Stress responses in mycobacteria

Mycobacterium tuberculosis is a successful pathogen that overcomes numerous challenges presented by the immune system of the host. This bacterium usually establishes a chronic infection in the host where it may silently persist inside a granuloma until, a failure in host defenses, leads to manifestation of the disease. None of the conventional anti-tuberculosis drugs are able to target these persisting bacilli. Development of drugs against such persisting bacilli is a constant challenge since the physiology of these dormant bacteria is still not understood at the molecular level. Some evidence suggests that the in vivo environment encountered by the persisting bacteria is anoxic and nutritionally starved. Based on these assumptions, anaerobic and starved cultures are used as models to study the molecular basis of dormancy. This review outlines the problem of persistence of M. tuberculosis and the various in vitro models used to study mycobacterial latency. The basis of selecting the nutritional starvation model has been outlined here. Also, the choice of M. smegmatis as a model suitable for studying mycobacterial latency is discussed. Lastly, general issues related to oxidative stress and bacterial responses to it have been elaborated. We have also discussed general control of OxyR-mediated regulation and emphasized the processes which manifest in the absence of functional OxyR in the bacteria. Lastly, a new class of protein called Dps has been reviewed for its important role in protecting DNA under stress.     

Role of N and C-terminal tails in DNA binding and assembly in Dps: structural studies of Mycobacterium smegmatis Dps deletion mutants

Mycobacterium smegmatis Dps degrades spontaneously into a species in which 16 C-terminal residues are cleaved away. A second species, in which all 26 residues constituting the tail were deleted, was cloned, expressed and purified. The first did not bind DNA but formed dodecamers like the native protein, while the second did not bind to DNA and failed to assemble into dodecamers, indicating a role in assembly also for the tail. In the crystal structure of the species without the entire C-terminal tail the molecule has an unusual open decameric structure resulting from the removal of two adjacent subunits from the original dodecameric structure of the native form. A Dps dodecamer could assemble with a dimer or one of two trimers (trimer-A and trimer-B) as intermediate. Trimer-A is the intermediate species in the M. smegmatis protein. Estimation of the surface area buried on trimerization indicates that association within trimer-B is weak. It weakens further when the C-terminal tail is removed, leading to the disruption of the dodecameric structure. Thus, the C-terminal tail has a dual role, one in DNA binding and the other in the assembly of the dodecamer. M. smegmatis Dps also has a short N-terminal tail. A species with nine N-terminal residues deleted formed trimers but not dodecamers in solution, unlike wild-type M. smegmatis Dps, under the same conditions. Unlike in solution, the N-terminal mutant forms dodecamers in the crystal. In native Dps, the N-terminal stretch of one subunit and the C-terminal stretch of a neighboring subunit lock each other into ordered positions. The deletion of one stretch results in the disorder of the other. This disorder appears to result in the formation of a trimeric species of the N-terminal deletion mutant contrary to the indication provided by the native structure. The ferroxidation site is intact in the mutants.     

Bimodal protection of DNA by Mycobacterium smegmatis DNA-binding protein from stationary phase cells

Some members of the DNA-binding protein from stationary phase cells (Dps) family of proteins have been shown to play an important role in protecting microorganisms from oxidative or nutritional stress. Dps homologs have been identified in various bacteria such as Escherichia coli, Bacillus subtilis, and Listeria innocua. Recently we have reported the presence of a Dps homolog, Ms-Dps, in Mycobacterium smegmatis. Ms-Dps was found to have a nonspecific DNA binding ability. Here we have detected two stable oligomeric forms of Ms-Dps in vitro, a trimeric and a dodecameric form. Interestingly, the conversion of Dps from a trimeric to a dodecameric form takes place upon incubation at 37 degrees C for 12 h. These two oligomeric forms differ in their DNA binding properties. The dodecameric form is capable of DNA binding and forming large crystalline arrays with DNA, whereas the trimeric form cannot do so. However, even in the absence of DNA binding, the trimeric form has the capacity to protect the DNA against Fenton's-mediated damage. The protection is afforded by the ferroxidase activity of the trimer. However, the trimeric form cannot protect DNA from DNaseI attack, for which a direct physical shielding of DNA by the dodecamer is required. Thus we suggest that Ms-Dps provides a bimodal protection of DNA by its two different oligomeric forms.     

