Mycobacterium tuberculosis protease MarP activates a peptidoglycan hydrolase during acid stress.

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 RipA's 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 Mtb's survival in acidic conditions.

Mycobacterium tuberculosis releases an antacid that remodels phagosomes.

Mycobacterium tuberculosis (Mtb) is the world's most deadly pathogen. Unlike less virulent mycobacteria, Mtb produces 1-tuberculosinyladenosine (1-TbAd), an unusual terpene nucleoside of unknown function. In the present study 1-TbAd has been shown to be a naturally evolved phagolysosome disruptor. 1-TbAd is highly prevalent among patient-derived Mtb strains, where it is among the most abundant lipids produced. Synthesis of TbAd analogs and their testing in cells demonstrate that their biological action is dependent on lipid linkage to the 1-position of adenosine, which creates a strong conjugate base. Furthermore, C20 lipid moieties confer passage through membranes. 1-TbAd selectively accumulates in acidic compartments, where it neutralizes the pH and swells lysosomes, obliterating their multilamellar structure. During macrophage infection, a 1-TbAd biosynthesis gene (Rv3378c) confers marked phagosomal swelling and intraphagosomal inclusions, demonstrating an essential role in regulating the Mtb cellular microenvironment. Although macrophages kill intracellular bacteria through phagosome acidification, Mtb coats itself abundantly with antacid.

Disruption of an M. tuberculosis membrane protein causes a magnesium-dependent cell division defect and failure to persist in mice.

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 ß-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.

Interruption of mycothiol synthesis and intracellular redox status impact iron-regulated reporter activation in Mycobacterium smegmatis.

Iron scavenging is required for full virulence of mycobacterial pathogens. During infection, the host immune response restricts mycobacterial access to iron, which is essential for bacterial respiration and DNA synthesis. The Mycobacterium tuberculosis iron-dependent regulator (IdeR) responds to changes in iron accessibility by repressing iron-uptake genes when iron is available. In contrast, iron-uptake gene transcription is induced when iron is depleted. The ideR gene is essential in M. tuberculosis and is required for bacterial growth. To further study how iron regulates transcription, wee developed an iron responsive reporter system that relies on an IdeR-regulated promoter to drive Cre and loxP mediated recombination in Mycobacterium smegmatis. Recombination leads to the expression of an antibiotic resistance gene so that mutations that activate the IdeR-regulated promoter can be selected. A transposon library in the background of this reporter system was exposed to media containing iron and hemin, and this resulted in the selection of mutants in the antioxidant mycothiol synthesis pathway. We validated that inactivation of the mycothiol synthesis gene mshA results in increased recombination and increased IdeR-regulated promoter activity in the reporter system. Further, we show that vitamin C, which has been shown to oxidize iron through the Fenton reaction, can decrease promoter activity in the mshA mutant. We conclude that the intracellular redox state balanced by mycothiol can alter IdeR activity in the presence of iron.IMPORTANCEMycobacterium smegmatis is a tractable organism to study mycobacterial gene regulation. We used M. smegmatis to construct a novel recombination-based reporter system that allows for the selection of mutations that deregulate a promoter of interest. Transposon mutagenesis and insertion sequencing (TnSeq) in the recombination reporter strain identified genes that impact iron regulated promoter activity in mycobacteria. We found that the mycothiol synthesis gene mshA is required for IdeR mediated transcriptional regulation by maintaining intracellular redox balance. By affecting the oxidative state of the intracellular environment, mycothiol can modulate iron-dependent transcriptional activity. Taken more broadly, this novel reporter system can be used in combination with transposon mutagenesis to identify genes that are required by Mycobacterium tuberculosis to overcome temporary or local changes in iron availability during infection.

Reframing antimicrobial resistance as a continuous spectrum of manifestations.

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.

Multiform antimicrobial resistance from a metabolic mutation.

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.

Building Walls: Work That Never Ends.

Fluorescent amino acid analogs have proven to be useful tools for studying the dynamics of peptidoglycan metabolism. García-Heredia and colleagues showed that their route of incorporation differs depending on the adjunct fluorophore and applied this property to investigate mycobacterial peptidoglycan synthesis and remodeling with heightened granularity.

Persistent Mycobacterium tuberculosis infection in mice requires PerM for successful cell division.

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.

Distinct Spatiotemporal Dynamics of Peptidoglycan Synthesis between Mycobacterium smegmatis and Mycobacterium tuberculosis.

