Dynamin-like proteins mediate extracellular vesicle secretion in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) secretes extracellular vesicles (EVs) containing a variety of proteins, lipoproteins, and lipoglycans. While emerging evidence suggests that EVs contribute to tuberculosis pathogenesis, the factors and molecular mechanisms involved in mycobacterial EV production have not been identified. In this study, we use a genetic approach to identify Mtb proteins that mediate vesicle release in response to iron limitation and antibiotic exposure. We uncover a critical role for the isoniazid-induced, dynamin-like proteins, IniA and IniC, in mycobacterial EV biogenesis. Further characterization of a Mtb iniA mutant shows that the production of EVs enables intracellular Mtb to export bacterial components into the extracellular environment to communicate with host cells and potentially modulate the immune response. The findings advance our understanding of the biogenesis and functions of mycobacterial EVs and provide an avenue for targeting vesicle production in vivo.

Redox Cycling Dioxonaphthoimidazoliums Disrupt Iron Homeostasis in Mycobacterium bovis Bacillus Calmette-Guérin.

The dioxonaphthoimidazolium scaffold is a novel, highly bactericidal redox cycling antituberculosis chemotype that is reliant on the respiratory enzyme Type II NADH dehydrogenase (NDH2) for the generation of reactive oxygen species (ROS). Here, we employed Mycobacterium bovis Bacillus Calmette-Guérin (M. bovis BCG) reporter strains to show that ROS generated by the redox cycler SA23 simulated an iron deficient state in the bacteria, which led to a compensatory increase in the expression of the iron acquisition mbtB gene while collaterally reducing the expression of the iron storage bfrB gene. Exacerbating the iron deficiency via the inclusion of an iron chelator or aggravating oxidative stress by deploying a catalase (KatG) loss-of-function mutant strain enhanced the activity of SA23, whereas a combined approach of treating the katG mutant strain with an iron chelator led to even greater gains in activity. Our results support the notion that the activity of SA23 pivots on a vicious cycle of events that involve the derailment of iron homeostasis toward greater acquisition of the metal, overwhelmed oxidative stress defenses due to enhanced Fenton reactivity, and, ultimately, self-inflicted death. Hence, we posit that redox cyclers that concurrently perturb the iron equilibrium and cellular respiration are well-positioned to be potent next-generation anti-tubercular drugs. IMPORTANCE Cellular respiration in mycobacteria is a potentially rich target space for the discovery of novel drug entities. Here, we show that a redox cycling bactericidal small molecule that selectively activates a respiratory complex in mycobacteria has the surprising effect of disrupting iron homeostasis. Our results support the notion that the disruption of cellular respiration is a potent driver of reactive oxygen species (ROS) generation by the redox cycling molecule. Mycobacteria respond by acquiring iron to restore the levels depleted by the prevailing oxidizing conditions, which inadvertently trigger the compensatory acquisition of the metal. This leads to overwhelmed oxidative stress defenses and yet more iron depletion. For organisms that are unable to break out of this pernicious cycle of events, cell death is the inevitable outcome. Hence, aberrant ROS production by a redox cycling bactericidal agent inflicts a plethora of damaging effects on mycobacteria, including the derailment of iron homeostasis.

The Iron Response of Mycobacterium tuberculosis and Its Implications for Tuberculosis Pathogenesis and Novel Therapeutics.

Most pathogenic bacteria require iron for growth. However, this metal is not freely available in the mammalian host. Due to its poor solubility and propensity to catalyze the generation of reactive oxygen species, host iron is kept in solution bound to specialized iron binding proteins. Access to iron is an important factor in the outcome of bacterial infections; iron limitation frequently induces virulence and drives pathogenic interactions with host cells. Here, we review the response of Mycobacterium tuberculosis to changes in iron availability, the relevance of this response to TB pathogenesis, and its potential for the design of new therapeutic interventions.

Extracellular vesicles in the context of Mycobacterium tuberculosis infection.