Structural studies on the second Mycobacterium smegmatis Dps: invariant and variable features of structure, assembly and function

A second DNA binding protein from stationary-phase cells of Mycobacterium smegmatis (MsDps2) has been identified from the bacterial genome. It was cloned, expressed and characterised and its crystal structure was determined. The core dodecameric structure of MsDps2 is the same as that of the Dps from the organism described earlier (MsDps1). However, MsDps2 possesses a long N-terminal tail instead of the C-terminal tail in MsDps1. This tail appears to be involved in DNA binding. It is also intimately involved in stabilizing the dodecamer. Partly on account of this factor, MsDps2 assembles straightway into the dodecamer, while MsDps1 does so on incubation after going through an intermediate trimeric stage. The ferroxidation centre is similar in the two proteins, while the pores leading to it exhibit some difference. The mode of sequestration of DNA in the crystalline array of molecules, as evidenced by the crystal structures, appears to be different in MsDps1 and MsDps2, highlighting the variability in the mode of Dps-DNA complexation. A sequence search led to the identification of 300 Dps molecules in bacteria with known genome sequences. Fifty bacteria contain two or more types of Dps molecules each, while 195 contain only one type. Some bacteria, notably some pathogenic ones, do not contain Dps. A sequence signature for Dps could also be derived from the analysis.     

Estimation of Förster's distance between two ends of Dps protein from mycobacteria: distance heterogeneity as a function of oligomerization and DNA binding

Dps protein (DNA binding Protein from Starved Cells) from Mycobacterium smegmatis (Ms-Dps) is known to undergo an in vitro irreversible oligomeric transition from trimer to dodecamer. This transition helps the protein to provide for bimodal protection to the bacterial DNA from the free radical and Fenton mediated damages in the stationary state. The protein exists as a stable trimer, when purified from E. coli cells transformed with an over-expression plasmid. Both trimer as well as dodecamer are known to exhibit ferroxidation activity, thus removing toxic hydroxyl radicals in vivo, whereas iron accumulation and non-sequence specific DNA binding activity are found in dodecamer only. This seems to be aided by the positively charged long C-terminal tail of the protein. We used frequency domain phase-modulation fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET) to monitor this oligomeric switch from a trimer to a dodecamer and to elucidate the structure of DNA–Dps dodecamer complex. As Ms-Dps is devoid of any Cysteine residues, a Serine is mutated to Cysteine (S169C) at a position adjacent to the putative DNA binding domain. This Cysteine is subsequently labeled with fluorescent probe and another probe is placed at the N-terminus, as crystal structure of the protein reveals several side-chain interactions between these two termini, and both are exposed towards the surface of the protein. Here, we report the Förster's distance distribution in the trimer and the dodecamer in the presence and absence of DNA. Through discrete lifetime analysis of the probes tagged at the respective regions in the macromolecule, coupled with Maximum Entropy Method (MEM) analysis, we show that the dodecamer, upon DNA binding shows conformational heterogeneity in overall structure, perhaps mediated by a non-specific DNA–protein interaction. On the other hand, the nature of DNA–Dps interaction is not known and several models exist in literature. We show here with the help of fluorescence anisotropy measurements of labeled DNA having different length and unlabeled native dodecameric protein that tandem occupation of DNA binding sites by a series of Dps molecules perhaps guide the tight packing of Dps over DNA backbone.     

Identification of a regulated alkaline phosphatase,a cell surface-associated lipoprotein,in Mycobacterium smegmatis

Although alkaline phosphatases are common in a wide variety of bacteria, there has been no prior evidence for alkaline phosphatases in Mycobacterium smegmatis. Here we report that transposon insertions in the pst operon, encoding homologues of an inorganic phosphate transporter, leads to constitutive expression of a protein with alkaline phosphatase activity. DNA sequence analysis revealed that M. smegmatis does indeed have a phoA gene that shows high homology to other phoA genes. The M. smegmatis phoA gene was shown to be induced by phosphate starvation and thus negatively regulated by the pst operon. Interestingly, the putative M. smegmatis PhoA has a hydrophobic N-terminal domain which resembles a lipoprotein signal sequence. The M. smegmatis PhoA was demonstrated to be an exported protein associated with the cell surface. Furthermore, immunoprecipitation of PhoA from [(14)C]acetate-labeled M. smegmatis cell lysates demonstrated that this phosphatase is a lipoprotein.     

