Zimmerman et&#xa0;al., 2017</xref>). In addition to these roles, ethambutol&#x2019;s clinical success may be due to its synergistic interaction with rifampicin (<xref ref-type="bibr" rid="B33">Cokol et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B101">McNeil et&#xa0;al., 2019</xref>). Rifampicin is a hydrophobic, high molecular weight compound for which the mycobacterial envelope serves as a permeability barrier (<xref ref-type="bibr" rid="B166">Xu et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B101">McNeil et&#xa0;al., 2019</xref>; <xref ref-type="bibr" rid="B90">Li et&#xa0;al., 2022</xref>). By disrupting proper formation of arabinogalactan, ethambutol promotes more efficient uptake of rifampicin to exert its bactericidal effect (<xref ref-type="bibr" rid="B33">Cokol et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B101">McNeil et&#xa0;al., 2019</xref>).</p>
<p>Despite the long-standing appreciation that bacterial surface structures can impede antibiotic uptake, the physiochemical details of this phenomenon are not fully understood in mycobacteria. Chemical-genetic studies have shown that mAGP-related mutants in Mtb are hypersusceptible to certain antibiotics but not others, suggesting that the cell envelope is a relevant barrier for certain drugs such as rifampicin and bedaquiline, but not other drugs like linezolid (<xref ref-type="bibr" rid="B40">Davis et&#xa0;al., 2014</xref>; <xref ref-type="bibr" rid="B83">Larrouy-Maumus et&#xa0;al., 2016</xref>; <xref ref-type="bibr" rid="B90">Li et&#xa0;al., 2022</xref>). In the latter scenario it is unclear whether there are specific importers for these drugs (<xref ref-type="bibr" rid="B126">Rempel et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B90">Li et&#xa0;al., 2022</xref>) or whether drug diffusion is unaffected by the envelope. While compound size certainly seems to negatively affect uptake beyond a certain threshold (<xref ref-type="bibr" rid="B40">Davis et&#xa0;al., 2014</xref>; <xref ref-type="bibr" rid="B90">Li et&#xa0;al., 2022</xref>), the physiochemical properties that allow some compounds but not others to easily traverse the Mtb envelope are not fully established. Future chemical-genetic studies could be focused on profiling mAGP-associated mutants against a physiochemically diverse set of antitubercular compounds, or in practice any compounds for which uptake can be quantitatively monitored, to identify which chemical scaffolds are efficiently blocked by the Mtb envelope (<xref ref-type="bibr" rid="B40">Davis et&#xa0;al., 2014</xref>; <xref ref-type="bibr" rid="B169">Zgurskaya and Rybenkov, 2019</xref>). This approach can be achieved using existing genome-scale CRISPRi libraries (<xref ref-type="bibr" rid="B18">Bosch et&#xa0;al., 2021</xref>). Alternatively, more targeted libraries (degron or CRISPRi) can be generated to specifically target mAGP-associated genes and profile the susceptibilities of each mutant. These screens could help reveal which physiochemical properties are associated with the ability or inability to traverse the mycobacterial envelope and help to define the &#x201c;rules&#x201d; of drug uptake in mycobacteria (<xref ref-type="bibr" rid="B40">Davis et&#xa0;al., 2014</xref>; <xref ref-type="bibr" rid="B83">Larrouy-Maumus et&#xa0;al., 2016</xref>; <xref ref-type="bibr" rid="B169">Zgurskaya and Rybenkov, 2019</xref>; <xref ref-type="bibr" rid="B172">Zhao et&#xa0;al., 2020</xref>).</p>
<p>At the same time, such screens could also reveal how different molecular structures within the envelope serve as a barrier to antibiotic uptake. Although often viewed as a unitary structure, the mAGP network is remarkably complex and disrupting different components of this structure may differentially sensitize Mtb to particular compounds. For example, knockdown of many arabinogalactan and mycolic acid biosynthetic enzymes seems to potentiate the activity of bedaquiline (<xref ref-type="bibr" rid="B94">Lupien et&#xa0;al., 2018</xref>; <xref ref-type="bibr" rid="B90">Li et&#xa0;al., 2022</xref>). However, for reasons that remain unclear, this effect is not observed as strongly with disruption of peptidoglycan biosynthetic enzymes (<xref ref-type="bibr" rid="B90">Li et&#xa0;al., 2022</xref>). Are these differential phenotypes simply a result of genetic redundancy in peptidoglycan biosynthesis, or do they reflect some degree of barrier specificity for different envelope structures?</p>
<p>While chemical-genetic interactions can help inform which physiochemical properties and potentially which envelope structures are most important for intrinsic drug resistance, there are several limitations. For example, many antitubercular compounds target envelope biosynthesis either directly (<xref ref-type="bibr" rid="B11">Banerjee et&#xa0;al., 1994</xref>; <xref ref-type="bibr" rid="B55">Goude et&#xa0;al., 2009</xref>) or indirectly (<xref ref-type="bibr" rid="B143">Stover et&#xa0;al., 2000</xref>; <xref ref-type="bibr" rid="B148">Thiede et&#xa0;al., 2022</xref>). Let&#x2019;s assume that a CRISPRi knockdown strain against the essential arabinogalactan biosynthetic enzyme <italic>dprE1</italic> renders Mtb more sensitive to a given compound. There are several potential explanations to explain this interaction. First, DprE1 or another target involved in arabinogalactan biosynthesis may be the direct target of the screened compound (<xref ref-type="bibr" rid="B79">Kumar et&#xa0;al., 2018</xref>). Second, lack of <italic>dprE1</italic> activity may weaken the arabinogalactan layer sufficiently to increase envelope permeability and compound uptake. Third, the chemical-genetic interaction may be independent of compound uptake and reflect a more mechanism-specific collateral vulnerability associated with <italic>dprE1</italic> inhibition and arabinogalactan biosynthesis perturbation (<xref ref-type="bibr" rid="B158">Wang et&#xa0;al., 2019</xref>). Therefore, care should be exercised when interpreting chemical-genetic interactions and such studies should be coupled with mass spectrometry drug-uptake quantification to differentiate between these various possibilities (<xref ref-type="bibr" rid="B40">Davis et&#xa0;al., 2014</xref>; <xref ref-type="bibr" rid="B121">Planck and Rhee, 2021</xref>). Further, structural and/or biochemical approaches can be used to identify the target of a particular compound, helping to differentiate between a direct or an indirect mechanism for a specific chemical-genetic interaction (<xref ref-type="bibr" rid="B114">Pellecchia et&#xa0;al., 2002</xref>; <xref ref-type="bibr" rid="B171">Zhang et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B110">Ottavi et&#xa0;al., 2022</xref>).</p>
