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.

The adenylyl cyclase Rv2212 modifies the proteome and infectivity of Mycobacterium bovis BCG

All organisms have the capacity to sense and respond to environmental changes. These signals often involve the use of second messengers such as cyclic adenosine monophosphate (cAMP). This second messenger is widely distributed among organisms and coordinates gene expression related with pathogenesis, virulence, and environmental adaptation. Genomic analysis in Mycobacterium tuberculosis has identified 16 adenylyl cyclases (AC) and one phosphodiesterase, which produce and degrade cAMP, respectively. To date, ten AC have been biochemically characterized and only one (Rv0386) has been found to be important during murine infection with M. tuberculosis. Here, we investigated the impact of hsp60-driven Rv2212 gene expression in Mycobacterium bovis Bacillus Calmette-Guerin (BCG) during growth in vitro, and during macrophage and mice infection. We found that hsp60-driven expression of Rv2212 resulted in an increased capacity of replication in murine macrophages but an attenuated phenotype in lungs and spleen when administered intravenously in mice. Furthermore, this strain displayed an altered proteome mainly affecting proteins associated with stress conditions (bfrB, groEL-2, DnaK) that could contribute to the attenuated phenotype observed in mice.

Intracellular activities of Salmonella enterica in murine dendritic cells.

Dendritic cells (DC) efficiently phagocytose invading bacteria, but fail to kill intracellular pathogens such as Salmonella enterica serovar Typhimurium (S. Typhimurium). We analysed the intracellular fate of Salmonella in murine bone marrow-derived DC (BM-DC). The intracellular proliferation and subcellular localization were investigated for wild-type S. Typhimurium and mutants deficient in Salmonella pathogenicity island 2 (SPI2), a complex virulence factor that is essential for systemic infections in the murine model and intracellular survival and replication in macrophages. Using a segregative plasmid to monitor intracellular cell division, we observed that, in BM-DC, S. Typhimurium represents a static, non-dividing population. In BM-DC, S. Typhimurium resides in a membrane-bound compartment that has acquired late endosomal markers. However, these bacteria respond to intracellular stimuli, because induction of SPI2 genes was observed. S. Typhimurium within DC are also able to translocate a virulence protein into their host cells. SPI2 function was not required for intracellular survival in DC, but we observed that the maturation of the Salmonella-containing vesicle is different in DC infected with wild-type bacteria and a strain deficient in SPI2. Our observations indicate that S. Typhimurium in DC are able to modify normal processes of their host cells.

NOD2-Nitric Oxide-responsive MicroRNA-146a Activates Sonic Hedgehog Signaling to Orchestrate Inflammatory Responses in Murine Model of Inflammatory Bowel Disease

Background: Genetic variants of NOD2 are linked to inflammatory bowel disease (IBD) etiology. Results: DSS model of colitis in wild-type and inducible nitric-oxide synthase (iNOS) null mice revealed that NOD2-iNOS/NO-responsive microRNA-146a targets NUMB gene facilitating Sonic hedgehog (SHH) signaling. Conclusion: miR-146a-mediated NOD2-SHH signaling regulates gut inflammation. Significance: Identification of novel regulators of IBD provides new insights into pathophysiology and development of new therapy concepts. Inflammatory bowel disease (IBD) is a debilitating chronic inflammatory disorder of the intestine. The interactions between enteric bacteria and genetic susceptibilities are major contributors of IBD etiology. Although genetic variants with loss or gain of NOD2 functions have been linked to IBD susceptibility, the mechanisms coordinating NOD2 downstream signaling, especially in macrophages, during IBD pathogenesis are not precisely identified. Here, studies utilizing the murine dextran sodium sulfate model of colitis revealed the crucial roles for inducible nitric-oxide synthase (iNOS) in regulating pathophysiology of IBDs. Importantly, stimulation of NOD2 failed to activate Sonic hedgehog (SHH) signaling in iNOS null macrophages, implicating NO mediated cross-talk between NOD2 and SHH signaling. NOD2 signaling up-regulated the expression of a NO-responsive microRNA, miR-146a, that targeted NUMB gene and alleviated the suppression of SHH signaling. In vivo and ex vivo studies confirmed the important roles for miR-146a in amplifying inflammatory responses. Collectively, we have identified new roles for miR-146a that established novel cross-talk between NOD2-SHH signaling during gut inflammation. Potential implications of these observations in therapeutics could increase the possibility of defining and developing better regimes to treat IBD pathophysiology.

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- ) 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- -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- -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- -induced NO production, and they highlight the critical role of nirC as a virulence gene.