The relA homolog of Mycobacterium smegmatis affects cell appearance, viability, and gene expression

The modification of metabolic pathways to allow for a dormant lifestyle appears to be an important feature for the survival of pathogenic bacteria within their host. One regulatory mechanism for persistent Mycobacterium tuberculosis infections is the stringent response. In this study, we analyze the stringent response of a nonpathogenic, saprophytic mycobacterial species, Mycobacterium smegmatis. The use of M. smegmatis as a tool for studying the mycobacterial stringent response was demonstrated by measuring the expression of two M. tuberculosis genes, hspX and eis, in M. smegmatis in the presence and absence of rel(Msm). The stringent response plays a role in M. smegmatis cellular and colony formation that is suggestive of changes in the bacterial cell wall structure.     

Reassessment of protein stability, DNA binding, and protection of Mycobacterium smegmatis Dps

The structure and function of Mycobacterium smegmatis Dps (DNA-binding proteins from starved cells) and of the protein studied by Gupta and Chatterji, in which the C terminus that is used for binding DNA contains a histidine tag, have been characterized in parallel. The native dodecamer dissociated reversibly into dimers above pH 7.5 and below pH 6.0, with apparent pK(a) values of approximately 7.65 and 4.75; at pH approximately 4.0, dimers formed monomers. Based on structural analysis, the two dissociation steps have been attributed to breakage of the salt bridges between Glu(157) and Arg(99) located at the 3-fold symmetry axes and to protonation of Asp(66) hydrogen-bonded to Lys(36) across the dimer interface, respectively. The C-terminal tag did not affect subunit dissociation, but altered DNA binding dramatically. At neutral pH, protonation of the histidine tag promoted DNA condensation, whereas in the native C terminus, compensation of negative and positive charges led to DNA binding without condensation. This different mode of interaction with DNA has important functional consequences as indicated by the failure of the native protein to protect DNA from DNase-mediated cleavage and by the efficiency of the tagged protein in doing so as a result of DNA sequestration in the condensates. Chemical protection of DNA from oxidative damage is realized by Dps proteins in a multistep iron oxidation/uptake/mineralization process. Dimers have a decreased protection efficiency due to disruption of the dodecamer internal cavity, where iron is deposited and mineralized after oxidation at the ferroxidase center.     

Biocrystalline structures in the nucleoids of the stationary and dormant prokaryotic cells

Compaction and biocrystallization of the nucleoid are presently considered as a necessary and important stage in the transformation of the cell ultrastructure during change of microbial cultures strategies from growth to survival. Nucleoid biocrystallization in the stationary phase cells is achieved due to structural regularity of the DNA complexes with the histone-like Dps protein. Our experiments with Escherichia coli mutants, overproducers of the Dps protein, confirmed nucleoid biocrystallization in the late stationary phase cells. Since nucleoid biocrystallization was revealed in E. сoli cells without Dps overproduction at late stages of starvation, it is constitutive in the cycle of development of microbial populations. The present work concentrated on detection of the nucleoid biocrystalline structure in (1) long-starved (21 day in the chemostat mode) bacterial cells (genera Arthrobacter and Pseudomonas), (2) dormant ametabolic (anabiotic) cells of such prokaryotes as archaea and non-spore-forming bacteria, (3) endospores of bacilli, (4) streptomycete exospores, and (5) in the cells surviving in permafrost for (2?3 Ma). The topics discussed include nucleoid biocrystallization as a necessary stage of maturation of the dormant microbial cells providing for survival and preservation of the species, dynamics of nucleoid biocrystallization during maturation of the dormant cells, and its possible role for the preservation of genetic information in the case of autolysis of most of the cells in a developing culture.     

