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To fight antimicrobial resistance (AMR), we must recognize and target all its manifestations. In this review, we briefly summarize the history that led to recognition of the various manifestations of AMR in bacterial pathogens and the ways in which they interrelate. We emphasize the importance of distinguishing between AMR arising from genetic alterations versus induction of endogenous machinery in response to environmental triggers, including - paradoxically - stresses from host immunity and antimicrobial therapy. We present an integrated view of AMR by reframing it as a spectrum of phenotypes within a continuous three-dimensional space defined by the growth rate, prevalence, and kill rate of cells displaying AMR. Finally, we reflect on strategies that may help stem the emergence of AMR.

Disruption of a metabolic pathway causes tolerance, high persistence, and MIC-shifted resistance to diverse antibiotics., A critical challenge for microbiology and medicine is how to cure infections by bacteria that survive antibiotic treatment by persistence or tolerance. Seeking mechanisms behind such high survival, we developed a forward-genetic method for efficient isolation of high-survival mutants in any culturable bacterial species. We found that perturbation of an essential biosynthetic pathway (arginine biosynthesis) in a mycobacterium generated three distinct forms of resistance to diverse antibiotics, each mediated by induction of WhiB7: high persistence and tolerance to kanamycin, high survival upon exposure to rifampicin, and minimum inhibitory concentration–shifted resistance to clarithromycin. As little as one base change in a gene that encodes, a metabolic pathway component conferred multiple forms of resistance to multiple antibiotics with different targets. This extraordinary resilience may help explain how substerilizing exposure to one antibiotic in a regimen can induce resistance to others and invites development of drugs targeting the mediator of multiform resistance, WhiB7.

Bacterial chaperones ClpB and DnaK, homologs of the respective eukaryotic heat shock proteins Hsp104 and Hsp70, are essential in the reactivation of toxic protein aggregates that occur during translation or periods of stress. In the pathogen Mycobacterium tuberculosis (Mtb), the protective effect of chaperones extends to survival in the presence of host stresses, such as protein-damaging oxidants. However, we lack a full understanding of the interplay of Hsps and other stress response genes in mycobacteria. Here, we employ genome-wide transposon mutagenesis to identify the genes that support clpB function in Mtb. In addition to validating the role of ClpB in Mtb's response to oxidants, we show that HtpG, a homolog of Hsp90, plays a distinct role from ClpB in the proteotoxic stress response. While loss of neither clpB nor htpG is lethal to the cell, loss of both through genetic depletion or small molecule inhibition impairs recovery after exposure to host-like stresses, especially reactive nitrogen species. Moreover, defects in cells lacking clpB can be complemented by overexpression of other chaperones, demonstrating that Mtb's stress response network depends upon finely tuned chaperone expression levels. These results suggest that inhibition of multiple chaperones could work in concert with host immunity to disable Mtb.

Insufficient development of new antibiotics and the rising resistance of bacteria to those that we have are putting the world at risk of losing the most widely curative class of medicines currently available. Preventing deaths from antimicrobial resistance (AMR) will require exploiting emerging knowledge not only about genetic AMR conferred by horizontal gene transfer or de novo mutations but also about phenotypic AMR, which lacks a stably heritable basis. This Review summarizes recent advances and continuing limitations in our understanding of AMR and suggests approaches for combating its clinical consequences, including identification of previously unexploited bacterial targets, new antimicrobial compounds, and improved combination drug regimens.

The ability of Mycobacterium tuberculosis (Mtb) to persist in its host is central to the pathogenesis of tuberculosis, yet the underlying mechanisms remain incompletely defined. PerM, an integral membrane protein, is required for persistence of Mtb in mice. Here, we show that perM deletion caused a cell division defect specifically during the chronic phase of mouse infection, but did not affect Mtb’s cell replication during acute infection. We further demonstrate that PerM is required for cell division in chronically infected mice and in vitro under host-relevant stresses because it is part of the mycobacterial divisome and stabilizes the essential divisome protein FtsB. These data highlight the importance of sustained cell division for Mtb persistence, define condition-specific requirements for cell division and reveal that survival of Mtb during chronic infection depends on a persistence divisome.