The production of extracellular vesicles (EVs) has emerged as an important process in bacterial biology and host-pathogen interactions. Like many other bacteria, mycobacteria, including Mycobacterium tuberculosis (Mtb), the causative agent of human tuberculosis (TB), produces EVs in vitro and in vivo. These membrane-enclosed nanoparticles enable Mtb to secrete hydrophobic molecules, proteins, lipids and glycolipids in a concentrated and protected manner and engage in remote interactions with the host. The nature of the material secreted in mycobacterial EVs, the functional attributes of these vesicles and their potential as protective antigens have stimulated great interest in the mycobacterial field. Although the field of EVs in mycobacterial infections is developing, it has already uncovered a whole new dimension for Mtb-host interactions potentially relevant to TB pathogenesis. In this mini-review, we discuss the current evidence supporting an important role of mycobacterial EVs in modulating cellular immune response, the challenges and recent advances in understanding the mechanisms of vesicle biogenesis and the implications for development of new preventive and therapeutic tools against TB.

Identificación de Helicobacter pylori por medio de la coloración especial de Warthin-Starry en biopsias de pacientes con gastritis crónica folicular, previamente negativas en la tinción de hematoxilina-eosina / Warthin-Starry stain identification of Helicobacter pylori in biopsies of patients who previously tested negative in hematoxylin-eosin staining for follicular gastritis

Resumen Las técnicas empleadas para la detección del Helicobacter pylori (H. pylori) son no invasivas e invasivas. En estas últimas, la presencia del H. pylori se determina a partir de la tinción de hematoxilina-eosina (HE), prueba rutinaria, mientras que en pocas ocasiones se aplica la tinción de Warthin-Starry (WS) como coloración especial. Objetivo: identificar la presencia de H. pylori por medio de la coloración especial de la WS en biopsias de pacientes con gastritis crónica folicular, previamente negativas en la tinción HE. Materiales y métodos: se desarrolló un estudio de tipo descriptivo transversal, en un período de 12 meses. Se tomaron los bloques de parafina de las muestras de la mucosa gástrica de pacientes con diagnóstico de gastritis crónica e hiperplasia folicular. Además, se extrajo un corte histológico del mismo bloque, al cual se le aplicó HE y se determinó la presencia o ausencia de H. pylori. Así, de estar ausente, se tomó del mismo bloque un corte adicional y se aplicó WS. Esto se evaluó con el fin de identificar la existencia o no del bacilo. Resultados: se recolectaron 314 muestras; 209 fueron negativas y 105 fueron positivas para HE. El 45 % (94) de estas muestras fueron positivas respecto a la presencia del bacilo, al aplicar la segunda coloración, y el 55 % (115) de las muestras persistieron negativas. Conclusión: el hallazgo de H. pylori es significativamente alto al aplicar la coloración de WS a muestras cuyo estudio histológico evidenció la ausencia del bacilo en biopsias de la mucosa gástrica, especialmente en muestras con escasa cantidad de bacterias.

Abstract Non-invasive and invasive techniques can be used for detection of Helicobacter pylori. An invasive technique identifies the bacteria through routine hematoxylin-eosin staining. Warthin-Starry stain is rarely used. Objective: Our objective was to identify H. pylori by Warthin-Starry staining of patient's biopsies with chronic follicular gastritis who had previously tested negative in hematoxylin-eosin staining. Materials and methods: This is a descriptive, cross-sectional descriptive study that was carried out over a period of 12 months. The study examined paraffin blocks of samples taken from the gastric mucosa of patients diagnosed with chronic gastritis and follicular hyperplasia. A histological section was extracted from a block and tested with hematoxylin-eosin staining for the presence or absence of H. pylori. If absent, an additional cut was taken from the same block and Warthin-Starry staining was used to retest for the presence of the bacteria. Results: Of the 314 samples collected, 209 tested negative, and 105 tested positive for H. pylori when hematoxylin-eosin staining was used. Of the 209 negative samples, 45% (94) tested positive when Warthin Starry stain was used, and 55% (115) still tested negative. Conclusion: Findings of H. pylori are significantly higher when Warthin Starry stain was used to test samples whose previous histological study had evidenced an absence of the bacillus, especially in samples with a small amount of bacteria.

Effect of Iron Supplementation on the Outcome of Non-Progressive Pulmonary Mycobacterium tuberculosis Infection.

