SOCS3 expression induced by PIM2 requires PKC and PI3K signaling

Initiation of proinflammatory host immunity in response to infection represents as a key event in effective control and containment of the pathogen at the site of infection as well as in elicitation of robust immune memory responses. In the current investigation, we demonstrate that an integral cell wall antigen of the mycobacterial envelope, Phosphatidyl-myo-inositol dimannosides (PIM2) triggers Suppressor of cytokine signaling (SOCS) 3 expression in macrophages in a Toll-like receptor 2 (TLR2)-MyD88 dependent manner. Data derived from signaling perturbations suggest the involvement of phosphoinositide-3 kinase (PI3K) and protein kinase C (PKC) signaling pathways during PIM2 induced SOCS3 expression. Further, pharmacological inhibition of ERK1/2, but not of p38 MAP kinase or JNK abrogated the induced expression of SOCS3. The PIM2 induced activation of ERK1/2 was dependent on the activation of PI3K or PKC signaling which in turn regulated p65 nuclear factor -kappa B (NF-kappa B) nuclear translocation. Overall, current study delineates the role for PI3K-PKC axis and ERK1/2 signaling as key signaling events during PIM2 induced SOCS3 expression in macrophages.

Salmonella Typhimurium: Insight into the multi-faceted role of the LysR-type transcriptional regulators in Salmonella

The LysR-type transcriptional regulators (LTTRs) are widely distributed in various genera of prokaryotes LTTRs are DNA binding proteins that can positively or negatively regulate target gene expression and can also repress their own transcription Salmonella enterica comprises a group of Gram-negative bacteria capable of causing clinical syndromes that range from self-limiting diarrhoea to severe fibrinopurulent necrotizing enteritis and life threatening systemic disease. The survival and replication of Salmonella in macrophages and in infected host is brought about by the means of various two component regulatory systems, transporters and other virulence islands In Salmonella genome the existence of 44 LTTRs has been documented These LTTRs regulate bacterial stress response. systemic virulence in mice and also many virulence determinants in vitro. Here we focus on the findings that elucidate the structure and function of the LTTRs in Salmonella and discuss the importance of these LTTRs in making Salmonella a Successful pathogen...

Prediction of protein-protein interactions between Helicobacter pylori and a human host

A lack of information on protein-protein interactions at the host-pathogen interface is impeding the understanding of the pathogenesis process. A recently developed, homology search-based method to predict protein-protein interactions is applied to the gastric pathogen, Helicobacter pylori to predict the interactions between proteins of H. pylori and human proteins in vitro. Many of the predicted interactions could potentially occur between the pathogen and its human host during pathogenesis as we focused mainly on the H. pylori proteins that have a transmembrane region or are encoded in the pathogenic island and those which are known to be secreted into the human host. By applying the homology search approach to protein-protein interaction databases DIP and iPfam, we could predict in vitro interactions for a total of 623 H. pylori proteins with 6559 human proteins. The predicted interactions include 549 hypothetical proteins of as yet unknown function encoded in the H. pylori genome and 13 experimentally verified secreted proteins. We have recognized 833 interactions involving the extracellular domains of transmembrane proteins of H. pylori. Structural analysis of some of the examples reveals that the interaction predicted by us is consistent with the structural compatibility of binding partners. Examples of interactions with discernible biological relevance are discussed.

Characterization of the restriction enzyme-like endonuclease encoded by the Entamoeba histolytica non-long terminal repeat retrotransposon EhLINE1

