Caspase-mediated cleavage of the exosome subunit PM/Scl-75 during apoptosis

Recent studies have implicated the dying cell as a potential reservoir of modified autoantigens that might initiate and drive systemic autoimmunity in susceptible hosts. A number of subunits of the exosome, a complex of 3'→5' exoribonucleases that functions in a variety of cellular processes, are recognized by the so-called anti-PM/Scl autoantibodies, found predominantly in patients suffering from an overlap syndrome of myositis and scleroderma. Here we show that one of these subunits, PM/Scl-75, is cleaved during apoptosis. PM/Scl-75 cleavage is inhibited by several different caspase inhibitors. The analysis of PM/Scl-75 cleavage by recombinant caspase proteins shows that PM/Scl-75 is efficiently cleaved by caspase-1, to a smaller extent by caspase-8, and relatively inefficiently by caspase-3 and caspase-7. Cleavage of the PM/Scl-75 protein occurs in the C-terminal part of the protein at Asp369 (IILD³⁶⁹↓G), and at least a fraction of the resulting N-terminal fragments of PM/Scl-75 remains associated with the exosome. Finally, the implications of PM/Scl-75 cleavage for exosome function and the generation of anti-PM/Scl-75 autoantibodies are discussed.     

Human neutrophil clearance of bacterial pathogens triggers anti-microbial γδ T cell responses in early infection

Human blood Vγ9/Vδ2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vγ9/Vδ2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8). In turn, Vγ9/Vδ2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN)-γ and tumor necrosis factor (TNF)-α. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), requires cell-cell contact of Vγ9/Vδ2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1), and results in a TNF-α dependent proliferation of Vγ9/Vδ2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting γδ T cell responses in vitro. Patients with acute peritoneal-dialysis (PD)-associated bacterial peritonitis--characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity--show a selective activation of local Vγ9/Vδ2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The γδ T cell-driven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of γδ T cells and TNF-α and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive γδ T cells in early infection and suggest novel diagnostic and therapeutic approaches.     

Influenza vaccination for immunocompromised patients: systematic review and meta-analysis from a public health policy perspective

Background

Immunocompromised patients are vulnerable to severe or complicated influenza infection. Vaccination is widely recommended for this group. This systematic review and meta-analysis assesses influenza vaccination for immunocompromised patients in terms of preventing influenza-like illness and laboratory confirmed influenza, serological response and adverse events.

Methodology/Principal Findings

Electronic databases and grey literature were searched and records were screened against eligibility criteria. Data extraction and risk of bias assessments were performed in duplicate. Results were synthesised narratively and meta-analyses were conducted where feasible. Heterogeneity was assessed using I² and publication bias was assessed using Begg''s funnel plot and Egger''s regression test. Many of the 209 eligible studies included an unclear or high risk of bias. Meta-analyses showed a significant effect of preventing influenza-like illness (odds ratio [OR] = 0.23; 95% confidence interval [CI] = 0.16–0.34; p<0.001) and laboratory confirmed influenza infection (OR = 0.15; 95% CI = 0.03–0.63; p = 0.01) through vaccinating immunocompromised patie nts compared to placebo or unvaccinated controls. We found no difference in the odds of influenza-like illness compared to vaccinated immunocompetent controls. The pooled odds of seroconversion were lower in vaccinated patients compared to immunocompetent controls for seasonal influenza A(H1N1), A(H3N2) and B. A similar trend was identified for seroprotection. Meta-analyses of seroconversion showed higher odds in vaccinated patients compared to placebo or unvaccinated controls, although this reached significance for influenza B only. Publication bias was not detected and narrative synthesis supported our findings. No consistent evidence of safety concerns was identified.

Conclusions/Significance

Infection prevention and control strategies should recommend vaccinating immunocompromised patients. Potential for bias and confounding and the presence of heterogeneity mean the evidence reviewed is generally weak, although the directions of effects are consistent. Areas for further research are identified.     

Control of CydB and GltA1 expression by the SenX3 RegX3 two component regulatory system of Mycobacterium tuberculosis

Two component regulatory systems are used widely by bacteria to coordinate changes in global gene expression profiles in response to environmental signals. The SenX3-RegX3 two component system of Mycobacterium tuberculosis has previously been shown to play a role in virulence and phosphate-responsive control of gene expression. We demonstrate that expression of SenX3-RegX3 is controlled in response to growth conditions, although the absolute changes are small. Global gene expression profiling of a RegX3 deletion strain and wild-type strain in different culture conditions (static, microaerobic, anaerobic), as well as in an over-expressing strain identified a number of genes with changed expression patterns. Among those were genes previously identified as differentially regulated in aerobic culture, including ald (encoding alanine dehydrogenase) cyd,encoding a subunit of the cytochrome D ubiquinol oxidase, and gltA1, encoding a citrate synthase. Promoter activity in the upstream regions of both cydB and gltA1 was altered in the RegX3 deletion strain. DNA-binding assays confirmed that RegX3 binds to the promoter regions of ald, cydB and gltA1 in a phosphorylation-dependent manner. Taken together these data suggest a direct role for the SenX-RegX3 system in modulating expression of aerobic respiration, in addition to its role during phosphate limitation.     

