Dps Functions as a Key Player in Bacterial Iron Homeostasis.

Iron plays a vital role in the maintenance of life, being central to various cellular processes, from respiration to gene regulation. It is essential for iron to be stored in a nontoxic and readily available form. DNA binding proteins under starvation (Dps) belong to the ferritin family of iron storage proteins and are adept at storing iron in their hollow protein shells. Existing solely in prokaryotes, these proteins have the additional functions of DNA binding and protection from oxidative stress. Iron storage proteins play a functional role in storage, release, and transfer of iron and therefore are central to the optimal functioning of iron homeostasis. Here we review the multifarious properties of Dps through relevant biochemical and structural studies with a focus on iron storage and ferroxidation. We also examine the role of Dps as a possible candidate as an iron donor to iron-sulfur (Fe-S) clusters, which are ubiquitous to many biological processes.

C-4-Modified Isotetrones Prevent Biofilm Growth and Persister Cell Resuscitation in Mycobacterium smegmatis.

Hyperphosphorylated nucleotide (p)ppGpp, synthesized by Rel protein, regulates the stringent response pathway responsible for biofilm and persister cell growth in mycobacteria. The discovery of vitamin C as an inhibitor of Rel protein activities raises the prospect of tetrone lactones to prevent such pathways. The closely related isotetrone lactone derivatives are identified herein as inhibitors of the above processes in a mycobacterium. Synthesis and biochemical evaluations show that an isotetrone possessing phenyl substituent at C-4 inhibit the biofilm formation at 400 µg mL-1, 84 h post-exposure, followed by moderate inhibition by the isotetrone possessing the p-hydroxyphenyl substituent. The latter isotetrone inhibits the growth of persister cells at 400 µg mL-1 f.c. when monitored for 2 weeks, under PBS starvation. Isotetrones also potentiate the inhibition of antibiotic-tolerant regrowth of cells by ciprofloxacin (0.75 µg mL-1) and thus act as bioenhancers. Molecular dynamics studies show that isotetrone derivatives bind to the RelMsm protein more efficiently than vitamin C at a binding site possessing serine, threonine, lysine, and arginine.

Aza-Michael promoted glycoconjugation of PETIM dendrimers and selectivity in mycobacterial growth inhibitions.

The benign nature of aza-Michael addition reaction in aqueous solutions is demonstrated herein to conduct a direct glycoconjugation of amine-terminated poly(ether imine) (PETIM) dendrimers. Zero to three generations of dendrimers, possessing up to 16 amine functionalities at their peripheries, undergo aza-Michael reaction with unsaturated sugar vinyl sulfoxide in aq. MeOH solutions and afford the corresponding dendrimers modified with multiple glycosyl moieties at the periphery. First order kinetics of the glycoconjugation is monitored at varying temperatures and the rate constants are observed to be 60-508 s-1, for zero and first generation dendrimers. The antibacterial effects of amine-terminated dendrimers and the corresponding glycoconjugates are studied across Gram-positive, Gram-negative and acid-fast bacteria. Among the species, M. smegmatis and M. tuberculosis showed the greatest growth inhibition effect at micromolar concentrations, for the native amine-terminated and the corresponding glycoconjugated dendrimers. Quantitative assays are performed to adjudge the inhibition efficacies of dendrimers and the glycoconjugates. Selectivity to inhibit M. smegmatis and M. tuberculosis growth, and minimal effects on other bacterial species by dendrimers and glycoconjugates are emphasized.

Regulatory mechanisms of c-di-AMP synthase from Mycobacterium smegmatis revealed by a structure: Function analysis.