<p>Moreover, growth of Mtb in axenic culture often ignores two key components of the mycobacterial cell surface. PDIMs are a family of lipids involved in Mtb virulence, with over 1% of the Mtb genome dedicated to PDIM biosynthetic genes (<xref ref-type="bibr" rid="B151">Trivedi et&#xa0;al., 2005</xref>; <xref ref-type="bibr" rid="B46">Domenech and Reed, 2009</xref>; <xref ref-type="bibr" rid="B127">Rens et&#xa0;al., 2021</xref>). Because of the metabolic costs associated with synthesizing PDIMs and the fact that these lipids are not only dispensable in standard axenic culture but can restrict permeability of culture carbon sources (<xref ref-type="bibr" rid="B9">Ates et&#xa0;al., 2015</xref>; <xref ref-type="bibr" rid="B156">Wang et&#xa0;al., 2020</xref>), lab-grown Mtb frequently sustains loss of function mutations in PDIM biosynthetic enzymes (<xref ref-type="bibr" rid="B46">Domenech and Reed, 2009</xref>). The lack of PDIMs has been associated with increased sensitivity to drugs and altered nutrient uptake, consistent with PDIMs being a relevant permeability barrier in Mtb (<xref ref-type="bibr" rid="B141">Soetaert et&#xa0;al., 2015</xref>; <xref ref-type="bibr" rid="B156">Wang et&#xa0;al., 2020</xref>). Therefore, care should be taken to assess the role of PDIM in compound uptake. Lastly, mycobacteria are frequently cultured in the presence of detergent to prevent cell clumping. Detergents act by stripping the mycobacterial capsule, which may influence Mtb&#x2019;s small molecule permeability (<xref ref-type="bibr" rid="B142">Stokes et&#xa0;al., 2004</xref>; <xref ref-type="bibr" rid="B72">Kalscheuer et&#xa0;al., 2019</xref>). Therefore, as with PDIM, confirmation of relevant chemical-genetic interactions should be performed under conditions in which the Mtb capsule is intact (e.g. infection models, detergent-free plates, etc.).</p>
<p>The Mtb envelope is not a static structure and is influenced by the growth environment of the bacteria (<xref ref-type="bibr" rid="B132">Sarathy et&#xa0;al., 2013</xref>; <xref ref-type="bibr" rid="B83">Larrouy-Maumus et&#xa0;al., 2016</xref>; <xref ref-type="bibr" rid="B76">Koh et&#xa0;al., 2022</xref>). For example, Sarathy et&#xa0;al. demonstrated that non-replicating and nutrient-starved bacteria display greatly reduced drug uptake, likely through a cell wall remodeling process that is not entirely understood (<xref ref-type="bibr" rid="B38">Cunningham and Spreadbury, 1998</xref>; <xref ref-type="bibr" rid="B165">Wu et&#xa0;al., 2016</xref>). Perturbing envelope integrity may play a greater role in increasing compound uptake in Mtb grown under these conditions than standard replicating conditions (<xref ref-type="bibr" rid="B132">Sarathy et&#xa0;al., 2013</xref>). Further, Koh et&#xa0;al. showed that rifampicin is less effective when Mtb is grown on the <italic>in vivo-</italic>relevant carbon source cholesterol as a result of modifications to the Mtb envelope (<xref ref-type="bibr" rid="B76">Koh et&#xa0;al., 2022</xref>). This effect could be specifically reversed through selective cell envelope disruption. These studies highlight the importance of performing chemical-genetic screens in host-relevant carbon sources and stress conditions. This will help identify environments in which successful cell envelope disruption will facilitate antibiotic entry.</p>
<p>Having a more complete understanding of the mycobacterial envelope as a barrier to antibiotic uptake will pave the way for several important applications. First, this knowledge can be used to better predict synergistic drug combinations and inform the preclinical testing of new combination therapies. For example, several studies have shown that bedaquiline can be potentiated by inhibiting proper mAGP synthesis (<xref ref-type="bibr" rid="B85">Lechartier et&#xa0;al., 2012</xref>; <xref ref-type="bibr" rid="B94">Lupien et&#xa0;al., 2018</xref>; <xref ref-type="bibr" rid="B90">Li et&#xa0;al., 2022</xref>). Part of the success of the bedaquiline, pretomanid, and linezolid (BPaL) combination (<xref ref-type="bibr" rid="B35">Conradie et&#xa0;al., 2020</xref>) may be due to the disruption of mycolic acids by pretomanid (<xref ref-type="bibr" rid="B143">Stover et&#xa0;al., 2000</xref>; <xref ref-type="bibr" rid="B99">Manjunatha et&#xa0;al., 2009</xref>), resulting in increased bedaquiline uptake. This synergy would likely extend to pre-clinical DprE1 inhibitors (<xref ref-type="bibr" rid="B85">Lechartier et&#xa0;al., 2012</xref>; <xref ref-type="bibr" rid="B94">Lupien et&#xa0;al., 2018</xref>). Second, knowledge of the genetic regulation of cell envelope synthesis will allow for the rational prioritization of target-based drug discovery candidates. Ideally, these studies will identify targets for which inhibition not only leads to bacterial death but also potentiates the uptake and activity of other drugs. Third, as mentioned above, knowledge of the physiochemical &#x201c;rules&#x201d; that allow compounds to traverse the mAGP will help direct medicinal chemistry efforts to improve compound uptake. Lastly, this knowledge can be used to identify mechanism-specific synergies that target different components of the mAGP complex. Isoniazid and ethambutol have been used together in first-line TB therapy for decades but have been shown to be slightly antagonistic or additive at best (<xref ref-type="bibr" rid="B33">Cokol et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B82">Larkins-Ford et&#xa0;al., 2021</xref>). Chemical-genetic profiling of cell envelope-targeting compounds may help to reveal other targets, either in the same pathway or parallel pathways, that will act synergistically, thus optimizing the therapeutic potential of this highly vulnerable chemical complex.</p>
</sec>
<sec id="s4">
<title>Efflux pumps as the next line of defense against antibiotics</title>
