Priyanka Das

Engagement of TLR Signaling as Adjuvant: towards Smarter Vaccine and Beyond

Toll like receptors (TLRs) are a family of conserved pattern recognition receptors that recognizes specific microbial patterns and allow the cell to distinguish between self and non-self materials. The very property of the TLRs to link innate and adaptive immunity offers a novel prospect to develop vaccines engaging TLR signaling. The presence of TLR ligands as adjuvant in conjunction with the vaccine is shown to increase the efficacy and response to the immunization with a particular antigen. For infectious as well as for noninfectious diseases, TLR activation have been used in both established and experimental vaccines. The choice of the TLR agonist to be used, the subsequent efficacy and the safety profile of the vaccine is thus a crucial step in vaccine development. Recent studies also suggest the involvement of other non-TLR immune receptors to control vaccine immunogenicity. Here we focus on the findings dealing with TLR ligands as adjuvant and discuss the importance of these studies to develop an optimal vaccine.

Modulation of the Arginase Pathway in the Context of Microbial Pathogenesis: A Metabolic Enzyme Moonlighting as an Immune Modulator

Arginine is a crucial amino acid that serves to modulate the cellular immune response during infection. Arginine is also a common substrate for both inducible nitric oxide synthase (iNOS) and arginase. The generation of nitric oxide from arginine is responsible for efficient immune response and cytotoxicity of host cells to kill the invading pathogens. On the other hand, the conversion of arginine to ornithine and urea via the arginase pathway can support the growth of bacterial and parasitic pathogens. The competition between iNOS and arginase for arginine can thus contribute to the outcome of several parasitic and bacterial infections. There are two isoforms of vertebrate arginase, both of which catalyze the conversion of arginine to ornithine and urea, but they differ with regard to tissue distribution and subcellular localization. In the case of infection with Mycobacterium, Leishmania, Trypanosoma, Helicobacter, Schistosoma, and Salmonella spp., arginase isoforms have been shown to modulate the pathology of infection by various means. Despite the existence of a considerable body of evidence about mammalian arginine metabolism and its role in immunology, the critical choice to divert the host arginine pool by pathogenic organisms as a survival strategy is still a mystery in infection biology.

Novel role of the nitrite transporter NirC in Salmonella pathogenesis: SPI2-dependent suppression of inducible nitric oxide synthase in activated macrophages

Activation of macrophages by interferon gamma (IFN-gamma) and the subsequent production of nitric oxide (NO) are critical for the host defence against Salmonella enterica serovar Typhimurium infection. We report here the inhibition of IFN-gamma-induced NO production in RAW264.7 macrophages infected with wild-type Salmonella. This phenomenon was shown to be dependent on the nirC gene, which encodes a potential nitrite transporter. We observed a higher NO output from IFN-gamma-treated macrophages infected with a nirC mutant of Salmonella. The nirC mutant also showed significantly decreased intracellular proliferation in a NO-dependent manner in activated RAW264.7 macrophages and in liver, spleen and secondary lymph nodes of mice, which was restored by complementing the gene in trans. Under acidified nitrite stress, a twofold more pronounced NO-mediated repression of SPI2 was observed in the nirC knockout strain compared to the wild-type. This enhanced SPI2 repression in the nirC knockout led to a higher level of STAT-1 phosphorylation and inducible nitric oxide synthase (iNOS) expression than seen with the wild-type strain. In iNOS knockout mice, the organ load of the nirC knockout strain was similar to that of the wild-type strain, indicating that the mutant is exclusively sensitive to the host nitrosative stress. Taken together, these results reveal that intracellular Salmonella evade killing in activated macrophages by downregulating IFN-gamma-induced NO production, and they highlight the critical role of nirC as a virulence gene.

Visiting the cell biology of Salmonella infection

Salmonella, a Gram-negative facultative intracellular pathogen is capable of infecting vast array of hosts. The striking ability of Salmonella to overcome every hurdle encountered in the host proves that they are true survivors. In the host, Salmonella infects various cell types and needs to survive and replicate by countering the defense mechanism of the specific cell. In this review, we will summarize the recent insights into the cell biology of Salmonella infection. Here, we will focus on the findings that deal with the specific mechanism of various cell types to control Salmonella infection. Further, the survival strategies of the pathogen in response to the host immunity will also be discussed in detail. Better understanding of the mechanisms by which Salmonella evade the host defense system and establish pathogenesis will be critical in disease management.