Cyclic-di-nucleotide-based secondary messengers regulate various physiological functions, including stress responses in bacteria. Cyclic diadenosine monophosphate (c-di-AMP) has recently emerged as a crucial second messenger with implications in processes including osmoregulation, antibiotic resistance, biofilm formation, virulence, DNA repair, ion homeostasis, and sporulation, and has potential therapeutic applications. The contrasting activities of the enzymes diadenylate cyclase (DAC) and phosphodiesterase (PDE) determine the equilibrium levels of c-di-AMP. Although c-di-AMP is suspected of playing an essential role in the pathophysiology of bacterial infections and in regulating host-pathogen interactions, the mechanisms of its regulation remain relatively unexplored in mycobacteria. In this report, we biochemically and structurally characterize the c-di-AMP synthase (MsDisA) from Mycobacterium smegmatis. The enzyme activity is regulated by pH and substrate concentration; conditions of significance in the homoeostasis of c-di-AMP levels. Substrate binding stimulates conformational changes in the protein, and pApA and ppApA are synthetic intermediates detectable when enzyme efficiency is low. Unlike the orthologous Bacillus subtilis enzyme, MsDisA does not bind to, and its activity is not influenced in the presence of DNA. Furthermore, we have determined the cryo-EM structure of MsDisA, revealing asymmetry in its structure in contrast to the symmetric crystal structure of Thermotoga maritima DisA. We also demonstrate that the N-terminal minimal region alone is sufficient and essential for oligomerization and catalytic activity. Our data shed light on the regulation of mycobacterial DisA and possible future directions to pursue.

An Overview of Dps: Dual Acting Nanovehicles in Prokaryotes with DNA Binding and Ferroxidation Properties.

DNA binding proteins under starvation (Dps) are proteins belonging to the ferritin family with the capacity for DNA binding, in addition to iron storage and ferroxidation. Present only in the prokaryotes, these multifaceted proteins have been assigned with a number of roles, from pathogenesis to nucleoid condensation and protection. They have a significant role in protecting the cells from free radical assaults, indirectly by sequestration of iron and by directly binding to the DNA. Due to their symmetry, stability and biomineralization capacity, these proteins have ever increasing potential applications in biotechnology and drug delivery. This chapter tries to bring together all these aspects of Dps in the view of current understanding and older perspectives by studies of our group as well as other experts in the field.

Validation of Omega Subunit of RNA Polymerase as a Functional Entity.

The bacterial RNA polymerase (RNAP) is a multi-subunit protein complex (α2ßß'ω σ) containing the smallest subunit, ω. Although identified early in RNAP research, its function remained ambiguous and shrouded with controversy for a considerable period. It was shown before that the protein has a structural role in maintaining the conformation of the largest subunit, ß', and its recruitment in the enzyme assembly. Despite evolutionary conservation of ω and its role in the assembly of RNAP, E. coli mutants lacking rpoZ (codes for ω) are viable due to the association of the global chaperone protein GroEL with RNAP. To get a better insight into the structure and functional role of ω during transcription, several dominant lethal mutants of ω were isolated. The mutants showed higher binding affinity compared to that of native ω to the α2ßß' subassembly. We observed that the interaction between α2ßß' and these lethal mutants is driven by mostly favorable enthalpy and a small but unfavorable negative entropy term. However, during the isolation of these mutants we isolated a silent mutant serendipitously, which showed a lethal phenotype. Silent mutant of a given protein is defined as a protein having the same sequence of amino acids as that of wild type but having mutation in the gene with alteration in base sequence from more frequent code to less frequent one due to codon degeneracy. Eventually, many silent mutants were generated to understand the role of rare codons at various positions in rpoZ. We observed that the dominant lethal mutants of ω having either point mutation or silent in nature are more structured in comparison to the native ω. However, the silent code's position in the reading frame of rpoZ plays a role in the structural alteration of the translated protein. This structural alteration in ω makes it more rigid, which affects the plasticity of the interacting domain formed by ω and α2ßß'. Here, we attempted to describe how the conformational flexibility of the ω helps in maintaining the plasticity of the active site of RNA polymerase. The dominant lethal mutant of ω has a suppressor mapped near the catalytic center of the ß' subunit, and it is the same for both types of mutants.

Pleiotropic Effects of Bacterial Small Alarmone Synthetases: Underscoring the Dual-Domain Small Alarmone Synthetases in Mycobacterium smegmatis.