Summary Vancomycin-resistant Enterococcus (VRE) are highly antibiotic-resistant and readily transmissible pathogens that cause severe infections in hospitalized patients. We discovered that lithocholic acid (LCA), a secondary bile acid prevalent in the cecum and colon of mice and humans, impairs separation of growing VRE diplococci, causing the formation of long chains and increased biofilm formation. Divalent cations reversed this LCA-induced switch to chaining and biofilm formation. Experimental evolution in the presence of LCA yielded mutations in the essential two-component kinase yycG/walK and three-component response regulator liaR that locked VRE in diplococcal mode, impaired biofilm formation, and increased susceptibility to the antibiotic daptomycin. These mutant VRE strains were deficient in host colonization because of their inability to compete with intestinal microbiota. This morphotype switch presents a potential non-bactericidal therapeutic target that may help clear VRE from the intestines of dominated patients, as occurs frequently during hematopoietic stem cell transplantation.

Mycobacterium tuberculosis (Mtb) can persist in the human host in a latent state for decades, in part because it has the ability to withstand numerous stresses imposed by host immunity. Prior studies have established the essentiality of the periplasmic protease MarP for Mtb to survive in acidified phagosomes and establish and maintain infection in mice. However, the proteolytic substrates of MarP that mediate these phenotypes were unknown. Here, we used biochemical methods coupled with supravital chemical probes that facilitate imaging of nascent peptidoglycan to demonstrate that during acid stress MarP cleaves the peptidoglycan hydrolase RipA, a process required for RipA9s activation. Failure of RipA processing in MarP‐deficient cells leads to cell elongation and chain formation, a hallmark of progeny cell separation arrest. Our results suggest that sustaining peptidoglycan hydrolysis, a process required for cell elongation, separation of progeny cells, and cell wall homeostasis in growing cells, may also be essential for Mtb9s survival in acidic conditions.

INTRODUCTION Mycobacterium tuberculosis (Mtb) is the leading global cause of lethal infection in humans and accounts for the largest number of drug-resistant infections by a single bacterial pathogen. Resistance is particularly high against the most widely prescribed tuberculosis (TB) drug, isoniazid. Isoniazid blocks synthesis of mycolates, ultralong-chain fatty acids that provide structure to the waxy coat that surrounds Mtb cells and are incorporated into some of its virulence lipids. There is currently no known method to block the synthesis of both mycolates and nonmycolate-containing virulence lipids of Mtb at a single point of control. One such control point is phosphopantetheinyl transferase (PptT). PptT transfers 4′-phosphopantetheine (Ppt) from coenzyme A (CoA) to acyl carrier proteins (ACPs) that synthesize the lipids critical to Mtb structural integrity and virulence. RATIONALE TB drug discovery often begins with whole-cell, high-throughput screens that yield compounds that kill Mtb by unknown means. Selection of Mtb mutants resistant to these compounds can indicate candidate targets of the active compound, but experimental validation is required to confirm the functionally relevant target, which is often an enzyme. A suitable target must be essential in vivo, such that its inhibition precludes development of TB in animal models, but also “vulnerable,” meaning that a pharmacologically attainable level of inhibition should be lethal to Mtb within a patient. The inhibitor should act only on Mtb, and resistance should be rare. RESULTS Screening a chemical library revealed an amidino-urea compound called “8918” that kills Mtb, including drug-resistant clinical isolates. 8918 inhibits Mtb in mice and spares other bacteria, yeast, and mammalian cells. Rare Mtb mutants resistant to 8918 bore a point mutation in the PptT gene rv2794c, altering an amino acid residue overlying the Ppt-binding pocket of PptT. When Mtb carried the mutant allele as an extra copy of rv2794c, the Mtb was protected from 8918. 8918 inhibited recombinant PptT, albeit noncompetitively and incompletely. The impact of 8918 on the Mtb metabolome and lipids was consistent with inhibition of PptT in the intact cell. A crystal structure of the PptT-8918 complex at 1.8-Å resolution confirmed that 8918 binds within the Ppt binding pocket, adjacent to the phosphoadenosine phosphate portion of CoA. Intact CoA remained in the PptT-8918 complex, but the Ppt arm was displaced, decreasing but not abolishing PptT’s catalytic activity. Strains of Mtb producing reduced amounts of PptT became hypersensitive to 8918. It was puzzling that even partial inhibition of PptT killed Mtb. We observed that mutants with disruption of rv2795c, a gene encoding a hypothetical protein, were also highly resistant to 8918. Recombinant Rv2795c protein hydrolyzed Ppt from a mycolate-building holo-ACP that is a substrate for PptT. The action of this Ppt hydrolase (PptH) resembled that of nonhomologous enzymes called ACP hydrolases that remove Ppt from ACPs in vitro but whose physiological function is unknown. CONCLUSION We identified a small molecule that kills Mtb by inhibiting PptT, demonstrating that a key enzyme in CoA metabolism is a viable target for TB drug development. Even partial inhibition of PptT is toxic to Mtb, likely because PptH synergizes with the inhibitor by undoing the PptT reaction. PptT and PptH are co-regulated by translation from the same operon, and thus Mtb cannot respond to inhibition of PptT by making more PptT without also generating more PptH. The joint functioning of PptT and PptH suggests that Mtb closely regulates the activation of ACPs. The transcriptional co-regulation and constitutive function of both members of the PptT-PptH couple suggests that a posttranslational signal that impairs PptT more than PptH could allow Mtb to rapidly reverse a prior commitment to synthesis of its metabolically most costly lipids.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains one of the world’s deadliest infectious diseases and urgently requires new antibiotics to treat drug-resistant strains and to decrease the duration of therapy. During infection, Mtb encounters numerous stresses associated with host immunity, including hypoxia, reactive oxygen and nitrogen species, mild acidity, nutrient starvation, and metal sequestration and intoxication. The Mtb proteostasis network, composed of chaperones, proteases, and a eukaryotic-like proteasome, provides protection from stresses and chemistries of host immunity by maintaining the integrity of the mycobacterial proteome. In this Review, we explore the proteostasis network as a noncanonical target for antibacterial drug discovery.

