ESX-3 secretion system in Mycobacterium: An overview.

Mycobacteria are microorganisms distributed in the environment worldwide, and some of them, such as Mycobacterium tuberculosis or M. leprae, are pathogenic. The hydrophobic mycobacterial cell envelope has low permeation and bacteria need to export products across their structure. Mycobacteria possess specialized protein secretion systems, such as the Early Secretory Antigenic Target 6 secretion (ESX) system. Five ESX loci have been described in M. tuberculosis, called ESX-1 to ESX-5. The ESX-3 secretion system has been associated with mycobacterial metabolism and growth. The locus of this system is highly conserved across mycobacterial species. Metallo-proteins regulate negative ESX-3 transcription in high conditions of iron and zinc. Moreover, this secretion system is part of an antioxidant regulatory pathway linked to Zinc. EccA3, EccB3, EccC3, EccD3, and EccE3 are components of the ESX-3 secretion machinery, whereas EsxG-EsxH, PE5-PPE4, and PE15-PPE20 are proteins secreted by this system. In addition, EspG3 and MycP3 are complementary proteins involved in transport and proteolysis respectively. This system is associated to mycobacterial virulence by releasing the bacteria from the phagosome and inhibiting endomembrane damage response. Furthermore, components of this system inhibit the host immune response by reducing the recognition of M. tuberculosis-infected cells. The components of the ESX-3 secretion system play a role in drug resistance and cell wall integrity. Moreover, the expression data of this system indicated that external and internal factors affect ESX-3 locus expression. This review provides an overview of new findings on the ESX-3 secretion system, its regulation, expression, and functions.

Serum level of Selenium, Zinc, and Vitamin C and their relation to the clinical spectrum of leprosy.

INTRODUCTION: Leprosy is a chronic infectious disease with many risk factors including inadequate nutrient intake and nutritional deficiencies, which affect the immune system, and influence leprosy progression. OBJECTIVES: To elucidate the relation between the serum level of zinc, vitamin C, and selenium and the clinical spectrum of leprosy. METHODOLOGY: A case control study included 100 leprotic patients (50 multibacillary and 50 paucibacillary) and 100 age and sex matched controls. Vitamin C was measured by ELISA, zinc was measured by using centronic colorimetric spectrophotometry, and selenium was measured by Inductivity Coupled Plasma Optical Emission Spectroscopy technique. RESULTS: Zinc and Vitamin C levels were significantly lower in paucibacillary (mean ± SD = 89.86 ± 20.712 and 2.52 ± 1.27 respectively) and multibacillary (mean ± SD = 81.41 ± 18.61 and 1.98 ± 0.59 respectively) than in controls (mean ± SD = 107.34 ± 3.98 and 4.95 ± 2.45 respectively) (p value < 0.001) with no significant difference between paucibacillary and multibacillary patients (p value = 0.142 and = 0.066 respectively). Selenium level showed no significant difference between the three groups (p value > 0.05) (mean ± SD = 51.27 ± 42.61 in paucibacillary, 47.54 ± 30.21 in multibacillary, and 44.07 ± 46.58 in controls). CONCLUSIONS: Lower serum levels of zinc and vitamin C in leprosy patients may be a result of disease pathogenesis or related to the antioxidants based treatment. It might also present prior to the disease onset due to malnutrition that may have accelerated the development of leprosy.

Conditional DnaB Protein Splicing Is Reversibly Inhibited by Zinc in Mycobacteria.

Inteins, as posttranslational regulatory elements, can tune protein function to environmental changes by conditional protein splicing (CPS). Translated as subdomains interrupting host proteins, inteins splice to scarlessly join flanking sequences (exteins). We used DnaB-intein1 (DnaBi1) from a replicative helicase of Mycobacterium smegmatis to build a kanamycin intein splicing reporter (KISR) that links splicing of DnaBi1 to kanamycin resistance. Using expression in heterologous Escherichia coli, we observed phenotypic classes of various levels of splicing-dependent resistance (SDR) and related these to the insertion position of DnaBi1 within the kanamycin resistance protein (KanR). The KanR-DnaBi1 construct demonstrating the most stringent SDR was used to probe for CPS of DnaB in the native host environment, M. smegmatis We show here that zinc, important during mycobacterial pathogenesis, inhibits DnaB splicing in M. smegmatis Using an in vitro reporter system, we demonstrated that zinc potently and reversibly inhibited DnaBi1 splicing, as well as splicing of a comparable intein from Mycobacterium leprae Finally, in a 1.95 Å crystal structure, we show that zinc inhibits splicing through binding to the very cysteine that initiates the splicing reaction. Together, our results provide compelling support for a model whereby mycobacterial DnaB protein splicing, and thus DNA replication, is responsive to environmental zinc.IMPORTANCE Inteins are present in a large fraction of prokaryotes and localize within conserved proteins, including the mycobacterial replicative helicase DnaB. In addition to their extensive protein engineering applications, inteins have emerged as environmentally responsive posttranslational regulators of the genes that encode them. While several studies have shown compelling evidence of conditional protein splicing (CPS), examination of splicing in the native host of the intein has proven to be challenging. Here, we demonstrated through a number of measures, including the use of a splicing-dependent sensor capable of monitoring intein activity in the native host, that zinc is a potent and reversible inhibitor of mycobacterial DnaB splicing. This work also expands our knowledge of site selection for intein insertion within nonnative proteins, demonstrating that splicing-dependent host protein activation correlates with proximity to the active site. Additionally, we surmise that splicing regulation by zinc has mycobacteriocidal and CPS application potential.