The genome of the human pathogen Entamoeba histolytica, a primitive protist, contains non-long terminal repeat retrotransposable elements called EhLINEs. These encode reverse transcriptase and endonuclease required for retrotransposition. The endonuclease shows sequence similarity with bacterial restriction endonucleases. Here we report the salient enzymatic features of one such endonuclease. The kinetics of an EhLINE1-encoded endonuclease catalyzed reaction, determined under steady-state and single-turnover conditions, revealed a significant burst phase followed by a slower steady-state phase, indicating that release of product could be the slower step in this reaction. For circular supercoiled DNA the K-m was 2.6 x 10-8 m and the k(cat) was 1.6 x 10-2 sec-1. For linear E. histolytica DNA substrate the K-m and k(cat) values were 1.3 x 10-8 m and 2.2 x 10-4 sec-1 respectively. Single-turnover reaction kinetics suggested a noncooperative mode of hydrolysis. The enzyme behaved as a monomer. While Mg2+ was required for activity, 60% activity was seen with Mn2+ and none with other divalent metal ions. Substitution of PDX12-14D (a metal-binding motif) with PAX(12-14)D caused local conformational change in the protein tertiary structure, which could contribute to reduced enzyme activity in the mutated protein. The protein underwent conformational change upon the addition of DNA, which is consistent with the known behavior of restriction endonucleases. The similarities with bacterial restriction endonucleases suggest that the EhLINE1-encoded endonuclease was possibly acquired from bacteria through horizontal gene transfer. The loss of strict sequence specificity for nicking may have been subsequently selected to facilitate spread of the retrotransposon to intergenic regions of the E. histolytica genome.

The Role of UPF0157 in the Folding of M-tuberculosis Dephosphocoenzyme A Kinase and the Regulation of the Latter by CTP

Background: Targeting the biosynthetic pathway of Coenzyme A (CoA) for drug development will compromise multiple cellular functions of the tubercular pathogen simultaneously. Structural divergence in the organization of the penultimate and final enzymes of CoA biosynthesis in the host and pathogen and the differences in their regulation mark out the final enzyme, dephosphocoenzyme A kinase (CoaE) as a potential drug target. Methodology/Principal Findings: We report here a complete biochemical and biophysical characterization of the M. tuberculosis CoaE, an enzyme essential for the pathogen's survival, elucidating for the first time the interactions of a dephosphocoenzyme A kinase with its substrates, dephosphocoenzyme A and ATP; its product, CoA and an intrinsic yet novel inhibitor, CTP, which helps modulate the enzyme's kinetic capabilities providing interesting insights into the regulation of CoaE activity. We show that the mycobacterial enzyme is almost 21 times more catalytically proficient than its counterparts in other prokaryotes. ITC measurements illustrate that the enzyme follows an ordered mechanism of substrate addition with DCoA as the leading substrate and ATP following in tow. Kinetic and ITC experiments demonstrate that though CTP binds strongly to the enzyme, it is unable to participate in DCoA phosphorylation. We report that CTP actually inhibits the enzyme by decreasing its Vmax. Not surprisingly, a structural homology search for the modeled mycobacterial CoaE picks up cytidylmonophosphate kinases, deoxycytidine kinases, and cytidylate kinases as close homologs. Docking of DCoA and CTP to CoaE shows that both ligands bind at the same site, their interactions being stabilized by 26 and 28 hydrogen bonds respectively. We have also assigned a role for the universal Unknown Protein Family 0157 (UPF0157) domain in the mycobacterial CoaE in the proper folding of the full length enzyme. Conclusions/Significance: In view of the evidence presented, it is imperative to assign a greater role to the last enzyme of Coenzyme A biosynthesis in metabolite flow regulation through this critical biosynthetic pathway.

Location of Pathogenic Bacteria during Persistent Infections: Insights from an Analysis Using Game Theory

Bacterial persistent infections are responsible for a significant amount of the human morbidity and mortality. Unlike acute bacterial infections, it is very difficult to treat persistent bacterial infections (e.g. tuberculosis). Knowledge about the location of pathogenic bacteria during persistent infection will help to treat such conditions by designing novel drugs which can reach such locations. In this study, events of bacterial persistent infections were analyzed using game theory. A game was defined where the pathogen and the host are the two players with a conflict of interest. Criteria for the establishment of Nash equilibrium were calculated for this game. This theoretical model, which is very simple and heuristic, predicts that during persistent infections pathogenic bacteria stay in both intracellular and extracellular compartments of the host. The result of this study implies that a bacterium should be able to survive in both intracellular and extracellular compartments of the host in order to cause persistent infections. This explains why persistent infections are more often caused by intracellular pathogens like Mycobacterium and Salmonella. Moreover, this prediction is in consistence with the results of previous experimental studies.

