Beatrice Saviola

Isolation of acid-inducible genes of Mycobacterium tuberculosis with the use of recombinase-based in vivo expression technology

ABSTRACT A better understanding of mycobacterial gene regulation under certain stress conditions (e.g., low pH) may provide insight into mechanisms of adaptation during infection. To identify mycobacterial promoters induced at low pH, we adapted the recombinase-based in vivo expression technology (RIVET) promoter trap system for use with mycobacteria. Our results show that the TnpR recombinase of transposon γδ is active in Mycobacterium smegmatis and Mycobacterium tuberculosis. We developed a method to perform sequential double selection with mycobacteria by using RIVET, with a kanamycin preselection and a sucrose postselection. A library of M. tuberculosis DNA inserted upstream of tnpR was created, and using the double selection, we identified two promoters which are upregulated at low pH. The promoter regions drive the expression of a gene encoding a putative lipase, lipF (Rv3487c), as well as a PE-PGRS gene, Rv0834c, in a pH-dependent manner in both M. smegmatis and M. tuberculosis. The acid inducibility of lipF and Rv0834c was independent of the stress response sigma factor, SigF, as acid induction of the two genes in an M. tuberculosis sigF mutant strain was similar to that in the wild-type strain. No induction of lipF or Rv0834c was observed during infection of J774 murine macrophages, an observation which is in agreement with previous reports on the failure of phagosomes containing M. tuberculosis to acidify.

Glutathione and infection.

BACKGROUND The tripeptide γ-glutamylcysteinylglycine or glutathione (GSH) has demonstrated protective abilities against the detrimental effects of oxidative stress within the human body, as well as protection against infection by exogenous microbial organisms. SCOPE OF REVIEW In this review we describe how GSH works to modulate the behavior of many cells including the cells of the immune system, augmenting the innate and the adaptive immunity as well as conferring protection against microbial, viral and parasitic infections. This article unveils the direct antimicrobial effects of GSH in controlling Mycobacterium tuberculosis (M. tb) infection within macrophages. In addition, we summarize the effects of GSH in enhancing the functional activity of various immune cells such as natural killer (NK) cells and T cells resulting in inhibition in the growth of M. tb inside monocytes and macrophages. Most importantly we correlate the decreased GSH levels previously observed in individuals with pulmonary tuberculosis (TB) with an increase in the levels of pro-inflammatory cytokines which aid in the growth of M. tb. MAJOR CONCLUSIONS In conclusion, this review provides detailed information on the protective integral effects of GSH along with its therapeutic effects as they relate to the human immune system and health. GENERAL SIGNIFICANCE It is important to note that the increases in the levels of pro-inflammatory cytokines are not only detrimental to the host due to the sequel that follow such as fever and cachexia, but also due to the alteration in the functions of immune cells. The additional protective effects of GSH are evident after sequel that follows the depletion of this antioxidant. This is evident in a condition such as Cystic Fibrosis (CF) where an increased oxidant burden inhibits the clearance of the affecting organism and results in oxidant-induced anti-protease inhibition. GSH has a similar protective effect in protozoans as it does in human cells. Thus GSH is integral to the survival of some of the protozoans because some protozoans utilize the compound trypanothione [T(SH)2] as their main antioxidant. T(SH)2 in turn requires GSH for its production. Hence a decrease in the levels of GSH (by a known inhibitor such as buthionine sulfoximine [BSO] can have adverse effects of the protozoan parasites. This article is part of a Special Issue entitled Cellular functions of glutathione.

Glutathione supplementation improves macrophage functions in HIV.

In this study, we determined the effects of glutathione (GSH)-enhancing agents in restoring the levels of GSH in isolated macrophages from individuals with HIV infection thereby resulting in improved control of Mycobacterium tuberculosis. Our results indicate that treatment with N-acetyl cysteine or a liposomal formulation of glutathione (lGSH) resulted in replenishment of reduced also known as free GSH (rGSH), and correlated with a decrease in the intracellular growth of M. tuberculosis. Finally, we observed differences in the amount of the catalytic subunit of glutamine-cysteine ligase (GCLC), glutathione synthase, and glutathione reductase present in macrophages derived from healthy and HIV-infected individuals. These changes correlated with changes in free radicals as well as rGSH levels. Our results indicate that HIV infection leads to increased production of free radicals and decreased production of GCLC resulting in depletion of rGSH and this may lead, in part, to the loss of innate immune function observed in HIV patients. These findings represent a novel mechanism for control of M. tuberculosis infection, and a possible supplement to current HIV treatments.