SummaryMycobacterium tuberculosis (Mtb) defends itself against host immunity and chemotherapy at several levels, including the repair or degradation of irreversibly oxidized proteins (IOPs). To investigate how Mtb deals with IOPs that can neither be repaired nor degraded, we used new chemical and biochemical probes and improved image analysis algorithms for time-lapse microscopy to reveal a defense against stationary phase stress, oxidants, and antibiotics—the sequestration of IOPs into aggregates in association with the chaperone ClpB, followed by the asymmetric distribution of aggregates within bacteria and between their progeny. Progeny born with minimal IOPs grew faster and better survived a subsequent antibiotic stress than their IOP-burdened sibs. ClpB-deficient Mtb had a marked recovery defect from stationary phase or antibiotic exposure and survived poorly in mice. Treatment of tuberculosis might be assisted by drugs that cripple the pathway by which Mtb buffers, sequesters, and asymmetrically distributes IOPs.

Vancomycin resistant Enterococcus (VRE) is a highly antibiotic-resistant and readily transmissible pathogen that causes severe infections in hospitalized patients. We discovered that lithocholic acid (LCA), a secondary bile acid prevalent in the cecum and colon of mice and humans, impaired separation of growing VRE diplococci causing the formation of long chains, increasing biofilm formation and enhancing VRE’s ability to colonize the host intestine. Divalent cations reversed the switch to chaining and biofilm formation. Experimental evolution in the presence of LCA yielded mutations in yycG/walK and liaR that locked VRE in diplococcal mode, impaired biofilm formation and increased susceptibility to Daptomycin. These strains were deficient in host colonization specifically by affecting VRE’s ability to compete with intestinal microbiota. This morphotype switch presents a non-bactericidal therapeutic target that may help to clear VRE from the intestines of dominated patients, as occurs frequently during hematopoietic stem cell transplantation.

We identified N,C-capped dipeptides that are selective for the Mycobacterium tuberculosis proteasome over human constitutive and immunoproteasomes. Differences in S3 and S1 binding pockets appeared to account for species-selectivity. The inhibitors are able to penetrate mycobacteria and kill non-replicating M. tuberculosis under nitrosative stress.

Rifampicin, which inhibits bacterial RNA polymerase, provides one of the most effective treatments for tuberculosis. Inhibition of the transcription termination factor Rho is used to treat some bacterial infections, but its importance varies across bacteria. Here we show that Rho of Mycobacterium tuberculosis functions to both define the 3′ ends of mRNAs and silence substantial fragments of the genome. Brief inactivation of Rho affects over 500 transcripts enriched for genes of foreign DNA elements and bacterial virulence factors. Prolonged inactivation of Rho causes extensive pervasive transcription, a genome-wide increase in antisense transcripts, and a rapid loss of viability of replicating and non-replicating M. tuberculosis in vitro and during acute and chronic infection in mice. Collectively, these data suggest that inhibition of Rho may provide an alternative strategy to treat tuberculosis with an efficacy similar to inhibition of RNA polymerase., The transcription termination factor Rho is essential for growth in some bacteria but not in others. Here, Botella et al. show that Rho inactivation causes extensive pervasive transcription and loss of viability of the pathogen Mycobacterium tuberculosis both in vitro and in a mouse model of infection.

