Quorum sensing (QS) is a key regulator of virulence and biofilm formation in Pseudomonas aeruginosa and other medically relevant bacteria. Aqueous extracts of six plants, Conocarpus erectus, Chamaesyce hypericifolia, Callistemon viminalis, Bucida buceras, Tetrazygia bicolor, and Quercus virginiana, were examined in this study for their effects on P. aeruginosa virulence factors and the QS system. C. erectus, B. buceras, and C. viminalis caused a significant inhibition of LasA protease, LasB elastase, pyoverdin production, and biofilm formation. Additionally, each plant presented a distinct effect profile on the las and rhl QS genes and their respective signaling molecules, suggesting that different mechanisms are responsible for efficacy. Extracts of all plants caused the inhibition of QS genes and QS-controlled factors, with marginal effects on bacterial growth, suggesting that the quorum-quenching mechanisms are unrelated to static or cidal effects.Pneumonia due to microbial infections is a major cause of morbidity and mortality in immunocompromised patients. Pseudomonas aeruginosa hails as the leading pathogen among patients with cystic fibrosis, diffused panbronchitis, and chronic obstructive pulmonary disease (16,29,37). In addition, P. aeruginosa remains one of the major causes of nosocomial infections (10). The success of this organism is largely due to the production of a myriad of virulence factors (including LasA protease, LasB elastase, pyoverdin, pyocyanin, and alginate) and its ability to form intractable biofilms (38).Expression of many of the virulence factors in P. aeruginosa is controlled by a quorum-sensing (QS) system (59), an intercellular communication scheme in which bacteria are able to detect the population density (via signaling molecules and receptors) and control gene expression accordingly (55). P. aeruginosa elaborates two main sets of QS systems: lasI-lasR and rhlI-rhlR (55). LasI and RhlI are synthetases that manufacture the autoinducer signaling molecules N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) and N-butanoyl-L-homoserine lactone (BHL), respectively. These molecules diffuse out into the environment, and when they reach a putative threshold concentration, they activate the receptors lasR and rhlR. These receptors, in turn, coordinate the regulation of pathogenicity. A third signal, the Pseudomonas quinolone signal, also plays an integral role in the QS system (50). This secondary metabolite of P. aeruginosa is incorporated into the QS hierarchy in times of cell stress (43). This intricate communication system of P. aeruginosa is mirrored in many gramnegative pathogenic bacteria, where it coordinates the regulation of virulence, including motility, biofilm formation, and toxin production (18,19,21,48,54).The misuse and abuse of antibiotics in pharmacotherapy have led to the development of widespread resistance in the target organism. The failure of existing antibiotics to control infection makes it crucial to find alternatives to currently available drugs. Since pathogenici...

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In members of the family Enterobacteriaceae, ampC, which encodes a ␤-lactamase, is regulated by an upstream, divergently transcribed gene, ampR. However, in Pseudomonas aeruginosa, the regulation of ampC is not understood. In this study, we compared the characteristics of a P. aeruginosa ampR mutant, PAOampR, with that of an isogenic ampR ؉ parent. The ampR mutation greatly altered AmpC production. In the absence of antibiotic, PAOampR expressed increased basal ␤-lactamase levels. However, this increase was not followed by a concomitant increase in the P ampC promoter activity. The discrepancy in protein and transcription analyses led us to discover the presence of another chromosomal AmpR-regulated ␤-lactamase, PoxB. We found that the expression of P. aeruginosa ampR greatly altered the ␤-lactamase production from ampC and poxB in Escherichia coli: it up-regulated AmpC but down-regulated PoxB activities. In addition, the constitutive P ampR promoter activity in PAOampR indicated that AmpR did not autoregulate in the absence or presence of inducers. We further demonstrated that AmpR is a global regulator because the strain carrying the ampR mutation produced higher levels of pyocyanin and LasA protease and lower levels of LasB elastase than the wild-type strain. The increase in LasA levels was positively correlated with the P lasA , P lasI , and P lasR expression. The reduction in the LasB activity was positively correlated with the P rhlR expression. Thus, AmpR plays a dual role, positively regulating the ampC, lasB, and rhlR expression levels and negatively regulating the poxB, lasA, lasI, and lasR expression levels.

Protein kinase C-θ (PKC-θ) translocates to the center of the immunological synapse, but the underlying mechanism and its importance in T cell activation are unknown. We found that the PKC-θ V3 domain is necessary and sufficient for IS localization mediated by Lck-dependent association with CD28. We identified a conserved proline-rich motif in V3 required for CD28 association and IS localization. CD28 association was essential for PKC-θ-mediated downstream signaling and TH2 and TH17, but not TH1, differentiation. Ectopic V3 expression sequestered PKC-θ from the IS and interfered with its functions. These results identify a unique mode of CD28 signaling, establish a molecular basis for the IS localization of PKC-θ, and implicate V3-based “decoys” as therapeutic modalities for T cell-mediated inflammatory diseases.