The human response to Mycobacterium tuberculosis (Mtb) infection is affected by the availability of iron (Fe), which is necessary for proper immune cell function and is essential for the growth and virulence of bacteria. Increase in host Fe levels promotes Mtb growth and tuberculosis (TB) pathogenesis, while Fe-supplementation to latently infected, asymptomatic individuals is a significant risk factor for disease reactivation. However, the effect of Fe-supplementation on the host immunity during latent Mtb infection remains unclear, due partly to the paucity in availability of animal models that recapitulate key pathophysiological features seen in humans. We have demonstrated that rabbits can develop non-progressive latency similar to infected humans. In this study, using this model we have evaluated the effect of Fe-supplementation on the bacterial growth, disease pathology, and immune response. Systemic and lung Fe parameters, gene expression profile, lung bacterial burden, and disease pathology were determined in the Mtb-infected/Fe- or placebo-supplemented rabbits. Results show that Fe-supplementation to Mtb-infected rabbits did not significantly change the hematocrit and Hb levels, although it elevated total Fe in the lungs. Expression of selected host iron- and immune-response genes in the blood and lungs was perturbed in Mtb-infected/Fe-supplemented rabbits. Iron-supplementation during acute or chronic stages of Mtb infection did not significantly affect the bacterial burden or disease pathology in the lungs. Data presented in this study is of significant relevance for current public health policies on Fe-supplementation therapy given to anemic patients with latent Mtb infection.

Mycobacterial extracellular vesicles and host pathogen interactions.

Mycobacteria, like other bacteria, archaea and eukaryotic cells, naturally release extracellular vesicles (EVs) to interact with their environment. EVs produced by pathogenic bacteria are involved in many activities including cell-cell communication, immunomodulation, virulence and cell survival. Although EVs released by thick cell wall microorganisms like mycobacteria were recognized only recently, studies of Mycobacterium tuberculosis EVs already point to their important roles in host pathogen interactions, opening exciting new areas of investigation. This minireview will summarize the current understanding of mycobacterial EV biology and roles in pathogenesis and will discuss their potential therapeutic applications.

The Capacity of Mycobacterium tuberculosis To Survive Iron Starvation Might Enable It To Persist in Iron-Deprived Microenvironments of Human Granulomas.

This study was conducted to investigate the role of iron deprivation in the persistence of Mycobacterium tuberculosis We present evidence of iron restriction in human necrotic granulomas and demonstrate that under iron starvation M. tuberculosis persists, refractive to antibiotics and capable of restarting replication when iron is made available. Transcriptomics and metabolomic analyses indicated that the persistence of M. tuberculosis under iron starvation is dependent on strict control of endogenous Fe utilization and is associated with upregulation of pathogenicity and intrinsic antibiotic resistance determinants. M. tuberculosis mutants compromised in their ability to survive Fe starvation were identified. The findings of this study advance the understanding of the physiological settings that may underpin the chronicity of human tuberculosis (TB) and are relevant to the design of effective antitubercular therapies.IMPORTANCE One-third of the world population may harbor persistent M. tuberculosis, causing an asymptomatic infection that is refractory to treatment and can reactivate to become potentially lethal tuberculosis disease. However, little is known about the factors that trigger and maintain M. tuberculosis persistence in infected individuals. Iron is an essential nutrient for M. tuberculosis growth. In this study, we show, first, that in human granulomas the immune defense creates microenvironments in which M. tuberculosis likely experiences drastic Fe deprivation and, second, that Fe-starved M. tuberculosis is capable of long-term persistence without growth. Together, these observations suggest that Fe deprivation in the lung might trigger a state of persistence in M. tuberculosis and promote chronic TB. We also identified vulnerabilities of iron-restricted persistent M. tuberculosis, which can be exploited for the design of new antitubercular therapies.

Functions and importance of mycobacterial extracellular vesicles.

The release of cellular factors by means of extracellular vesicles (EVs) is conserved in archaea, bacteria, and eukaryotes. EVs are released by growing bacteria as part of their interaction with their environment and, for pathogenic bacteria, constitute an important component of their interactions with the host. While EVs released by gram-negative bacteria have been extensively studied, the vesicles released by thick cell wall microorganisms like mycobacteria were recognized only recently and are less well understood. Nonetheless, studies of mycobacterial EVs have already suggested roles in pathogenesis, opening exciting new avenues of research aimed at understanding their biogenesis and potential use in antitubercular strategies. In this minireview, we discuss the discovery of mycobacterial vesicles, the current understanding of their nature, content, regulation, and possible functions, as well as their potential therapeutic applications.