<p>The selective permeability of the mycobacterial envelope collaborates with additional mechanisms to promote intrinsic drug resistance (<xref ref-type="bibr" rid="B108">Nikaido, 1994</xref>). Once a chemical compound traverses the mAGP, it can encounter another line of defense in the form of drug efflux pumps (<xref ref-type="bibr" rid="B119">Piddock et&#xa0;al., 2000</xref>; <xref ref-type="bibr" rid="B40">Davis et&#xa0;al., 2014</xref>; <xref ref-type="bibr" rid="B84">Laws et&#xa0;al., 2022</xref>). Efflux pumps are transmembrane proteins that facilitate the transport of small molecules out of the periplasm and/or the cytosol (<xref ref-type="bibr" rid="B15">Blair et&#xa0;al., 2015</xref>; <xref ref-type="bibr" rid="B152">Venter et&#xa0;al., 2015</xref>; <xref ref-type="bibr" rid="B19">Boyer et&#xa0;al., 2022</xref>). Mtb encodes several dozen putative and validated drug efflux pumps, which have been comprehensively reviewed by Laws et&#xa0;al. (<xref ref-type="bibr" rid="B84">Laws et&#xa0;al., 2022</xref>). Some efflux pumps, such as those of the ATP Binding Cassette (ABC) family, are regulated by ATP hydrolysis (<xref ref-type="bibr" rid="B20">Braibant et&#xa0;al., 2000</xref>; <xref ref-type="bibr" rid="B113">Pasca et&#xa0;al., 2004</xref>), whereas major facilitator superfamily (MFS) efflux pumps are regulated by proton-induced conformational changes (<xref ref-type="bibr" rid="B89">Li et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B84">Laws et&#xa0;al., 2022</xref>). Other pumps, such as those of the resistance-nodulation-cell-division (RND) superfamily rely on a drug-proton antiporter mechanism (<xref ref-type="bibr" rid="B152">Venter et&#xa0;al., 2015</xref>; <xref ref-type="bibr" rid="B89">Li et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B84">Laws et&#xa0;al., 2022</xref>). The efflux pumps of Mtb vary greatly in their compound specificity with some having a single validated transported substrate and others having many substrates (<xref ref-type="bibr" rid="B92">Liu et&#xa0;al., 2019</xref>).</p>
<p>The clinical importance of drug efflux in Mtb has been well established. For example, the MmpS5/L5 efflux pump (RND superfamily) has been shown to be active against several drugs including bedaquiline and clofazimine (<xref ref-type="bibr" rid="B58">Hartkoorn et&#xa0;al., 2014</xref>; <xref ref-type="bibr" rid="B22">Briffotaux et&#xa0;al., 2017</xref>). Expression of the <italic>mmpS5/L5</italic> operon is negatively regulated by the transcriptional repressor MmpR (Rv0678) (<xref ref-type="bibr" rid="B22">Briffotaux et&#xa0;al., 2017</xref>). Loss of function mutations in <italic>rv0678</italic> result in constitutive MmpS5/L5 expression and confer acquired drug resistance to bedaquiline, representing a significant complication to the long-term success of this new TB drug (<xref ref-type="bibr" rid="B43">de Vos et&#xa0;al., 2019</xref>; <xref ref-type="bibr" rid="B109">Nimmo et&#xa0;al., 2020</xref>). Interestingly, for reasons that remain unclear, some clinical Mtb strains harbor loss-of-function <italic>rv0678</italic> mutations that pre-date the clinical use of bedaquiline (<xref ref-type="bibr" rid="B154">Villellas et&#xa0;al., 2017</xref>). The presence of these mutations could reflect earlier clinical exposure to clofazimine or other drugs. Another example is Rv1258c (Tap), an MFS efflux pump active against several antituberculars including streptomycin and rifampicin (<xref ref-type="bibr" rid="B3">Adams et&#xa0;al., 2011</xref>; <xref ref-type="bibr" rid="B4">Adams et&#xa0;al., 2019</xref>; <xref ref-type="bibr" rid="B92">Liu et&#xa0;al., 2019</xref>). Tap expression is activated by the transcription factor <italic>whiB7</italic>. WhiB7 or genes involved in its regulation can in turn sustain mutations that result in constitutive activation of the WhiB7 regulon, including Tap, to promote acquired drug resistance (<xref ref-type="bibr" rid="B124">Reeves et&#xa0;al., 2013</xref>; <xref ref-type="bibr" rid="B135">Schrader et&#xa0;al., 2021</xref>; <xref ref-type="bibr" rid="B90">Li et&#xa0;al., 2022</xref>). Thus, efflux pumps like MmpS5/L5 and Tap promote intrinsic resistance in Mtb and can further be augmented by mutation to promote acquired drug resistance.</p>
<p>Most validated or putative efflux pumps in Mtb are poorly characterized, but it is likely that efflux pumps beyond MmpS5/L5 and Tap contribute to intrinsic drug resistance in Mtb (<xref ref-type="bibr" rid="B125">Remm et&#xa0;al., 2022</xref>). An in-depth characterization of these under-studied efflux pumps is much needed (<xref ref-type="bibr" rid="B144">Szumowski et&#xa0;al., 2012</xref>). To facilitate this characterization, one could systematically generate underexpression and overexpression strains for all predicted Mtb efflux pumps. For example, a small, targeted CRISPRi library (<xref ref-type="bibr" rid="B18">Bosch et&#xa0;al., 2021</xref>) could be generated that contains knockdown strains for all validated and predicted efflux pumps. This library could be treated with a wide range of antitubercular compounds to determine which mutants display reduced fitness under which drug treatment conditions. Some efflux pumps may overlap in the types of compounds transported (<xref ref-type="bibr" rid="B140">Smith and Blair, 2014</xref>). To address this possibility, combinatorial libraries in which multiple efflux pump genes are simultaneously silenced (<xref ref-type="bibr" rid="B164">Wong and Rock, 2021</xref>; <xref ref-type="bibr" rid="B90">Li et&#xa0;al., 2022</xref>) may help to identify functional redundancies between efflux pumps. Further, because some efflux pumps may not be highly expressed under standard lab conditions (<xref ref-type="bibr" rid="B56">Gupta et&#xa0;al., 2010</xref>; <xref ref-type="bibr" rid="B3">Adams et&#xa0;al., 2011</xref>), knocking down the corresponding gene may not produce a phenotype. To overcome this, a parallel pool of barcoded overexpression strains for each efflux pump could be generated and screened against the same panel of antitubercular compounds (<xref ref-type="bibr" rid="B62">Hicks et&#xa0;al., 2018</xref>). Lastly, as in the case for MmpS5/L5 and Tap, one could interrogate the increasing amount of Mtb clinical strain genome sequencing available to identify predicted efflux pumps or their regulators under positive selection. Should such evidence exist, it seems reasonable to predict that the relevant selective pressure is antibiotics, although other mechanisms cannot be ruled out.</p>
<p>In designing these experiments, it will be important to carefully curate the list of predicted efflux pumps. For example, several ABC proteins have been annotated as efflux pumps even though these proteins lack transmembrane helices (<xref ref-type="bibr" rid="B49">Duan et&#xa0;al., 2019</xref>; <xref ref-type="bibr" rid="B84">Laws et&#xa0;al., 2022</xref>). Two such previously annotated efflux pumps, Rv1473 and EttA, turn out to indeed influence drug activity in Mtb but have nothing to do with efflux and rather are ATP-dependent regulators of the ribosome (<xref ref-type="bibr" rid="B137">Sharkey et&#xa0;al., 2016</xref>; <xref ref-type="bibr" rid="B37">Cui et&#xa0;al., 2022</xref>; <xref ref-type="bibr" rid="B90">Li et&#xa0;al., 2022</xref>). Furthermore, genetic studies of efflux pumps should be validated with biochemical approaches to unambiguously demonstrate drug efflux activity.</p>