Peptidoglycan (PG), a polymer cross-linked by d-amino acid-containing peptides, is an essential component of the bacterial cell wall. We found that a fluorescent d-alanine analog (FDAA) incorporates chiefly at one of the two poles in Mycobacterium smegmatis but that polar dominance varies as a function of the cell cycle in Mycobacterium tuberculosis: immediately after cytokinesis, FDAAs are incorporated chiefly at one of the two poles, but just before cytokinesis, FDAAs are incorporated comparably at both. These observations suggest that mycobacterial PG-synthesizing enzymes are localized in functional compartments at the poles and septum and that the capacity for PG synthesis matures at the new pole in M. tuberculosis Deeper knowledge of the biology of mycobacterial PG synthesis may help in discovering drugs that disable previously unappreciated steps in the process.IMPORTANCE People are dying all over the world because of the rise of antimicrobial resistance to medicines that could previously treat bacterial infections, including tuberculosis. Here, we used fluorescent d-alanine analogs (FDAAs) that incorporate into peptidoglycan (PG)-the synthesis of which is an attractive drug target-combined with high- and super-resolution microscopy to investigate the spatiotemporal dynamics of PG synthesis in M. smegmatis and M. tuberculosis FDAA incorporation predominates at one of the two poles in M. smegmatis In contrast, while FDAA incorporation into M. tuberculosis is also polar, there are striking variations in polar dominance as a function of the cell cycle. This suggests that enzymes involved in PG synthesis are localized in functional compartments in mycobacteria and that M. tuberculosis possesses a mechanism for maturation of the capacity for PG synthesis at the new pole. This may help in discovering drugs that cripple previously unappreciated steps in the process.

Stressed mycobacteria use the chaperone ClpB to sequester irreversibly oxidized proteins asymmetrically within and between cells.

Mycobacterium 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.

Target-based screen against a periplasmic serine protease that regulates intrabacterial pH homeostasis in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) maintains its intrabacterial pH (pHIB) near neutrality in the acidic environment of phagosomes within activated macrophages. A previously reported genetic screen revealed that Mtb loses this ability when the mycobacterial acid resistance protease (marP) gene is disrupted. In the present study, a high throughput screen (HTS) of compounds against the protease domain of MarP identified benzoxazinones as inhibitors of MarP. A potent benzoxazinone, BO43 (6-chloro-2-(2'-methylphenyl)-4H-1,3-benzoxazin-4-one), acylated MarP and lowered Mtb's pHIB and survival during incubation at pH 4.5. BO43 had similar effects on MarP-deficient Mtb, suggesting the existence of additional target(s). Reaction of an alkynyl-benzoxazinone, BO43T, with Mycobacterium bovis variant bacille Calmette-Guérin (BCG) followed by click chemistry with azido-biotin identified both the MarP homologue and the high temperature requirement A1 (HtrA1) homologue, an essential protein. Thus, the chemical probe identified through a target-based screen not only reacted with its intended target in the intact cells but also implicated an additional enzyme that had eluded a genetic screen biased against essential genes.

Metallobiology of host-pathogen interactions: an intoxicating new insight.

Iron, zinc and copper, among others, are transition metals with multiple biological roles that make them essential elements for life. Beyond the strict requirement of transition metals by the vertebrate immune system for its proper functioning, novel mechanisms involving direct metal intoxication of microorganisms are starting to be unveiled as important components of the immune system, in particular against Mycobacterium tuberculosis. In parallel, metal detoxification systems in bacteria have been recently characterized as crucial microbial virulence determinants. Here, we will focus on these exciting advancements implicating copper- and zinc-mediated microbial poisoning as a novel innate immune mechanism against microbial pathogens, shedding light on an emerging field in the metallobiology of host-pathogen interactions.

Mycobacterial p(1)-type ATPases mediate resistance to zinc poisoning in human macrophages.

Mycobacterium tuberculosis thrives within macrophages by residing in phagosomes and preventing them from maturing and fusing with lysosomes. A parallel transcriptional survey of intracellular mycobacteria and their host macrophages revealed signatures of heavy metal poisoning. In particular, mycobacterial genes encoding heavy metal efflux P-type ATPases CtpC, CtpG, and CtpV, and host cell metallothioneins and zinc exporter ZnT1, were induced during infection. Consistent with this pattern of gene modulation, we observed a burst of free zinc inside macrophages, and intraphagosomal zinc accumulation within a few hours postinfection. Zinc exposure led to rapid CtpC induction, and ctpC deficiency caused zinc retention within the mycobacterial cytoplasm, leading to impaired intracellular growth of the bacilli. Thus, the use of P(1)-type ATPases represents a M. tuberculosis strategy to neutralize the toxic effects of zinc in macrophages. We propose that heavy metal toxicity and its counteraction might represent yet another chapter in the host-microbe arms race.

A murine DC-SIGN homologue contributes to early host defense against Mycobacterium tuberculosis.

The C-type lectin dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN) mediates the innate immune recognition of microbial carbohydrates. We investigated the function of this molecule in the host response to pathogens in vivo, by generating mouse lines lacking the DC-SIGN homologues SIGNR1, SIGNR3, and SIGNR5. Resistance to Mycobacterium tuberculosis was impaired only in SIGNR3-deficient animals. SIGNR3 was expressed in lung phagocytes during infection, and interacted with M. tuberculosis bacilli and mycobacterial surface glycoconjugates to induce secretion of critical host defense inflammatory cytokines, including tumor necrosis factor (TNF). SIGNR3 signaling was dependent on an intracellular tyrosine-based motif and the tyrosine kinase Syk. Thus, the mouse DC-SIGN homologue SIGNR3 makes a unique contribution to protection of the host against a pulmonary bacterial pathogen.