Roles of Salmonella enterica serovar Typhimurium encoded Peptidase N during systemic infection of Ifnγ(-/-) mice.

Pathogen encoded peptidases are known to be important during infection; however, their roles in modulating host responses in immunocompromised individuals are not well studied. The roles of S. typhimurium (WT) encoded Peptidase N (PepN), a major aminopeptidase and sole M1 family member, was studied in mice lacking Interferon-γ (IFNγ), a cytokine important for immunity. S. typhimurium lacking pepN (ΔpepN) displays enhanced colony forming units (CFU) compared to WT in peripheral organs during systemic infection in C57BL/6 mice. However, Ifnγ(-/-) mice show higher CFU compared to C57BL/6 mice, resulting in lower fold differences between WT and ΔpepN. Concomitantly, reintroduction of pepN in ΔpepN (ΔpepN/pepN) reduces CFU, demonstrating pepN-dependence. Interestingly, expression of a catalytically inactive PepN (ΔpepN/E298A) also lowers CFU, demonstrating that the decrease in CFU is independent of the catalytic activity of PepN. In addition, three distinct differences are observed between infection of C57BL/6 and Ifnγ(-/-) mice: First, serum amounts of TNFα and IL1β post infection are significantly lower in Ifnγ(-/-) mice. Second, histological analysis of C57BL/6 mice reveals that damage in spleen and liver upon infection with WT or ΔpepN is greater compared to ΔpepN/pepN or ΔpepN/E298A. On the other hand, Ifnγ(-/-) mice are highly susceptible to organ damage by all strains of S. typhimurium used in this study. Finally, greater survival of C57BL/6, but not Ifnγ(-/-) mice, is observed upon infection with ΔpepN/pepN or ΔpepN/E298A. Overall, the roles of the host encoded IFNγ during infection with S. typhimurium strains with varying degrees of virulence are highlighted.

Surface chemical and adsorption studies using Thiobacillus ferrooxidans with reference to bacterial adhesion to sulfide minerals

Adhesion of Thiobacillus ferrooxidans to pyrite and chalcopyrite in relation to its importance in bioleaching and bioflotation has been studied. Electrokinetic studies as well as FT-IR spectra suggest that the surface chemistry of Thiobacillus ferrooxidans depends on bacterial growth conditions. Sulfur-,Pyrite- and chalcopyrite-grown Thiobacillus ferrooxidans were found to be relatively more hydrophobic. The altered surface chemistry of Thiobacillus ferrooxidans was due to secretion of newer and specific proteinaceous compounds. The adsorption density corresponds to a monolayer coverage in a horizontal orientation of the cells. The xanthate flotation of pyrite in presence of Thiobacillus ferrooxidans is strongly depressed where as the cells have insignificant effect on chalcopyrite flotation. This study demonstrate that: (a)Thiobacillus ferrooxidans cells can be used for selective flotation of chalcopyrite from pyrite and importantly at natural pH values. (b)Sulfur-grown cells exhibits higher leaching kinetics than ferrous ion-grown cells.

Hypoxia-Mediated Impairment of the Mitochondrial Respiratory Chain Inhibits the Bactericidal Activity of Macrophages

In infected tissues oxygen tensions are low. As innate immune cells have to operate under these conditions, we analyzed the ability of macrophages (M phi) to kill Escherichia coli or Staphylococcus aureus in a hypoxic microenvironment. Oxygen restriction did not promote intracellular bacterial growth but did impair the bactericidal activity of the host cells against both pathogens. This correlated with a decreased production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates. Experiments with phagocyte NADPH oxidase (PHOX) and inducible NO synthase (NOS2) double-deficient M phi revealed that in E. coli- or S. aureus-infected cells the reduced antibacterial activity during hypoxia was either entirely or partially independent of the diminished PHOX and NOS2 activity. Hypoxia impaired the mitochondrial activity of infected M phi. Inhibition of the mitochondrial respiratory chain activity during normoxia (using rotenone or antimycin A) completely or partially mimicked the defective antibacterial activity observed in hypoxic E. coli-or S. aureus-infected wild-type M phi, respectively. Accordingly, inhibition of the respiratory chain of S. aureus-infected, normoxic PHOX-/- NOS2(-/-) M phi further raised the bacterial burden of the cells, which reached the level measured in hypoxic PHOX-/- NOS2(-/-) M phi cultures. Our data demonstrate that the reduced killing of S. aureus or E. coli during hypoxia is not simply due to a lack of PHOX and NOS2 activity but partially or completely results from an impaired mitochondrial antibacterial effector function. Since pharmacological inhibition of the respiratory chain raised the generation of ROI but nevertheless phenocopied the effect of hypoxia, ROI can be excluded as the mechanism underlying the antimicrobial activity of mitochondria.