The nucleotide alarmone (p)ppGpp, signaling the stringent response, is known for more than 5 decades. The cellular turnover of the alarmone is regulated by RelA/SpoT homolog (RSH) superfamily of enzymes. There are long RSHs (RelA, SpoT, and Rel) and short RSHs [small alarmone synthetases (SAS) and small alarmone hydrolases (SAH)]. Long RSHs are multidomain proteins with (p)ppGpp synthesis, hydrolysis, and regulatory functions. Short RSHs are single-domain proteins with a single (p)ppGpp synthesis/hydrolysis function with few exceptions having two domains. Mycobacterial RelZ is a dual-domain SAS with RNase HII and the (p)ppGpp synthetase activity. SAS is known to impact multiple cellular functions independently and in accordance with the long RSH. Few SAS in bacteria including RelZ synthesize pGpp, the third small alarmone, along with the conventional (p)ppGpp. SAS can act as an RNA-binding protein for the negative allosteric inhibition of (p)ppGpp synthesis. Here, we initially recap the important features and molecular functions of different SAS that are previously characterized to understand the obligation for the "alarmone pool" produced by the long and short RSHs. Then, we focus on the RelZ, especially the combined functions of RNase HII and (p)ppGpp synthesis from a single polypeptide to connect with the recent findings of SAS as an RNA-binding protein. Finally, we conclude with the possibilities of using single-stranded RNA (ssRNA) as an additional therapeutic strategy to combat the persistent infections by inhibiting the redundant (p)ppGpp synthetases.

Small-Molecule Inhibition of Bacterial Biofilm.

Antibiotic resistance is a massive and serious threat to human welfare and healthcare. Apart from being genetically resistant to antibiotics, the other important mechanism by which bacteria can evade antibiotics is multidrug tolerance. Here cells enter into a transiently nongrowing phase, and as a result, latent infection remains inside the host, causing disease recurrence. Biofilm-derived antibiotic tolerance and persister formation of the pathogenic bacteria inside the host remain a serious issue of treatment failure and recurrent chronic infection in the case of all major pathogens. As a result, new chemotherapeutic agents are sought that specifically inhibit biofilm formation or maturation as well as cause the dispersion of mature biofilms, thus allowing the conventional drugs to kill sensitive cells residing inside. This mini-review attempts to analyze different small-molecule-based chemical approaches that have been used to enable bacterial biofilm inhibition at different steps of maturation.

Tracking the homeostasis of second messenger cyclic-di-GMP in bacteria.

Cyclic-di-GMP (c-di-GMP) is an important second messenger in bacteria which regulates the bacterial transition from motile to sessile phase and also plays a major role in processes such as cell division, exopolysaccharide synthesis, and biofilm formation. Due to its crucial role in dictating the bacterial phenotype, the synthesis and hydrolysis of c-di-GMP is tightly regulated via multiple mechanisms. Perturbing the c-di-GMP homeostasis affects bacterial growth and survival, so it is necessary to understand the underlying mechanisms related to c-di-GMP metabolism. Most techniques used for estimating the c-di-GMP concentration lack single-cell resolution and do not provide information about any heterogeneous distribution of c-di-GMP inside cells. In this review, we briefly discuss how the activity of c-di-GMP metabolising enzymes, particularly bifunctional proteins, is modulated to maintain c-di-GMP homeostasis. We further highlight how fluorescence-based methods aid in understanding the spatiotemporal regulation of c-di-GMP signalling. Finally, we discuss the blind spots in our understanding of second messenger signalling and outline how they can be addressed in the future.

RelZ-Mediated Stress Response in Mycobacterium smegmatis: pGpp Synthesis and Its Regulation.

