Role of the Mycobacterium tuberculosis ESX-4 Secretion System in Heme Iron Utilization and Pore Formation by PPE Proteins.

Mycobacterium tuberculosis (Mtb) is transmitted through aerosols and primarily colonizes within the lung. The World Health Organization estimates that Mtb kills ~1.4 million people every year. A key aspect that makes Mtb such a successful pathogen is its ability to overcome iron limitation mounted by the host immune response. In our previous studies, we have shown that Mtb can utilize iron from heme, the largest source of iron in the human host, and that it uses two redundant heme utilization pathways. In this study, we show that the ESX-4 type VII secretion system (T7SS) is necessary for extracellular heme uptake into the Mtb cell through both heme utilization pathways. ESX-4 influences the secretion of the culture filtrate proteins Rv0125 and Rv1085c, which are also necessary for efficient heme utilization. We also discovered that deletion of the alternative sigma factor SigM significantly reduced Mtb heme utilization through both pathways and predict that SigM is a global positive regulator of core heme utilization genes of both pathways. Finally, we present the first direct evidence that some mycobacterial PPE (proline-proline-glutamate motif) proteins of the PPE protein family are pore-forming membrane proteins. Altogether, we identified core components of both Mtb Heme utilization pathways that were previously unknown and identified a novel channel-forming membrane protein of Mtb. IMPORTANCE M. tuberculosis (Mtb) is completely dependent on iron acquisition in the host to cause disease. The largest source of iron for Mtb in the human host is heme. Here, we show that the ancestral ESX-4 type VII secretion system is required for the efficient utilization of heme as a source of iron, which is an essential nutrient. This is another biological function identified for ESX-4 in Mtb, whose contribution to Mtb physiology is poorly understood. A most exciting finding is that some mycobacterial PPE (proline-proline-glutamate motif) proteins that have been implicated in the nutrient acquisition are membrane proteins that can form channels in a lipid bilayer. These observations have far-reaching implications because they support an emerging theme that PPE proteins can function as channel proteins in the outer mycomembrane for nutrient acquisition. Mtb has evolved a heme uptake system that is drastically different from all other known bacterial heme acquisition systems.

Exogenously Scavenged and Endogenously Synthesized Heme Are Differentially Utilized by Mycobacterium tuberculosis.

Heme is both an essential cofactor and an abundant source of nutritional iron for the human pathogen Mycobacterium tuberculosis. While heme is required for M. tuberculosis survival and virulence, it is also potentially cytotoxic. Since M. tuberculosis can both synthesize and take up heme, the de novo synthesis of heme and its acquisition from the host may need to be coordinated in order to mitigate heme toxicity. However, the mechanisms employed by M. tuberculosis to regulate heme uptake, synthesis, and bioavailability are poorly understood. By integrating ratiometric heme sensors with mycobacterial genetics, cell biology, and biochemistry, we determined that de novo-synthesized heme is more bioavailable than exogenously scavenged heme, and heme availability signals the downregulation of heme biosynthetic enzyme gene expression. Ablation of heme synthesis does not result in the upregulation of known heme import proteins. Moreover, we found that de novo heme synthesis is critical for survival from macrophage assault. Altogether, our data suggest that mycobacteria utilize heme from endogenous and exogenous sources differently and that targeting heme synthesis may be an effective therapeutic strategy to treat mycobacterial infections. IMPORTANCE Mycobacterium tuberculosis infects ~25% of the world's population and causes tuberculosis (TB), the second leading cause of death from infectious disease. Heme is an essential metabolite for M. tuberculosis, and targeting the unique heme biosynthetic pathway of M. tuberculosis could serve as an effective therapeutic strategy. However, since M. tuberculosis can both synthesize and scavenge heme, it was unclear if inhibiting heme synthesis alone could serve as a viable approach to suppress M. tuberculosis growth and virulence. The importance of this work lies in the development and application of genetically encoded fluorescent heme sensors to probe bioavailable heme in M. tuberculosis and the discovery that endogenously synthesized heme is more bioavailable than exogenously scavenged heme. Moreover, it was found that heme synthesis protected M. tuberculosis from macrophage killing, and bioavailable heme in M. tuberculosis is diminished during macrophage infection. Altogether, these findings suggest that targeting M. tuberculosis heme synthesis is an effective approach to combat M. tuberculosis infections.