Diet and nutrition: An important risk factor in leprosy.

Leprosy, once considered as poor man's disease may cause severe neurological complications and physical disabilities. Classification of leprosy depends upon the cell mediated and humoral immune responses of the host, from tuberculoid to lepromatous stage. Current therapy to prevent the disease is not only very lengthy but also consists of expensive multiple antibiotics in combination. Treatment and the duration depend on the bacillary loads, from six months in paucibacillary to a year in multibacillary leprosy. Although as per WHO recommendations, these antibiotics are freely available but still out of reach to patients of many rural areas of the world. In this review, we have focused on the nutritional aspect during the multi-drug therapy of leprosy along with the role of nutrition, particularly malnutrition, on susceptibility of Mycobacterium leprae and development of clinical symptoms. We further discussed the diet plan for the patients and how diet plans can affect the immune responses during the disease.

Toenail concentrations of zinc, selenium and nickel in patients with chronic recurrent warts: A pilot two-group comparative study.

BACKGROUND: Normal immune functioning requires sufficient levels of trace elements including zinc and selenium, while elements such as nickel can be immunotoxic. AIM: To assess long-term abnormalities in zinc, selenium and nickel levels in patients with chronic recurrent warts. METHODS: Toenail samples were taken from 28 patients with chronic recurrent warts and 30 apparently healthy matching controls were analysed. Toenail concentrations of zinc, selenium and nickel were measured using inductively-coupled plasma-optical emission spectroscopy. RESULTS: Selenium levels were significantly higher in patients than in controls (P = 0.03). Levels of trace elements did not correlate with the number or duration of warts. Toenail nickel levels in all subjects were higher than globally reported values. LIMITATIONS: A small sample size and the absence of regional reference ranges for concentrations of trace elements in toenails. CONCLUSION: Zinc does not seem to be involved in the chronicity of warts, and it is unclear if selenium has a protective role against warts. Our finding of high concentrations of nickel in both patients and controls raises concerns about environmental exposure.

Evidences for zinc (II) and copper (II) ion interactions with Mycobacterium leprae HSP18: Effect on its structure and chaperone function.

Mycobacterium leprae uptakes various bivalent metal ions via different transporters in host species. Uptake of Cu2+ and Zn2+ are essential for generation of superoxide dismutases and catalases, which provide defense against reactive oxygen species mediated death of this pathogen in macrophages. Furthermore, it has also been noticed that levels of different bivalent metal ions (Ca2+, Mg2+, Cu2+ and Zn2+) in blood serum are altered in leprotic patients. Mycobacterium leprae HSP18 is an immunodominant antigen which helps in growth and survival of Mycobacterium leprae in host species. A possible link can exist between HSP18 and aberration of bivalent metal ion homeostasis. Therefore, we investigated the interaction of these four bivalent metal ions with HSP18 and found that the protein only interacts with Zn2+ and Cu2+. Such association process is reversible and moderately high affinity in nature with unit binding stoichiometry. Theoretical studies revealed that the most probable site for Zn2+-binding lies in the N-terminal domain; While, the same for Cu2+-binding lies in the "α-crystallin domain" of HSP18. Binding of Zn2+/Cu2+ to HSP18 brings about subtle changes in the secondary and tertiary structure of HSP18 but are distinctly opposite in nature. While Zn2+ causes oligomeric association, Cu2+ leads to oligomeric dissociation of HSP18. Structural stability, surface hydrophobicity and chaperone activity of HSP18 are enhanced on Zn2+ binding, while all of them are reduced upon Cu2+ binding. Altogether, metal ions binding to HSP18 regulate its function which may have far reaching effect on the survival and pathogenicity of Mycobacterium leprae in host species.

Influence of a Former Mining Area in the Heavy Metals Concentrations in Blood of Free-Living Mediterranean Pond Turtles (Mauremys leprosa).