<p>Given the role of drug efflux in intrinsic and acquired Mtb drug resistance, there has been considerable interest in developing efflux pump inhibitors (EPIs) to potentiate TB drug regimens. Numerous small molecule EPIs, including both natural products and synthetic compounds, have been described (<xref ref-type="bibr" rid="B144">Szumowski et&#xa0;al., 2012</xref>; <xref ref-type="bibr" rid="B97">Machado et&#xa0;al., 2016</xref>; <xref ref-type="bibr" rid="B84">Laws et&#xa0;al., 2022</xref>). Many of these EPIs show broad activity against numerous efflux pumps and appear to act in a relatively non-specific manner by disrupting membrane energetics (<xref ref-type="bibr" rid="B2">Adams et&#xa0;al., 2014</xref>; <xref ref-type="bibr" rid="B131">Ruth et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B84">Laws et&#xa0;al., 2022</xref>). Indeed, many EPIs have antimycobacterial activity as single agents, which could reflect synthetic lethality of multi-efflux pump inhibition or an efflux pump independent mode of action, e.g. disruption of membrane energetics (<xref ref-type="bibr" rid="B29">Chen et&#xa0;al., 2018</xref>; <xref ref-type="bibr" rid="B125">Remm et&#xa0;al., 2022</xref>). The pleiotropic consequence of disrupting membrane energetics may confound the interpretation how EPIs potentiate the activity of other TB drugs (<xref ref-type="bibr" rid="B6">Amaral and Viveiros, 2017</xref>; <xref ref-type="bibr" rid="B29">Chen et&#xa0;al., 2018</xref>). Despite their unclear mode of action, some EPIs may have potential for use in TB therapy. For example, there are several studies showing that the antipsychotic drug thioridazine has direct antitubercular activity (<xref ref-type="bibr" rid="B5">Amaral et&#xa0;al., 2001</xref>; <xref ref-type="bibr" rid="B1">Abbate et&#xa0;al., 2012</xref>; <xref ref-type="bibr" rid="B120">Pieroni et&#xa0;al., 2015</xref>). Thioridazine also displays synergy with other drugs, possibly by altering membrane potential and reducing drug efflux (<xref ref-type="bibr" rid="B32">Coelho et&#xa0;al., 2015</xref>; <xref ref-type="bibr" rid="B97">Machado et&#xa0;al., 2016</xref>; <xref ref-type="bibr" rid="B131">Ruth et&#xa0;al., 2020</xref>). Although there is no evidence of direct efflux pump inhibition by thioridazine, its ability to disrupt membrane potential may lower cellular ATP levels, thereby limiting the activity of ATP-dependent efflux pumps. Alternatively, or in addition, thioridazine-mediated disruption of proton gradients may alter the ability of MFS efflux pumps to undergo proton-induced conformational changes or of RND family efflux pumps to carry out drug-proton antiport exchange.</p>
<p>Ultimately, while non-specific EPIs may have clinical value, specific and selective EPIs could help augment TB treatment. However, substantial advances in our understanding of efflux pump specificity and structure may be required to identify such compounds. Until then, more generic EPIs, especially those that have stand-alone antitubercular activity, may be of utility for TB treatment (<xref ref-type="bibr" rid="B1">Abbate et&#xa0;al., 2012</xref>; <xref ref-type="bibr" rid="B129">Rodrigues et&#xa0;al., 2020</xref>).</p>
</sec>
<sec id="s5">
<title>Beyond drug uptake and efflux: Cytosolic mechanisms of intrinsic antibiotic resistance</title>
<p>An intrepid compound has traversed the mycobacterial envelope, avoided efflux, and is ready to engage its target. What next? Once again, Mtb is well-equipped with numerous cytosolic mechanisms of intrinsic drug resistance. Once again, Mtb is well-equipped with numerous cytosolic mechanisms of intrinsic drug resistance (<xref ref-type="fig" rid="f2"><bold>Figure&#xa0;2</bold></xref>). (<xref ref-type="bibr" rid="B15">Blair et&#xa0;al., 2015</xref>). Not surprisingly, these processes tend to be more drug-specific than selective envelope permeability and efflux, and based on current knowledge are most frequently seen with the antituberculars which target the ribosome (<xref ref-type="bibr" rid="B162">Wilson, 2014</xref>). Some of the most well-studied mechanisms of cytosolic intrinsic drug resistance in Mtb are listed in <xref ref-type="table" rid="T1">
<bold>Table&#xa0;1</bold>
</xref>.</p>
<fig id="f2" position="float">
<label>Figure&#xa0;2</label>
<caption>
<p>The many layers of intrinsic antibiotic resistance in Mtb. Illustration of intrinsic resistance factors at the Mtb cell surface and inside the cytosol. CAP, capsule; PDIM, phthiocerol dimycocerosates; MOM, mycobacterial outer membrane; AG, arabinogalactan; PG, peptidoglycan; PM, plasma membrane; STR, streptomycin; BDQ, bedaquiline; AMK, amikacin; Ac, acetylation modification (CH<sub>3</sub>CO); CLR, clarithromycin; CH<sub>3</sub>, methylation of ribosomal RNA; LZD, linezolid; FQ, fluoroquinolone; NAD, nicotinamide adenine dinucleotide (depicted as a drug adduct); NMN, nicotinamide mononucleotide; AMP, adenosine monophosphate. Figure graphics were generated using Biorender software.</p>
</caption>
<graphic mimetype="image" mime-subtype="tiff" xlink:href="fcimb-12-997283-g002.tif"/>
</fig>
<table-wrap id="T1" position="float">
<label>Table&#xa0;1</label>
<caption>
<p>Cytosolic intrinsic drug resistance factors of Mtb.</p>
</caption>
<table frame="hsides">
<tbody>
<tr>
<td valign="top" align="left">
<bold>Resistance Gene</bold>
</td>
<td valign="top" align="left">
<bold>Protection specificity</bold>
</td>
<td valign="top" align="left">
<bold>Mechanism</bold>
</td>
<td valign="top" align="left">
<bold>Reference</bold>
</td>
</tr>
<tr>
<td valign="top" align="left">
<italic>whiB7</italic> (<italic>rv3197A</italic>)</td>
<td valign="top" align="left">Ribosome-targeting antibiotics</td>
<td valign="top" align="left">Transcription of other resistance factors</td>
<td valign="top" align="left">(<xref ref-type="bibr" rid="B106">Morris et&#xa0;al., 2005</xref>)</td>
</tr>
<tr>
<td valign="top" align="left">
<italic>erm(37)</italic> (<italic>rv1988</italic>)</td>
<td valign="top" align="left">Macrolides, lincosamides, streptogramin B antibiotics</td>
<td valign="top" align="left">Methylation of the 23S rRNA drug binding site</td>