A Novel Approach To Identify Inhibitors of Iron Acquisition Systems of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen that has been declared by the World Health Organization as a "priority 1 critical pathogen" needing immediate new strategies for chemotherapy. During infection, P. aeruginosa uses redundant mechanisms to acquire ferric, heme (Hm), or ferrous iron from the host to survive and colonize. Significant efforts have been undertaken to develop siderophore blockers to inhibit ferric iron acquisition by P. aeruginosa, but there is a lack of inhibitors that can block Hm or ferrous iron acquisition by P. aeruginosa. We developed and employed a targeted high-throughput screen (HTS) and identified a molecule(s) that can specifically inhibit the Hm and ferrous iron acquisition systems of P. aeruginosa. Our targeted approach relies on screening a small-molecule library against P. aeruginosa under three growth conditions, where the only variable was the iron source (ferric, Hm, or ferrous iron). Each condition served as a counterscreen for the other, and we identified molecules that inhibit the growth of P. aeruginosa in the presence of only Hm or ferrous iron. Our data indicate that econazole, bithionate, and raloxifene inhibit the growth of P. aeruginosa in the presence of Hm and that oxyquinoline inhibits the growth of P. aeruginosa in the presence of ferrous iron. These iron-specific inhibitors do not interfere with the activity of meropenem, a commercial antipseudomonal, and can also increase meropenem activity. In conclusion, we present a proof of concept of a successful targeted conditional screening method by which we can identify specific iron acquisition inhibitors. This approach is highly adaptable and can easily be extended to any other pathogen. IMPORTANCE Since acquiring iron is paramount to P. aeruginosa's survival and colonization in the human host, developing novel strategies to block the access of P. aeruginosa to host iron will allow us to starve it of an essential nutrient. P. aeruginosa uses siderophore, heme, or ferrous iron uptake systems to acquire iron in the human host. We have developed a novel approach through which we can directly identify molecules that can prevent P. aeruginosa from utilizing heme or ferrous iron. This approach overcomes the need for the in silico design of molecules and identifies structurally diverse biologically active inhibitor molecules. This screening approach is adaptable and can be extended to any pathogen. Since Gram-negative pathogens share many similarities in iron acquisition at both the mechanistic and molecular levels, our screening approach presents a significant opportunity to develop novel broad-spectrum iron acquisition inhibitors of Gram-negative pathogens.

Heme and hemoglobin utilization by Mycobacterium tuberculosis.

Iron is essential for growth of Mycobacterium tuberculosis (Mtb), but most iron in the human body is stored in heme within hemoglobin. Here, we demonstrate that the substrate-binding protein DppA of the inner membrane Dpp transporter is required for heme and hemoglobin utilization by Mtb. The 1.27 Å crystal structure of DppA shows a tetrapeptide bound in the protein core and a large solvent-exposed crevice for heme binding. Mutation of arginine 179 in this cleft eliminates heme binding to DppA and prevents heme utilization by Mtb. The outer membrane proteins PPE36 and PPE62 are also required for heme and hemoglobin utilization, indicating that these pathways converge at the cell surface of Mtb. Albumin, the most abundant blood protein, binds heme specifically and bypasses the requirements for PPE36, PPE62 and Dpp. Thus, our study reveals albumin-dependent and -independent heme uptake pathways, highlighting the importance of iron acquisition from heme for Mtb.

A Protein Complex from Human Milk Enhances the Activity of Antibiotics and Drugs against Mycobacterium tuberculosis.

