Protective Efficacy of BCG Vaccine against Mycobacterium leprae and Non-Tuberculous Mycobacterial Infections.

The genus mycobacterium includes several species that are known to cause infections in humans. The microorganisms are classified into tuberculous and non-tuberculous based on their morphological characteristics, defined by the dynamic relationship between the host defenses and the infectious agent. Non-tuberculous mycobacteria (NTM) include all the species of mycobacterium other than the ones that cause tuberculosis (TB). The group of NTM contains almost 200 different species and they are found in soil, water, animals-both domestic and wild-milk and food products, and from plumbed water resources such as sewers and showerhead sprays. A systematic review of Medline between 1946 and 2014 showed an 81% decline in TB incidence rates with a simultaneous 94% increase in infections caused by NTM. Prevalence of infections due to NTM has increased relative to infections caused by TB owing to the stringent prevention and control programs in Western countries such as the USA and Canada. While the spread of typical mycobacterial infections such as TB and leprosy involves human contact, NTM seem to spread easily from the environment without the risk of acquiring from a human contact except in the case of M. abscessus in patients with cystic fibrosis, where human transmission as well as transmission through fomites and aerosols has been recorded. NTM are opportunistic in their infectious processes, making immunocompromised individuals such as those with other systemic infections such as HIV, immunodeficiencies, pulmonary disease, or usage of medications such as long-term corticosteroids/TNF-α inhibitors more susceptible. This review provides insight on pathogenesis, treatment, and BCG vaccine efficacy against M. leprae and some important NTM infections.

Over expression of the constitutive expression refractory Mycobacterium tuberculosis Rv3488 protein with an acid-inducible expression system.

As inducible protein expression plasmids available for mycobacterial species are limited, here we demonstrate the utility of an acid-inducible promoter driving gene and subsequent protein expression of a difficult to express protein. We wanted to assess the use of an acid inducible promoter expression system to produce proteins in a mycobacterial system, specifically proteins that when overexpressed interfere with mycobacterial cell growth. Overexpression of those types of proteins would require a tightly regulated promoter system. We employed the Mycobacterium tuberculosis lipF minimal acid-inducible promoter (mpr) which had previously been shown to be upregulated by acidic stress >100 X and to have a low basal level of expression in the absence of acidic stress. It is active from pH 4.3 up to pH 6.4 making this an acid range that is compatible with mycobacterial growth or survival and active at acidic ranges encountered in vivo within acidified phagosomes of macrophages. We therefore cloned the M. tuberculosis gene Rv3488, whose product had been unable to be expressed constitutively, into a plasmid downstream of the lipF mpr promoter and overexpressed this gene in the presence of acidic pH in Mycobacterium smegmatis. Sustained overexpression of the gene leads to inhibition of replication of mycobacterial cells as well as inhibition of carotenoid pigment synthesis, while short-term overexpression leads to appropriate protein expression.

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

OBJECTIVE: In 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. RESULTS: As 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.

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.

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.

Acidochromogenicity is a common characteristic in nontuberculous mycobacteria.

BACKGROUND: An 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. RESULTS: We 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. CONCLUSIONS: Mycobacterial 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.

The era of 'omics unlimited.

Determining the primary sequences of informational macromolecules is no longer a limiting factor for our ability to completely understand the biological functioning of cells and organisms. Similarly, our understanding of transcriptional regulation (transcriptomics) has been greatly enhanced by the availability of microarrays. Our next hurdle is to learn the biochemical functions of all the gene products (proteomics) and the totality of all the interactions among them (interactomics). Using traditional biochemical methods, this will take a very long time. More efficient methods are needed to address these questions, or at least to suggest possible candidates for further testing. High-resolution imaging using molecule-specific tags will reveal details of cellular architecture that are expected to provide additional insights and clues about the interactions and functions of many gene products. Computer modeling of macromolecular structures and functional systems will be of key importance. We present here a brief historical and futuristic perspective of genomics and some of its other 'omics offshoots in the post-genomic era.

The lipF promoter of Mycobacterium tuberculosis is upregulated specifically by acidic pH but not by other stress conditions.

The lipF gene of Mycobacterium tuberculosis has been implicated in pathogenesis and its promoter has been shown to be upregulated by acidic stress. To further define the acidic pH that upregulates the lipF promoter from M. tuberculosis and to establish that it is specifically upregulated by acid stress and not by other environmental stresses, promoter expression levels were measured under a variety of conditions. The conditions measured were pH, temperature, oxidative stress, and hypoxic stress.

Mutational analysis of the -10 region from the Mycobacterium tuberculosis lipF promoter.

The promoter driving expression of the Mycobacterium tuberculosis gene lipF (Rv3487c) had previously been identified as being upregulated by acidic stress. Subsequently a 59 base pair (bp) acid inducible minimal promoter region was isolated in which a putative -10 region was identified. In this study we use mutational analysis to investigate the -10 region of the lipF promoter. Mutations within this region lead to a dramatic decrease of promoter activity, while a mutation outside of this region does not affect promoter activity.

Phage L5 integrating vectors are present within the Mycobacterial Cell in an equilibrium between integrated and excised states.

Integrating mycobacterial plasmids containing the phage L5 attachment site (attP) are able to insert into the mycobacterial chromosome attB site. Plasmids containing the attP site and chromosome containing the attB site are present in equilibrium between the inserted and the excised states in the presence of the phage L5 integrase.

Determination of the minimal acid-inducible promoter region of the lipF gene from Mycobacterium tuberculosis.

The Mycobacterium tuberculosis gene lipF, Rv3487c, is transcriptionally upregulated by exposure to acidic growth media. We previously identified a 477 base pair (bp) region of DNA 147 bp upstream of lipF that is transcriptionally upregulated by exposure to growth media at pH 4.5 [Saviola, B., Woolwine, S., Bishai, W. R., 2003. Isolation of acid-inducible genes of Mycobacterium tuberculosis with the use of recombinase-based in vivo expression technology. Infect. Immun. 71, 1379-1388]. In this study we truncate the lipF promoter region first from the 3' DNA end and then from the 5' DNA end. The truncated promoter regions were placed upstream of the gene for the green fluorescent protein (gfp) and each promoter region was analyzed in Mycobacterium smegmatis for its ability to undergo transcriptional upregulation in response to acid stress. A minimal acid-inducible promoter region was identified and is located between -515 bp and -573 bp with respect to the start site of translation of lipF. The 59 bp minimal promoter region is a defined DNA sequence that confers full promoter activity that is transcriptionally upregulated in response to acid stress. Primer extension analysis was performed on acid-induced M. smegmatis bearing the minimal promoter region fused to gfp and revealed a start site of transcription specifically upregulated by acid stress corresponding to -511 bp upstream of lipF with respect to the start of translation.

Method to integrate multiple plasmids into the mycobacterial chromosome.

In order to create a system in which two independent plasmids can be integrated into a mycobacterial chromosome, a mycobacterial plasmid was constructed containing the phage attachment site attP from the mycobacteriophage L5 genome and additionally containing the bacterial attachment site, attB. This plasmid will integrate into the mycobacterial chromosome via recombination of the plasmid-borne attP site with the chromosomal attB site in the presence of a mycobacterial vector carrying the L5 integrase (int) gene. The integrated plasmid has a plasmid-borne attB site that is preserved and will accept the integration of additional mycobacterial plasmids containing the L5 attP site. This system should be useful in the construction of novel mycobacterial strains. In particular, this system provides a method by which several recombinant antigens or reporter constructs can be sequentially inserted into a mycobacterial strain and subsequently tested.

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

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 gammadelta 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.