The molecular basis of how Borrelia burgdorferi (Bb), the Lyme disease spirochete, maintains itself in nature via a complex life cycle in ticks and mammals is poorly understood. Outer surface ...(lipo)protein A (OspA) of Bb has been the most intensively studied of all borrelial molecular constituents, and hence, much has been speculated about the potential role(s) of OspA in the life cycle of Bb. However, the precise function of OspA (along with that of its close relative and operonic partner, outer surface lipoprotein B OspB) heretofore has not been directly determined, due primarily to the inability to generate an OspA/B-deficient mutant from a virulent strain of Bb. In this study, we created an OspA/B-deficient mutant of an infectious human isolate of Bb (strain 297) and found that OspA/B function was not required for either Bb infection of mice or accompanying tissue pathology. However, OspA/B function was essential for Bb colonization of and survival within tick midguts, events crucial for sustaining Bb in its natural enzootic life cycle.
The ability of dendritic cells (DC) to initiate immune responses in naive T cells is dependent upon a maturation process that allows the cells to develop their potent Ag-presenting capacity. Although ...immature DC can be derived in vitro by treatment of peripheral blood monocytes with GM-CSF and IL-4, additional signals such as those provided by TNF-alpha, CD40 ligand, or LPS are required for complete maturation and maximum APC function. Because we recently found that microbial lipoproteins can activate monocytes and DC through Toll-like receptor (TLR) 2, we also investigated whether lipoproteins can drive DC maturation. Immature DC were cultured with or without lipoproteins and were monitored for expression of cell surface markers indicative of maturation. Stimulation with lipopeptides increased expression of CD83, MHC class II, CD80, CD86, CD54, and CD58, and decreased CD32 expression and endocytic activity; these lipopeptide-matured DC also displayed enhanced T cell stimulatory capacity in MLR, as measured by T cell proliferation and IFN-gamma secretion. The lipid moiety of the lipopeptide was found to be essential for induction of maturation. Preincubation of maturing DC with an anti-TLR2 blocking Ab before addition of lipopeptide blocked the phenotypic and functional changes associated with DC maturation. These results demonstrate that lipopeptides can stimulate DC maturation via TLR2, providing a mechanism by which products of bacteria can participate in the initiation of an immune response.
The mammalian innate immune system retains from Drosophila a family of homologous Toll-like receptors (TLRs) that mediate responses to microbial ligands. Here, we show that TLR2 activation leads to ...killing of intracellular Mycobacterium tuberculosis in both mouse and human macrophages, through distinct mechanisms. In mouse macrophages, bacterial lipoprotein activation of TLR2 leads to a nitric oxide-dependent killing of intracellular tubercle bacilli, but in human monocytes and alveolar macrophages, this pathway was nitric oxide-independent. Thus, mammalian TLRs respond (as Drosophila Toll receptors do) to microbial ligands and also have the ability to activate antimicrobial effector pathways at the site of infection.
Borrelia burgdorferi (Bb), the agent of Lyme disease, exists in nature through a complex enzootic life cycle that involves both ticks and mammals. As Bb transitions between its two diverse niches, ...profound adaptive changes occur that are reflected in differential patterns of gene expression, particularly involving lipoprotein genes. Using a mutagenesis approach, we show that Rrp2 (gene BB0763), one of the proteins predicted by the Bb genome (www.tigr.org) to be a response regulator of a two-component sensory transduction system, is a pivotal regulator governing the expression of major membrane lipoproteins such as OspC, DbpA, and Mlp8, as well as many other mammalian infection-associated immunogens of Bb. Sequence analysis additionally suggested that Rrp2 is a bacterial enhancer-binding protein, essential for σ54-dependent gene activation. Mutagenesis of a key amino acid residue within a putative activation domain revealed that Rrp2 controlled lipoprotein expression by governing the expression of the alternative$\sigma\!-\!factor\>\sigma^s$in a σ54-dependent manner. We therefore propose a signal transduction pathway involving Rrp2, σ54, and σs, which in concert control the expression of key lipoproteins and other infection-associated immunogens in Bb.
The generation of cell-mediated immunity against many infectious pathogens involves the production of interleukin-12 (IL-12), a key signal of the innate immune system. Yet, for many pathogens, the ...molecules that induce IL-12 production by macrophages and the mechanisms by which they do so remain undefined. Here it is shown that microbial lipoproteins are potent stimulators of IL-12 production by human macrophages, and that induction is mediated by Toll-like receptors (TLRs). Several lipoproteins stimulated TLR-dependent transcription of inducible nitric oxide synthase and the production of nitric oxide, a powerful microbicidal pathway. Activation of TLRs by microbial lipoproteins may initiate innate defense mechanisms against infectious pathogens.