Unveiling the role of Dps in the organization of mycobacterial nucleoid

In order to preserve genetic information in stress conditions, bacterial DNA is organized into higher order nucleoid structure. In this paper, with the help of Atomic Force Microscopy, we show the different structural changes in mycobacterial nucleoid at different points of growth in the presence of different concentrations of glucose in the medium. We also observe that in Mycobacterium smegmatis, two different Dps proteins (Dps1 and Dps2) promote two types of nucleoid organizations. At the late stationary phase, under low glucose availability, Dps1 binds to DNA to form a very stable toroid structure. On the other hand, under the same condition, Dps2-DNA complex forms an incompletely condensed toroid and finally forms a further stable coral reef structure in the presence of RNA. This coral reef structure is stable in high concentration of bivalent ion like Mg(2+).     

Structure, function and regulation of the DNA-binding protein Dps and its role in acid and oxidative stress resistance in Escherichia coli: a review

Dps, the DNA‐binding protein from starved cells, is capable of providing protection to cells during exposure to severe environmental assaults; including oxidative stress and nutritional deprivation. The structure and function of Dps have been the subject of numerous studies and have been examined in several bacteria that possess Dps or a structural/functional homologue of the protein. Additionally, the involvement of Dps in stress resistance has been researched extensively as well. The ability of Dps to provide multifaceted protection is based on three intrinsic properties of the protein: DNA binding, iron sequestration, and its ferroxidase activity. These properties also make Dps extremely important in iron and hydrogen peroxide detoxification and acid resistance as well. Regulation of Dps expression in E. coli is complex and partially dependent on the physiological state of the cell. Furthermore, it is proposed that Dps itself plays a role in gene regulation during starvation, ultimately making the cell more resistant to cytotoxic assaults by controlling the expression of genes necessary for (or deleterious to) stress resistance. The current review focuses on the aforementioned properties of Dps in E. coli, its prototypic organism. The consequences of elucidating the protective mechanisms of this protein are far‐reaching, as Dps homologues have been identified in over 1000 distantly related bacteria and Archaea. Moreover, the prevalence of Dps and Dps‐like proteins in bacteria suggests that protection involving DNA and iron sequestration is crucial and widespread in prokaryotes.     

Expression of mycobacterial cell division protein, FtsZ, and dormancy proteins, DevR and Acr, within lung granulomas throughout guinea pig infection

The ability of Mycobacterium tuberculosis to persist in a dormant state is a hallmark of tuberculosis. An insight into the expression of mycobacterial proteins will contribute to our understanding of bacterial physiology in vivo. To this end, the expression of FtsZ, Acr and DevR was assessed in the lung granulomas of guinea pigs infected with M. tuberculosis. Antigen immunostaining was then compared with the detection of acid-fast bacilli (AFB) and mycobacterial DNA. Surprisingly, immunostaining for all three antigens was observed throughout the course of infection; maximum expression of all antigens was noted at 20 weeks of infection. The intensity of immunostaining correlated well with the presence of intact bacteria, suggesting that mycobacterial antigens in the extracellular fraction have a short half-life; in contrast to protein, extracellular bacterial DNA was found to be more stable. Immunostaining for bacterial division and dormancy markers could not clearly distinguish between replicating and non-replicating organisms during the course of infection. The detection of Acr and DevR from 4 weeks onwards indicates that the dormancy proteins are expressed from early on in infection. Both antigen staining and DNA detection from intact bacilli were useful for detecting intact mycobacteria in the absence of AFB.     

The crystal structures of Lactococcus lactis MG1363 Dps proteins reveal the presence of an N-terminal helix that is required for DNA binding

Dps proteins play a major role in the protection of bacterial DNA from damage by reactive oxygen species. Previous studies have implicated the extended lysine-containing N-terminal regions of Dps subunits in DNA binding, but this part of the structure has not previously been observed crystallographically. Here the structures of two Dps proteins (DpsA and DpsB) from Lactococcus lactis MG1363 reveal for the first time the presence of an N-terminal alpha helix that extends from the core of the Dps subunit. Consequently, the N-terminal helices are displayed in parallel pairs on the exterior of the dodecameric Dps assemblies. Both DpsA and DpsB bind DNA. Deletion of the DpsA N-terminal helix impaired DNA binding. The N-terminal Lys residues of Escherichia coli Dps have been implicated in DNA binding. Replacement of the lactococcal DpsA Lys residues 9, 15 and 16 by Glu did not inhibit DNA binding. However, DNA binding was inhibited by EDTA, suggesting a role for cations in DNA binding. In contrast to E. coli, Bacillus brevis and Mycobacterium smegmatis Dps:DNA complexes, in which DNA interacts with crystalline Dps phases, L. lactis DNA:Dps complexes appeared as non-crystalline aggregates of protein and DNA in electron micrographs.     