Mycobacterium tuberculosis, the causative agent of human tuberculosis (TB), has surpassed HIV/AIDS as the leading cause of death from a single infectious agent. The increasing occurrence of drug-resistant strains has become a major challenge for health care systems and, in some cases, has rendered TB untreatable. However, the development of new TB drugs has been plagued with high failure rates and costs. Alternative strategies to increase the efficacy of current TB treatment regimens include host-directed therapies or agents that make M. tuberculosis more susceptible to existing TB drugs. In this study, we show that HAMLET, an α-lactalbumin-oleic acid complex derived from human milk, has bactericidal activity against M. tuberculosis HAMLET consists of a micellar oleic acid core surrounded by a shell of partially denatured α-lactalbumin molecules and unloads oleic acid into cells upon contact with lipid membranes. At sublethal concentrations, HAMLET potentiated a remarkably broad array of TB drugs and antibiotics against M. tuberculosis For example, the minimal inhibitory concentrations of rifampin, bedaquiline, delamanid, and clarithromycin were decreased by 8- to 16-fold. HAMLET also killed M. tuberculosis and enhanced the efficacy of TB drugs inside macrophages, a natural habitat of M. tuberculosis Previous studies showed that HAMLET is stable after oral delivery in mice and nontoxic in humans and that it is possible to package hydrophobic compounds in the oleic acid core of HAMLET to increase their solubility and metabolic stability. The potential of HAMLET and other liprotides as drug delivery and sensitization agents in TB chemotherapy is discussed here.

Regulation of Escherichia coli Pathogenesis by Alternative Sigma Factor N.

σN (also σ54) is an alternative sigma factor subunit of the RNA polymerase complex that regulates the expression of genes from many different ontological groups. It is broadly conserved in the Eubacteria with major roles in nitrogen metabolism, membrane biogenesis, and motility. σN is encoded as the first gene of a five-gene operon including rpoN (σN), ptsN, hpf, rapZ, and npr that has been genetically retained among species of Escherichia, Shigella, and Salmonella. In an increasing number of bacteria, σN has been implicated in the control of genes essential to pathogenic behavior, including those involved in adherence, secretion, immune subversion, biofilm formation, toxin production, and resistance to both antimicrobials and biological stressors. For most pathogens how this is achieved is unknown. In enterohemorrhagic Escherichia coli (EHEC) O157, Salmonella enterica, and Borrelia burgdorferi, regulation of virulence by σN requires another alternative sigma factor, σS, yet the model by which σN-σS virulence regulation is predicted to occur is varied in each of these pathogens. In this review, the importance of σN to bacterial pathogenesis is introduced, and common features of σN-dependent virulence regulation discussed. Emphasis is placed on the molecular mechanisms underlying σN virulence regulation in E. coli O157. This includes a review of the structure and function of regulatory pathways connecting σN to virulence expression, predicted input signals for pathway stimulation, and the role for cognate σN activators in initiation of gene systems determining pathogenic behavior.

PPE Surface Proteins Are Required for Heme Utilization by Mycobacterium tuberculosis.

Iron is essential for replication of Mycobacterium tuberculosis, but iron is efficiently sequestered in the human host during infection. Heme constitutes the largest iron reservoir in the human body and is utilized by many bacterial pathogens as an iron source. While heme acquisition is well studied in other bacterial pathogens, little is known in M. tuberculosis To identify proteins involved in heme utilization by M. tuberculosis, a transposon mutant library was screened for resistance to the toxic heme analog gallium(III)-porphyrin (Ga-PIX). Inactivation of the ppe36, ppe62, and rv0265c genes resulted in resistance to Ga-PIX. Growth experiments using isogenic M. tuberculosis deletion mutants showed that PPE36 is essential for heme utilization by M. tuberculosis, while the functions of PPE62 and Rv0265c are partially redundant. None of the genes restored growth of the heterologous M. tuberculosis mutants, indicating that the proteins encoded by the genes have separate functions. PPE36, PPE62, and Rv0265c bind heme as shown by surface plasmon resonance spectroscopy and are associated with membranes. Both PPE36 and PPE62 proteins are cell surface accessible, while the Rv0265c protein is probably located in the periplasm. PPE36 and PPE62 are, to our knowledge, the first proline-proline-glutamate (PPE) proteins of M. tuberculosis that bind small molecules and are involved in nutrient acquisition. The absence of a virulence defect of the ppe36 deletion mutant indicates that the different iron acquisition pathways of M. tuberculosis may substitute for each other during growth and persistence in mice. The emerging model of heme utilization by M. tuberculosis as derived from this study is substantially different from those of other bacteria. IMPORTANCE: Tuberculosis is caused by Mycobacterium tuberculosis and is a devastating disease affecting eight million people each year. Iron is an essential nutrient for replication of M. tuberculosis in the human host. More than 70% of iron in the human body is bound in heme. Not surprisingly, many bacterial pathogens, including M. tuberculosis, are able to acquire iron from heme. However, the mechanism of heme uptake by M. tuberculosis is poorly understood. We have identified two novel surface proteins that bind heme and are required for heme utilization by M. tuberculosis These findings constitute a major advancement of our understanding of iron acquisition by M. tuberculosis and show that M. tuberculosis has evolved heme uptake systems different from the paradigms established by other bacteria.

