Analysis of intracellular expressed proteins of Mycobacterium tuberculosis clinical isolates

Background

Tuberculosis (TB) is the most threatening infectious disease globally. Although progress has been made to reduce global incidence of TB, emergence of multidrug resistant (MDR) TB threatens to undermine these advances. To combat the disease, novel intervention strategies effective against drug resistant and sensitive subpopulations of M. tuberculosis are urgently required as adducts in the present treatment regimen. Using THP-1 cells we have analyzed and compared the global protein expression profile of broth-cultured and intraphagosomally grown drug resistant and sensitive M.tuberculosis clinical isolates.

Results

On comparing the two dimensional (2-DE) gels, many proteins were found to be upregulated/expressed during intracellular state which were identified by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). Four proteins (adenosylhomocysteinase, aspartate carbomyltransferase, putatitive thiosulfate sulfurtransferase and universal stress protein) were present in both intracellular MDR and sensitive isolates and three of these belonged to intermediary metabolism and respiration category. Two proteins (alanine dehydrogenase and adenosine kinase) of intracellular MDR isolate and two (glucose-6-phosphate isomerase and ATP synthase epsilon chain) of intracellular sensitive isolate belonged to intermediary metabolism and respiration category. One protein (Peroxidase/Catalase) of intracellular MDR and three (HSPX, 14 kDa antigen and 10 kDa chaperonin) of sensitive isolate belonged to virulence, detoxification and adaptation category. ESAT-6 of intracellular MDR belonged to cell wall and cell processes category. Two proteins (Antigen 85-C and Antigen 85-A) of intracellular sensitive isolate were involved in lipid metabolism while probable peptidyl-prolyl cis-trans isomerase A was involved in information pathways. Four (Rv0635, Rv1827, Rv0036c and Rv2032) of intracellular MDR and two proteins (Rv2896c and Rv2558c) of sensitive isolate were hypothetical proteins which were functionally characterized using bioinformatic tools. Bioinformatic findings revealed that the proteins encoded by Rv0036, Rv2032c, Rv0635, Rv1827 and Rv2896c genes are involved in cellular metabolism and help in intracellular survival.

Conclusions

Mass spectrometry and bioinformatic analysis of both MDR and sensitive isolates of M. tuberculosis during intraphagosomal growth showed that majority of commonly upregulated/expressed proteins belonged to the cellular metabolism and respiration category. Inhibitors of the metabolic enzymes/intermediate can therefore serve as suitable drug targets against drug-resistant and sensitive subpopulations of M. tuberculosis.     

Phytoremediation potential of Helianthus annuus L in sewage-irrigated Indo-Gangetic alluvial soils

The study of phytoremediation potential of Helianthus annuus L was conducted in the sewage-irrigated Indo-Gangetic alluvial soils, India. Calcium @ 1.0% and Zn @ 40 ppm enhanced the yield of H. annuus L and minimized the toxicity of Cr in the investigated soils. The study indicated that H. annuus L is highly sensitive to Cr and Zn in terms of metallic pollution; and may be used as indicator plant. For Cr-phytoremediation, humic acid treatment @ 500 mL/acre induced the Cr-accumulation in roots (p < 0.007) and in shoots (p < 0.015), which was recorded 3.21 and 3.16 mg/kg in root and shoot of H. annuus L, respectively. We suggest that H. annuus L fulfils the necessary condition for efficiently increasing species bioaccumulation after soil treatment with humic acid in Cr-polluted sewage-irrigated soils through soil- plant rhizospheric processes.     

Structural basis of the chromodomain of Cbx3 bound to methylated peptides from histone h1 and G9a

Background

HP1 proteins are highly conserved heterochromatin proteins, which have been identified to be structural adapters assembling a variety of macromolecular complexes involved in regulation of gene expression, chromatin remodeling and heterochromatin formation. Much evidence shows that HP1 proteins interact with numerous proteins including methylated histones, histone methyltransferases and so on. Cbx3 is one of the paralogues of HP1 proteins, which has been reported to specifically recognize trimethylated histone H3K9 mark, and a consensus binding motif has been defined for the Cbx3 chromodomain.

Methodology/Principal Findings

Here, we found that the Cbx3 chromodomain can bind to H1K26me2 and G9aK185me3 with comparable binding affinities compared to H3K9me3. We also determined the crystal structures of the human Cbx3 chromodomain in complex with dimethylated histone H1K26 and trimethylated G9aK185 peptides, respectively. The complex structures unveil that the Cbx3 chromodomain specifically bind methylated histone H1K26 and G9aK185 through a conserved mechanism.

Conclusions/Significance

The Cbx3 chromodomain binds with comparable affinities to all of the methylated H3K9, H1K26 and G9aK185 peptides. It is suggested that Cbx3 may regulate gene expression via recognizing both histones and non-histone proteins.     

