Loss of SET1/COMPASS methyltransferase activity reduces lifespan and fertility in Caenorhabditis elegans.

Changes in histone post-translational modifications are associated with aging through poorly defined mechanisms. Histone 3 lysine 4 (H3K4) methylation at promoters is deposited by SET1 family methyltransferases acting within conserved multiprotein complexes known as COMPASS. Previous work yielded conflicting results about the requirement for H3K4 methylation during aging. Here, we reassessed the role of SET1/COMPASS-dependent H3K4 methylation in Caenorhabditis elegans lifespan and fertility by generating set-2(syb2085) mutant animals that express a catalytically inactive form of SET-2, the C. elegans SET1 homolog. We show that set-2(syb2085) animals retain the ability to form COMPASS, but have a marked global loss of H3K4 di- and trimethylation (H3K4me2/3). Reduced H3K4 methylation was accompanied by loss of fertility, as expected; however, in contrast to earlier studies, set-2(syb2085) mutants displayed a significantly shortened, not extended, lifespan and had normal intestinal fat stores. Other commonly used set-2 mutants were also short-lived, as was a cfp-1 mutant that lacks the SET1/COMPASS chromatin-targeting component. These results challenge previously held views and establish that WT H3K4me2/3 levels are essential for normal lifespan in C. elegans.

Caenorhabditis elegans semi-automated liquid screen reveals a specialized role for the chemotaxis gene cheB2 in Pseudomonas aeruginosa virulence.

Pseudomonas aeruginosa is an opportunistic human pathogen that causes infections in a variety of animal and plant hosts. Caenorhabditis elegans is a simple model with which one can identify bacterial virulence genes. Previous studies with C. elegans have shown that depending on the growth medium, P. aeruginosa provokes different pathologies: slow or fast killing, lethal paralysis and red death. In this study, we developed a high-throughput semi-automated liquid-based assay such that an entire genome can readily be scanned for virulence genes in a short time period. We screened a 2,200-member STM mutant library generated in a cystic fibrosis airway P. aeruginosa isolate, TBCF10839. Twelve mutants were isolated each showing at least 70% attenuation in C. elegans killing. The selected mutants had insertions in regulatory genes, such as a histidine kinase sensor of two-component systems and a member of the AraC family, or in genes involved in adherence or chemotaxis. One mutant had an insertion in a cheB gene homologue, encoding a methylesterase involved in chemotaxis (CheB2). The cheB2 mutant was tested in a murine lung infection model and found to have a highly attenuated virulence. The cheB2 gene is part of the chemotactic gene cluster II, which was shown to be required for an optimal mobility in vitro. In P. aeruginosa, the main player in chemotaxis and mobility is the chemotactic gene cluster I, including cheB1. We show that, in contrast to the cheB2 mutant, a cheB1 mutant is not attenuated for virulence in C. elegans whereas in vitro motility and chemotaxis are severely impaired. We conclude that the virulence defect of the cheB2 mutant is not linked with a global motility defect but that instead the cheB2 gene is involved in a specific chemotactic response, which takes place during infection and is required for P. aeruginosa pathogenicity.

Caenorhabditis elegans SET1/COMPASS Maintains Germline Identity by Preventing Transcriptional Deregulation Across Generations.

Chromatin regulators contribute to the maintenance of the germline transcriptional program. In the absence of SET-2, the Caenorhabditis elegans homolog of the SET1/COMPASS H3 Lys4 (H3K4) methyltransferase, animals show transgenerational loss of germline identity, leading to sterility. To identify transcriptional signatures associated with progressive loss of fertility, we performed expression profiling of set-2 mutant germlines across generations. We identify a subset of genes whose misexpression is first observed in early generations, a step we refer to as priming; their misexpression then further progresses in late generations, as animals reach sterility. Analysis of misregulated genes shows that down-regulation of germline genes, expression of somatic transcriptional programs, and desilencing of the X-chromosome are concurrent events leading to loss of germline identity in both early and late generations. Upregulation of transcription factor LIN-15B, the C/EBP homolog CEBP-1, and TGF-ß pathway components strongly contribute to loss of fertility, and RNAi inactivation of cebp-1 and TGF-ß/Smad signaling delays the onset of sterility, showing they individually contribute to maintenance of germ cell identity. Our approach therefore identifies genes and pathways whose misexpression actively contributes to the loss of germ cell fate. More generally, our data shows how loss of a chromatin regulator in one generation leads to transcriptional changes that are amplified over subsequent generations, ultimately leading to loss of appropriate cell fate.

Histone Methylation and Memory of Environmental Stress.

Cellular adaptation to environmental stress relies on a wide range of tightly controlled regulatory mechanisms, including transcription. Changes in chromatin structure and organization accompany the transcriptional response to stress, and in some cases, can impart memory of stress exposure to subsequent generations through mechanisms of epigenetic inheritance. In the budding yeast Saccharomyces cerevisiae, histone post-translational modifications, and in particular histone methylation, have been shown to confer transcriptional memory of exposure to environmental stress conditions through mitotic divisions. Recent evidence from Caenorhabditis elegans also implicates histone methylation in transgenerational inheritance of stress responses, suggesting a more widely conserved role in epigenetic memory.

Dissecting the machinery that introduces disulfide bonds in Pseudomonas aeruginosa.