Mycobacteria, metals, and the macrophage.

Mycobacterium tuberculosis is a facultative intracellular pathogen that thrives inside host macrophages. A key trait of M. tuberculosis is to exploit and manipulate metal cation trafficking inside infected macrophages to ensure survival and replication inside the phagosome. Here, we describe the recent fascinating discoveries that the mammalian immune system responds to infections with M. tuberculosis by overloading the phagosome with copper and zinc, two metals which are essential nutrients in small quantities but are toxic in excess. M. tuberculosis has developed multi-faceted resistance mechanisms to protect itself from metal toxicity including control of uptake, sequestration inside the cell, oxidation, and efflux. The host response to infections combines this metal poisoning strategy with nutritional immunity mechanisms that deprive M. tuberculosis from metals such as iron and manganese to prevent bacterial replication. Both immune mechanisms rely on the translocation of metal transporter proteins to the phagosomal membrane during the maturation process of the phagosome. This review summarizes these recent findings and discusses how metal-targeted approaches might complement existing TB chemotherapeutic regimens with novel anti-infective therapies.

σ(N) -dependent control of acid resistance and the locus of enterocyte effacement in enterohemorrhagic Escherichia coli is activated by acetyl phosphate in a manner requiring flagellar regulator FlhDC and the σ(S) antagonist FliZ.

In enterohemorrhagic Escherichia coli (EHEC), sigma factor N (σ(N)) regulates glutamate-dependent acid resistance (GDAR) and the locus of enterocyte effacement (LEE); discrete genetic systems that are required for transmission and virulence of this intestinal pathogen. Regulation of these systems requires nitrogen regulatory protein C, NtrC, and is a consequence of NtrC-σ(N) -dependent reduction in the activity of sigma factor S (σ(S)). This study elucidates pathway components and stimuli for σ(N)-directed regulation of GDAR and the LEE in EHEC. Deletion of fliZ, the product of which reduces σ(S) activity, phenocopied rpoN (σ(N)) and ntrC null strains for GDAR and LEE control, acid resistance, and adherence. Upregulation of fliZ by NtrC-σ(N) was shown to be indirect and required an intact flagellar regulator flhDC. Activation of flhDC by NtrC-σ(N) and FlhDC-dependent regulation of GDAR and the LEE was dependent on σ(N)-promoter flhDP 2 , and a newly described NtrC upstream activator sequence. Addition of ammonium chloride significantly altered expression of GDAR and LEE, acid resistance, and adherence, independently of rpoN, ntrC, and the NtrC sensor kinase, ntrB. Altering the availability of NtrC phosphodonor acetyl phosphate by growth without glucose, with acetate addition, or by deletion of acetate kinase ackA, abrogated NtrC-σ(N)-dependent control of flhDC, fliZ, GDAR, and the LEE.