lac Repressor Is an Antivirulence Factor of Salmonella enterica: Its Role in the Evolution of Virulence in Salmonella

The genus Salmonella includes many pathogens of great medical and veterinary importance. Bacteria belonging to this genus are very closely related to those belonging to the genus Escherichia. lacZYA operon and lacI are present in Escherichia coli, but not in Salmonella enterica. It has been proposed that Salmonella has lost lacZYA operon and lacI during evolution. In this study, we have investigated the physiological and evolutionary significance of the absence of lacI in Salmonella enterica. Using murine model of typhoid fever, we show that the expression of Lacl causes a remarkable reduction in the virulence of Salmonella enterica. Lacl also suppresses the ability of Salmonella enterica to proliferate inside murine macrophages. Microarray analysis revealed that Lacl interferes with the expression of virulence genes of Salmonella pathogenicity island 2. This effect was confirmed by RT-PCR and Western blot analysis. Interestingly, we found that SBG0326 of Salmonella bongori is homologous to lacI of Escherichia coli. Salmonella bongori is the only other species of the genus Salmonella and it lacks the virulence genes of Salmonella pathogenicity island 2. Overall, our results demonstrate that Lacl is an antivirulence factor of Salmonella enterica and suggest that absence of lacI has facilitated the acquisition of virulence genes of Salmonella pathogenicity island 2 in Salmonella enterica making it a successful systemic pathogen.

Molecular characterisation of ABC transporter type FtsE and FtsX proteins of Mycobacterium tuberculosis.

Elicitation of drug resistance and various survival strategies inside host macrophages have been the hallmarks of Mycobacterium tuberculosis as a successful pathogen. ATP Binding Cassette (ABC) transporter type proteins are known to be involved in the efflux of drugs in bacterial and mammalian systems. FtsE, an ABC transporter type protein, in association with the integral membrane protein FtsX, is involved in the assembly of potassium ion transport proteins and probably of cell division proteins as well, both of which being relevant to tubercle bacillus. In this study, we cloned ftsE gene of M. tuberculosis, overexpressed and purified. The recombinant MtFtsE-6xHis protein and the native MtFtsE protein were found localized on the membrane of E. coli and M. tuberculosis cells, respectively. MtFtsE-6xHis protein showed ATP binding in vitro, for which the K42 residue in the Walker A motif was found essential. While MtFtsE-6xHis protein could partially complement growth defect of E. coli ftsE temperature-sensitive strain MFT1181, co-expression of MtFtsE and MtFtsX efficiently complemented the growth defect, indicating that the MtFtsE and MtFtsX proteins might be performing an associated function. MtFtsE and MtFtsX-6xHis proteins were found to exist as a complex on the membrane of E. coli cells co-expressing the two proteins.

Role of neutrophils in murine salmonellosis

Gastrointestinal infections with Salmonella enterica serovars have different clinical outcomes that range from localized inflammation to a life-threatening systemic disease in the case of typhoid fever. Using a mouse model of systemic salmonellosis, we investigated the contribution of neutrophils to the innate immune defense against Salmonella after oral infection. Neutrophil infiltration was dependent on the bacterial burden in various infected organs (Peyer's patches, mesenteric lymph nodes, spleen, and liver). However, the massive infiltration of neutrophils did not allow clearance of an infection with wild-type Salmonella, presumably due to protection of intracellular Salmonella against neutrophil activities. A Salmonella mutant strain deficient in Salmonella pathogenicity island 2 (SPI2) was able to infect systemic sites, but its replication was highly restricted and it did not cause detectable attraction of neutrophils. Neutrophil depletion by antibody treatment of mice did not restore the virulence of SPI2 or auxotrophic mutant strains, supporting the hypothesis that attenuation of the strains is not due to greater susceptibility to neutrophil killing. Our observations reveal that neutrophils have completely different roles during systemic salmonellosis and localized gastrointestinal infections. In the latter conditions, rapid neutrophil attraction efficiently prevents the spread of the pathogen, whereas the neutrophil influx is delayed during systemic infections and cannot protect against lethal bacteremia.