Use of sulfated linked cyclitols as heparan sulfate mimetics to probe the heparin/heparan sulfate binding specificity of proteins

Heparin and heparan sulfate (HS) are structurally diverse glycosaminoglycans (GAG) that are known to interact, via unique structural motifs, with a wide range of functionally distinct proteins and modulate their biological activity. To define the GAG motifs that interact with proteins, we assessed the ability of 15 totally synthetic HS mimetics to interact with 10 functionally diverse proteins that bind heparin/HS. The HS mimetics consisted of cyclitol-based pseudo-sugars coupled by linkers of variable chain length, flexibility, orientation, and hydrophobicity, with variations in sulfation also being introduced into some molecules. Three of the proteins tested, namely hepatocyte growth factor, eotaxin, and elastase, failed to interact with any of the sulfated linked cyclitols. In contrast, each of the remaining seven proteins tested exhibited a unique reactivity pattern with the panel of HS mimetics, with tetrameric cyclitols linked by different length alkyl chains being particularly informative. Thus, compounds with short alkyl spacers (2-3 carbon atoms) effectively blocked the interaction of fibroblast growth factor-1 (FGF-1) and lipoprotein lipase with heparin/HS, whereas longer chain spacers (7-10 carbon atoms) were required for optimal inhibition of FGF-2 and vascular endothelial growth factor binding. This effect was most pronounced with the chemokine, interleukin-8, where alkyl-linked tetrameric cyclitols were essentially inactive unless a spacer of >7 carbon atoms was used. The heparin-inhibitable enzymes heparanase and cathepsin G also displayed characteristic inhibition patterns, cathepsin G interacting promiscuously with most of the sulfated cyclitols but heparanase activity being inhibited most effectively by HS mimetics that structurally resemble a sulfated pentasaccharide. These data indicate that a simple panel of HS mimetics can be used to probe the HS binding specificity of proteins, with the position of anionic groups in the HS mimetics being critical.     

Functional demonstration of reverse transsulfuration in the Mycobacterium tuberculosis complex reveals that methionine is the preferred sulfur source for pathogenic Mycobacteria

Methionine can be used as the sole sulfur source by the Mycobacterium tuberculosis complex although it is not obvious from examination of the genome annotation how these bacteria utilize methionine. Given that genome annotation is a largely predictive process, key challenges are to validate these predictions and to fill in gaps for known functions for which genes have not been annotated. We have addressed these issues by functional analysis of methionine metabolism. Transport, followed by metabolism of (35)S methionine into the cysteine adduct mycothiol, demonstrated the conversion of exogenous methionine to cysteine. Mutational analysis and cloning of the Rv1079 gene showed it to encode the key enzyme required for this conversion, cystathionine gamma-lyase (CGL). Rv1079, annotated metB, was predicted to encode cystathionine gamma-synthase (CGS), but demonstration of a gamma-elimination reaction with cystathionine as well as the gamma-replacement reaction yielding cystathionine showed it encodes a bifunctional CGL/CGS enzyme. Consistent with this, a Rv1079 mutant could not incorporate sulfur from methionine into cysteine, while a cysA mutant lacking sulfate transport and a methionine auxotroph was hypersensitive to the CGL inhibitor propargylglycine. Thus, reverse transsulfuration alone, without any sulfur recycling reactions, allows M. tuberculosis to use methionine as the sole sulfur source. Intracellular cysteine was undetectable so only the CGL reaction occurs in intact mycobacteria. Cysteine desulfhydrase, an activity we showed to be separable from CGL/CGS, may have a role in removing excess cysteine and could explain the ability of M. tuberculosis to recycle sulfur from cysteine, but not methionine.     

ChlR1 is required for loading papillomavirus E2 onto mitotic chromosomes and viral genome maintenance

Autonomously replicating DNA viruses must evade mitotic checkpoints and actively partition their genomes to maintain persistent infection. The E2 protein serves these functions by tethering papillomavirus episomes to mitotic chromosomes; however, the mechanism remains unresolved. We show that E2 binds ChlR1, a DNA helicase that plays a role in sister chromatid cohesion. The E2 mutation W130R fails to bind ChlR1 and correspondingly does not associate with mitotic chromosomes. Viral genomes encoding this E2 mutation are not episomally maintained in cell culture. Notably, E2 W130R binds Brd4, which reportedly acts as a mitotic tether, indicating this interaction is insufficient for E2 association with mitotic chromosomes. RNAi-induced depletion of ChlR1 significantly reduced E2 localization to mitotic chromosomes. These studies provide compelling evidence that ChlR1 association is required for loading the papillomavirus E2 protein onto mitotic chromosomes and represents a kinetochore-independent mechanism for viral genome maintenance and segregation.