Characterization of the twin-arginine translocase secretion system of Mycobacterium smegmatis

The twin-arginine translocation (TAT) system secretes fully folded proteins that contain a twin-arginine motif within their signal sequence across the cytoplasmic membrane in bacteria. Using a green fluorescent protein fused with a TAT signal sequence, we demonstrated that Mycobacterium smegmatis contains a TAT system. By inactivating individual genes, we showed that three genes (tatA, tatB, and tatC) are required for a functional TAT system in M. smegmatis. The tat mutants exhibited a decreased growth rate and altered colony morphology compared to the parent strain. Comparison of the secreted proteins of the deltatatC and parent strain by two-dimensional polyacrylamide gel electrophoresis revealed an alteration in the secretion of at least five proteins, and one of the major TAT-dependent secreted proteins was identified as beta-lactamase (BlaS). The genome of M. smegmatis was analyzed with the TATFIND program, and 49 putative TAT substrates were identified, including the succinate transporter DctP. Because disruption of the TAT secretion system has a direct effect on the physiology of M. smegmatis and homologs of the TAT proteins are also present in the genome of Mycobacterium tuberculosis, the TAT secretion system or its substrates may be good candidates for drug or vaccine development.     

Spatial organization of Dps and DNA–Dps complexes

DNA co-crystallization with Dps family proteins is a fundamental mechanism, which preserves DNA in bacteria from harsh conditions. Though many aspects of this phenomenon are well characterized, the spatial organization of DNA in DNA–Dps co-crystals is not completely understood, and existing models need further clarification. To advance in this problem we have utilized atomic force microscopy (AFM) as the main structural tool, and small-angle X-scattering (SAXS) to characterize Dps as a key component of the DNA-protein complex. SAXS analysis in the presence of EDTA indicates a significantly larger radius of gyration for Dps than would be expected for the core of the dodecamer, consistent with the N-terminal regions extending out into solution and being accessible for interaction with DNA. In AFM experiments, both Dps protein molecules and DNA–Dps complexes adsorbed on mica or highly oriented pyrolytic graphite (HOPG) surfaces form densely packed hexagonal structures with a characteristic size of about 9 nm. To shed light on the peculiarities of DNA interaction with Dps molecules, we have characterized individual DNA–Dps complexes. Contour length evaluation has confirmed the non-specific character of Dps binding with DNA and revealed that DNA does not wrap Dps molecules in DNA–Dps complexes. Angle analysis has demonstrated that in DNA–Dps complexes a Dps molecule contacts with a DNA segment of ~6 nm in length. Consideration of DNA condensation upon complex formation with small Dps quasi-crystals indicates that DNA may be arranged along the rows of ordered protein molecules on a Dps sheet.     

Molecular mechanism of in vitro oligomerization of Dps from Mycobacterium smegmatis: mutations of the residues identified by "interface cluster" analysis

The irreversible dodecamerization of native Dps trimers from Mycobacterium smegmatis, in vitro, is known to be directly associated with the bimodal function of this protein. Hence it is important to explore this pathway at the molecular level. Two types of trimers, Trimer A (tA) and Trimer B (tB), can be derived from the dodecamer due to the inherent 3-fold symmetry of the spherical crystal structure. These derived trimers were expressed as protein structure graphs (PSGs) using the computed interaction strength among the residues. Interface clusters which were identified from PSGs allowed us to convincingly predict E146 and F47 for further mutation studies. Various single and double mutants were constructed and characterized. We were finally able to generate a single mutant F47E impaired in dodecamerization and a double mutant E146AF47E as native monomer in solution. These two observed results suggest that the two trimers are important for dodecamerization and that the residues selected are important for the structural stability of the protein in vitro.     