Stringent response is a conserved stress response mechanism in which bacteria employ the second messengers guanosine tetraphosphate and guanosine pentaphosphate [collectively termed (p)ppGpp] to reprogram their cellular processes under stress. In mycobacteria, these alarmones govern a multitude of cellular phenotypes, such as cell division, biofilm formation, antibiotic tolerance, and long-term survival. Mycobacterium smegmatis possesses the bifunctional RelMsm as a (p)ppGpp synthetase and hydrolase. In addition, it contains a short alarmone synthetase MS_RHII-RSD (renamed RelZ), which contains an RNase H domain in tandem with the (p)ppGpp synthetase domain. The physiological functions of RelMsm have been well documented, but there is no clear picture about the cellular functions of RelZ in M. smegmatis RelZ has been implicated in R-loop induced stress response due to its unique domain architecture. In this study, we elucidate the differential substrate utilization pattern of RelZ compared to that of RelMsm We unveil the ability of RelZ to use GMP as a substrate to synthesize pGpp, thereby expanding the repertoire of second messengers known in mycobacteria. We have demonstrated that the pGpp synthesis activity of RelZ is negatively regulated by RNA and pppGpp. Furthermore, we investigated its role in biofilm formation and antibiotic tolerance. Our findings highlight the complex role played by the RelZ in cellular physiology of M. smegmatis and sheds light upon its functions distinct from those of RelMsmIMPORTANCE Bacteria utilize nucleotide messengers to survive the hostile environmental conditions and the onslaught of attacks within the host. The second messengers guanosine tetraphosphate and pentaphosphate [(p)ppGpp] have a profound impact on the long-term survival, biofilm formation, antibiotic tolerance, virulence, and pathogenesis of bacteria. Therefore, understanding the stress response mechanism regulated by (p)ppGpp is essential for discovering inhibitors of stress response and potential drug targets. Mycobacterium smegmatis contains two (p)ppGpp synthetases: RelMsm and RelZ. Our study unravels the novel regulatory mechanisms of RelZ activity and its role in mediating antibiotic tolerance. We further reveal its ability to synthesize novel second messenger pGpp, which may have regulatory roles in mycobacteria.

Unraveling the Role of Silent Mutation in the ω-Subunit of Escherichia coli RNA Polymerase: Structure Transition Inhibits Transcription.

The bacterial RNA polymerase is a multi-subunit enzyme complex composed of six subunits, α2ßß'σω. The function of this enzyme is to transcribe the DNA base sequence to the RNA intermediate, which is ultimately translated to protein. Though the contribution of each subunit in RNA synthesis has been clearly elucidated, the role of the smallest ω-subunit is still unclear despite several studies. Recently, a study on a dominant negative mutant of rpoZ has been reported in which the mutant was shown to render the RNA polymerase defective in transcription initiation (ω6, N60D) and gave an insight on the function of ω in RNA polymerase. Serendipitously, we also obtained a silent mutant, and the mutant was found to be lethal during the isolation of toxic mutants. The primary focus of this study is to understand the mechanistic details of this lethality. Isolated ω shows a predominantly unstructured circular dichroism profile and becomes α-helical in the enzyme complex. This structural transition is perhaps the reason for this lack of function. Subsequently, we generated several silent mutants of ω to investigate the role of codon bias and the effect of rare codons with respect to their position in rpoZ. Not all silent mutations affect the structure. RNA polymerase when reconstituted with structurally altered silent mutants of ω is transcriptionally inactive. The CodonPlus strain, which has surplus tRNA, was used to assess for the rescue of the phenotype in lethal silent mutants.

Sugar Vinyl Sulfoxide Glycoconjugation of Peptides and Lysozyme: Abrogation of Proteolysis at the Lysine Sites.

We describe a glycoconjugation strategy in which a sugar vinyl sulfoxide, acting as Michael donor, reacts efficiently with amine nucleophiles arising from the lysine side chain in peptides and proteins, at physiological pH and temperature. The method permits glycoconjugation of the lysine residues present in lysozyme with the sugar vinyl sulfoxide. The glycoconjugation of the protein abrogates the trypsin-mediated proteolysis at the lysine sites. The modified protein catalyzes digestion of the Gram-negative Escherichia coli cell wall and retains the same antimicrobial property as the native lysozyme.

Mannopyranoside Glycolipids Inhibit Mycobacterial and Biofilm Growth and Potentiate Isoniazid Inhibition Activities in M.†smegmatis.