MntR(Rv2788): a transcriptional regulator that controls manganese homeostasis in Mycobacterium tuberculosis.

The pathogenic mycobacterium Mycobacterium tuberculosis encodes two members of the DtxR/MntR family of metalloregulators, IdeR and SirR. IdeR represses gene expression in response to ferrous iron, and we here demonstrate that SirR (Rv2788), although also annotated as an iron-dependent repressor, functions instead as a manganese-dependent transcriptional repressor and is therefore renamed MntR. MntR regulates transporters that promote manganese import and genes that respond to metal ion deficiency such as the esx3 system. Repression of manganese import by MntR is essential for survival of M. tuberculosis under conditions of high manganese availability, but mntR is dispensable during infection. In contrast, manganese import by MntH and MntABCD was found to be indispensable for replication of M. tuberculosis in macrophages. These results suggest that manganese is limiting in the host and that interfering with import of this essential metal may be an effective strategy to attenuate M. tuberculosis.

Vaccination with an Attenuated Ferritin Mutant Protects Mice against Virulent Mycobacterium tuberculosis.

Mycobacterium tuberculosis the causative agent of tuberculosis affects millions of people worldwide. New tools for treatment and prevention of tuberculosis are urgently needed. We previously showed that a ferritin (bfrB) mutant of M. tuberculosis has altered iron homeostasis and increased sensitivity to antibiotics and to microbicidal effectors produced by activated macrophages. Most importantly, M. tuberculosis lacking BfrB is strongly attenuated in mice, especially, during the chronic phase of infection. In this study, we examined whether immunization with a bfrB mutant could confer protection against subsequent infection with virulent M. tuberculosis in a mouse model. The results show that the protection elicited by immunization with the bfrB mutant is comparable to BCG vaccination with respect to reduction of bacterial burden. However, significant distinctions in the disease pathology and host genome-wide lung transcriptome suggest improved containment of Mtb infection in animals vaccinated with the bfrB mutant, compared to BCG. We found that downmodulation of inflammatory response and enhanced fibrosis, compared to BCG vaccination, is associated with the protective response elicited by the bfrB mutant.

The mycobacterial iron-dependent regulator IdeR induces ferritin (bfrB) by alleviating Lsr2 repression.

Emerging evidence indicates that precise regulation of iron (Fe) metabolism and maintenance of Fe homeostasis in Mycobacterium tuberculosis (Mtb) are essential for its survival and proliferation in the host. IdeR is a central transcriptional regulator of Mtb genes involved in Fe metabolism. While it is well understood how IdeR functions as a repressor, how it induces transcription of a subset of its targets is still unclear. We investigated the molecular mechanism of IdeR-mediated positive regulation of bfrB, the gene encoding the major Fe-storage protein of Mtb. We found that bfrB induction by Fe required direct interaction of IdeR with a DNA sequence containing four tandem IdeR-binding boxes located upstream of the bfrB promoter. Results of in vivo and in vitro transcription assays identified a direct repressor of bfrB, the histone-like protein Lsr2. IdeR counteracted Lsr2-mediated repression in vitro, suggesting that IdeR induces bfrB transcription by antagonizing the repressor activity of Lsr2. Together, these results elucidate the main mechanism of bfrB positive regulation by IdeR and identify Lsr2 as a new factor contributing to Fe homeostasis in mycobacteria.

Role for Mycobacterium tuberculosis membrane vesicles in iron acquisition.

Mycobacterium tuberculosis releases membrane vesicles packed with molecules that can modulate the immune response. Because environmental conditions often influence the production and content of bacterial vesicles, this study examined M. tuberculosis microvesicles released under iron limitation, a common condition faced by pathogens inside the host. The findings indicate that M. tuberculosis increases microvesicle production in response to iron restriction and that these microvesicles contain mycobactin, which can serve as an iron donor and supports replication of iron-starved mycobacteria. Consequently, the results revealed a role of microvesicles in iron acquisition in M. tuberculosis, which can be critical for survival in the host.