PIM2 Induced COX-2 and MMP-9 Expression in Macrophages Requires PI3K and Notch1 Signaling

Activation of inflammatory immune responses during granuloma formation by the host upon infection of mycobacteria is one of the crucial steps that is often associated with tissue remodeling and breakdown of the extracellular matrix. In these complex processes, cyclooxygenase-2 (COX-2) plays a major role in chronic inflammation and matrix metalloproteinase-9 (MMP-9) significantly in tissue remodeling. In this study, we investigated the molecular mechanisms underlying Phosphatidyl-myo-inositol dimannosides (PIM2), an integral component of the mycobacterial envelope, triggered COX-2 and MMP-9 expression in macrophages. PIM2 triggers the activation of Phosphoinositide-3 Kinase (PI3K) and Notch1 signaling leading to COX-2 and MMP-9 expression in a Toll-like receptor 2 (TLR2)-MyD88 dependent manner. Notch1 signaling perturbations data demonstrate the involvement of the cross-talk with members of PI3K and Mitogen activated protein kinase pathway. Enforced expression of the cleaved Notch1 in macrophages induces the expression of COX-2 and MMP-9. PIM2 triggered significant p65 nuclear factor-kappa B (NF-kappa B) nuclear translocation that was dependent on activation of PI3K or Notch1 signaling. Furthermore, COX-2 and MMP-9 expression requires Notch1 mediated recruitment of uppressor of Hairless (CSL) and NF-kappa B to respective promoters. Inhibition of PIM2 induced COX-2 resulted in marked reduction in MMP-9 expression clearly implicating the role of COX-2 dependent signaling events in driving the MMP-9 expression. Taken together, these data implicate PI3K and Notch1 signaling as obligatory early proximal signaling events during PIM2 induced COX-2 and MMP-9 expression in macrophages.

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.

Nitric Oxide Is Involved in Mycobacterium bovis Bacillus Calmette-Guerin-Activated Jagged1 and Notch1 Signaling.

Pathogenic mycobacteria have evolved unique strategies to survive within the hostile environment of macrophages. Modulation of key signaling cascades by NO, generated by the host during infection, assumes critical importance in overall cell-fate decisions. We show that NO is a critical factor in Mycobacterium bovis bacillus Calmette-Guérin-mediated Notch1 activation, as the generation of activated Notch1 or expression of Notch1 target genes matrix metalloproteinase-9 (MMP-9) or Hes1 was abrogated in macrophages derived from inducible NO synthase (iNOS) knockout (iNOS(-/-)), but not from wild-type, mice. Interestingly, expression of the Notch1 ligand Jagged1 was compromised in M. bovis bacillus Calmette-Guérin-stimulated iNOS(-/-) macrophages, and loss of Jagged1 expression or Notch1 signaling could be rescued by NO donors. Signaling perturbations or genetic approaches implicated that robust expression of MMP-9 or Hes1 required synergy and cross talk between TLR2 and canonical Notch1-PI3K cascade. Further, CSL/RBP-Jk contributed to TLR2-mediated expression of MMP-9 or Hes1. Correlative evidence shows that, in a murine model for CNS tuberculosis, this mechanism operates in vivo only in brains derived from WT but not from iNOS(-/-) mice. Importantly, we demonstrate the activation of Notch1 signaling in vivo in granulomatous lesions in the brains of Mycobacterium tuberculosis-infected human patients with tuberculous meningitis. Current investigation identifies NO as a pathological link that modulates direct cooperation of TLR2 with Notch1-PI3K signaling or Jagged1 to regulate specific components of TLR2 responses. These findings provide new insights into mechanisms by which Notch1, TLR2, and NO signals are integrated in a cross talk that modulates a defined set of effector functions in macrophages.

Human alveolar T lymphocyte responses to Mycobacterium tuberculosis antigens: role for CD4+ and CD8+ cytotoxic T cells and relative resistance of alveolar macrophages to lysis.

T cell-mediated cytotoxicity against Mycobacterium tuberculosis (MTB)-infected macrophages may be a major mechanism of specific host defense, but little is known about such activities in the lung. Thus, the capacity of alveolar lymphocyte MTB-specific cell lines (AL) and alveolar macrophages (AM) from tuberculin skin test-positive healthy subjects to serve as CTL and target cells, respectively, in response to MTB (H37Ra) or purified protein derivative (PPD) was investigated. Mycobacterial Ag-pulsed AM were targets of blood CTL activity at E:T ratios of > or = 30:1 (51Cr release assay), but were significantly more resistant to cytotoxicity than autologous blood monocytes. PPD- plus IL-2-expanded AL and blood lymphocytes were cytotoxic for autologous mycobacterium-stimulated monocytes at E:T ratios of > or = 10:1. The CTL activity of lymphocytes expanded with PPD was predominantly class II MHC restricted, whereas the CTL activity of lymphocytes expanded with PPD plus IL-2 was both class I and class II MHC restricted. Both CD4+ and CD8+ T cells were enriched in BL and AL expanded with PPD and IL-2, and both subsets had mycobacterium-specific CTL activity. Such novel cytotoxic responses by CD4+ and CD8+ T cells may be a major mechanism of defense against MTB at the site of disease activity.