Molecular typing of fecal eukaryotic microbiota of human infants and their respective mothers

The micro-eukaryotic diversity from the human gut was investigated using universal primers directed towards 18S rRNA gene, fecal samples being the source of DNA. The subjects in this study included two breast-fed and two formula-milk-fed infants and their mothers. The study revealed that the infants did not seem to harbour any microeukaryotes in their gut. In contrast, there were distinct eukaryotic microbiota present in the mothers. The investigation is the first of its kind in the comparative study of the human feces to reveal the presence of micro-eukaryotic diversity variance in infants and adults from the Indian subcontinent. The micro-eukaryotes encountered during the investigation include known gut colonizers like Blastocystis and some fungi species. Some of these micro-eukaryotes have been speculated to be involved in clinical manifestations of various diseases. The study is an attempt to highlight the importance of micro-eukaryotes in the human gut.     

Bacterial cell surface damage due to centrifugal compaction

Centrifugal damage has been known to alter bacterial cell surface properties and interior structures, including DNA. Very few studies exist on bacterial damage caused by centrifugation because of the difficulty in relating centrifugation speed and container geometry to the damage caused. Here, we provide a simple, versatile method of analysis for describing the compaction of bacteria during centrifugation based on a proposed centrifugation coefficient, C. Values of C can be related to different bacterial cell surface properties. Changing the geometry of the centrifugation container or centrifugation speeds changed the value of C significantly. Initial deposition rates of Staphylococcus aureus ATCC 12600 to a glass surface decayed exponentially from 4,217 to 1,478 cm⁻² s⁻¹ with increasing C, while the proportion of staphylococci with a zeta potential of around −15 mV decreased from 97 to 58%. These surface-sensitive parameters were used independently to derive a critical centrifugation coefficient (0.040), above which centrifugation was considered to impact the outcome of surface-sensitive experiments due to cell surface damage. The critical centrifugation coefficient could successfully predict staphylococcal cell surface damage, i.e., a significant change in initial deposition rate or zeta potential distribution, in 84% of all cases included here, whereas the centrifugation speed could predict damage in only 58% of all cases. Moreover, controlling the centrifugation coefficient within narrow limits over a series of experiments yielded 43% smaller standard deviations in initial staphylococcal deposition rates than with centrifugation at fixed speeds for replicate experiments.     

Adhesive bond stiffness of Staphylococcus aureus with and without proteins that bind to an adsorbed fibronectin film

Staphylococcus aureus is known to cause biomaterial-associated infections of implants and devices once it has breached the skin and mucosal barriers. Adhesion is the initial step in the development of a biomaterial-associated infection, and strategies to prevent staphylococcal adhesion and thus biomaterial-associated infections require understanding of the adhesive bond. The aim of this study was to compare the adhesive bond stiffnesses of two S. aureus strains with and without fibronectin-binding proteins (FnBPs) adhering to a fibronectin-coated quartz crystal microbalance (QCM) sensor surface on the basis of a coupled- resonance model. Both fibronectin adsorption and staphylococcal adhesion were accompanied by negative frequency shifts, regardless of the absence or presence of FnBPs on the staphylococcal cell surfaces. This is the opposite of the positive frequency shifts often observed for other bacterial strains adhering to bare sensor surfaces. Most likely, adhering staphylococci sink into and deform the adsorbed protein layer, creating stiff binding with the sensor surface due to an increased bacterium-substratum contact area. S. aureus 8325-4 possesses FnBPs and yields less negative frequency shifts (Δf) that are further away from the zero-crossing frequency than S. aureus DU5883. This suggests that FnBPs on S. aureus 8325-4 create a stiffer bond to the fibronectin coating than has been observed for S. aureus DU5883. Due to a limited window of observation, as defined by the available resonance frequencies in QCM, we could not determine exact stiffness values.     

Functional role of the sarcin-ricin loop of the 23S rRNA in the elongation cycle of protein synthesis

The sarcin-ricin loop (SRL) is one of the longest conserved sequences in the 23S ribosomal RNA. The SRL has been accepted as crucial for the activity of the ribosome because it is targeted by cytotoxins such as α-sarcin and ricin that completely abolish translation. Nevertheless, the precise functional role of the SRL in translation is not known. Recent biochemical and structural studies indicate that the SRL is critical for triggering GTP hydrolysis on elongation factor Tu (EF-Tu) and elongation factor G (EF-G). To determine the functional role of the SRL in the elongation stage of protein synthesis, we analyzed mutations in the SRL that are known to abolish protein synthesis and are lethal to cells. Here, we show that the SRL is not critical for GTP hydrolysis on EF-Tu and EF-G. The SRL also is not essential for peptide bond formation. Our results, instead, suggest that the SRL is crucial for anchoring EF-G on the ribosome during mRNA-tRNA translocation.