Salmonella pathogenicity island 2 mediates protection of intracellular Salmonella from reactive nitrogen intermediates.

Salmonella typhimurium causes an invasive disease in mice that has similarities to human typhoid. A type III protein secretion system encoded by Salmonella pathogenicity island 2 (SPI2) is essential for virulence in mice, as well as survival and multiplication within macrophages. Reactive nitrogen intermediates (RNI) synthesized by inducible nitric oxide synthase (iNOS) are involved in the control of intracellular pathogens, including S. typhimurium. We studied the effect of Salmonella infection on iNOS activity in macrophages. Immunofluorescence microscopy demonstrated efficient colocalization of iNOS with bacteria deficient in SPI2 but not wild-type Salmonella, and suggests that the SPI2 system interferes with the localization of iNOS and Salmonella. Furthermore, localization of nitrotyrosine residues in the proximity was observed for SPI2 mutant strains but not wild-type Salmonella, indicating that peroxynitrite, a potent antimicrobial compound, is excluded from Salmonella-containing vacuoles by action of SPI2. Altered colocalization of iNOS with intracellular Salmonella required the function of the SPI2-encoded type III secretion system, but not of an individual "Salmonella translocated effector." Inhibition of iNOS increased intracellular proliferation of SPI2 mutant bacteria and, to a lesser extent, of wild-type Salmonella. The defect in systemic infection of a SPI2 mutant strain was partially restored in iNOS(-/-) mice. In addition to various strategies to detoxify RNI or repair damage due to RNI, avoidance of colocalization with RNI is important in adaptation of a pathogen to an intracellular life style.

An adaptive drug delivery design using neural networks for effective treatment of infectious diseases: A simulation study

An adaptive drug delivery design is presented in this paper using neural networks for effective treatment of infectious diseases. The generic mathematical model used describes the coupled evolution of concentration of pathogens, plasma cells, antibodies and a numerical value that indicates the relative characteristic of a damaged organ due to the disease under the influence of external drugs. From a system theoretic point of view, the external drugs can be interpreted as control inputs, which can be designed based on control theoretic concepts. In this study, assuming a set of nominal parameters in the mathematical model, first a nonlinear controller (drug administration) is designed based on the principle of dynamic inversion. This nominal drug administration plan was found to be effective in curing "nominal model patients" (patients whose immunological dynamics conform to the mathematical model used for the control design exactly. However, it was found to be ineffective in curing "realistic model patients" (patients whose immunological dynamics may have off-nominal parameter values and possibly unwanted inputs) in general. Hence, to make the drug delivery dosage design more effective for realistic model patients, a model-following adaptive control design is carried out next by taking the help of neural networks, that are trained online. Simulation studies indicate that the adaptive controller proposed in this paper holds promise in killing the invading pathogens and healing the damaged organ even in the presence of parameter uncertainties and continued pathogen attack. Note that the computational requirements for computing the control are very minimal and all associated computations (including the training of neural networks) can be carried out online. However it assumes that the required diagnosis process can be carried out at a sufficient faster rate so that all the states are available for control computation.

Disease burden due to Streptococcus dysgalactiae subsp equisimilis (group G and C streptococcus) is higher than that due to Streptococcus pyogenes among Mumbai school children