Alveolar macrophages as accessory cells for human gamma delta T cells activated by Mycobacterium tuberculosis.

Alveolar macrophages form the first line of defense against inhaled droplets containing Mycobacterium tuberculosis by controlling mycobacterial growth and regulating T cell responses. CD4+ and gamma delta T cells, two major T cell subsets activated by M. tuberculosis, require accessory cells for activation. However, the ability of alveolar macrophages to function as accessory cells for T cell activation remains controversial. We sought to determine the ability of alveolar macrophages to serve as accessory cells for resting (HLA-DR-, IL-2R-) and activated (HLA-DR+, IL-2R+) gamma delta T cells in response to M. tuberculosis and its Ag, and to compare accessory cell function for gamma delta T cells of alveolar macrophages and blood monocytes obtained from the same donor. Alveolar macrophages were found to serve as accessory cells for both resting and activated gamma delta T cells in response to M. tuberculosis Ag. At high alveolar macrophage to T cell ratios (> 3:1), however, expansion of resting gamma delta T cells was inhibited by alveolar macrophages. The inhibition of resting gamma delta T cells by alveolar macrophages was dose-dependent, required their presence during the first 24 h, and was partially overcome by IL-2. Alveolar macrophages did not inhibit activated gamma delta T cells even at high accessory cell to T cell ratios, and alveolar macrophages functioned as well as monocytes as accessory cells. Monocytes were not inhibitory for either resting or activated gamma delta T cells. These findings support the following model. In the normal alveolus the alveolar macrophage to T cell ratio is > or = 9:1, and therefore the threshold for resting gamma delta T cell activation is likely to be high. Once a nonspecific inflammatory response occurs, such as after invasion by M. tuberculosis, this ratio is altered, favoring gamma delta T cell activation by alveolar macrophages.

Curcumin Increases the Pathogenicity of Salmonella enterica Serovar Typhimurium in Murine Model

Curcumin has gained immense importance for its vast therapeutic and prophylactic applications. Contrary to this, our study reveals that it regulates the defense pathways of Salmonella enterica serovar Typhimurium ( S. Typhimurium) to enhance its pathogenicity. In a murine model of typhoid fever, we observed higher bacterial load in Peyer's,patches, mesenteric lymph node, spleen and liver, when infected with curcumin-treated Salmonella. Curcumin increased the resistance of S. Typhimurium against antimicrobial agents like antimicrobial peptides, reactive oxygen and nitrogen species. This increased tolerance might be attributed to the up-regulation of genes involved in resistance against antimicrobial peptides - pmrD and pmrHFIJKLM and genes with antioxidant function - mntH, sodA and sitA. We implicate that iron chelation property of curcumin have a role in regulating mntH and sitA. Interestingly, we see that the curcumin-mediated modulation of pmr genes is through the PhoPQ regulatory system. Curcumin downregulates SPI1 genes, required for entry into epithelial cells and upregulates SPI2 genes required to intracellular survival. Since it is known that the SPI1 and SPI2 system can be regulated by the PhoPQ system, this common regulator could explain curcumin's mode of action. This data urges us to rethink the indiscriminate use of curcumin especially during Salmonella outbreaks.

Salmonella enterica Serovar Typhimurium Lacking hfq Gene Confers Protective Immunity against Murine Typhoid

Salmonella enterica is an important enteric pathogen and its various serovars are involved in causing both systemic and intestinal diseases in humans and domestic animals. The emergence of multidrug-resistant strains of Salmonella leading to increased morbidity and mortality has further complicated its management. Live attenuated vaccines have been proven superior over killed or subunit vaccines due to their ability to induce protective immunity. Of the various strategies used for the generation of live attenuated vaccine strains, focus has gradually shifted towards manipulation of virulence regulator genes. Hfq is a RNA chaperon which mediates the binding of small RNAs to the mRNA and assists in post-transcriptional gene regulation in bacteria. In this study, we evaluated the efficacy of the Salmonella Typhimurium Dhfq strain as a candidate for live oral vaccine in murine model of typhoid fever. Salmonella hfq deletion mutant is highly attenuated in cell culture and animal model implying a significant role of Hfq in bacterial virulence. Oral immunization with the Salmonella hfq deletion mutant efficiently protects mice against subsequent oral challenge with virulent strain of Salmonella Typhimurium. Moreover, protection was induced upon both multiple as well as single dose of immunizations. The vaccine strain appears to be safe for use in pregnant mice and the protection is mediated by the increase in the number of CD4(+) T lymphocytes upon vaccination. The levels of serum IgG and secretory-IgA in intestinal washes specific to lipopolysaccharide and outer membrane protein were significantly increased upon vaccination. Furthermore, hfq deletion mutant showed enhanced antigen presentation by dendritic cells compared to the wild type strain. Taken together, the studies in murine immunization model suggest that the Salmonella hfq deletion mutant can be a novel live oral vaccine candidate.