Role of Cytokines and Chemokines in HIV Infection

Human immunodeficiency virus (HIV) is the cause of acquired immunodeficiency syndrome (AIDS). Blood monocytes and resident macrophages are important in vivo cell targets for HIV infection and their role in AIDS pathogenesis are well documented. These cells of innate immune defenses usually survive HIV infection, serve as a major virus reservoir, and function as immunoregulatory cells through secretion of several pro-inflammatory cytokines and chemokines in response to HIV infection, thereby recruiting and activating new target cells for the virus, including CD4+ T cells. This review describes the alterations in the synthesis of host cytokines and chemokines following HIV infection thereby favoring successful survival of the virus inside the host and enhancing the susceptibility of the host to opportunistic infections.

Acidochromogenicity is a common characteristic in nontuberculous mycobacteria

BackgroundAn acidic environment is something likely encountered by mycobacteria in the environment or in a human host. Previously mycobacterial species had been known to produce carotenoid pigments in response to light or constitutively.ResultsWe have tested the ability of various mycobacteria to grow on solid agar plates of differing acidity, and have shown that many species of mycobacteria previously thought to not produce pigment are pigmented when exposed to acidic stress. The Mycobacterium smegmatis promoter region upstream of the genes homologous to those of other mycobacterial species known to code for proteins involved in carotenoid biosynthesis was found to be upregulated under acidic stress.ConclusionsMycobacterial species can produce pigment in response to conditions not previously known to induce chromogenicity in mycobacteria. In addition many mycobacterial species previously thought to not produce pigment are actually chromogenic under acidic conditions.

Induction of the acid inducible lipF promoter is reversibly inhibited in pH ranges of pH 4.2-4.0

ObjectiveIn the human body pathogenic mycobacteria encounter low pH within the phagosomes of macrophages where they reside after being internalized by the host cell. Low pH within macrophages has been shown to induce expression of a variety of genes within these bacteria. It had been previously observed that the Mycobacterium tuberculosis lipF promoter is transcriptionally upregulated between pHs 4.5–6.4 in Mycobacterium smegmatis, with an upper pH limit of 6.4 capable of promoter induction. To better understand the parameters of acid induced gene expression, we sought to determine the lower pH limit capable of lipF promoter induction.ResultsAs we had already determined an upper pH limit, we determine here that there is a lower limit of pH’s capable of upregulating the lipF promoter, with pH below 4.3 not positively upregulating the promoter. At non-inducing pH 4.2 the bacterial cells remain viable in the absence of acid induced lipF promoter upregulation and subsequent exposure to acid pH 5.0 results in lipF promoter upregulation. There appears to be a lower limit of pH capable of upregulating lipF promoter expression and this limit is not due to cell death.

Pigments and Pathogenesis

In recent years pigments have been identified in human nutrition to have a positive effect on human health and reduction to oxidative stress exposure. In the media it has become common wisdom that colourful food is naturally better to consume for humans and animals. Now recently it has been shown that pigments aid microbial species as well, and conversely these microbial pigments may result in more morbidity and mortality for the human host infected by these colourful microbes. Similar pigments that are available for consumption in food are also present in many bacterial species. Presumably these pigments aid the bacteria in their survival in the environment and within a human or animal host. Importantly, interference with the production of certain microbial pigments results in some bacterial strains that are more susceptible to environmental stressors and the host immune system. These studies seem to indicate a role of pigments for in vivo survival by microbial species.

Response of Mycobacterial Species to an Acidic Environment

Bacteria must be able to respond to a host of environmental stresses. The ability is vital if bacteria are to withstand insults both of an external location as well as during infection of an animal host. Many investigators study these responses individually in order to better understand pathogenic processes. Desiccation, response to UV light, heat, and cold stress can be encountered in the external environment. Low oxygen tension, heat stress, oxidative stress, nitrosidative stress, and acidic stress are all environments that can be encountered upon infection in vivo of a host. This review focuses on acidity as a stress which can be important in mycobacterial pathogenesis. Acidity can be found in such environments as acidic water and soil and can affect mycobacteria in an animal host. Mycobacteria encounter acidic stress at sites of inflammation and within phagosomes of macrophages. Exposure to acidic stress in the external environment may prime mycobacteria to upregulate genes involved in pathogenesis. Upregulation in response to acidic stress may prime mycobacteria to be more resistant to other stresses and to be more able to persist in vivo.