Phosphorylation of Enoyl-Acyl Carrier Protein Reductase InhA Impacts Mycobacterial Growth and Survival

InhA, the primary target for the first line anti-tuberculosis drug isoniazid, is a key enzyme of the fatty-acid synthase II system involved in mycolic acid biosynthesis in Mycobacterium tuberculosis. In this study, we show that InhA is a substrate for mycobacterial serine/threonine protein kinases. Using a novel approach to validate phosphorylation of a substrate by multiple kinases in a surrogate host (Escherichia coli), we have demonstrated efficient phosphorylation of InhA by PknA, PknB, and PknH, and to a lower extent by PknF. Additionally, the sites targeted by PknA/PknB have been identified and shown to be predominantly located at the C terminus of InhA. Results demonstrate in vivo phosphorylation of InhA in mycobacteria and validate Thr-266 as one of the key sites of phosphorylation. Significantly, our studies reveal that the phosphorylation of InhA by kinases modulates its biochemical activity, with phosphorylation resulting in decreased enzymatic activity. Co-expression of kinase and InhA alters the growth dynamics of Mycobacterium smegmatis, suggesting that InhA phosphorylation in vivo is an important event in regulating its activity. An InhA-T266E mutant, which mimics constitutive phosphorylation, is unable to rescue an M. smegmatis conditional inhA gene replacement mutant, emphasizing the critical role of Thr-266 in mediating post-translational regulation of InhA activity. The involvement of various serine/threonine kinases in modulating the activity of a number of enzymes of the mycolic acid synthesis pathway, including InhA, accentuates the intricacies of mycobacterial signaling networks in parallel with the changing environment.     

An Acid-loading Chloride Transport Pathway in the Intraerythrocytic Malaria Parasite,Plasmodium falciparum

The intraerythrocytic malaria parasite exerts tight control over its ionic composition. In this study, a combination of fluorescent ion indicators and ³⁶Cl⁻ flux measurements was used to investigate the transport of Cl⁻ and the Cl⁻-dependent transport of “H⁺-equivalents” in mature (trophozoite stage) parasites, isolated from their host erythrocytes. Removal of extracellular Cl⁻, resulting in an outward [Cl⁻] gradient, gave rise to a cytosolic alkalinization (i.e. a net efflux of H⁺-equivalents). This was reversed on restoration of extracellular Cl⁻. The flux of H⁺-equivalents was inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid and, when measured in ATP-depleted parasites, showed a pronounced dependence on the pH of the parasite cytosol; the flux was low at cytosolic pH values < 7.2 but increased steeply with cytosolic pH at values > 7.2. ³⁶Cl⁻ influx measurements revealed the presence of a Cl⁻ uptake mechanism with characteristics similar to those of the Cl⁻-dependent H⁺-equivalent flux. The intracellular concentration of Cl⁻ in the parasite was estimated to be ∼48 mm in situ. The data are consistent with the intraerythrocytic parasite having in its plasma membrane a 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid-sensitive transporter that, under physiological conditions, imports Cl⁻ together with H⁺-equivalents, resulting in an intracellular Cl⁻ concentration well above that which would occur if Cl⁻ ions were distributed passively in accordance with the parasite''s large, inwardly negative membrane potential.     

Neutrophil elastase and myeloperoxidase regulate the formation of neutrophil extracellular traps

Neutrophils release decondensed chromatin termed neutrophil extracellular traps (NETs) to trap and kill pathogens extracellularly. Reactive oxygen species are required to initiate NET formation but the downstream molecular mechanism is unknown. We show that upon activation, neutrophil elastase (NE) escapes from azurophilic granules and translocates to the nucleus, where it partially degrades specific histones, promoting chromatin decondensation. Subsequently, myeloperoxidase synergizes with NE in driving chromatin decondensation independent of its enzymatic activity. Accordingly, NE knockout mice do not form NETs in a pulmonary model of Klebsiella pneumoniae infection, which suggests that this defect may contribute to the immune deficiency of these mice. This mechanism provides for a novel function for serine proteases and highly charged granular proteins in the regulation of chromatin density, and reveals that the oxidative burst induces a selective release of granular proteins into the cytoplasm through an unknown mechanism.     