Deorphanization of Malonyl CoA:ACP Transacylase Drug Target in Plasmodium falciparum (PfFabD) Using Bacterial Antagonists: A Piggyback' Approach for Antimalarial Drug Discovery

Quest for new drug targets in Plasmodium sp. has underscored malonyl CoA:ACP transacylase (PfFabD) of fatty acid biosynthetic pathway in apicoplast. In this study, a piggyback approach was employed for the receptor deorphanization using inhibitors of bacterial FabD enzymes. Due to the lack of crystal structure, theoretical model was constructed using the structural details of homologous enzymes. Sequence and structure analysis has localized the presence of two conserved pentapeptide motifs: GQGXG and GXSXG and five key invariant residues viz., Gln109, Ser193, Arg218, His305 and Gln354 characteristic of FabD enzyme. Active site mapping of PfFabD using substrate molecules has disclosed the spatial arrangement of key residues in the cavity. As structurally similar molecules exhibit similar biological activities, signature pharmacophore fingerprints of FabD antagonists were generated using 0D-3D descriptors for molecular similarity-based cluster analysis and to correlate with their binding profiles. It was observed that antagonists showing good geometrical fitness score were grouped in cluster-1, whereas those exhibiting high binding affinities in cluster-2. This study proves important to shed light on the active site environment to reveal the hotspot for binding with higher affinity and to narrow down the virtual screening process by searching for close neighbors of the active compounds.

A Rhodanine Derivative CCR-11 Inhibits Bacterial Proliferation by Inhibiting the Assembly and GTPase Activity of FtsZ

A perturbation of FtsZ assembly dynamics has been shown to inhibit bacterial cytokinesis. In this study, the antibacterial activity of 151 rhodanine compounds was assayed using Bacillus subtilis cells. Of 151 compounds, eight strongly inhibited bacterial proliferation at 2 mu M. Subsequently, we used the elongation of B. subtilis cells as a secondary screen to identify potential FtsZ-targeted antibacterial agents. We found that three compounds significantly increased bacterial cell length. One of the three compounds, namely, CCR-11 (E)-2-thioxo-5-({3-(trifluoromethyl)phenyl]furan-2-yl}methylene) thiazolidin-4-one], inhibited the assembly and GTPase activity of FtsZ in vitro. CCR-11 bound to FtsZ with a dissociation constant of 1.5 +/- 0.3 mu M. A docking analysis indicated that CCR-11 may bind to FtsZ in a cavity adjacent to the T7 loop and that short halogen oxygen, H-bonding, and hydrophobic interactions might be important for the binding of CCR-11 with FtsZ. CCR-11 inhibited the proliferation of B. subtilis cells with a half-maximal inhibitory concentration (IC50) of 1.2 +/- 0.2 mu M and a minimal inhibitory concentration of 3 mu M. It also potently inhibited proliferation of Mycobacterium smegmatis cells. Further, CCR-11 perturbed Z-ring formation in B. subtilis cells; however, it neither visibly affected nucleoid segregation nor altered the membrane integrity of the cells. CCR-11 inhibited HeLa cell proliferation with an IC50 value of 18.1 +/- 0.2,mu M (similar to 15 x IC50 of B. subtilis cell proliferation). The results suggested that CCR-11 inhibits bacterial cytokinesis by inhibiting FtsZ assembly, and it can be used as a lead molecule to develop FtsZ-targeted antibacterial agents.

Comparative Transcriptomics of H. pylori Strains AM5, SS1 and Their hpyAVIBM Deletion Mutants: Possible Roles of Cytosine Methylation

Helicobacter pylori is an important human pathogen and one of the most successful chronic colonizers of the human body. H. pylori uses diverse mechanisms to modulate its interaction with the host in order to promote chronic infection and overcome host immune response. Restriction-modification genes are a major part of strain-specific genes present in H. pylori. The role of N-6 -adenine methylation in bacterial gene regulation and virulence is well established but not much is known about the effect of C-5 -cytosine methylation on gene expression in prokaryotes. In this study, it was observed by microarray analysis and RT-PCR, that deletion of an orphan C-5 -cytosine methyltransferase, hpyAVIBM in H. pylori strains AM5and SS1 has a significant effect on the expression of number of genes belonging to motility, adhesion and virulence. AM Delta DhpyAVIBM mutant strain has a different LPS profile and is able to induce high IL-8 production compared to wild-type. hpyAVIBM from strain 26695 is able to complement mutant SS1 and AM5 strains. This study highlights a possible significance of cytosine methylation in the physiology of H. pylori.