Lipomannan and lipoarabinomannan are integral components of the mycobacterial cell wall. Earlier studies demonstrated that synthetic arabinan and arabinomannan glycolipids acted as inhibitors of mycobacterial growth, in addition to exhibiting inhibitory activities of mycobacterial biofilm. Herein, it is demonstrated that synthetic mannan glycolipids are better inhibitors of mycobacterial growth, whereas lipoarabinomannan has a higher inhibition efficiency to biofilm. Syntheses of mannan glycolipids with a graded number of mannan moieties and an arabinomannan glycolipid are conducted by chemical methods and subsequent mycobacterial growth and biofilm inhibition studies are conducted on Mycobacterium smegmatis. Growth inhibition of (73±3) % is observed with a mannose trisaccharide containing a glycolipid, whereas this glycolipid did not promote biofilm inhibition activity better than that of arabinomannan glycolipid. The antibiotic supplementation activities of glycolipids on growth and biofilm inhibitions are evaluated. Increases in growth and biofilm inhibitions are observed if the antibiotic is supplemented with glycolipids, which leads to a significant reduction of inhibition concentrations of the antibiotic.

4-Hydroxy-2-pyridone Derivatives and the δ-pyrone Isostere as Novel Agents Against Mycobacterium smegmatis Biofilm Inhibitors.

BACKGROUND: The treatment of a bacterial infection when the bacterium is growing in a biofilm is a vexed issue. This is because the bacteria in a biofilm behaves differently compared to the individual planktonic free-form. As a result, traditional antibacterial agents lose their activity. OBJECTIVE: Presently, there are not many drugs that are effective against bacteria growing in biofilms. Based on literature reports, we have sought to develop novel derivatives of 4-hydroxy-2- pyridone as both antimycobacterial and antibiofilm agents. METHODS: The pyridone derivatives were synthesized by reacting 4-hydroxy-6-methyl-2H-pyran-2- one with appropriate amines and followed by reaction with substituted phenyl isocyanates as reported in the literature. RESULTS: Four compounds in this series significantly inhibit the growth and formation of biofilm by Mycobacterium smegmatis (mc2 155 strain) at 50 µg/ml. Further, in silico evaluation of the ADME parameters shows that these compounds possess good drug-like properties and have the potential to be developed both as antibiofilm and as oral antimycobacterial agents. CONCLUSION: This finding is of significance as presently very few small molecules are known to inhibit biofilm formation in mycobacteria. These compounds are unique in the sense that they are more potent against Mycobacterium smegmatis in the biofilm state compared to the planktonic form.

Substrate-induced domain movement in a bifunctional protein, DcpA, regulates cyclic di-GMP turnover: Functional implications of a highly conserved motif.

In eubacteria, cyclic di-GMP (c-di-GMP) signaling is involved in virulence, persistence, motility and generally orchestrates multicellular behavior in bacterial biofilms. Intracellular c-di-GMP levels are maintained by the opposing activities of diguanylate cyclases (DGCs) and cognate phosphodiesterases (PDEs). The c-di-GMP homeostasis in Mycobacterium smegmatis is supported by DcpA, a conserved, bifunctional protein with both DGC and PDE activities. DcpA is a multidomain protein whose GAF-GGDEF-EAL domains are arranged in tandem and are required for these two activities. To gain insight into how interactions among these three domains affect DcpA activity, here we studied its domain dynamics using real-time FRET. We demonstrate that substrate binding in DcpA results in domain movement that prompts a switch from an "open" to a "closed" conformation and alters its catalytic activity. We found that a single point mutation in the conserved EAL motif (E384A) results in complete loss of the PDE activity of the EAL domain and in a significant decrease in the DGC activity of the GGDEF domain. Structural analyses revealed multiple hydrophobic and aromatic residues around Cys579 that are necessary for proper DcpA folding and maintenance of the active conformation. On the basis of these observations and taking into account additional bioinformatics analysis of EAL domain-containing proteins, we identified a critical putatively conserved motif, GCXXXQGF, that plays an important role in c-di-GMP turnover. We conclude that a substrate-induced conformational switch involving movement of a loop containing a conserved motif in the bifunctional diguanylate cyclase-phosphodiesterase DcpA controls c-di-GMP turnover in M. smegmatis.

