The incidence and prevalence of non-tuberculous mycobacterial (NTM) infections have been increasing worldwide and lately led to an emerging public health problem. Among rapidly growing NTM,
is the ...most pathogenic and drug resistant opportunistic germ, responsible for disease manifestations ranging from "curable" skin infections to only "manageable" pulmonary disease. Challenges in
treatment stem from the bacteria's high-level innate resistance and comprise long, costly and non-standardized administration of antimicrobial agents, poor treatment outcomes often related to adverse effects and drug toxicities, and high relapse rates. Drug resistance in
is conferred by an assortment of mechanisms. Clinically acquired drug resistance is normally conferred by mutations in the target genes. Intrinsic resistance is attributed to low permeability of
cell envelope as well as to (multi)drug export systems. However, expression of numerous enzymes by
, which can modify either the drug-target or the drug itself, is the key factor for the pathogen's phenomenal resistance to most classes of antibiotics used for treatment of other moderate to severe infectious diseases, like macrolides, aminoglycosides, rifamycins, β-lactams and tetracyclines. In 2009, when
genome sequence became available, several research groups worldwide started studying
antibiotic resistance mechanisms. At first, lack of tools for
genetic manipulation severely delayed research endeavors. Nevertheless, the last 5 years, significant progress has been made towards the development of conditional expression and homologous recombination systems for
. As a result of recent research efforts, an erythromycin ribosome methyltransferase, two aminoglycoside acetyltransferases, an aminoglycoside phosphotransferase, a rifamycin ADP-ribosyltransferase, a β-lactamase and a monooxygenase were identified to frame the complex and multifaceted intrinsic resistome of
, which clearly contributes to complications in treatment of this highly resistant pathogen. Better knowledge of the underlying mechanisms of drug resistance in
could improve selection of more effective chemotherapeutic regimen and promote development of novel antimicrobials which can overwhelm the existing resistance mechanisms. This article reviews the currently elucidated molecular mechanisms of antibiotic resistance in
, with a focus on its drug-target-modifying and drug-modifying enzymes.
The gastric lamina propria of mice that have been experimentally infected with the pathobiont Helicobacter pylori hosts a dense network of myeloid cells that includes BATF3-dependent CD103+ dendritic ...cells (DCs). We show here that CD103+ DCs are strictly required for gastric Th1 responses to H. pylori and for H. pylori infection control. A similar dependence of type 1 immunity on CD103+ DCs is observed in a Mycobacterium bovis BCG infection model, and in a syngeneic colon cancer model. Strikingly, we find that not only the expansion and/or recruitment of Th1 cells, but also of peripherally induced, neuropilin-negative regulatory T-cells to sites of infection requires BATF3-dependent DCs. A shared feature of the examined models is the strongly reduced production of the chemokines and CXCR3 ligands CXCL9, 10 and 11 in BATF3-deficient mice. The results implicate BATF3-dependent DCs in the recruitment of CXCR3+ effector and regulatory T-cells to target tissues and in their local expansion.
inhibits autophagy to promote its survival in host cells. However, the molecular mechanisms by which
inhibits autophagy are poorly understood. Here, we report a previously unknown mechanism in which
...phosphoribosyltransferase (
PRT) inhibits autophagy in an mTOR, negative regulator of autophagy, independent manner by inducing histone hypermethylation (H3K9me2/3) at the
and
promoters by activating p38-MAPK- and EHMT2 methyltransferase-dependent signaling pathways. Additionally, we find that
PRT induces EZH2 methyltransferase-dependent H3K27me3 hypermethylation and reduces histone acetylation modifications (H3K9ac and H3K27ac) by upregulating histone deacetylase 3 to inhibit autophagy. In summary, this is the first demonstration that
inhibits autophagy by inducing histone hypermethylation in autophagy-related genes to promote intracellular bacterial survival.