Under-representation of intrinsic terminators across bacterial genomic islands: Rho as a principal regulator of xenogenic DNA expression

Two transcription termination mechanisms - intrinsic and Rho-dependent - have evolved in bacteria. The Rho factor occurs in most bacterial lineages, and has been hypothesized to play a global regulatory role. Genome-wide studies using microarray, 2D-gel electrophoresis and ChIP-chip provided evidence that Rho serves to silence transcription from horizontally acquired genes and prophages in Escherichia coli K-12, implicating the factor to be a part of the ``cellular immune mechanism'' protecting against deleterious phages and aberrant gene expression from acquired xenogenic DNA. We have investigated this model by adopting an alternate in silico approach and have extended the study to other species. Our analysis shows that several genomic islands across diverse phyla have under-representation of intrinsic terminators, similar to that experimentally observed in E. coli K-12. This implies that Rho-dependent termination is the predominant process operational in these islands and that silencing of foreign DNA is a conserved function of Rho. From the present analysis, it is evident that horizontally acquired islands have lost intrinsic terminators to facilitate Rho-dependent termination. These results underscore the importance of Rho as a conserved, genome-wide sentinel that regulates potentially toxic xenogenic DNA. (C) 2012 Elsevier B.V. All rights reserved.

Differential modulation of intracellular survival of cytosolic and vacuolar pathogens by curcumin

Curcumin, a principal component of turmeric, acts as an immunomodulator regulating the host defenses in response to a diseased condition. The role of curcumin in controlling certain infectious diseases is highly controversial. It is known to alleviate symptoms of Helicobacter pylori infection and exacerbate that of Leishmania infection. We have evaluated the role of curcumin in modulating the fate of various intracellular bacterial pathogens. We show that pretreatment of macrophages with curcumin attenuates the infections caused by Shigella flexneri (clinical isolates) and Listeria monocytogenes and aggravates those caused by Salmonella enterica serovar Typhi CT18 (a clinical isolate), Salmonella enterica serovar Typhimurium, Staphylococcus aureus, and Yersinia enterocolitica. Thus, the antimicrobial nature of curcumin is not a general phenomenon. It modulated the intracellular survival of cytosolic (S. flexneri and L. monocytogenes) and vacuolar (Salmonella spp., Y. enterocolitica, and S. aureus) bacteria in distinct ways. Through colocalization experiments, we demonstrated that curcumin prevented the active phagosomal escape of cytosolic pathogens and enhanced the active inhibition of lysosomal fusion by vacuolar pathogens. A chloroquine resistance assay confirmed that curcumin retarded the escape of the cytosolic pathogens, thus reducing their inter- and intracellular spread. We have demonstrated that the membrane-stabilizing activity of curcumin is crucial for its differential effect on the virulence of the bacteria.

Curcumin reduces the antimicrobial activity of ciprofloxacin against Salmonella Typhimurium and Salmonella Typhi

Typhoidal and non-typhoidal infection by Salmonella is a serious threat to human health. Ciprofloxacin is the last drug of choice to clear the infection. Ciprofloxacin, a gyrase inhibitor, kills bacteria by inducing chromosome fragmentation, SOS response and reactive oxygen species (ROS) in the bacterial cell. Curcumin, an active ingredient from turmeric, is a major dietary molecule among Asians and possesses medicinal properties. Our research aimed at investigating whether curcumin modulates the action of ciprofloxacin. We investigated the role of curcumin in interfering with the antibacterial action of ciprofloxacin in vitro and in vivo. RTPCR, DNA fragmentation and confocal microscopy were used to investigate the modulation of ciprofloxacin-induced SOS response, DNA damage and subsequent filamentation by curcumin. Chemiluminescence and nitroblue tetrazolium reduction assays were performed to assess the interference of curcumin with ciprofloxacin-induced ROS. DNA binding and cleavage assays were done to understand the rescue of ciprofloxacin-mediated gyrase inhibition by curcumin. Curcumin interferes with the action of ciprofloxacin thereby increasing the proliferation of Salmonella Typhi and Salmonella Typhimurium in macrophages. In a murine model of typhoid fever, mice fed with curcumin had an increased bacterial burden in the reticuloendothelial system and succumbed to death faster. This was brought about by the inhibition of ciprofloxacin-mediated downstream signalling by curcumin. The antioxidant property of curcumin is crucial in protecting Salmonella against the oxidative burst induced by ciprofloxacin or interferon (IFN), a pro-inflammatory cytokine. However, curcumin is unable to rescue ciprofloxacin-induced gyrase inhibition. Curcumins ability to hinder the bactericidal action of ciprofloxacin and IFN might significantly augment Salmonella pathogenesis.