Shape and Flexibility in the Titin 11-Domain Super-Repeat

Titin is a giant protein of striated muscle with important roles in the assembly, intracellular signalling and passive mechanical properties of sarcomeres. The molecule consists principally of ∼ 300 immunoglobulin and fibronectin domains arranged in a chain more than 1 μm long. The isoform-dependent N-terminal part of the molecule forms an elastic connection between the end of the thick filament and the Z-line. The larger, constitutively expressed C-terminal part is bound to the thick filament. Through most of the thick filament part, the immunoglobulin and fibronectin domains are arranged in a repeating pattern of 11 domains termed the ‘large super-repeat’. There are 11 contiguous copies of the large super-repeat making up a segment of the molecule nearly 0.5 μm long. We have studied a set of two-domain and three-domain recombinant fragments from the large super-repeat region by electron microscopy, synchrotron X-ray solution scattering and analytical ultracentrifugation, with the goal of reconstructing the overall structure of this part of titin. The data illustrate different average conformations in different domain pairs, which correlate with differences in interdomain linker lengths. They also illustrate interdomain bending and flexibility around average conformations. Overall, the data favour a helical conformation in the super-repeat. They also suggest that this region of titin is dimerised when bound to the thick filament.     

Genome-Wide Transposon Mutagenesis Identifies a Role for Host Neuroendocrine Stress Hormones in Regulating the Expression of Virulence Genes in Salmonella