The objective of this study was to assess metal exposure in the Mediterranean Pond Turtle (Mauremys leprosa) inhabiting a watercourse in an ancient mining district polluted by different metals ("Rambla de Las Moreras", southeastern Spain) and included in the Ramsar Convention. For this purpose, mercury (Hg), lead (Pb), copper (Cu), zinc (Zn) and cadmium (Cd) were analysed in blood samples from 42 M. leprosa healthy adults (27 males and 13 females). The highest median concentrations were found for Zn, followed by Cu, Pb, Hg and Cd (366, 33, 9, 0.83 and 0.14 µg/dL, respectively). Although the literature regarding toxic metals in freshwater turtles is relatively scarce, Pb may cause deleterious effects in our population. In general, males presented higher levels than females, which could be due to maternal transfer during egg formation. The significant correlations between Cu-Cd and Cu-Hg suggest the implication of an efficient mechanism of detoxification involving metallothioneins.

Micronutrients influencing the immune response in leprosy / Micronutrientes que influyen en la respuesta immune en la lepra

La lepra es una enfermedad infecciosa crónica causada por el Mycobacterium leprae, un bacilo intracelular de transmisión aérea. La enfermedad afecta la piel y los nervios periféricos y causa secuelas neurológicas. El bacilo se multiplica lentamente en el hospedador y posiblemente la enfermedad ocurre por el mal funcionamiento de la respuesta inmunitaria del hospedador. Esta revisión aborda el papel de algunos micronutrientes específicos en la respuesta inmunitaria, tales como las vitaminas A, D, E, C, el cinc y el selenio, detallando sus mecanismos de acción en las enfermedades infecciosas y en la lepra. La respuesta inmunitaria a los patógenos libera sustancias nocivas que producen lesión tisular. Esta revisión también aborda cómo una menor cantidad de antioxidantes puede contribuir a un aumento del estrés oxidativo y a complicaciones de las enfermedades infecciosas y la lepra. Puesto que los micronutrientes poseen un efecto regulador de la respuesta inmunitaria innata y adaptativa, es importante un equilibrio perfecto de sus concentraciones para mejorar la respuesta inmunitaria frente a los patógenos (AU)

Leprosy is a chronic infectious disease caused by Mycobacterium leprae, an intracellular bacillus of airborne transmission. The disease affects the skin and peripheral nerves and can cause neurological sequelae. The bacillusmultiplies slowly in the host and the disease probably occurs due to malfunctioning in host immune response. This review addresses the role of some specific micronutrients in the immune response, such as Vitamins A, D, E, C, Zinc and Selenium, detailing their mechanisms of actions in infectious diseases, and in leprosy. The immune response to pathogens releases harmful substances, which lead to tissue damage. This review discusses how a decreased level of antioxidants may contribute to an increased oxidative stress and complications of infectious diseases and leprosy. As the nutrients have a regulatory effect in the innate and adaptative immune responses, a perfect balance in their concentrations is important to improve the immune response against the pathogens (AU)

Solution structure of the Mycobacterium tuberculosis EsxG·EsxH complex: functional implications and comparisons with other M. tuberculosis Esx family complexes.

Mycobacterium tuberculosis encodes five type VII secretion systems that are responsible for exporting a number of proteins, including members of the Esx family, which have been linked to tuberculosis pathogenesis and survival within host cells. The gene cluster encoding ESX-3 is regulated by the availability of iron and zinc, and secreted protein products such as the EsxG·EsxH complex have been associated with metal ion acquisition. EsxG and EsxH have previously been shown to form a stable 1:1 heterodimeric complex, and here we report the solution structure of the complex, which features a core four-helix bundle decorated at both ends by long, highly flexible, N- and C-terminal arms that contain a number of highly conserved residues. Despite clear similarities in the overall backbone fold to the EsxA·EsxB complex, the structure reveals some striking differences in surface features, including a potential protein interaction site on the surface of the EsxG·EsxH complex. EsxG·EsxH was also found to contain a specific Zn(2+) binding site formed from a cluster of histidine residues on EsxH, which are conserved across obligate mycobacterial pathogens including M. tuberculosis and Mycobacterium leprae. This site may reflect an essential role in zinc ion acquisition or point to Zn(2+)-dependent regulation of its interaction with functional partner proteins. Overall, the surface features of both the EsxG·EsxH and the EsxA·EsxB complexes suggest functions mediated via interactions with one or more target protein partners.

From Zn to Mn: the study of novel manganese-binding groups in the search for new drugs against tuberculosis.