<td valign="top" align="left">(<xref ref-type="bibr" rid="B98">Madsen et&#xa0;al., 2005</xref>)</td>
</tr>
<tr>
<td valign="top" align="left">
<italic>eis</italic> (<italic>rv2416c</italic>)</td>
<td valign="top" align="left">Amikacin and kanamycin</td>
<td valign="top" align="left">Aminoglycoisde acetylation and inactivation</td>
<td valign="top" align="left">(<xref ref-type="bibr" rid="B168">Zaunbrecher et&#xa0;al., 2009</xref>)</td>
</tr>
<tr>
<td valign="top" align="left">
<italic>hflX</italic> (<italic>rv2725c</italic>)</td>
<td valign="top" align="left">Macrolides, lincosamides</td>
<td valign="top" align="left">Rescue of stalled ribosomes</td>
<td valign="top" align="left">(<xref ref-type="bibr" rid="B130">Rudra et&#xa0;al., 2020</xref>)</td>
</tr>
<tr>
<td valign="top" align="left">
<italic>ocrA (rv1473)</italic>
</td>
<td valign="top" align="left">Oxazolidinones and phenicols</td>
<td valign="top" align="left">Drug displacement from ribosome</td>
<td valign="top" align="left">(<xref ref-type="bibr" rid="B137">Sharkey et&#xa0;al., 2016</xref>; <xref ref-type="bibr" rid="B7">Antonelli et&#xa0;al., 2018</xref>; <xref ref-type="bibr" rid="B90">Li et&#xa0;al., 2022</xref>)</td>
</tr>
<tr>
<td valign="top" align="left">
<italic>smpB/ssr</italic> (<italic>rv3100c/ssr</italic>)</td>
<td valign="top" align="left">Oxazolidinones, phenicols, clarithromycin</td>
<td valign="top" align="left">Rescue of stalled ribosomes</td>
<td valign="top" align="left">(<xref ref-type="bibr" rid="B90">Li et&#xa0;al., 2022</xref>)</td>
</tr>
<tr>
<td valign="top" align="left">
<italic>mfpAB</italic> (<italic>rv3361c/rv3362c</italic>)</td>
<td valign="top" align="left">Fluoroquinolones</td>
<td valign="top" align="left">DNA mimicry, protection of DNA gyrase from FQs</td>
<td valign="top" align="left">(<xref ref-type="bibr" rid="B60">Hegde et&#xa0;al., 2005</xref>; <xref ref-type="bibr" rid="B146">Tao et&#xa0;al., 2013</xref>)</td>
</tr>
<tr>
<td valign="top" align="left">
<italic>cinA</italic> (<italic>rv1901</italic>)</td>
<td valign="top" align="left">Isoniazid, ethionamide, nitroimidazoles</td>
<td valign="top" align="left">Cleavage of drug-NAD adducts</td>
<td valign="top" align="left">(<xref ref-type="bibr" rid="B157">Wang et&#xa0;al., 2011</xref>; <xref ref-type="bibr" rid="B78">Kreutzfeldt et&#xa0;al., 2022</xref>)</td>
</tr>
</tbody>
</table>
</table-wrap>
<p>The different layers of intrinsic cytosolic resistance can all be seen within the <italic>whiB7</italic> pathway. WhiB7 is a transcription factor that senses translational stalling which can be triggered by ribosome stress during drug treatment, host-derived stressors, and poorly characterized metabolic changes (<xref ref-type="bibr" rid="B106">Morris et&#xa0;al., 2005</xref>; <xref ref-type="bibr" rid="B24">Burian et&#xa0;al., 2013</xref>). During unstressed conditions, <italic>whiB7</italic> expression is low due to upstream ORF (uORF)-mediated transcription attenuation (<xref ref-type="bibr" rid="B86">Lee et&#xa0;al., 2022</xref>). Translation of the uORF in the <italic>whiB7</italic> 5&#x2019; leader fails to prevent formation of a Rho-independent terminator, resulting in transcription termination prior to transcription of the <italic>whiB7</italic> ORF. However, stalled translation of the uORF promotes formation of an antiterminator, resulting in high-level transcription of the <italic>whiB7</italic> ORF. This subsequently further activates transcription from the <italic>whiB7</italic> promoter and those of the WhiB7 regulon genes. Among the WhiB7 regulon is <italic>tap</italic>, the multidrug efflux pump described in the previous section. Furthermore, WhiB7 promotes the transcription of several cytosolic resistance factors including <italic>eis</italic>, an aminoglycoside acetyltransferase that can chemically modify and inactivate amikacin and kanamycin (drug modification) (<xref ref-type="bibr" rid="B168">Zaunbrecher et&#xa0;al., 2009</xref>). Another WhiB7 regulon gene is <italic>erm(37)</italic>, a ribosomal RNA methyltransferase which modifies the macrolide binding site on the 23S rRNA to prevent drug binding (target modification) (<xref ref-type="bibr" rid="B98">Madsen et&#xa0;al., 2005</xref>). Moreover, WhiB7 promotes transcription of <italic>hflX</italic>, a ribosome recycling factor that can help to rescue stalled ribosomes (target rescue) (<xref ref-type="bibr" rid="B130">Rudra et&#xa0;al., 2020</xref>). This WhiB7 pathway presumably evolved in an ancestral soil-dwelling actinobacterium that encountered ribosome-targeting antibiotics in its environment.</p>
</sec>
<sec id="s6">
<title>Expanding our knowledge of cytosolic intrinsic resistance factors &amp; how to overcome them</title>
<p>Compared to the hundreds of genes which contribute to intrinsic resistance by regulating cell envelope processes (<xref ref-type="bibr" rid="B166">Xu et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B90">Li et&#xa0;al., 2022</xref>), there are many fewer known instances of resistance factors in the Mtb cytosol. This can likely be explained by the fact that cell envelope-associated intrinsic resistance factors are likely to be pleiotropic by preventing the uptake of many diverse compounds, whereas cytosolic resistance factors are likely to be specific to a particular drug or class of drugs. The relative paucity of known cytosolic resistance factors may also be explained by the limited scope of the drugs that have been screened in chemical-genetic studies. Future chemical-genetic screening efforts could focus on comprehensively defining the intrinsic &#x201c;resistome&#x201d; for a larger panel of antitubercular drugs.</p>
<p>We posit that there is merit to performing chemical-genetic profiling on FDA approved drugs with detectable but limited antitubercular activity. Although in some cases this lack of potency may be explained by poor drug uptake (which could potentially be improved by mAGP disruption) or alteration of a specific molecular target, in some cases it may be the result of a specific intrinsic resistance factor. This is the case with macrolides which are ineffective against Mtb due to <italic>whiB7</italic>-mediated expression of <italic>erm(37)</italic> (<xref ref-type="bibr" rid="B106">Morris et&#xa0;al., 2005</xref>). In instances where <italic>whiB7</italic> has been mutationally inactivated, clarithromycin displays potent activity against Mtb (<xref ref-type="bibr" rid="B159">Warit et&#xa0;al., 2015</xref>; <xref ref-type="bibr" rid="B90">Li et&#xa0;al., 2022</xref>). Using the macrolide paradigm, where <italic>whiB7</italic> is central to Mtb&#x2019;s intrinsic resistance, there may be parallel cases where one or several factors are responsible for limiting the activity of a given drug. Ultimately, by defining the intrinsic &#x201c;resistome&#x201d; for these compounds it may allow for three potential follow up strategies to advance their potential use in the clinic.</p>