Micro-shock wave assisted bacterial transformation

A gene is a unit of heredity in a living organism. It normally resides on a stretch of DNA that codes for a type of protein or for an RNA chain that has a function in the organism. All living things depend on genes, as they specify all proteins and functional RNA chains. Genes hold the information to build and maintain an organism’s cells and pass genetic traits to offspring. The gene has to be transferred to bacteria or eukaryotic cells for basic and applied molecular biology studies. Bacteria can uptake exogenous genetic material by three ways: conjugation, transduction and transformation. Genetic material is naturally transferred to bacteria in case of conjugation and transferred through bacteriophage in transduction. Transformation is the acquisition of exogenous genetic material through cell wall. The ability of bacteria of being transformed is called competency and those bacteria which have competency are competent cells. Divalent Calcium ions can make the bacteria competent and a heat shock can cause the bacteria to uptake DNA. But the heat shock method cannot be used for all the bacteria. In electroporation, a brief electric shock with an electric field of 10-20kV/cmmakes pores in the cell wall, facilitates the DNA to enter into the bacteria. Microprecipitates, microinjection, liposomes, and biological vectors are also used to transfer polar molecules like DNA into host cells.

Interferon-gamma- and glucocorticoid-mediated pathways synergize to enhance death of CD4(+) CD8(+) thymocytes during Salmonella enterica serovar Typhimurium infection

Thymic atrophy is known to occur during infections; however, there is limited understanding of its causes and of the cross-talk between different pathways. This study investigates mechanisms involved in thymic atrophy during a model of oral infection by Salmonella enterica serovar Typhimurium (S.typhimurium). Significant death of CD4+CD8+ thymocytes, but not of single-positive thymocytes or peripheral lymphocytes, is observed at later stages during infection with live, but not heat-killed, bacteria. The death of CD4+CD8+ thymocytes is Fas-independent as shown by infection studies with lpr mice. However, apoptosis occurs with lowering of mitochondrial potential and higher caspase-3 activity. The amounts of cortisol, a glucocorticoid, and interferon- (IFN-), an inflammatory cytokine, increase upon infection. To investigate the functional roles of these molecules, studies were performed using Ifn/ mice together with RU486, a glucocorticoid receptor antagonist. Treatment of C57BL/6 mice with RU486 does not affect colony-forming units (CFU), amounts of IFN- and mouse survival; however, there is partial rescue in thymocyte death. Upon infection, Ifn/ mice display higher CFU and lower survival but more surviving thymocytes are recovered. However, there is no difference in cortisol amounts in C57BL/6 and Ifn/ mice. Importantly, the number of CD4+CD8+ thymocytes is significantly higher in Ifn/ mice treated with RU486 along with lower caspase-3 activity and mitochondrial damage. Hence, endogenous glucocorticoid and IFN--mediated pathways are parallel but synergize in an additive manner to induce death of CD4+CD8+ thymocytes during S.typhimurium infection. The implications of this study for host responses during infection are discussed.

The bystander effect in optically trapped red blood cells due to Plasmodium falciparum infection

In a previous study of the properties of red blood cells (RBC) trapped in an optical tweezers trap, an increase in the spectrum of Brownian fluctuations for RBCs from a Plasmodium falciparum culture (due to increased rigidity) compared with normal RBCs was measured. A bystander effect was observed, whereby RBCs actually hosting the parasite had an effect on the physical properties of remaining non-hosting RBCs. The distribution of corner frequency (f(c)) in the power spectrum of single RBCs held in an optical tweezers trap was studied. Two tests were done to confirm the bystander effect. In the first, RBCs from an infected culture were separated into hosting and non-hosting RBCs. In the second, all RBCs were removed from the infected culture, and normal RBCs were incubated in the spent medium. The trapping environment was the same for all measurements so only changes in the properties of RBCs were measured. In the first experiment, a similar and statistically significant increase was measured both for hosting and non-hosting RBCs. In the second experiment, normal RBCs incubated in spent medium started to become rigid after a few hours and showed complete changes (comparable with RBCs from the infected culture) after 24 h. These experiments provide direct evidence of medium-induced changes in the properties of RBCs in an infected culture, regardless of whether the RBCs actually host the parasite.