RESUMO
The surfaces of many Gram-negative bacteria are decorated with soluble proteins anchored to the outer membrane via an acylated N-terminus; these proteins are referred to as surface lipoproteins or SLPs. In Neisseria meningitidis, SLPs such as transferrin-binding protein B (TbpB) and factor-H binding protein (fHbp) are essential for host colonization and infection because of their essential roles in iron acquisition and immune evasion, respectively. Recently, we identified a family of outer membrane proteins called Slam (Surface lipoprotein assembly modulator) that are essential for surface display of neisserial SLPs. In the present study, we performed a bioinformatics analysis to identify 832 Slam related sequences in 638 Gram-negative bacterial species. The list included several known human pathogens, many of which were not previously reported to possess SLPs. Hypothesizing that genes encoding SLP substrates of Slams may be present in the same gene cluster as the Slam genes, we manually curated neighboring genes for 353 putative Slam homologs. From our analysis, we found that 185 (~52%) of the 353 putative Slam homologs are located adjacent to genes that encode a protein with an N-terminal lipobox motif. This list included genes encoding previously reported SLPs in Haemophilus influenzae and Moraxella catarrhalis, for which we were able to show that the neighboring Slams are necessary and sufficient to display these lipoproteins on the surface of Escherichia coli. To further verify the authenticity of the list of predicted SLPs, we tested the surface display of one such Slam-adjacent protein from Pasteurella multocida, a zoonotic pathogen. A robust Slam-dependent display of the P. multocida protein was observed in the E. coli translocation assay indicating that the protein is a Slam-dependent SLP. Based on multiple sequence alignments and domain annotations, we found that an eight-stranded beta-barrel domain is common to all the predicted Slam-dependent SLPs. These findings suggest that SLPs with a TbpB-like fold are found widely in Proteobacteria where they exist with their interaction partner Slam. In the future, SLPs found in pathogenic bacteria can be investigated for their role in virulence and may also serve as candidates for vaccine development.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Bactérias Gram-Negativas/genética , Lipoproteínas/genética , Lipoproteínas/isolamento & purificação , Antígenos de Bactérias/imunologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/isolamento & purificação , Escherichia coli/genética , Haemophilus influenzae/genética , Humanos , Evasão da Resposta Imune , Moraxella catarrhalis/genética , Família Multigênica , Neisseria meningitidis/genética , Pasteurella multocida/genética , Proteobactérias/genética , Alinhamento de Sequência , Proteína B de Ligação a Transferrina/imunologiaRESUMO
Clinical and epidemiological synergy exists between the globally important sexually transmitted infections, gonorrhea and HIV. Neisseria gonorrhoeae, which causes gonorrhea, is particularly adept at driving HIV-1 expression, but the molecular determinants of this relationship remain undefined. N. gonorrhoeae liberates a soluble factor that potently induces expression from the HIV-1 LTR in coinfected cluster of differentiation 4-positive (CD4(+)) T lymphocytes, but this factor is not a previously described innate effector. A genome-wide mutagenesis approach was undertaken to reveal which component(s) of N. gonorrhoeae induce HIV-1 expression in CD4(+) T lymphocytes. A mutation in the ADP-heptose biosynthesis gene, hldA, rendered the bacteria unable to induce HIV-1 expression. The hldA mutant has a truncated lipooligosaccharide structure, contains lipid A in its outer membrane, and remains bioactive in a TLR4 reporter-based assay but did not induce HIV-1 expression. Mass spectrometry analysis of extensively fractionated N. gonorrhoeae-derived supernatants revealed that the LTR-inducing fraction contained a compound having a mass consistent with heptose-monophosphate (HMP). Heptose is a carbohydrate common in microbes but is absent from the mammalian glycome. Although ADP-heptose biosynthesis is common among Gram-negative bacteria, and heptose is a core component of most lipopolysaccharides, N. gonorrhoeae is peculiar in that it effectively liberates HMP during growth. This N. gonorrhoeae-derived HMP activates CD4(+) T cells to invoke an NF-κB-dependent transcriptional response that drives HIV-1 expression and viral production. Our study thereby shows that heptose is a microbial-specific product that is sensed as an innate immune agonist and unveils the molecular link between N. gonorrhoeae and HIV-1.
Assuntos
Coinfecção/imunologia , Gonorreia , Infecções por HIV , HIV-1/enzimologia , Heptoses/imunologia , Neisseria gonorrhoeae/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/microbiologia , Linfócitos T CD4-Positivos/virologia , Feminino , Gonorreia/imunologia , Gonorreia/microbiologia , Gonorreia/virologia , Infecções por HIV/imunologia , Infecções por HIV/microbiologia , Infecções por HIV/virologia , Repetição Terminal Longa de HIV/genética , HIV-1/imunologia , Heptoses/genética , Heptoses/metabolismo , Humanos , Células Jurkat , Masculino , Neisseria gonorrhoeae/imunologia , Receptor 5 Toll-Like/imunologiaRESUMO
Transgenic models are invaluable tools for researching retinal degenerative disease mechanisms. However, they are time-consuming and expensive to generate and maintain. We have developed an alternative to transgenic rodent models of retinal degeneration using transgenic Xenopus laevis. We have optimized this system to allow rapid analysis of transgene effects in primary transgenic animals, thereby providing an alternative to establishing transgenic lines, and simultaneously allowing rigorous comparisons between the effects of different transgenes.
Assuntos
Animais Geneticamente Modificados/genética , Modelos Animais de Doenças , Degeneração Retiniana/genética , Xenopus laevis/genética , Animais , Humanos , TransgenesRESUMO
X-linked juvenile retinoschisis is a heritable condition of the retina in males caused by mutations in the RS1 gene. Still, the cellular function and retina-specific expression of RS1 are poorly understood. To address the latter issue, we characterized the minimal promoter driving expression of RS1 in the retina. Binding site prediction, site-directed mutagenesis, and reporter assays suggest an essential role of two nearby cone-rod homeobox (CRX)-responsive elements (CRE) in the proximal -177/+32 RS1 promoter. Chromatin immunoprecipitation associates the RS1 promoter in vivo with CRX, the coactivators CBP, P300, GCN5 and acetylated histone H3. Transgenic Xenopus laevis expressing a green fluorescent protein (GFP) reporter under the control of RS1 promoter sequences show that the -177/+32 fragment drives GFP expression in photoreceptors and bipolar cells. Mutating either of the two conserved CRX binding sites results in strongly decreased RS1 expression. Despite the presence of sequence motifs in the promoter, NRL and NR2E3 appear not to be essential for RS1 expression. Together, our in vitro and in vivo results indicate that two CRE sites in the minimal RS1 promoter region control retinal RS1 expression and establish CRX as a key factor driving this expression.