IdeR is required for iron homeostasis and virulence in Mycobacterium tuberculosis.

Iron is an essential but potentially harmful nutrient, poorly soluble in aerobic conditions, and not freely available in the human host. To acquire iron, bacteria have evolved high affinity iron acquisition systems that are expressed under iron limitation often in conjunction with virulence determinants. Because excess iron can be dangerous, intracellular iron must be tightly controlled. In mycobacteria, IdeR functions as a global iron dependent transcriptional regulator, but because inactivation of ideR is lethal for Mycobacterium tuberculosis, it has not been possible to use genetics to fully characterize this protein's function or examine the requirement of iron regulation during tuberculosis infection. In this work, a conditional M. tuberculosis ideR mutant was generated and used to study the basis of IdeR's essentiality. This investigation uncovered positive regulation of iron storage as a critical aspect of IdeR's function in regular culture and a prominent factor for survival under stresses associated with life in macrophages. Moreover, this study demonstrates that IdeR is indispensable in the mouse model of tuberculosis, thereby linking iron homeostasis to virulence in M. tuberculosis.

The ESX-3 secretion system is necessary for iron and zinc homeostasis in Mycobacterium tuberculosis.

ESX-3 is one of the five type VII secretion systems encoded by the Mycobacterium tuberculosis genome. We recently showed the essentiality of ESX-3 for M. tuberculosis viability and proposed its involvement in iron and zinc metabolism. In this study we confirmed the role of ESX-3 in iron uptake and its involvement in the adaptation to low zinc environment in M. tuberculosis. Moreover, we unveiled functional differences between the ESX-3 roles in M. tuberculosis and M. smegmatis showing that in the latter ESX-3 is only involved in the adaptation to iron and not to zinc restriction. Finally, we also showed that in M. tuberculosis this secretion system is essential for iron and zinc homeostasis not only in conditions in which the concentrations of these metals are limiting but also in metal sufficient conditions.

A ferritin mutant of Mycobacterium tuberculosis is highly susceptible to killing by antibiotics and is unable to establish a chronic infection in mice.

Iron is an essential, elusive, and potentially toxic nutrient for most pathogens, including Mycobacterium tuberculosis. Due to the poor solubility of ferric iron under aerobic conditions, free iron is not found in the host. M. tuberculosis requires specialized iron acquisition systems to replicate and cause disease. It also depends on a strict control of iron metabolism and intracellular iron levels to prevent iron-mediated toxicity. Under conditions of iron sufficiency, M. tuberculosis represses iron acquisition and induces iron storage, suggesting an important role for iron storage proteins in iron homeostasis. M. tuberculosis synthesizes two iron storage proteins, a ferritin (BfrB) and a bacterioferritin (BfrA). The individual contributions of these proteins to the adaptive response of M. tuberculosis to changes in iron availability are not clear. By generating individual knockout strains of bfrA and bfrB, the contribution of each one of these proteins to the maintenance of iron homeostasis was determined. The effect of altered iron homeostasis, resulting from impaired iron storage, on the resistance of M. tuberculosis to in vitro and in vivo stresses was examined. The results show that ferritin is required to maintain iron homeostasis, whereas bacterioferritin seems to be dispensable for this function. M. tuberculosis lacking ferritin suffers from iron-mediated toxicity, is unable to persist in mice, and, most importantly, is highly susceptible to killing by antibiotics, showing that endogenous oxidative stress can enhance the antibiotic killing of this important pathogen. These results are relevant for the design of new therapeutic strategies against M. tuberculosis.

Lipidomic discovery of deoxysiderophores reveals a revised mycobactin biosynthesis pathway in Mycobacterium tuberculosis.