Drug resistance in Mycobacterium tuberculosis is a global problem, with major consequences for treatment and public health systems. As the emergence and spread of drug-resistant tuberculosis ...epidemics is largely influenced by the impact of the resistance mechanism on bacterial fitness, we wished to investigate whether compensatory evolution occurs in drug-resistant clinical isolates of M. tuberculosis. By combining information from molecular epidemiology studies of drug-resistant clinical M. tuberculosis isolates with genetic reconstructions and measurements of aminoglycoside susceptibility and fitness in Mycobacterium smegmatis, we have reconstructed a plausible pathway for how aminoglycoside resistance develops in clinical isolates of M. tuberculosis. Thus, we show by reconstruction experiments that base changes in the highly conserved A-site of 16S rRNA that: (i) cause aminoglycoside resistance, (ii) confer a high fitness cost and (iii) destabilize a stem-loop structure, are associated with a particular compensatory point mutation that restores rRNA secondary structure and bacterial fitness, while maintaining to a large extent the drug-resistant phenotype. The same types of resistance and associated mutations can be found in M. tuberculosis in clinical isolates, suggesting that compensatory evolution contributes to the spread of drug-resistant tuberculosis disease.
Post‐translational modification of proteins with prokaryotic ubiquitin‐like protein (Pup) is the bacterial equivalent of ubiquitination in eukaryotes. Mycobacterial pupylation is a two‐step process ...in which the carboxy‐terminal glutamine of Pup is first deamidated by Dop (deamidase of Pup) before ligation of the generated γ‐carboxylate to substrate lysines by the Pup ligase PafA. In this study, we identify a new feature of the pupylation system by demonstrating that Dop also acts as a depupylase in the Pup proteasome system in vivo and in vitro. Dop removes Pup from substrates by specific cleavage of the isopeptide bond. Depupylation can be enhanced by the unfolding activity of the mycobacterial proteasomal ATPase Mpa.
Bacteria modify proteins with a prokaryotic ubiquitin‐like protein (Pup). Here the enzyme that removes Pup in a process analogous to deubiquitination has been identified.
Synthetic biology provides insight into natural gene-network dynamics and enables assembly of engineered transcription circuitries for production of difficult-to-access therapeutic molecules. In ...Mycobacterium tuberculosis EthR binds to a specific operator (OethR) thereby repressing ethA and preventing EthA-catalyzed conversion of the prodrug ethionamide, which increases the resistance of the pathogen to this last-line-of-defense treatment. We have designed a synthetic mammalian gene circuit that senses the EthR-OethR interaction in human cells and produces a quantitative reporter gene expression readout. Challenging of the synthetic network with compounds of a rationally designed chemical library revealed 2-phenylethyl-butyrate as a nontoxic substance that abolished EthR's repressor function inside human cells, in mice, and within M. tuberculosis where it triggered derepression of ethA and increased the sensitivity of this pathogen to ethionamide. The discovery of antituberculosis compounds by using synthetic mammalian gene circuits may establish a new line of defense against multidrug-resistant M. tuberculosis.
Lipoproteins are important components of the mycobacterial cell envelope due to their function in cell wall homeostasis and bacterial virulence. They are post-translationally modified with lipid- and ...glycosyl-residues in various species and interference with acylation or glycosylation leads to reduced growth and attenuated virulence in
. Lipoproteins are also expressed in the emerging and highly drug resistant pathogen
which frequently affects the lungs of patients with chronic pulmonary disease or cystic fibrosis. We investigated post-translational modification, acylation and glycosylation, of heterologously expressed (
LppX and Mpt83) and endogenous (SodC) lipoproteins at the molecular level in
and identified MAB_1122c as protein
-mannosyltransferase (Pmt). Both, heterologous and endogenous lipoproteins carried a characteristic lipid anchor with palmitic acid (C16), palmitoleic acid (C16:1), oleic acid (C18), or tuberculostearic acid (C19) modifications. Multiple hexose-moieties were detected in the
-terminal region of the model lipoproteins expressed in
. Conservation of lipoprotein glycosylation in
and
was revealed and points toward the existence of an
-glycosylation motif or other regulatory mechanisms regarding this post-translational modification. Deletion of MAB_1122c prevented glycosylation and affected susceptibility to specific antibiotics which are large or target peptidoglycan synthesis and to lysozyme. Cell envelope permeability of
Δ
was increased and mutant bacteria showed reduced survival inside macrophages. The results provide a link between post-translational modification of lipoproteins and the permeability of the mycobacterial cell envelope which stresses the importance of lipoproteins as components of this complex structure.