In most eubacteria, apicomplexans, and most plants, including the causal agents for diseases such as malaria, leprosy, and tuberculosis, the methylerythritol phosphate pathway is the route for the biosynthesis of the C(5) precursors to the essential isoprenoid class of compounds. Owing to their absence in humans, the enzymes of the methylerythritol phosphate pathway have become attractive targets for drug discovery. This work investigates a new class of inhibitors against the second enzyme of the pathway, 1-deoxy-D-xylulose 5-phosphate reductoisomerase. Inhibition of this enzyme may involve the chelation of a crucial active site Mn ion, and the metal-chelating moieties studied here have previously been shown to be successful in application to the zinc-dependent metalloproteinases. Quantum mechanics and docking calculations presented in this work suggest the transferability of these metal-chelating compounds to Mn-containing 1-deoxy-D-xylulose 5-phosphate reductoisomerase enzyme, as a promising starting point to the development of potent inhibitors.

Zinc: role in immunity, oxidative stress and chronic inflammation.

PURPOSE OF REVIEW: Zinc is essential for multiple cellular functions including immunity. Many investigators have used zinc supplementation in an attempt to affect the outcome of various diseases. These efforts were aimed at either supporting immunity by zinc administration or correcting the zinc dependent immune functions in zinc deficient individuals. RECENT FINDINGS: In this review, recent findings of zinc supplementation in various diseases have been presented. Beneficial therapeutic response of zinc supplementation has been observed in the diarrhea of children, chronic hepatitis C, shigellosis, leprosy, tuberculosis, pneumonia, acute lower respiratory tract infection, common cold, and leishmaniasis. Zinc supplementation was effective in decreasing incidences of infections in the elderly, in patients with sickle cell disease (SCD) and decreasing incidences of respiratory tract infections in children. Zinc supplementation has prevented blindness in 25% of the elderly individuals with dry type of AMD. Zinc supplementation was effective in decreasing oxidative stress and generation of inflammatory cytokines such as TNF-alpha and IL-1beta in elderly individuals and patients with SCD. SUMMARY: Zinc supplementation has been successfully used as a therapeutic and preventive agent for many conditions. Zinc functions as an intracellular signal molecule for immune cells.

Trace element levels in alopecia areata.

BACKGROUND: Alopecia areata (AA) is a recurrent, nonscarring type of hair loss considered to be an autoimmune process. Though its etiopathology is not fully understood, there are claims that imbalance of trace elements may trigger the onset of AA. AIM: The aim of the present study was to assess the levels of zinc, copper, and magnesium in the serum of AA patients. METHODS: Fifty AA patients (34 men and 16 women), and fifty age and sex matched healthy control subjects were studied. Samples were analyzed using atomic absorption spectrometric methods. RESULTS: Serum zinc levels were significantly decreased (P < 0.05) in AA patients whose disease was extensive, prolonged, and resistant to treatment, whereas serum copper and magnesium levels showed insignificant rise compared to controls. CONCLUSION: We conclude that copper and magnesium levels are not altered in AA, but the decreased zinc levels found in our study may merit further investigation of the relationship.

Impact of the discovery of human zinc deficiency on health.

The essentiality of zinc was recognized 46 years ago. Zinc deficiency resulting in growth retardation, hypogonadism, immune dysfunction and cognitive impairment affects nearly 2 billion subjects in the developing world. High phytate content of the cereal proteins consumed in the developing world, results in decreased availability of zinc for absorption. Zinc therapy has been very successful and life saving measure in patients with acrodermatitis enteropathica and Wilson's disease. Beneficial therapeutic responses of zinc supplementation have been ovserved in acute diarrhea in children, chronic hepatitis C, shigellosis, leprosy, leishmaniasis, and common cold. Zinc supplementation was effective in decreasing incidences of infection in elderly and patients with sickle cell disease. Zinc supplementation was effective in preventing blindness in 25% of the elderly with dry type of age related macular degeneration. Zinc supplementation in the elderly decreased oxidative stress and decreased generation of inflammatory cytokines. Zinc is an intracellular signaling molecule in monocytes, dendritic cells and macrophages and it plays an important role in cell-mediated immune functions and oxidative stress. Zinc is also an anti-inflammatory agent. These unique properties of zinc may have significant therapeutic benefits in several diseases in humans. In many diseases concurrent zinc deficiency may complicate the clinical features, affect adversely immunological status, increase oxidative stress and increase generation of inflammatory cytokines. Oxidative stress and chronic inflammation may play important causative roles in many chronic diseases, including atherosclerosis, several malignancies, neurological disorders, and auto-immune diseases. It is therefore, important that status of zinc is assessed and zinc deficiency corrected in these chronic diseases. A controlled clinical trial of zinc supplementation in these disorders in order to document the preventive and therapeutic effects of zinc is warranted.