Isoxyl (ISO) and thiacetazone (TAC), two prodrugs once used in the clinical treatment of tuberculosis, have long been thought to abolish Mycobacterium tuberculosis (M. tuberculosis) growth through the inhibition of mycolic acid biosynthesis, but their respective targets in this pathway have remained elusive. Here we show that treating M. tuberculosis with ISO or TAC results in both cases in the accumulation of 3-hydroxy C(18), C(20), and C(22) fatty acids, suggestive of an inhibition of the dehydratase step of the fatty-acid synthase type II elongation cycle. Consistently, overexpression of the essential hadABC genes encoding the (3R)-hydroxyacyl-acyl carrier protein dehydratases resulted in more than a 16- and 80-fold increase in the resistance of M. tuberculosis to ISO and TAC, respectively. A missense mutation in the hadA gene of spontaneous ISO- and TAC-resistant mutants was sufficient to confer upon M. tuberculosis high level resistance to both drugs. Other mutations found in hypersusceptible or resistant M. tuberculosis and Mycobacterium kansasii isolates mapped to hadC. Mutations affecting the non-essential mycolic acid methyltransferases MmaA4 and MmaA2 were also found in M. tuberculosis spontaneous ISO- and TAC-resistant mutants. That MmaA4, at least, participates in the activation of the two prodrugs as proposed earlier is not supported by our biochemical evidence. Instead and in light of the known interactions of both MmaA4 and MmaA2 with HadAB and HadBC, we propose that mutations affecting these enzymes may impact the binding of ISO and TAC to the dehydratases.

Eukaryotic-like Ser/Thr protein kinases (STPKs) are present in many bacterial species, where they control various physiological and virulence processes by enabling microbial adaptation to specific environmental signals. PknJ is the only member of the 11 STPKs identified inMycobacterium tuberculosisthat still awaits characterization. Here we report that PknJ is a functional kinase that forms dimersin vitro, and contains a single transmembrane domain. Using a high-density peptide-chip-based technology, multiple potential mycobacterial targets were identified for PknJ. We confirmed PknJ-dependent phosphorylation of four of these targets: PknJ itself, which autophosphorylates at Thr168, Thr171and Thr173residues; the transcriptional regulator EmbR; the methyltransferase MmaA4/Hma involved in mycolic acid biosynthesis; and the dipeptidase PepE, whose encoding gene is located next topknJin the mycobacterial genome. Our results provide a number of candidate phospho-targets for PknJ and possibly other mycobacterial STPKs that could be studied to investigate the role of STPKs inM. tuberculosisphysiology and virulence.

For 4 decades, in vivo and in vitro studies have suggested that sulfoglycolipids (SGLs) play a role in the virulence or pathogenesis of the tubercle bacilli. However, the SGL structure and biosynthesis pathway remain only partially elucidated. Using the modern tools of structural analysis, including MALDI-time-of-flight MS, MS/MS, and two-dimensional NMR, we reevaluated the structure of the different SGL acyl (di-, tri-, and tetra-acylated) forms of the reference strain Mycobacterium tuberculosis H37Rv, as well as those produced by the mmpL8 knockout strains previously described to intracellularly accumulate di-acylated SGL. We report here the identification of new acyl forms: di-acylated SGL esterified by simple fatty acids only, as well as mono-acylated SGL bearing a hydroxyphthioceranoic acid, which were characterized in the wild-type strain. In a clinical strain, a complete family of mono-acylated SGLs was characterized in high abundance for the first time. For the mmpL8 mutant, SGLs were found to be esterified i) by an oxophthioceranoic acid, never observed so far, and ii) at nonconventional positions in the case of the unexpected tri-acylated forms. Our results further confirm the requirement of MmpL8 for the complete assembly of the tetra-acylated forms of SGL and also provide, by the discovery of new intermediates, insights in terms of the possible SGL biosynthetic pathways.