Lipoproteins of Gram-negative and Gram-positive bacteria carry a thioether-bound diacylglycerol but differ by a fatty acid amide bound to the α-amino group of the universally conserved cysteine. In ...Escherichia coli the N-terminal acylation is catalyzed by the N-acyltransferase Lnt. Using E. coli Lnt as a query in a BLASTp search, we identified putative lnt genes also in Gram-positive mycobacteria. The Mycobacterium tuberculosis lipoprotein LppX, heterologously expressed in Mycobacterium smegmatis, was N-acylated at the N-terminal cysteine, whereas LppX expressed in a M. smegmatis lnt::aph knock-out mutant was accessible for N-terminal sequencing. Western blot analyses of a truncated and tagged form of LppX indicated a smaller size of about 0.3 kDa in the lnt::aph mutant compared with the parental strain. Matrix-assisted laser desorption ionization time-of-flight/time-of-flight analyses of a trypsin digest of LppX proved the presence of the diacylglycerol modification in both strains, the parental strain and lnt::aph mutant. N-Acylation was found exclusively in the M. smegmatis parental strain. Complementation of the lnt::aph mutant with M. tuberculosis ppm1 restored N-acylation. The substrate for N-acylation is a C16 fatty acid, whereas the two fatty acids of the diacylglycerol residue were identified as C16 and C19:0 fatty acid, the latter most likely tuberculostearic acid. We demonstrate that mycobacterial lipoproteins are triacylated. For the first time to our knowledge, we identify Lnt activity in Gram-positive bacteria and assigned the responsible genes. In M. smegmatis and M. tuberculosis the open reading frames are annotated as MSMEG_3860 and M. tuberculosis ppm1, respectively.
Fidaxomicin (FDX) is a marketed antibiotic for the treatment of Clostridioides difficile infections (CDI). Fidaxomicin displays antibacterial properties against many Gram-positive bacteria, yet the ...application of this antibiotic is currently limited to treatment of CDI. Semisynthetic modifications present a promising strategy to improve its pharmacokinetic properties and also circumvent resistance development by broadening the structural diversity of the derivatives. Here, based on a rational design using cryo-EM structural analysis, we implement two strategic site-selective catalytic reactions with a special emphasis to study the role of the carbohydrate units. Site-selective introduction of various ester moieties on the noviose as well as a Tsuji-Trost type rhamnose cleavage allow the synthesis of novel fidaxomicin analogs with promising antibacterial activities against C. difficile and Mycobacterium tuberculosis.
Preprolipopoprotein diacylglyceryl transferase (Lgt) is the gating enzyme of lipoprotein biosynthesis, and it attaches a lipid structure to the N-terminal part of preprolipoproteins. Using Lgt from ...Escherichia coli in a BLASTp search, we identified the corresponding Lgt homologue in Mycobacterium tuberculosis and two homologous (MSMEG_3222 and MSMEG_5408) Lgt in Mycobacterium smegmatis. M. tuberculosis lgt was shown to be essential, but an M. smegmatis ΔMSMEG_3222 mutant could be generated. Using Triton X-114 phase separation and 14Cpalmitic acid incorporation, we demonstrate that MSMEG_3222 is the major Lgt in M. smegmatis. Recombinant M. tuberculosis lipoproteins Mpt83 and LppX are shown to be localized in the cell envelope of parental M. smegmatis but were absent from the cell membrane and cell wall in the M. smegmatis ΔMSMEG_3222 strain. In a proteomic study, 106 proteins were identified and quantified in the secretome of wild-type M. smegmatis, including 20 lipoproteins. All lipoproteins were secreted at higher levels in the ΔMSMEG_3222 mutant. We identify the major Lgt in M. smegmatis, show that lipoproteins lacking the lipid anchor are secreted into the culture filtrate, and demonstrate that M. tuberculosis lgt is essential and thus a validated drug target.
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