<p>One such strategy involves leveraging &#x201c;acquired drug sensitivities.&#x201d; Here, clinically prevalent mutations in intrinsic resistance factors may present therapeutic opportunities for drugs that are already approved for clinical uses outside of TB treatment. As described above, there are cases where Mtb clinical isolates sustain loss-of-function mutations in intrinsic resistance genes. In addition to the loss-of-function mutations in <italic>whiB7</italic>, there are several documented loss-of-function mutations in the <italic>mmpS5/L5</italic> efflux pump (<xref ref-type="bibr" rid="B103">Merker et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B90">Li et&#xa0;al., 2022</xref>). These mutations render Mtb hypersusceptible to bedaquiline and clofazimine. As the use of whole genome sequencing is expanded in clinical labs, we may be able to predict unique drug susceptibilities based on the genome sequence of the infecting strain (<xref ref-type="bibr" rid="B36">CRyPTIC Consortium, 2018</xref>; <xref ref-type="bibr" rid="B48">Doyle et&#xa0;al., 2018</xref>). This may be particularly useful for multidrug-resistant and extensively-drug resistant TB cases with limited treatment options. Until &#x201c;personalized&#x201d; TB treatment is more widely available, geographically concentrated sublineages may be targeted on the basis of their unique vulnerability to particular drugs (<xref ref-type="bibr" rid="B118">Phelan et&#xa0;al., 2019</xref>; <xref ref-type="bibr" rid="B90">Li et&#xa0;al., 2022</xref>).</p>
<p>Another strategy to potentiate antibiotic activity against Mtb is to specifically inhibit intrinsic resistance pathways. Developing small molecule inhibitors of intrinsic resistance factors may synergize with the drug of interest (<xref ref-type="bibr" rid="B64">Hugonnet et&#xa0;al., 2009</xref>; <xref ref-type="bibr" rid="B80">Kurz et&#xa0;al., 2016</xref>). The classic example of this can be seen with the use of beta lactamase inhibitors to prevent the degradation of beta lactam antibiotics. In addition to the Mtb beta lactamase, BlaC, two other well-characterized examples which modify drugs or drug adducts are Eis and CinA (<xref ref-type="bibr" rid="B168">Zaunbrecher et&#xa0;al., 2009</xref>; <xref ref-type="bibr" rid="B78">Kreutzfeldt et&#xa0;al., 2022</xref>). Many more drug-modifying enzymes likely remain to be discovered (<xref ref-type="bibr" rid="B168">Zaunbrecher et&#xa0;al., 2009</xref>; <xref ref-type="bibr" rid="B78">Kreutzfeldt et&#xa0;al., 2022</xref>). Small molecule discovery efforts could focus on identifying inhibitors of particular drug modifying enzymes. As such, inhibitors of Eis would likely potentiate aminoglycoside activity whereas inhibition of CinA would likely potentiate isoniazid and pretomanid activity. However, both <italic>eis</italic> and <italic>cinA</italic> are non-essential for Mtb growth under standard conditions and inhibitors of these enzymes may face difficulties in preclinical development because in monotherapy, they would be unlikely to have any antimycobacterial activity. Therefore, genetic strategies focusing on essential genes are ideal (degron libraries, CRISPRi) since these represent some of the most attractive drug targets. Essential genes that impart intrinsic drug resistance could be prioritized for target-based drug discovery in order to form synergistic drug combinations where both compounds have individual activity but work more efficiently in combination. One such target is the mycobacterial superoxide dismutase (<italic>sodA</italic>), an essential oxygen radical quenching enzyme. Our previous work identified that knockdown of <italic>sodA</italic> sensitizes Mtb to several different classes of drugs (<xref ref-type="bibr" rid="B90">Li et&#xa0;al., 2022</xref>). Inhibitors of <italic>sodA</italic> would likely synergize with other drugs while also producing a direct, antimycobacterial effect.</p>
<p>Finally, a comprehensive understanding of the Mtb intrinsic resistome may allow for the rational design of drug analogs that avoid specific intrinsic resistance mechanisms. There are several examples of this concept, where derivatives of a particular drug are recalcitrant to the resistance mechanisms that target the parent compound. For example, 3<sup>rd</sup> generation tetracyclines avoid the drug-displacing activity of TetM in certain Gram-positive species (<xref ref-type="bibr" rid="B68">Jenner et&#xa0;al., 2013</xref>). Similarly, ketolides are a family of macrolide derivatives that have been engineered to bind Erm-methylated bacterial ribosomes (<xref ref-type="bibr" rid="B27">Capobianco et&#xa0;al., 2000</xref>). The ketolide drug telithromycin is approved for the treatment of erythromycin-resistant <italic>S. pneumoniae</italic> infections (<xref ref-type="bibr" rid="B93">Lonks and Goldmann, 2005</xref>). This concept is further exemplified by the aminoglycosides in Mtb. Despite being structurally and chemically similar, the aminoglycoside acetyltransferase Eis seems to have activity against kanamycin and amikacin but not streptomycin (<xref ref-type="bibr" rid="B168">Zaunbrecher et&#xa0;al., 2009</xref>). Conversely, the drug efflux pump Tap is active against streptomycin but not amikacin (<xref ref-type="bibr" rid="B92">Liu et&#xa0;al., 2019</xref>; <xref ref-type="bibr" rid="B90">Li et&#xa0;al., 2022</xref>). Another example from Mtb is seen with the beta lactamase BlaC. BlaC efficiently degrades penicillins and cephalosporins, which likely explains the poor activity of these beta lactams against Mtb (<xref ref-type="bibr" rid="B64">Hugonnet et&#xa0;al., 2009</xref>; <xref ref-type="bibr" rid="B80">Kurz et&#xa0;al., 2016</xref>). However, carbapenem antibiotics such as meropenem are relatively recalcitrant to BlaC activity. Accordingly, meropenem has strong activity against Mtb and has shown promising results in early clinical trials (<xref ref-type="bibr" rid="B45">Diacon et&#xa0;al., 2016</xref>). Although meropenem is still paired with a beta lactamase inhibitor, the poor activity of BlaC in degrading meropenem likely explains its superior activity relative to other beta lactams. Although there are only a handful of published drug modifying enzymes in Mtb, this phenomenon is likely to be more common than is currently appreciated (<xref ref-type="bibr" rid="B160">Warrier et&#xa0;al., 2016</xref>; <xref ref-type="bibr" rid="B96">Luthra et&#xa0;al., 2018</xref>). More expansive metabolomic studies may help identify the modifications made to antibiotics within the Mtb periplasm and cytosol. A better understanding of intrinsic resistance mechanisms coupled with advances in structural biology and docking algorithms (<xref ref-type="bibr" rid="B111">Pagadala et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B71">Jumper et&#xa0;al., 2021</xref>) may facilitate the design highly specific drug analogs that circumvent the activity of particular intrinsic resistance proteins.</p>