Streptococcus pyogenes [group A streptococcus (GAS)], a human pathogen, and Streptococcus dysgalactiae subsp. equisimilis [human group G and C streptococcus (GGS/GCS)] are evolutionarily related, share the same tissue niche in humans, exchange genetic material, share up to half of their virulence-associated genes and cause a similar spectrum of diseases. Yet, GGS/GCS is often considered as a commensal bacterium and its role in streptococcal disease burden is under-recognized. While reports of the recovery of GGS/GCS from normally sterile sites are increasing, studies describing GGS/GCS throat colonization rates relative to GAS in the same population are very few. This study was carried out in India where the burden of streptococcal diseases, including rheumatic fever and rheumatic heart disease, is high. As part of a surveillance study, throat swabs were taken from 1504 children attending 7 municipal schools in Mumbai, India, during 2006-2008. GAS and GGS/GCS were identified on the basis of beta-haemolytic activity, carbohydrate group and PYR test, and were subsequently typed. The GGS/GCS carriage rate (1166/1504, 11%) was eightfold higher than the GAS carriage (22/1504, 1.5%) rate in this population. The 166 GGS/GCS isolates collected represented 21 different emm types (molecular types), and the 22 GAS isolates represented 15 different emm types. Although the rate of pharyngitis associated with GGS/GCS is marginally lower than with GAS, high rates of throat colonization by GGS/GCS underscore its importance in the pathogenesis of pharyngitis.

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.

Structural Ordering of Disordered Ligand-Binding Loops of Biotin Protein Ligase into Active Conformations as a Consequence of Dehydration

Mycobacterium tuberculosis (Mtb), a dreaded pathogen, has a unique cell envelope composed of high fatty acid content that plays a crucial role in its pathogenesis. Acetyl Coenzyme A Carboxylase (ACC), an important enzyme that catalyzes the first reaction of fatty acid biosynthesis, is biotinylated by biotin acetyl-CoA carboxylase ligase (BirA). The ligand-binding loops in all known apo BirAs to date are disordered and attain an ordered structure only after undergoing a conformational change upon ligand-binding. Here, we report that dehydration of Mtb-BirA crystals traps both the apo and active conformations in its asymmetric unit, and for the first time provides structural evidence of such transformation. Recombinant Mtb-BirA was crystallized at room temperature, and diffraction data was collected at 295 K as well as at 120 K. Transfer of crystals to paraffin and paratone-N oil (cryoprotectants) prior to flash-freezing induced lattice shrinkage and enhancement in the resolution of the X-ray diffraction data. Intriguingly, the crystal lattice rearrangement due to shrinkage in the dehydrated Mtb-BirA crystals ensued structural order of otherwise flexible ligand-binding loops L4 and L8 in apo BirA. In addition, crystal dehydration resulted in a shift of similar to 3.5 angstrom in the flexible loop L6, a proline-rich loop unique to Mtb complex as well as around the L11 region. The shift in loop L11 in the C-terminal domain on dehydration emulates the action responsible for the complex formation with its protein ligand biotin carboxyl carrier protein (BCCP) domain of ACCA3. This is contrary to the involvement of loop L14 observed in Pyrococcus horikoshii BirA-BCCP complex. Another interesting feature that emerges from this dehydrated structure is that the two subunits A and B, though related by a noncrystallographic twofold symmetry, assemble into an asymmetric dimer representing the ligand-bound and ligand-free states of the protein, respectively. In-depth analyses of the sequence and the structure also provide answers to the reported lower affinities of Mtb-BirA toward ATP and biotin substrates. This dehydrated crystal structure not only provides key leads to the understanding of the structure/function relationships in the protein in the absence of any ligand-bound structure, but also demonstrates the merit of dehydration of crystals as an inimitable technique to have a glance at proteins in action.

Effective treatment of infectious diseases: A nonlinear adaptive control theoretic approach

A nonlinear adaptive system theoretic approach is presented in this paper for effective treatment of infectious diseases that affect various organs of the human body. The generic model used does not represent any specific disease. However, it mimics the generic immunological dynamics of the human body under pathological attack, including the response to external drugs. From a system theoretic point of view, drugs can be interpreted as control inputs. Assuming a set of nominal parameters in the mathematical model, first a nonlinear controller is designed based on the principle of dynamic inversion. This treatment strategy was found to be effective in completely curing "nominal patients". However, in some cases it is ineffective in curing "realistic patients". This leads to serious (sometimes fatal) damage to the affected organ. To make the drug dosage design more effective, a model-following neuro-adaptive control design is carried out using neural networks, which are trained (adapted) online. From simulation studies, this adaptive controller is found to be effective in killing the invading microbes and healing the damaged organ even in the presence of parameter uncertainties and continuing pathogen attack.