Outer surface protein C (OspC) is a differentially expressed major surface lipoprotein of Borrelia burgdorferi. ospC is swiftly upregulated when spirochetes leave the Ixodes scapularis tick gut, ...migrate to the salivary gland, and exit the arthropod vector. Here we show that OspC strongly binds to the tick salivary gland, suggesting a role for OspC in spirochete adherence to this tissue. In vivo studies using a murine model of Lyme borreliosis showed that while OspC F(ab)(2) fragments did not influence either the viability of spirochetes or ospC gene expression, they did interfere with B. burgdorferi invasion of tick salivary glands. We then generated ospC knockout spirochetes in an infectious clone of B. burgdorferi and examined them within the vector. OspC-deficient or wild-type spirochetes persisted equally within the gut of unfed ticks and multiplied during the tick engorgement; however, unlike wild-type B. burgdorferi, the mutants were unable to invade salivary glands. Salivary gland colonization of OspC-deficient spirochetes was completely restored when this mutant was complemented in trans with a plasmid harboring the wild-type ospC gene. These studies conclusively demonstrate the importance of OspC in the invasion of tick salivary glands by B. burgdorferi, a critical step in the transmission of spirochetes from the arthropod vector to the mammalian host.
The expression and activation of Toll-like receptors (TLRs) was investigated in leprosy, a spectral disease in which clinical manifestations correlate with the type of immune response mounted toward ...Mycobacterium leprae. TLR2-TLR1 heterodimers mediated cell activation by killed M. leprae, indicating the presence of triacylated lipoproteins. A genome-wide scan of M. leprae detected 31 putative lipoproteins. Synthetic lipopeptides representing the 19-kD and 33-kD lipoproteins activated both monocytes and dendritic cells. Activation was enhanced by type-1 cytokines and inhibited by type-2 cytokines. In addition, interferon (IFN)-gamma and granulocyte-macrophage colony-stimulating factor (GM-CSF) enhanced TLR1 expression in monocytes and dendritic cells, respectively, whereas IL-4 downregulated TLR2 expression. TLR2 and TLR1 were more strongly expressed in lesions from the localized tuberculoid form (T-lep) as compared with the disseminated lepromatous form (L-lep) of the disease. These data provide evidence that regulated expression and activation of TLRs at the site of disease contribute to the host defense against microbial pathogens.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
RpoS and RpoN are two alternative sigma factors typically associated with general stress responses in bacteria. To date, there has been no experimental evidence that RpoS and RpoN can directly ...control the expression of one another. Herein, using a combined strategy of gene disruption and genetic complementation targeting rpoN and rpoS in Borrelia burgdorferi strain 297, we describe a regulatory network for B. burgdorferi. In this network, RpoN controls the expression of RpoS, which, in turn, governs the expression of two important membrane lipoproteins, outer surface protein C and decorin-binding protein A, and likely other proteins of B. burgdorferi. Our findings provide a foundation for elucidating further key regulatory networks that potentially impact many aspects of B. burgdorferi's parasitic strategy, host range, and virulence expression.
Mammalian Toll-like receptors (TLRs) are required for cell activation by bacterial lipoproteins (bLP) and LPS. Stimulation of monocytes with bLP and LPS results in a TLR-dependent induction of ...immunomodulatory genes leading to the production of pro-inflammatory cytokines. In this paper, we compared the expression and response of TLRs on monocytes and dendritic cells (DC). TLR2, but not TLR4, was detected on peripheral blood monocytes and DC, in lymphoid tissue CD1alpha+ DC as well as on in vitro monocyte-derived DC. Upon stimulation with bLP or LPS, monocytes produced IL-12 and IL-10 at similar levels, whereas monocyte-derived DC produced comparable levels of IL-12, but little IL-10. Greater than 90% of the bLP-induced production of IL-12 was blocked by anti-TLR2 mAb. Thus, DC express TLR2 and activation of this receptor by bLP provides an innate mechanism by which microbial pathogens preferentially activate cell-mediated immunity.
The paradigm for differential antigen expression in Borrelia burgdorferi, the agent of Lyme disease, is the reciprocal expression of its outer surface (lipo)proteins (Osp) A and C; as B. burgdorferi ...transitions from its arthropod vector into mammalian tissue, ospC is upregulated, and ospA is downregulated. In the current study, using B. burgdorferi cultivated under varying conditions in BSK‐H medium, we found that a decrease in pH, in conjunction with increases in temperature (e.g. 34°C or 37°C) and cell density, acted interdependently for the reciprocal expression of ospC and ospA. The lower pH (6.8), which induced the reciprocal expression of ospC and ospA in BSK‐H medium, correlated with a drop in pH from 7.4 to 6.8 of tick midgut contents during tick feeding. In addition to ospC and ospA, other genes were found to be regulated in reciprocal fashion. Such genes were either ospC‐like (e.g. ospF, mlp‐8 and rpoS) (group I) or ospA‐like (lp6.6 and p22) (group II); changes in expression occurred at the mRNA level. That the expression of rpoS, encoding a putative stress‐related alternative sigma factor (σs), was ospC‐like suggested that the expression of some of the group I genes may be controlled through σs. The combined results prompt a model that allows for predicting the regulation of other B. burgdorferi genes that may be involved in spirochaete transmission, virulence or mammalian host immune responses.
PDF