West Nile virus (WNV), a mosquito-borne flavivirus, has recently emerged in North America, and the elderly are particularly susceptible to severe neurological disease and death from infection with this virus. We have investigated the innate immune response of primary human macrophages to WNV in vitro and have found significant differences between the responsiveness of macrophages derived from younger donors and that from older donors. Binding of the glycosylated WNV envelope protein to the C-type lectin dendritic cell-specific intercellular adhesion molecule 3 (ICAM3) grabbing nonintegrin (DC-SIGN) leads to a reduction in the expression of Toll-like receptor 3 (TLR3) in macrophages from young donors via the signal transducer and activator of transcription 1 (STAT1)-mediated pathway. This signaling is impaired in the elderly, and the elevated levels of TLR3 result in an elevation of cytokine levels. This alteration of the innate immune response with aging may contribute to the permeability of the blood-brain barrier and suggests a possible mechanism for the increased severity of WNV infection in older individuals.

Polymorphonuclear leukocytes (PMN) are key in innate immunity but their role in viral pathogenesis is incompletely understood. In infection with West Nile virus (WNV), we found that expression of two PMN-attracting chemokines, Cxcl1 and Cxcl2, was rapidly and dramatically elevated in macrophages. PMN are rapidly recruited to the site of WNV infection in mice and support efficient replication of WNV. Mice depleted of PMN after WNV inoculation developed higher viremia and earlier death compared to the control group, suggesting a protective role for PMN. In contrast, when PMN were depleted prior to infection with WNV, and in mice deficient in Cxcr2, a chemokine receptor gene, viremia was reduced and survival was enhanced or delayed. Collectively, these data suggest that PMN have a biphasic response to WNV infection, serving as a reservoir for replication and dissemination in early infection and later contributing to viral clearance.

West Nile virus (WNV) is the most-common cause of mosquito-borne encephalitis in the United States. Invasion of the brain by WNV is influenced by viral and host factors, and the molecular mechanism underlying disruption of the blood-brain barrier is likely multifactorial. Here we show that matrix metalloproteinase 9 (MMP9) is involved in WNV entry into the brain by enhancing blood-brain barrier permeability. Murine MMP9 expression was induced in the circulation shortly after WNV infection, and the protein levels remained high even when viremia subsided. In the murine brain, MMP9 expression and its enzymatic activity were upregulated and MMP9 was shown to partly localize to the blood vessels. Interestingly, we also found that cerebrospinal fluid from patients suffering from WNV contained increased MMP9 levels. The peripheral viremia and expression of host cytokines were not altered in MMP9 ؊/؊ mice; however, these animals were protected from lethal WNV challenge. The resistance of MMP9 ؊/؊ mice to WNV infection correlated with an intact blood-brain barrier since immunoglobulin G, Evans blue leakage into brain, and type IV collagen degradation were markedly reduced in the MMP9 ؊/؊ mice compared with their levels in controls. Consistent with this, the brain viral loads, selected inflammatory cytokines, and leukocyte infiltrates were significantly reduced in the MMP9 ؊/؊ mice compared to their levels in wild-type mice. These data suggest that MMP9 plays a role in mediating WNV entry into the central nervous system and that strategies to interrupt this process may influence the course of West Nile encephalitis.Mosquito-borne flaviviruses are emerging as increasing threats to human health. For example, West Nile virus (WNV) is maintained worldwide in an enzootic cycle between the avian hosts and mosquito vectors. Since the outbreak in New York City in 1999, WNV has rapidly spread throughout North America, resulting in over 20,000 human cases through 2006, with a mortality rate of 3.9% (11). Infection is often asymptomatic, but high fever, meningitis, encephalitis, or acute flaccid paralysis may occur in 20 to 40% of infected individuals (11). Encephalitis is the major cause of death and is most common in the elderly (Centers for Disease Control and Prevention, Atlanta, GA).The mechanisms by which neurotropic flaviviruses enter the central nervous system (CNS) are not well understood. In theory, WNV can enter the brain through multiple pathways, including endothelial tight junctions, direct infection of endothelial cells, infected leukocytes that traffic to the CNS, infection of olfactory neurons, and/or direct axonal retrograde transport from infected peripheral neurons (7, 27, 28). It has recently been shown that increased peripheral viremia in Tolllike receptor 3 (TLR3)-deficient mice (TLR3 Ϫ/Ϫ ) did not result in increased viral entry into the brain. In contrast, TLR3 Ϫ/Ϫ mice were resistant to lethal WNV challenge owing to better blood-brain barrier (BBB) integrity (30), suggesting that WNV enters the brain primar...

Regulatory T cells (Treg cells), which maintain immune homeostasis and self-tolerance, form an immunological synapse (IS) with antigen-presenting cells (APCs). However, signaling events at the Treg IS remain unknown. Here we show that protein kinase C-η (PKC-η) associated with CTLA-4 and was recruited to the Treg IS. PKC-η-deficient Treg cells displayed defective suppressive activity, including suppression of tumor immunity but not autoimmune colitis. Phosphoproteomic analysis revealed an association between CTLA-4-PKC-η and the GIT-PIX-PAK complex, an IS-localized focal adhesion complex. Defective activation of this complex in PKC-η-deficient Treg cells was associated with reduced CD86 depletion from APCs by Treg cells. These results reveal a novel CTLA-4-PKC-η signaling axis required for contact-dependent suppression, implicating this pathway as a potential cancer immunotherapy target.