Lipoarabinomannan localization and abundance during growth of Mycobacterium smegmatis

Lipoarabinomannan (LAM) is a structurally heterogeneous amphipathic lipoglycan present in Mycobacterium spp. and other actinomycetes, which constitutes a major component of the cell wall and exhibits a wide spectrum of immunomodulatory effects. Analysis of Mycobacterium smegmatis subcellular fractions and spheroplasts showed that LAM and lipomannan (LM) were primarily found in a cell wall-enriched subcellular fraction and correlated with the presence (or absence) of the mycolic acids in spheroplast preparations, suggesting that LAM and LM are primarily associated with the putative outer membrane of mycobacteria. During the course of these studies significant changes in the LAM/LM content of the cell wall were noted relative to the age of the culture. The LAM content of the M. smegmatis cell wall was dramatically reduced as the bacilli approached stationary phase, whereas LM, mycolic acid, and arabinogalactan content appeared to be unchanged. In addition, cell morphology and acid-fast staining characteristics showed variations with growth phase of the bacteria. In the logarithmic phase, the bacteria were found to be classic rod-shaped acid-fast bacilli, while in the stationary phase M. smegmatis lost the characteristic rod shape and developed a punctate acid-fast staining pattern with carbolfuchsin. The number of viable bacteria was independent of LAM content and phenotype. Taken together, the results presented here suggest that LAM is primarily localized with the mycolic acids in the cell wall and that the cellular concentration of LAM in M. smegmatis is selectively modulated with the growth phase.     

Structure and Mechanism of Iron Translocation by a Dps Protein from Microbacterium arborescens

Dps (DNA protection during starvation) enzymes are a major class of dodecameric proteins that bacteria use to detoxify their cytosol through the uptake of reactive iron species. In the stationary growth phase of bacteria, Dps enzymes are primarily used to protect DNA by biocrystallization. To characterize the wild type Dps protein from Microbacterium arborescens that displays additional catalytic functions (amide hydrolysis and synthesis), we determined the crystal structure to a resolution of 2.05 Å at low iron content. The structure shows a single iron at the ferroxidase center coordinated by an oxo atom, one water molecule, and three ligating residues. An iron-enriched protein structure was obtained at 2 Å and shows the stepwise uptake of two hexahydrated iron atoms moving along channels at the 3-fold axis before a restriction site inside the channels requires removal of the hydration sphere. Supporting biochemical data provide insight into the regulation of this acylamino acid hydrolase. Moreover, the peroxidase activity of the protein was determined. The influence of iron and siderophores on the expression of acylamino acid hydrolase was monitored during several stages of cell growth. Altogether our data provide an interesting view of an unusual Dps-like enzyme evolutionarily located apart from the large Dps sequence clusters.     

Activity of Trifluoperazine against Replicating, Non-Replicating and Drug Resistant M. tuberculosis

Trifluoperazine, a knowm calmodulin antagonist, belongs to a class of phenothiazine compounds that have multiple sites of action in mycobacteria including lipid synthesis, DNA processes, protein synthesis and respiration. The objective of this study is to evaluate the potential of TFP to be used as a lead molecule for development of novel TB drugs by showing its efficacy on multiple drug resistant (MDR) Mycobacterium tuberculosis (M.tb) and non-replicating dormant M.tb. Wild type and MDR M.tb were treated with TFP under different growth conditions of stress like low pH, starvation, presence of nitric oxide and in THP-1 infection model. Perturbation in growth kinetics of bacilli at different concentrations of TFP was checked to determine the MIC of TFP for active as well as dormant bacilli. Results show that TFP is able to significantly reduce the actively replicating as well as non-replicating bacillary load. It has also shown inhibitory effect on the growth of MDR M.tb. TFP has shown enhanced activity against intracellular bacilli, presumably because phenothiazines are known to get accumulated in macrophages. This concentration was, otherwise, found to be non-toxic to macrophage in vitro. Our results show that TFP has the potential to be an effective killer of both actively growing and non-replicating bacilli including MDR TB. Further evaluation and in vivo studies with Trifluoperazine can finally help us know the feasibility of this compound to be used as either a lead compound for development of new TB drugs or as an adjunct in the current TB chemotherapy.