<p>Although a more chemically comprehensive screening effort may reveal unique and novel mechanisms of intrinsic drug resistance, there are some potential pitfalls to this approach. This chemical-genetics strategy would almost certainly fail for a drug like fosfomycin, a non-TB antibiotic which targets the peptidoglycan synthetic enzyme MurA. The lack of fosfomycin activity against Mtb is due to lack of conservation in the drug binding site, not the presence of a specific intrinsic resistance factor (<xref ref-type="bibr" rid="B42">de Smet et&#xa0;al., 1999</xref>). Compounds chosen to undergo chemical-genetic profiling should be rationally selected on the basis of target conservation if that information is available. Finally, we believe this strategy of chemical-genetic profiling is important for compounds early in pre-clinical development, especially those with poorly understood mechanisms. Defining the intrinsic resistome may provide insights regarding the molecular target of the compound and also its mode of action (i.e., downstream effects of activity). Following the logic described above, lead compounds may be optimized further through careful pairing in synergistic drug combinations and engineering to avoid the activity of specific resistance factors.</p>
</sec>
<sec id="s7">
<title>New genetic tools for chemical-genetic studies</title>
<p>So far we have primarily discussed chemical-genetic studies employing three main genetic techniques: transposon mutagenesis, regulated proteolysis, and CRISPRi. High-density transposon mutagenesis was first applied to Mtb almost two decades ago and it continues to be a rich genetic resource. Regulated proteolysis systems and CRISPRi are relatively new genetic tools for Mtb and present powerful strategies to investigate the role of essential genes. However, all three strategies have technical limitations. Continued innovation in mycobacterial genetics will be important to address some of the gaps in our knowledge of Mtb biology, especially intrinsic drug resistance. As mentioned above, many Mtb genes are not expressed at high levels during standard laboratory culture and thus loss-of-function genetic approaches may be insufficient to reveal a phenotype (<xref ref-type="bibr" rid="B134">Schnappinger et&#xa0;al., 2003</xref>; <xref ref-type="bibr" rid="B3">Adams et&#xa0;al., 2011</xref>). For example, genetic disruption of a macrophage-induced efflux pump may not sensitize Mtb to antibiotics in broth since it has a low level of expression under those conditions. However, a drug sensitivity phenotype would likely be observed in drug-treated macrophages. These sorts of chemical-genetic interactions could potentially be captured by screening drugs in complex environments that best mimic host-relevant conditions.</p>
<p>Alternatively, overexpression of that efflux pump in broth conditions would likely confer drug resistance. Gain-of-function genetics represents a complementary tool to capture chemical-genetic phenotypes that would be difficult to observe with loss-of-function techniques. However, high throughput mechanisms of gene activation do not yet exist in Mtb. Such techniques have been employed quite successfully in mammalian systems (<xref ref-type="bibr" rid="B63">Ho et&#xa0;al., 2020</xref>) and, with a lesser degree of success in other bacterial species (<xref ref-type="bibr" rid="B63">Ho et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B73">Kiattisewee et&#xa0;al., 2021</xref>). CRISPR activation (CRISPRa) is one such strategy in which a catalytically dead Cas9 is fused to a transcriptional activator (<xref ref-type="bibr" rid="B63">Ho et&#xa0;al., 2020</xref>). A specific guide RNA can be used to locate the transcription activating Cas9 to the promoter region of a specific gene or operon. Another option for systematic gene activation in bacteria is to use a transposon carrying a strong, outward-facing promoter (<xref ref-type="bibr" rid="B31">Coe et&#xa0;al., 2019</xref>). This system is less specific than CRISPRa and is confounded in many cases by simultaneous activation of one gene and disruption of an adjacent gene. Alternatively, with advances in DNA synthesis, barcoded overexpression plasmids could be built for individual Mtb genes or operons to systematically overexpress Mtb genes. If any of these techniques were employed successfully in Mtb, it may allow for the identification of chemical-genetic phenotypes missed by loss-of-function genetics, particularly for lowly expressed genes.</p>
<p>Lastly, new genetic techniques will be useful for chemical-genetic screens that rely on bacterial outgrowth. For example, Kreutzfeldt et&#xa0;al. (<xref ref-type="bibr" rid="B78">Kreutzfeldt et&#xa0;al., 2022</xref>) performed a TnSeq screen to identify mutants with reduced survival in isoniazid-treated macrophages, which involved outgrowth of the surviving bacteria on agar plates. Transposon mutagenesis is well suited for assays that involve bacterial outgrowth since it generally results in irreversible target gene disruption and does not require an &#x201c;off-switch.&#x201d; However, both CRISPRi and regulated proteolysis will likely have residual target knockdown after the removal of tetracycline, which may prevent or delay viable mutants for essential genes from successfully resuming growth (<xref ref-type="bibr" rid="B123">Qi et&#xa0;al., 2013</xref>). As such, these techniques may not be ideally suited, at least as currently implemented, for these types of screens. Therefore, next generation derivatives of both of these strategies that allow for an efficient &#x201c;off-switch&#x201d; will be paramount to screens relying on outgrowth such as those seeking to identify mutants with impaired survival during drug treatment.</p>