The identification of Mycobacterium tuberculosis genes necessary for persistence in vivo provides insight into bacterial biology as well as host defense strategies. We show that disruption of M. tuberculosis membrane protein PerM (Rv0955) resulted in an IFN-γ-dependent persistence defect in chronic mouse infection despite the mutant’s near normal growth during acute infection. The perM mutant required increased magnesium for replication and survival; incubation in low magnesium media resulted in cell elongation and lysis. Transcriptome analysis of the perM mutant grown in reduced magnesium revealed upregulation of cell division and cell wall biosynthesis genes, and live cell imaging showed PerM accumulation at the division septa in M. smegmatis. The mutant was acutely sensitive to β-lactam antibiotics, including specific inhibitors of cell division-associated peptidoglycan transpeptidase FtsI. Together, these data implicate PerM as a novel player in mycobacterial cell division and pathogenesis, and are consistent with the hypothesis that immune activation deprives M. tuberculosis of magnesium., Author Summary The success of Mycobacterium tuberculosis (Mtb) as a human pathogen is due to ability to persist in chronic infection, despite a robust adaptive immune response by the host. The mechanisms by which Mtb achieves this are, however, poorly understood. Here we show that a novel integral membrane protein, Rv0955/PerM, is essential for Mtb persistence during chronic mouse infection. The perM mutant required increased magnesium compared to wild type Mtb for replication and survival in culture and elongated in media with reduced magnesium concentration. Transcriptomic, electron microscopy and live cell imaging approaches provided evidence that PerM is involved in cell division. The survival defects of the perM mutant in reduced magnesium and during chronic mouse infection are consistent with the hypothesis that magnesium deprivation constitutes an IFN-γ dependent host defense strategy. This work also has potential clinical implications, as disruption of PerM renders Mtb susceptible to β-lactam antibiotics, which are commonly used to treat non-mycobacterial infections.

Tuberculosis (TB) is characterized by a tight interplay between Mycobacterium tuberculosis and host cells within granulomas. These cellular aggregates restrict bacterial spreading, but do not kill all the bacilli, which can persist for years. In-depth investigation of M. tuberculosis interactions with granuloma-specific cell populations are needed to gain insight into mycobacterial persistence, and to better understand the physiopathology of the disease. We have analyzed the formation of foamy macrophages (FMs), a granuloma-specific cell population characterized by its high lipid content, and studied their interaction with the tubercle bacillus. Within our in vitro human granuloma model, M. tuberculosis long chain fatty acids, namely oxygenated mycolic acids (MA), triggered the differentiation of human monocyte-derived macrophages into FMs. In these cells, mycobacteria no longer replicated and switched to a dormant non-replicative state. Electron microscopy observation of M. tuberculosis–infected FMs showed that the mycobacteria-containing phagosomes migrate towards host cell lipid bodies (LB), a process which culminates with the engulfment of the bacillus into the lipid droplets and with the accumulation of lipids within the microbe. Altogether, our results suggest that oxygenated mycolic acids from M. tuberculosis play a crucial role in the differentiation of macrophages into FMs. These cells might constitute a reservoir used by the tubercle bacillus for long-term persistence within its human host, and could provide a relevant model for the screening of new antimicrobials against non-replicating persistent mycobacteria., Author Summary Mycobacterium tuberculosis, the causative agent of tuberculosis, is responsible for dramatic health problems globally. It is estimated that this pathogen infects one-third of the human population and causes three million deaths annually. Most individuals remain asymptomatic for several years before developing an active disease. In such individuals, the bacilli are not cleared but rather persist in a dormant state. Major goals of TB research are to (i) understand how the bacilli remain alive for years within infected individuals, and (ii) find how to prevent their reactivation and hence clinical disease. During dormancy, most of the bacilli are confined to granulomas that consist of well-defined aggregates of different host immune cells. Granulomas prevent spreading of bacilli. In this study, we analyzed the role of a particular cell population found within granulomas, the “foamy macrophages”. These cells are filled with droplets of lipids, a well-known nutrient for persistent bacilli. We found that within these cells, the bacilli do not replicate, but remain alive and seem to internalize host lipids. The foamy macrophages might thus constitute a reservoir for persisting bacilli within their human host, and could provide a relevant model for screening of new antimicrobials against non-replicating persistent mycobacteria.