UNLABELLED: Disulfide bond formation is required for the folding of many bacterial virulence factors. However, whereas the Escherichia coli disulfide bond-forming system is well characterized, not much is known on the pathways that oxidatively fold proteins in pathogenic bacteria. Here, we report the detailed unraveling of the pathway that introduces disulfide bonds in the periplasm of the human pathogen Pseudomonas aeruginosa. The genome of P. aeruginosa uniquely encodes two DsbA proteins (P. aeruginosa DsbA1 [PaDsbA1] and PaDsbA2) and two DsbB proteins (PaDsbB1 and PaDsbB2). We found that PaDsbA1, the primary donor of disulfide bonds to secreted proteins, is maintained oxidized in vivo by both PaDsbB1 and PaDsbB2. In vitro reconstitution of the pathway confirms that both PaDsbB1 and PaDsbB2 shuttle electrons from PaDsbA1 to membrane-bound quinones. Accordingly, deletion of both P. aeruginosa dsbB1 (PadsbB1) and PadsbB2 is required to prevent the folding of several P. aeruginosa virulence factors and to lead to a significant decrease in pathogenicity. Using a high-throughput proteomic approach, we also analyzed the impact of PadsbA1 deletion on the global periplasmic proteome of P. aeruginosa, which allowed us to identify more than 20 new potential substrates of this major oxidoreductase. Finally, we report the biochemical and structural characterization of PaDsbA2, a highly oxidizing oxidoreductase, which seems to be expressed under specific conditions. By fully dissecting the machinery that introduces disulfide bonds in P. aeruginosa, our work opens the way to the design of novel antibacterial molecules able to disarm this pathogen by preventing the proper assembly of its arsenal of virulence factors. IMPORTANCE: The human pathogen Pseudomonas aeruginosa causes life-threatening infections in immunodepressed and cystic fibrosis patients. The emergence of P. aeruginosa strains resistant to all of the available antibacterial agents calls for the urgent development of new antibiotics active against this bacterium. The pathogenic power of P. aeruginosa is mediated by an arsenal of extracellular virulence factors, most of which are stabilized by disulfide bonds. Thus, targeting the machinery that introduces disulfide bonds appears to be a promising strategy to combat P. aeruginosa. Here, we unraveled the oxidative protein folding system of P. aeruginosa in full detail. The system uniquely consists of two membrane proteins that generate disulfide bonds de novo to deliver them to P. aeruginosa DsbA1 (PaDsbA1), a soluble oxidoreductase. PaDsbA1 in turn donates disulfide bonds to secreted proteins, including virulence factors. Disruption of the disulfide bond formation machinery dramatically decreases P. aeruginosa virulence, confirming that disulfide formation systems are valid targets for the design of antimicrobial drugs.

Type II-dependent secretion of a Pseudomonas aeruginosa DING protein.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that uses a wide range of protein secretion systems to interact with its host. Genes encoding the PAO1 Hxc type II secretion system are linked to genes encoding phosphatases (LapA/LapB). Microarray genotyping suggested that Pseudomonas aeruginosa clinical isolates, including urinary tract (JJ692) and blood (X13273) isolates, lacked the lapA/lapB genes. Instead, we show that they carry a gene encoding a protein of the PstS family. This protein, which we call LapC, also has significant similarities with LapA/LapB. LapC belongs to the family of DING proteins and displays the canonical DINGGG motif within its N terminus. DING proteins are members of a prokaryotic phosphate binding protein superfamily. We show that LapC is secreted in an Hxc-dependent manner and is under the control of the PhoB response regulator. The genetic organization hxc-lapC found in JJ692 and X13273 is similar to PA14, which is the most frequent P. aeruginosa genotype. While the role of LapA, LapB and LapC proteins remains unclear in P. aeruginosa pathogenesis, they are likely to be part of a phosphate scavenging or sensing system needed to survive and thrive when low phosphate environments are encountered within the host.

SpiC is required for secretion of Salmonella Pathogenicity Island 2 type III secretion system proteins.

Replication of Salmonella typhimurium in host cells depends in part on the action of the Salmonella Pathogenicity Island 2 (SPI-2) type III secretion system (TTSS), which translocates bacterial effector proteins across the membrane of the Salmonella-containing vacuole (SCV). We have shown previously that one activity of the SPI-2 TTSS is the assembly of a coat of F-actin in the vicinity of bacterial microcolonies. To identify proteins involved in SPI-2 dependent actin polymerization, we tested strains carrying mutations in each of several genes whose products are proposed to be secreted through the SPI-2 TTSS, for their ability to assemble F-actin around intracellular bacteria. We found that strains carrying mutations in either sseB, sseC, sseD or spiC were deficient in actin assembly. The phenotypes of the sseB-, sseC- and sseD- mutants can be attributed to their requirement for translocation of SPI-2 effectors. SpiC was investigated further in view of its proposed role as an effector. Transient expression of a myc::SpiC fusion protein in Hela cells did not induce any significant alterations to the host cell cytoskeleton, and failed to restore actin polymerization around intracellular spiC- mutant bacteria. However, the same protein did complement the mutant phenotype when expressed from a plasmid within bacteria. Furthermore, spiC was found to be required for SPI-2 mediated secretion of SseB, SseC and SseD in vitro. An antibody against SpiC detected the protein on immunoblots from total cell lysates of S. typhimurium expressing SpiC from a plasmid, but it was not detected in secreted fractions after exposure of cells to conditions that result in secretion of other SPI-2 effector proteins. Investigation of the trafficking of SCVs containing a spiC- mutant in macrophages revealed only a low level of association with the lysosomal marker cathepsin D, similar to that of wild-type bacteria. Together, these results show that SpiC is involved in the process of SPI-2 secretion and indicate that phenotypes associated with a spiC- mutant are caused by the inability of this strain to translocate effector proteins, thus calling for further investigation into the function(s) of this protein.