Sigma factor N, liaison to an ntrC and rpoS dependent regulatory pathway controlling acid resistance and the LEE in enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) is dependent on acid resistance for gastric passage and low oral infectious dose, and the locus of enterocyte effacement (LEE) for intestinal colonization. Mutation of rpoN, encoding sigma factor N (σ(N)), dramatically alters the growth-phase dependent regulation of both acid resistance and the LEE. This study reports on the determinants of σ(N)-directed acid resistance and LEE expression, and the underlying mechanism attributable to this phenotype. Glutamate-dependent acid resistance (GDAR) in TW14359ΔrpoN correlated with increased expression of the gadX-gadW regulatory circuit during exponential growth, whereas upregulation of arginine-dependent acid resistance (ADAR) genes adiA and adiC in TW14359ΔrpoN did not confer acid resistance by the ADAR mechanism. LEE regulatory (ler), structural (espA and cesT) and effector (tir) genes were downregulated in TW14359ΔrpoN, and mutation of rpoS encoding sigma factor 38 (σ(S)) in TW14359ΔrpoN restored acid resistance and LEE genes to WT levels. Stability, but not the absolute level, of σ(S) was increased in TW14359ΔrpoN; however, increased stability was not solely attributable to the GDAR and LEE expression phenotype. Complementation of TW14359ΔrpoN with a σ(N) allele that binds RNA polymerase (RNAP) but not DNA, did not restore WT levels of σ(S) stability, gadE, ler or GDAR, indicating a dependence on transcription from a σ(N) promoter(s) and not RNAP competition for the phenotype. Among a library of σ(N) enhancer binding protein mutants, only TW14359ΔntrC, inactivated for nitrogen regulatory protein NtrC, phenocopied TW14359ΔrpoN for σ(S) stability, GDAR and ler expression. The results of this study suggest that during exponential growth, NtrC-σ(N) regulate GDAR and LEE expression through downregulation of σ(S) at the post-translational level; likely by altering σ(S) stability or activity. The regulatory interplay between NtrC, other EBPs, and σ(N)-σ(S), represents a mechanism by which EHEC can coordinate GDAR, LEE expression and other cellular functions, with nitrogen availability and physiologic stimuli.

Shiga toxin 2 overexpression in Escherichia coli O157:H7 strains associated with severe human disease.

Variation in disease severity among Escherichia coli O157:H7 infections may result from differential expression of Shiga toxin 2 (Stx2). Eleven strains belonging to four prominent phylogenetic clades, including clade 8 strains representative of the 2006 U.S. spinach outbreak, were examined for stx2 expression by real-time PCR and western blot analysis. Clade 8 strains were shown to overexpress stx2 basally, and following induction with ciprofloxacin when compared to strains from clades 1-3. Differences in stx2 expression generally correlated with Stx2 protein levels. Single-nucleotide polymorphisms identified in regions upstream of stx2AB in clade 8 strains were largely absent in non-clade 8 strains. This study concludes that stx2 overexpression is common to strains from clade 8 associated with hemolytic uremic syndrome, and describes SNPs which may affect stx2 expression and which could be useful in the genetic differentiation of highly-virulent strains.

Crystallization of a non-steroidal anti-inflammatory drug from ethanol-water solution in presence of polymers: physicochemical characterization and release behaviour from suppositories.

Altered crystallization condition has been designed and adopted to a model non-steroidal anti-inflammatory drug, while crystallizing from ethanol-water solution in absence and presence of polymers such as Eudragit RS and ethylcellulose. To minimize the gastro-intestinal side effects nimesulide was considered as a model drug candidate for the development of suppository formulation. Physicochemical characteristics of the crystals were evaluated by Scanning Electron Microscopy (SEM). X-ray diffraction (XRD) and Fourier Transformed Infrared Spectroscopy (FT-IR). Smoothness and sharpness of the crystal have been decreased with increased concentration of a polymer. A little change in crystal habit and geometry has also been observed. Crystals are discrete in nature and more than 90% were in the range of 20-90 micron. The X-ray diffractions of nimesulide crystallized in absence of polymer and physical mixture of drug-polymer revealed fewer high intensity reflections when compared with the drug crystallized in presence of Eudragit RS, which testified a slight decreased ordering of crystal lattice in the latter. In presence of ethylcellulose, slightly increased ordering of crystal lattice was observed. No strong interactions were noticed as revealed by FT-IR spectroscopy. Drug dissolution rate from suppository formulations containing nimesulide crystallized in presence of polymer was found to delay as compared with the suppository prepared by nimesulide crystallized in absence of polymer.