The role of ω-subunit of Escherichia coli RNA polymerase in stress response.

ppGpp, an alarmone for stringent response, plays an important role in the reprogramming of the transcription complex at the time of stress. In Escherichia coli, ppGpp mediates its action by binding to at least two different sites on RNA polymerase (RNAP). One of the sites to which ppGpp binds to RNAP is at the ß'-ω interface; however, the underlying molecular mechanism and the physiological relevance of ppGpp binding to this site remain unclear. In this study, we have performed UV cross-linking experiments using 32 P azido-labeled ppGpp to probe its association with RNAP in the absence and presence of ω, and observed weaker binding of ppGpp to the RNAP without ω. Furthermore, we followed the binding kinetics of ppGpp to RNAP with and without ω by isothermal titration calorimetry and found it to be concurrent with the cross-linking results. Native ω is intrinsically disordered, and we have used a previously characterized structured mutant of ω, which affects the plasticity of the active site of RNAP. Results show that the flexibility conferred by the unstructured ω is a prerequisite for ppGpp binding to RNAP. We have analyzed the stress-associated phenotypes in an E. coli strain devoid of ω (∆rpoZ). ppGpp levels in ∆rpoZ strain were found to be similar to that of the wild-type strain. Interestingly, when the ∆rpoZ strain of E. coli was transferred after nutritional stress to an enriched media, the recovery of growth was compromised. We have identified a new phenotype of ∆rpoZ strain corresponding to defect in biofilm formation in minimal media.

Altered Distribution of RNA Polymerase Lacking the Omega Subunit within the Prophages along the Escherichia coli K-12 Genome.

The RNA polymerase (RNAP) of Escherichia coli K-12 is a complex enzyme consisting of the core enzyme with the subunit structure α2ßß'ω and one of the σ subunits with promoter recognition properties. The smallest subunit, omega (the rpoZ gene product), participates in subunit assembly by supporting the folding of the largest subunit, ß', but its functional role remains unsolved except for its involvement in ppGpp binding and stringent response. As an initial approach for elucidation of its functional role, we performed in this study ChIP-chip (chromatin immunoprecipitation with microarray technology) analysis of wild-type and rpoZ-defective mutant strains. The altered distribution of RpoZ-defective RNAP was identified mostly within open reading frames, in particular, of the genes inside prophages. For the genes that exhibited increased or decreased distribution of RpoZ-defective RNAP, the level of transcripts increased or decreased, respectively, as detected by reverse transcription-quantitative PCR (qRT-PCR). In parallel, we analyzed, using genomic SELEX (systemic evolution of ligands by exponential enrichment), the distribution of constitutive promoters that are recognized by RNAP RpoD holoenzyme alone and of general silencer H-NS within prophages. Since all 10 prophages in E. coli K-12 carry only a small number of promoters, the altered occupancy of RpoZ-defective RNAP and of transcripts might represent transcription initiated from as-yet-unidentified host promoters. The genes that exhibited transcription enhanced by RpoZ-defective RNAP are located in the regions of low-level H-NS binding. By using phenotype microarray (PM) assay, alterations of some phenotypes were detected for the rpoZ-deleted mutant, indicating the involvement of RpoZ in regulation of some genes. Possible mechanisms of altered distribution of RNAP inside prophages are discussed. IMPORTANCE The 91-amino-acid-residue small-subunit omega (the rpoZ gene product) of Escherichia coli RNA polymerase plays a structural role in the formation of RNA polymerase (RNAP) as a chaperone in folding the largest subunit (ß', of 1,407 residues in length), but except for binding of the stringent signal ppGpp, little is known of its role in the control of RNAP function. After analysis of genomewide distribution of wild-type and RpoZ-defective RNAP by the ChIP-chip method, we found alteration of the RpoZ-defective RNAP inside open reading frames, in particular, of the genes within prophages. For a set of the genes that exhibited altered occupancy of the RpoZ-defective RNAP, transcription was found to be altered as observed by qRT-PCR assay. All the observations here described indicate the involvement of RpoZ in recognition of some of the prophage genes. This study advances understanding of not only the regulatory role of omega subunit in the functions of RNAP but also the regulatory interplay between prophages and the host E. coli for adjustment of cellular physiology to a variety of environments in nature.