Arginase modulates Salmonella induced nitric oxide production in RAW264.7 macrophages and is required for Salmonella pathogenesis in mice model of infection

Arginine is a common substrate for both inducible nitric oxide synthase (iNOS) and arginase. The competition between iNOS and arginase for arginine contributes to the outcome of several parasitic and bacterial infections. Salmonella infection in macrophage cell line RAW264.7 induces iNOS. Because the availability of l-arginine is a major determinant for nitric oxide (NO) synthesis, we hypothesize that in the Salmonella infected macrophages NO production may be regulated by arginase. Here we report for the first time that Salmonella up-regulates arginase II but not arginase I isoform in RAW264.7 macrophages. Blocking arginase increases the substrate l-arginine availability to iNOS for production of more nitric oxide and perhaps peroxynitrite molecules in the infected cells allowing better killing of virulent Salmonella in a NO dependent manner. RAW264.7 macrophages treated with iNOS inhibitor Aminoguanidine reverts the attenuation in arginase-blocked condition. Further, the NO block created by Salmonella was removed by increasing concentration of l-arginine. The whole-mice system arginase I, although constitutive, is much more abundant than the inducible arginase II isoform. Inhibition of arginase activity in mice during the course of Salmonella infection reduces the bacterial burden and delays the disease outcome in a NO dependent manner.

Cationic Amino Acid Transporters and Salmonella Typhimurium ArgT Collectively Regulate Arginine Availability towards Intracellular Salmonella Growth

Cationic amino acid transporters (mCAT1 and mCAT2B) regulate the arginine availability in macrophages. How in the infected cell a pathogen can alter the arginine metabolism of the host remains to be understood. We reveal here a novel mechanism by which Salmonella exploit mCAT1 and mCAT2B to acquire host arginine towards its own intracellular growth within antigen presenting cells. We demonstrate that Salmonella infected bone marrow derived macrophages and dendritic cells show enhanced arginine uptake and increased expression of mCAT1 and mCAT2B. We show that the mCAT1 transporter is in close proximity to Salmonella containing vacuole (SCV) specifically by live intracellular Salmonella in order to access the macrophage cytosolic arginine pool. Further, Lysosome associated membrane protein 1, a marker of SCV, also was found to colocalize with mCAT1 in the Salmonella infected cell. The intra vacuolar Salmonella then acquire the host arginine via its own arginine transporter, ArgT for growth. The argT knockout strain was unable to acquire host arginine and was attenuated in growth in both macrophages and in mice model of infection. Together, these data reveal survival strategies by which virulent Salmonella adapt to the harsh conditions prevailing in the infected host cells.

The LysR-type transcriptional regulator Hrg counteracts phagocyte oxidative burst and imparts survival advantage to Salmonella enterica serovar Typhimurium

LysR-type transcriptional regulators (LTTRs) are one of the key players that help bacteria adapt to different environments. We have designated STM0952, a putative LTTR in Salmonella enterica serovar Typhimurium (S. Typhimurium), as hydrogen peroxide resistance gene (hrg). A hrg knockout mutant of S. Typhimurium was sensitive to oxidative products of the respiratory burst, specifically to H(2)O(2). The hrg mutant is profoundly attenuated in a murine model of infection and showed decreased intracellular proliferation in macrophages. It also induced increased amounts of reactive oxygen species and co-localization with gp91phox in the macrophage cell line, when compared to the wild-type. A strain overexpressing the hrg gene showed a survival advantage over the wild-type Salmonella under H(2)O(2)-induced stress. Microarray analysis suggested the presence of an Hrg regulon, which is required for resistance to the toxic oxidative products of the reticuloendothelial system.

TLR 9 Activation in Dendritic Cells Enhances Salmonella Killing and Antigen Presentation via Involvement of the Reactive Oxygen Species

Synthetic CpG containing oligodeoxynucleotide Toll like receptor-9 agonist (CpG DNA) activates innate immunity and can stimulate antigen presentation against numerous intracellular pathogens. It was observed that Salmonella Typhimurium growth can be inhibited by the CpG DNA treatment in the murine dendritic cells. This inhibitory effect was mediated by an increased reactive oxygen species production. In addition, it was noted that CpG DNA treatment of dendritic cells during Salmonella infection leads to an increased antigen presentation. Further this increased antigen presentation was dependent on the enhanced reactive oxygen species production elicited by Toll like receptor-9 activation. With the help of an exogenous antigen it was shown that Salmonella antigen could also be cross-presented in a better way by CpG induction. These data collectively indicate that CpG DNA enhance the ability of murine dendritic cells to contain the growth of virulent Salmonella through reactive oxygen species dependent killing.

Novel Relay Selection Rules for Average Interference-Constrained Cognitive AF Relay Networks

Cooperative relaying combined with selection exploits spatial diversity to significantly improve the performance of interference-constrained secondary users in an underlay cognitive radio (CR) network. However, unlike conventional relaying, the state of the links between the relay and the primary receiver affects the choice of the relay. Further, while the optimal amplify-and-forward (AF) relay selection rule for underlay CR is well understood for the peak interference-constraint, this is not so for the less conservative average interference constraint. For the latter, we present three novel AF relay selection (RS) rules, namely, symbol error probability (SEP)-optimal, inverse-of-affine (IOA), and linear rules. We analyze the SEPs of the IOA and linear rules and also develop a novel, accurate approximation technique for analyzing the performance of AF relays. Extensive numerical results show that all the three rules outperform several RS rules proposed in the literature and generalize the conventional AF RS rule.

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.