Bacterial sensing of environmental signals plays a key role in regulating virulence and mediating bacterium-host interactions. The sensing of the neuroendocrine stress hormones epinephrine (adrenaline) and norepinephrine (noradrenaline) plays an important role in modulating bacterial virulence. We used MudJ transposon mutagenesis to globally screen for genes regulated by neuroendocrine stress hormones in Salmonella enterica serovar Typhimurium. We identified eight hormone-regulated genes, including yhaK, iroC, nrdF, accC, yedP, STM3081, and the virulence-related genes virK and mig14. The mammalian α-adrenergic receptor antagonist phentolamine reversed the hormone-mediated effects on yhaK, virK, and mig14 but did not affect the other genes. The β-adrenergic receptor antagonist propranolol had no activity in these assays. The virK and mig14 genes are involved in antimicrobial peptide resistance, and phenotypic screens revealed that exposure to neuroendocrine hormones increased the sensitivity of S. Typhimurium to the antimicrobial peptide LL-37. A virK mutant and a virK mig14 double mutant also displayed increased sensitivity to LL-37. In contrast to enterohemorrhagic Escherichia coli (EHEC), we have found no role for the two-component systems QseBC and QseEF in the adrenergic regulation of any of the identified genes. Furthermore, hormone-regulated gene expression could not be blocked by the QseC inhibitor LED209, suggesting that sensing of hormones is mediated through alternative signaling pathways in S. Typhimurium. This study has identified a role for host-derived neuroendocrine stress hormones in downregulating S. Typhimurium virulence gene expression to the benefit of the host, thus providing further insights into the field of host-pathogen communication.Bacterial sensing of environmental signals plays a key role in regulating virulence gene expression and bacterium-host interactions. It is increasingly recognized that detection of host-derived molecules, such as the neuroendocrine stress hormones (catecholamines) epinephrine (adrenaline) and norepinephrine (noradrenaline), plays an important role in modulating bacterial virulence (29, 42).Physical and psychological stress has been linked to increased severity and susceptibility to infection in humans and other animals (23, 42), and epinephrine/norepinephrine levels are an important factor in this. Stress triggers an increase in plasma epinephrine levels (31), and plasma levels of epinephrine and norepinephrine have been reported to increase with patients suffering from postoperative sepsis compared to patients with no complications (32). Administration of norepinephrine and epinephrine to otherwise healthy subjects increases the severity of bacterial infections, including Clostridium perfringens in humans and enterohemorrhagic Escherichia coli (EHEC) in calves (42, 63, 65). Treatment with norepinephrine also increases the virulence of Salmonella enterica serovar Enteritidis in chicks and Salmonella enterica serovar Typhimurium in mice, with a substantial increase in bacterial numbers recovered from the cecum and liver in both cases (47, 65).Norepinephrine is found in large concentrations in the gut due to release by gastrointestinal neurones; indeed up to half the norepinephrine in the body may be produced in the enteric nervous system (ENS) (3). Epinephrine, while not normally found in the gut, is present in the bloodstream and is also produced by macrophages in response to bacteria-derived lipopolysaccharide (LPS) (12, 26). S. Typhimurium is an enteropathogen, can also cross the epithelial barrier to cause systemic infection, and will therefore encounter both these molecules in the normal infection cycle.Phenotypes induced by stress hormones in bacteria include increased adherence of EHEC to bovine intestinal mucosa (63), upregulation of type III secretion and Shiga toxin production in EHEC (22, 60), upregulation of type III secretion in Vibrio parahaemolyticus (51), increase in invasion of epithelial cells and breakdown of epithelial tight junctions by Campylobacter jejuni (15), affected motility and expression of iron uptake genes in S. Typhimurium (8, 9, 36), and modulated virulence in Borrelia burgdorferi (59). Epinephrine and norepinephrine can overcome the growth inhibition of many bacteria, including Salmonella, in serum-containing media (13, 43), due to the ability to act as a siderophore to facilitate iron uptake (13, 28, 47).Norepinephrine and epinephrine also interact with bacterial quorum-sensing (QS) systems. QS is a process of bacterial cell-cell communication in which each cell produces small signal molecules termed “autoinducers” (AIs), which regulate gene expression when a critical threshold concentration and therefore population density have been reached. QS affects diverse processes, including motility, virulence, biofilm formation, type III secretion, and luminescence (6, 64).The EHEC AI-3 QS system is important for motility and expression of the type III secretion system encoded by the locus of enterocyte effacement (LEE) (60). AI-3 sensing and signal transduction are mediated via the QseBC and QseEF two-component systems, respectively. Epinephrine and norepinephrine can substitute for AI-3, causing cross talk between the two signaling systems and induction of type III secretion and motility (57, 60). The sensor kinase QseC is autophosphorylated upon binding either epinephrine or norepinephrine (14), demonstrating the presence of adrenergic receptors in bacteria. These adrenergic phenotypes can also be blocked by the mammalian α- and β-adrenergic antagonists phentolamine and propranolol, although it should be noted that QseC is blocked only by the former (14, 60). This suggests the occurrence of cross talk between bacterial and mammalian cell signaling systems and the existence of multiple bacterial adrenergic sensors.To elucidate the role of host-derived stress hormones in the physiology and pathogenicity of S. Typhimurium, we used MudJ transposon mutagenesis to screen globally for epinephrine- and norepinephrine-regulated genes in S. Typhimurium.     

Foraging patterns and kleptoparasitism among three sympatric cormorants (Phalacrocorax spp.) from the Delhi region, North India

Cormorants, described as ‘foot-propelled pursuit divers’, constitute an important component of aquatic food webs and exhibit unique foraging behaviour patterns, which can be properly understood through a comparative study. Since, after a foraging dive they surface to ingest the prey, the intensity of kleptoparasitic attacks on the surface can have a major impact upon the net energetic gain for each individual. Inspite of the fact that cormorants and their habitats are severely threatened in India, their foraging behaviour has not been adequately studied. Such considerations prompted us to undertake field studies on three sympatric cormorants (Phalacrocorax spp.) at 60 different sites in the Delhi region of North India, during 2004–2007. By means of video photography, some key foraging parameters including group size, prey size and patterns of kleptoparasitic attacks were quantified. Along a loose body size gradient, we observed significant differences among the three species with respect to not only their preference for wetland size but also prey size. The frequency of a kleptoparasitic attacks depended upon the group size and foraging behaviour of each species. It was observed that several foraging bouts were abruptly terminated due to human disturbances, mostly at sites lying outside the protected areas. This observation points towards the need to conserve small waterbodies in the countryside, currently threatened by pollution and urbanization, for the benefit of a variety of waterbirds including cormorants.     