Vitamin C: A Natural Inhibitor of Cell Wall Functions and Stress Response in Mycobacteria.

Tuberculosis, caused by Mycobacterium tuberculosis, has re-emerged as a threat to human race. Conventional antibiotic treatments are failing due to different stress response strategies adopted by bacterial pathogens. Since time immemorial, Vitamin C is known to protect against pathogens by boosting immunity in humans. Recently, Vitamin C has been shown to directly kill M. tuberculosis including multiple drug-resistant strains by generation of oxidative radicals through Fenton's reaction. Concurrently, it inhibits (p)ppGpp-mediated stringent response thus effectively shutting down long-term survival and persistence in mycobacteria. Here, we have discussed historical perspective and recent evidences on Vitamin C-mediated inhibition of several key pathways of M. tuberculosis such as (p)ppGpp synthesis and mycobacterial cell wall function. Several cell wall components including mycolic acids are critical for mycobacterial virulence. We observed downregulation of various mycolic acids in M. smegmatis upon treatment with Vitamin C, and data have been presented here. Vitamin C has been shown to inhibit the biofilm growth as well as disrupt the formed biofilm in mycobacteria. Additionally, Vitamin C role in cell-mediated and humoral immunity has been elucidated. Vitamin C is toxic at high concentration; therefore we have proposed the idea of derivatizing Vitamin C in order to lower the minimal inhibition concentration (MIC) necessary to target M. tuberculosis.

Synthetic Arabinomannan Heptasaccharide Glycolipids Inhibit Biofilm Growth and Augment Isoniazid Effects in Mycobacterium smegmatis.

Biofilm formation, involving attachment to an adherent surface, is a critical survival strategy of mycobacterial colonies in hostile environmental conditions. Here we report the synthesis of heptasaccharide glycolipids based on mannopyranoside units anchored on to a branched arabinofuranoside core. Two types of glycolipids-2,3-branched and 2,5-branched-were synthesized and evaluated for their efficacies in inhibiting biofilm growth by the non-pathogenic mycobacterium variant Mycobacterium smegmatis. Biofilm formation was inhibited at a minimum biofilm growth inhibition concentration (MBIC) of 100 µg mL-1 in the case of the 2,5-branched heptasaccharide glycolipid. Further, we were able to ascertain that a combination of the drug isoniazid with the branched heptasaccharide glycolipid (50 µg mL-1 ) potentiates the drug, making it three times more effective, with an improved MBIC of 30 µg mL-1 . These studies establish that synthetic glycolipids not only act as inhibitors of biofilm growth, but also provide a synergistic effect when combined with significantly lowered concentrations of isoniazid to disrupt the biofilm structures of the mycobacteria.

A Mutation Directs the Structural Switch of DNA Binding Proteins under Starvation to a Ferritin-like Protein Cage.

Proteins of the ferritin family are ubiquitous in living organisms. With their spherical cage-like structures they are the iron storehouses in cells. Subfamilies of ferritins include 24-meric ferritins and bacterioferritins (maxiferritins), and 12-meric Dps (miniferritins). Dps safeguards DNA by direct binding, affording physical protection and safeguards from free radical-mediated damage by sequestering iron in its core. The maxiferritins can oxidize and store iron but cannot bind DNA. Here we show that a mutation at a critical interface in Dps alters its assembly from the canonical 12-mer to a ferritin-like 24-mer under crystallization. This structural switch was attributed to the conformational alteration of a highly conserved helical loop and rearrangement of the C-terminus. Our results demonstrate a novel concept of mutational switch between related protein subfamilies and corroborate the popular model for evolution by which subtle substitutions in an amino acid sequence lead to diversification among proteins.