To measure molecular changes underlying pathogen adaptation, we generated a searchable dataset of more than 12,000 mass spectrometry events, corresponding to lipids and small molecules that constitute a lipidome for Mycobacterium tuberculosis. Iron is essential for M. tuberculosis survival, and the organism imports this metal using mycobactin and carboxymycobactin siderophores. Detection of an unexpected siderophore variant and deletions of genes for iron scavenging has led to a revised mycobactin biosynthesis model. An organism-wide search of the M. tuberculosis database for hypothetical compounds predicted by this model led to the discovery of two families of previously unknown lipids, designated monodeoxymycobactins and monodeoxycarboxymycobactins. These molecules suggest a revised biosynthetic model that alters the substrates and order of action of enzymes through the mycobactin biosynthetic pathway. We tested this model genetically by solving M. tuberculosis lipidomes after deletion of the iron-dependent regulator (ideR), mycobactin synthase B (mbtB), or mycobactin synthase G (mbtG). These studies show that deoxymycobactins are actively regulated during iron starvation, and also define essential roles of MbtG in converting deoxymycobactins to mycobactin and in promoting M. tuberculosis growth. Thus, lipidomics is an efficient discovery tool that informs genetic relationships, leading to a revised general model for the biosynthesis of these virulence-conferring siderophores.

Examining the role of Rv2895c (ViuB) in iron acquisition in Mycobacterium tuberculosis.

Iron acquisition is essential for Mycobacterium tuberculosis (Mtb) virulence. Understanding the molecular mechanisms used by Mtb to scavenge iron during infection might reveal new targets for antimicrobial development. Rv2895c, a homolog of ViuB from Vibrio cholerae has been postulated to be involved in iron-siderophore uptake and utilization in Mtb. This study examines the requirement of Rv2895c for adaptation of Mtb to iron limitation. We show that Rv2895c is dispensable for normal replication of Mtb in iron deficient conditions and in human macrophages. Thus, contrary to the predictions of sequence analysis and in-vitro studies the genetic evidence indicates that in normal conditions Rv2895c is not required for iron acquisition in Mtb.

The Mycobacterium tuberculosis high-affinity iron importer, IrtA, contains an FAD-binding domain.

Iron is an essential nutrient not freely available to microorganisms infecting mammals. To overcome iron deficiency, bacteria have evolved various strategies including the synthesis and secretion of high-affinity iron chelators known as siderophores. The siderophores produced and secreted by Mycobacterium tuberculosis, exomycobactins, compete for iron with host iron-binding proteins and, together with the iron-regulated ABC transporter IrtAB, are required for the survival of M. tuberculosis in iron deficient conditions and for normal replication in macrophages and in mice. This study further characterizes the role of IrtAB in M. tuberculosis iron acquisition. Our results demonstrate a role for IrtAB in iron import and show that the amino terminus domain of IrtA is a flavin-adenine dinucleotide-binding domain essential for iron acquisition. These results suggest a model in which the amino terminus of IrtA functions to couple iron transport and assimilation.

Identification and functional characterization of the iron-dependent regulator (IdeR) of Mycobacterium avium subsp. paratuberculosis.

Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease in cattle and sheep, has unique iron requirements in that it is mycobactin-dependent for cultivation in vitro. The iron-dependent regulator (IdeR) is a well-characterized global regulator responsible for maintaining iron homeostasis in Mycobacterium tuberculosis (MTB). We identified an orthologous segment in the MAP genome, MAP2827, with >93 % amino acid identity to MTB IdeR. Electrophoretic mobility shift assays and DNase protection assays confirmed that MAP2827 binds the 19 bp consensus motif (iron box) on the MAP genome. Sequencing of MAP2827 from multiple isolates revealed a non-synonymous change (R91G) exclusive to sheep strains. Reporter gene assays and quantitative real-time RT-PCR assays in two diverse MAP strains and in an ideR deletion mutant of M. smegmatis (mc(2)155) suggested that both sheep MAP IdeR (sIdeR) and cattle MAP IdeR (cIdeR) repress mbtB transcription at high iron concentrations and relieve repression at low iron concentrations. On the other hand, bfrA (an iron storage gene) was upregulated by cIdeR when presented with MTB or the cattle MAP bfrA promoter, and was downregulated by sIdeR in the presence of MTB, or sheep or cattle MAP bfrA promoters, at high iron concentrations. The differential iron regulatory mechanisms between IdeR-regulated genes across strains may contribute to the differential growth or pathogenic characteristics of sheep and cattle MAP strains. Taken together, our study provides a possible reason for mycobactin dependency and suggests strong implications in the differential iron acquisition and storage mechanisms in MAP.