Synthesis, crystal structures and, antibacterial and antiproliferative activities in vitro of palladium(II) complexes of triphenylphosphine and thioamides

Palladium(II) complexes with triphenylphosphine (PPh₃) and thioamides of the general formulae, [Pd(L)₂(PPh₃)₂]Cl₂ and [Pd(L)₂(PPh₃)₂] have been prepared and characterized by elemental analysis, IR and NMR (¹H, ¹³C and ³¹P) methods, and two of them (trans-[Pd(PPh₃)₂(Dmtu)₂]Cl₂·(H₂O)(CH₃OH)_0.5 (1) and trans-[Pd(PPh₃)₂(Mpy)₂] (2)) by X-ray crystallography; where L = thiourea (Tu), methylthiourea (Metu), N,N′-dimethylthiourea (Dmtu), tetramethylthiourea (Tmtu), 2-mercaptopyridine (Mpy), 2-mercaptopyrimidine (Mpm) and thionicotinamide (Tna). The spectral data of the complexes are consistent with the sulfur coordination of thioamides to palladium(II). The crystal structures of the complexes show that (1) has ionic character consisting of [Pd(PPh₃)₂(Dmtu)₂]⁺² cations and uncoordinated Cl⁻ ions, while (2) is a neutral complex with Mpy behaving as anionic thiolate ligand. The coordination environment around palladium in (2) is nearly regular square-planar, while in (1) the trans angles show significant distortions from 180°. The complexes were screened for antibacterial effects, brine shrimps lethality bioassay and antitumor activity. These complexes showed significant activities in most of the cases against the tested bacteria as compared to that of a standard drug. Their antitumor activity against prostate cancer cells (PC3) is comparable with doxorubicin, together with no cytotoxic effects in brine shrimps lethality bioassay study.     

Potential chemoprevention of diethylnitrosamine-initiated and 2-acetylaminofluorene-promoted hepatocarcinogenesis by zerumbone from the rhizomes of the subtropical ginger (Zingiber zerumbet)

Zerumbone (ZER), a monosesquiterpene found in the subtropical ginger (Zingiber zerumbet Smith), possesses antiproliferative properties to several cancer cells lines, including the cervical, skin and colon cancers. In this study, the antitumourigenic effects of ZER were assessed in rats induced to develop liver cancer with a single intraperitoneal injection of diethylnitrosamine (DEN, 200 mg/kg) and dietary 2-acetylaminofluorene (AAF) (0.02%). The rats also received intraperitoneal ZER injections at 15, 30 or 60 mg/kg body wt. twice a week for 11 weeks, beginning week four post-DEN injection. The hepatocytes of positive control (DEN/AAF) rats were smaller with larger hyperchromatic nuclei than normal, showing cytoplasmic granulation and intracytoplasmic violaceous material, which were characteristics of hepatocarcinogenesis. Histopathological evaluations showed that ZER protects the rat liver from the carcinogenic effects of DEN and AAF. Serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (AP) and alpha-fetoprotein (AFP) were significantly lower (P < 0.05) in ZER-treated than untreated rats with liver cancer. The liver malondialdehyde (MDA) concentrations significantly (P < 0.05) increased in the untreated DEN/AAF rats indicating hepatic lipid peroxidation. There was also significant (P < 0.05) reduction in the hepatic tissue glutathione (GSH) concentrations. The liver sections of untreated DEN/AAF rats also showed abundant proliferating cell nuclear antigen (PCNA), while in ZER-treated rats the expression of this antigen was significantly (P < 0.05) lowered. By the TUNEL assay, there were significantly (P < 0.05) higher numbers of apoptotic cells in DEN/AAF rats treated with ZER than those untreated. Zerumbone treatment had also increased Bax and decreased Bcl-2 protein expression in the livers of DEN/AAF rats, which suggested increased apoptosis. Even after 11 weeks of ZER treatment, there was no evidence of abnormality in the liver of normal rats. This study suggests that ZER reduces oxidative stress, inhibits proliferation, induces mitochondria-regulated apoptosis, thus minimising DEN/AAF-induced carcinogenesis in rat liver. Therefore, ZER has great potential in the treatment of liver cancers.