</sec>
<sec id="s8">
<title>Exploring intrinsic resistance heterogeneity across diverse Mtb strains</title>
<p>
<italic>Mycobacterium tuberculosis</italic> displays a remarkable degree of genetic conservation across its major lineages and sublineages (<xref ref-type="bibr" rid="B77">Kremer et&#xa0;al., 1999</xref>). This is likely due to the recent evolutionary emergence of Mtb as well as the lack of horizontal gene transfer (<xref ref-type="bibr" rid="B16">Boritsch et&#xa0;al., 2016</xref>). However, there is a growing appreciation that the genetic differences that do exist between Mtb lineages, sublineages, and strains can have profound impacts on bacterial physiology and can influence virulence, immunogenicity, and drug resistance (<xref ref-type="bibr" rid="B122">Portevin et&#xa0;al., 2011</xref>; <xref ref-type="bibr" rid="B28">Carey et&#xa0;al., 2018</xref>). When measuring drug susceptibility profiles of clinical Mtb isolates, many groups have reported that clinical strains can have a wide range of minimum inhibitory concentrations (MIC) for some drugs (<xref ref-type="bibr" rid="B36">CRyPTIC Consortium, 2018</xref>; <xref ref-type="bibr" rid="B53">Farhat et&#xa0;al., 2019</xref>). Even strains that fall below the critical breakpoint for resistance can display a wide range of MIC values. Some of this MIC heterogeneity may be attributable to low-level acquired drug resistance mutations which are generally poorly understood (<xref ref-type="bibr" rid="B163">Wong et&#xa0;al., 2011</xref>; <xref ref-type="bibr" rid="B34">Colangeli et&#xa0;al., 2018</xref>; <xref ref-type="bibr" rid="B61">Hicks et&#xa0;al., 2020</xref>). However, MIC heterogeneity is also observed for new drugs with novel mechanisms of action and which have been used little (if at all) in the clinic (<xref ref-type="bibr" rid="B12">Bateson et&#xa0;al., 2022</xref>). Accordingly, barring unknown mechanisms of cross-resistance, there has been little or no selective pressure for the evolution of resistance towards these drugs.</p>
<p>There are at least two obvious explanations for MIC heterogeneity for new drugs. First, these differences could be explained by inter-strain differences in drug target vulnerability (<xref ref-type="bibr" rid="B28">Carey et&#xa0;al., 2018</xref>; <xref ref-type="bibr" rid="B18">Bosch et&#xa0;al., 2021</xref>). Using transposon sequencing, Carey et&#xa0;al. found differences in genetic essentiality between different Mtb isolates, with lineage 2 strains displaying a reduced reliance on the glyoxylate shunt (<xref ref-type="bibr" rid="B28">Carey et&#xa0;al., 2018</xref>). The authors could recapitulate this finding using a chemical inhibitor of malate synthase (GlcB), a key enzyme in this pathway. Further demonstrating this concept, recent work by Bosch et&#xa0;al. showed that the cytochrome C reductase gene, <italic>qcrB</italic>, displays enhanced genetic vulnerability in the lineage 2 strain, HN878, compared to the lineage 4 strain H37Rv (<xref ref-type="bibr" rid="B18">Bosch et&#xa0;al., 2021</xref>). Accordingly, HN878 is more susceptible to the QcrB inhibitor, Q203, than is H37Rv.</p>
<p>Alternatively, MIC heterogeneity may reflect differences in the levels of intrinsic drug resistance between Mtb strains. In cases where a particular strain is lacking an intrinsic resistance factor (i.e. <italic>whiB7 or mmpL5</italic>) or sustained mutations that result in its hyperactivitity, there can be a pronounced change in drug sensitivity with a clear genetic basis (<xref ref-type="bibr" rid="B159">Warit et&#xa0;al., 2015</xref>; <xref ref-type="bibr" rid="B154">Villellas et&#xa0;al., 2017</xref>; <xref ref-type="bibr" rid="B103">Merker et&#xa0;al., 2020</xref>; <xref ref-type="bibr" rid="B90">Li et&#xa0;al., 2022</xref>). However, in most cases it is difficult to pinpoint a genetic basis for the differences in intrinsic resistance levels between strains. Future studies aiming to map the genetic basis of intrinsic drug resistance could seek to define these mechanisms in representative Mtb clinical strains, beyond the most common lineage 4 reference strains H37Rv, Erdman, and CDC1551 (<xref ref-type="bibr" rid="B17">Borrell et&#xa0;al., 2019</xref>).</p>
</sec>
<sec id="s9" sec-type="conclusions">
<title>Conclusions</title>
<p>TB remains one of the most difficult infectious diseases to treat. Even strains that are classified as drug sensitive display a remarkably high level of intrinsic resistance to many categories of drugs. Effective strategies must be developed not only to treat drug-resistant TB, but also to treat drug-sensitive TB in a shorter amount of time and with lower relapse rates. To do so, we must identify mechanisms of intrinsic drug resistance in Mtb and find ways around them. The cell envelope remains the most well-characterized and, perhaps, most important contributor to intrinsic drug resistance in Mtb. Successful disruption of the mAGP complex is an established method of disarming intrinsic resistance and sensitizing Mtb to antibiotics. In addition to the envelope, Mtb also encodes many other factors that can block antibiotic action once a drug has entered the cell. Future chemical-genetic studies will be paramount in furthering our understanding of the many layers of intrinsic drug resistance that Mtb has against a diverse set of antibiotics. A thorough genetic dissection of intrinsic resistance in Mtb will hopefully pave the way for more prioritized target-based drug discovery and medicinal chemistry efforts to develop faster-acting TB treatment regimens.</p>
</sec>
<sec id="s10" sec-type="author-contributions">
<title>Author contributions</title>
<p>Literature review: NP and JR, Writing and editing of manuscript: NP and JR. All authors contributed to the article and approved the submitted version.</p>
</sec>
<sec id="s11" sec-type="funding-information">
<title>Funding</title>
<p>This work was supported by the Robertson Therapeutic Development Fund (JR), an NIH/NIAID New Innovator Award (1DP2AI14485001, JR), and a shared NIH TB research unit grant (U19AI162584, JR)</p>
</sec>
<sec id="s12" sec-type="COI-statement">
<title>Conflict of interest</title>
<p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>
</sec>
<sec id="s13" sec-type="disclaimer">
<title>Publisher&#x2019;s note</title>
<p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>
</sec>
</body>
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