The use of murine polyclonal anti-idiotypic antibodies as surrogate allergens in the diagnosis of Parthenium hypersensitivity

Background: Anti-idiotypic antibodies (Ab-2), which are the mirror images of idiotypic antibodies (Ab-1), may be useful as diagnostic reagents and for use as immunogen to induce antigen-specific immune responses. Methods and Results: To explore the biologic potential of Ab-2 as diagnostic reagents in allergic diseases, murine mouse (m) Ab-2 were raised by immunizing Balb/c mice with affinity purified rabbit (r) Ab-1 specific for the pollen of Parthenium hysterophorus, an allergenic weed that grows wild on the Indian subcontinent and in Australia, Mexico, and the southern United States. Affinity purified Parthenium-specific human (h)AB-1 could successfully inhibit the binding of mAb-2 to immobilized rAb-1. Further, Balb/c mice immunized with mAb-2 induced Parthenium-specific anti-anti-idiotypic IgE and IgG antibodies. Specificity of the Ab-2 was confirmed by the ability of Parthenium pollen extracts to inhibit the binding of allergen-specific IgE and IgG Ab-1 in the sera of patients with rhinitis to immobilized mAb-2. Parthenium-sensitive patients with rhinitis who had positive results on skin prick tests to Parthenium pollen extracts also responded with a positive skin reaction to mAb-2. Conclusion: Our data demonstrate that Parthenium-specific mAb-2 may be of value as surrogate allergens in allergen standardization and for in vitro diagnosis.

Intracellular Pathogen Sensor NOD2 Programs Macrophages to Trigger Notch1 Activation

Intracellular pathogen sensor, NOD2, has been implicated in regulation of wide range of anti-inflammatory responses critical during development of a diverse array of inflammatory diseases; however, underlying molecular details are still imprecisely understood. In this study, we demonstrate that NOD2 programs macrophages to trigger Notch1 signaling. Signaling perturbations or genetic approaches suggest signaling integration through cross-talk between Notch1-PI3K during the NOD2-triggered expression of a multitude of immunological parameters including COX-2/PGE(2) and IL-10. NOD2 stimulation enhanced active recruitment of CSL/RBP-Jk on the COX-2 promoter in vivo. Intriguingly, nitric oxide assumes critical importance in NOD2-mediated activation of Notch1 signaling as iNOS(-/-) macrophages exhibited compromised ability to execute NOD2-triggered Notch1 signaling responses. Correlative evidence demonstrates that this mechanism operates in vivo in brain and splenocytes derived from wild type, but not from iNOS(-/-) mice. Importantly, NOD2-driven activation of the Notch1-PI3K signaling axis contributes to its capacity to impart survival of macrophages against TNF-alpha or IFN-gamma-mediated apoptosis and resolution of inflammation. Current investigation identifies Notch1-PI3K as signaling cohorts involved in the NOD2-triggered expression of a battery of genes associated with anti-inflammatory functions. These findings serve as a paradigm to understand the pathogenesis of NOD2-associated inflammatory diseases and clearly pave a way toward development of novel therapeutics.

Augmentation of murine natural killer cell and antibody dependent cellular cytotoxicity activities by phyllanthus emblica, a new immunomodulator

When administered orally, Phyllanthus emblica, an excellent source of vitamin C (ascorbate), has been found to enhance natural killer (NK) cell activity and antibody dependent cellular cytotoxicity (ADCC) in syngeneic BALB/c mice, bearing Dalton's lymphoma ascites (DLA) tumor. P. emblica elicited a 2-fold increase in splenic NK cell activity on day 3 post tumor inoculation. Enhanced activity was highly significant on days 3, 5, 7 and 9 after tumor inoculation with respect to the untreated tumor bearing control. A significant enhancement in ADCC was documented on days 3, 7, 9, 11 and 13 in drug treated mice as compared to the control. An increase in life span (ILS) of 35% was recorded in tumor bearing mice treated with P. emblica. This increased survival was completely abrogated when NK cell and killer (K) cell activities were depleted either by cyclophosphamide or anti-asialo-GM, antibody treatment. These results indicate: (a) an absolute requirement for a functional NK cell or K cell population in order that P. emblica can exert its effect on tumor bearing animals, and (b) the antitumor activity of P. emblica is mediated primarily through the ability of the drug to augment natural cell mediated cytotoxicity.