Phylogenomics reveals a diverse Rickettsiales type IV secretion system.

With an obligate intracellular lifestyle, Alphaproteobacteria of the order Rickettsiales have inextricably coevolved with their various eukaryotic hosts, resulting in small, reductive genomes and strict dependency on host resources. Unsurprisingly, large portions of Rickettsiales genomes encode proteins involved in transport and secretion. One particular transporter that has garnered recent attention from researchers is the type IV secretion system (T4SS). Homologous to the well-studied archetypal vir T4SS of Agrobacterium tumefaciens, the Rickettsiales vir homolog (rvh) T4SS is characterized primarily by duplication of several of its genes and scattered genomic distribution of all components in several conserved islets. Phylogeny estimation suggests a single event of ancestral acquirement of the rvh T4SS, likely from a nonalphaproteobacterial origin. Bioinformatics analysis of over 30 Rickettsiales genome sequences illustrates a conserved core rvh scaffold (lacking only a virB5 homolog), with lineage-specific diversification of several components (rvhB1, rvhB2, and rvhB9b), likely a result of modifications to cell envelope structure. This coevolution of the rvh T4SS and cell envelope morphology is probably driven by adaptations to various host cells, identifying the transporter as an important target for vaccine development. Despite the genetic intractability of Rickettsiales, recent advancements have been made in the characterization of several components of the rvh T4SS, as well as its putative regulators and substrates. While current data favor a role in effector translocation, functions in DNA uptake and release and/or conjugation cannot at present be ruled out, especially considering that a mechanism for plasmid transfer in Rickettsia spp. has yet to be proposed.

Endotoxin tolerance dysregulates MyD88- and Toll/IL-1R domain-containing adapter inducing IFN-beta-dependent pathways and increases expression of negative regulators of TLR signaling.

Endotoxin tolerance reprograms cell responses to LPS by repressing expression of proinflammatory cytokines, while not inhibiting production of anti-inflammatory cytokines and antimicrobial effectors. Molecular mechanisms of induction and maintenance of endotoxin tolerance are incompletely understood, particularly with regard to the impact of endotoxin tolerization on signalosome assembly, activation of adaptor-kinase modules, and expression of negative regulators of TLR signaling in human cells. In this study, we examined LPS-mediated activation of MyD88-dependent and Toll-IL-1R-containing adaptor inducing IFN-beta (TRIF)-dependent pathways emanating from TLR4 and expression of negative regulators of TLR signaling in control and endotoxin-tolerant human monocytes. Endotoxin tolerization suppressed LPS-inducible TLR4-TRIF and TRIF-TANK binding kinase (TBK)1 associations, induction of TBK1 kinase activity, activation of IFN regulatory factor (IRF)-3, and expression of RANTES and IFN-beta. Tolerance-mediated dysregulation of the TLR4-TRIF-TBK1 signaling module was accompanied by increased levels of suppressor of IkappaB kinase-epsilon (SIKE) and sterile alpha and Armadillo motif-containing molecule (SARM). LPS-tolerant cells showed increased expression of negative regulators Toll-interacting protein (Tollip), suppressor of cytokine signaling (SOCS)-1, IL-1R-associated kinase-M, and SHIP-1, which correlated with reduced p38 phosphorylation, IkappaB-alpha degradation, and inhibited expression of TNF-alpha, IL-6, and IL-8. To examine functional consequences of increased expression of Tollip in LPS-tolerized cells, we overexpressed Tollip in 293/TLR4/MD-2 transfectants and observed blunted LPS-inducible activation of NF-kappaB and RANTES, while TNF-alpha responses were not affected. These data demonstrate dysregulation of TLR4-triggered MyD88- and TRIF-dependent signaling pathways and increased expression of negative regulators of TLR signaling in endotoxin-tolerant human monocytes.