The Legionella pneumophila effector DenR hijacks the host NRas proto-oncoprotein to downregulate MAPK signaling.

Small GTPases of the Ras subfamily are best known for their role as proto-oncoproteins, while their function during microbial infection has remained elusive. Here, we show that Legionella pneumophila hijacks the small GTPase NRas to the Legionella-containing vacuole (LCV) surface. A CRISPR interference screen identifies a single L. pneumophila effector, DenR (Lpg1909), required for this process. Recruitment is specific for NRas, while its homologs KRas and HRas are excluded from LCVs. The C-terminal hypervariable tail of NRas is sufficient for recruitment, and interference with either NRas farnesylation or S-acylation sites abrogates recruitment. Intriguingly, we detect markers of active NRas signaling on the LCV, suggesting it acts as a signaling platform. Subsequent phosphoproteomics analyses show that DenR rewires the host NRas signaling landscape, including dampening of the canonical mitogen-activated protein kinase pathway. These results provide evidence for L. pneumophila targeting NRas and suggest a link between NRas GTPase signaling and microbial infection.

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes.

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a multiplex, randomized CRISPR interference sequencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii. Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes.

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a multiplex, randomized CRISPR interference sequencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44 Legionella pneumophila virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with L. pneumophila bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. lpg2888 and lpg3000 were particularly fascinating for their apparent redundant functions during L. pneumophila human macrophage infection, while lpg3000 alone was essential for L. pneumophila virulence in the amoeban host Acanthamoeba castellanii. Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.

A multiplex CRISPR interference tool for virulence gene interrogation in Legionella pneumophila.

Catalytically inactive dCas9 imposes transcriptional gene repression by sterically precluding RNA polymerase activity at a given gene to which it was directed by CRISPR (cr)RNAs. This gene silencing technology, known as CRISPR interference (CRISPRi), has been employed in various bacterial species to interrogate genes, mostly individually or in pairs. Here, we developed a multiplex CRISPRi platform in the pathogen Legionella pneumophila capable of silencing up to ten genes simultaneously. Constraints on precursor-crRNA expression were overcome by combining a strong promoter with a boxA element upstream of a CRISPR array. Using crRNAs directed against virulence protein-encoding genes, we demonstrated that CRISPRi is fully functional not only during growth in axenic media, but also during macrophage infection, and that gene depletion by CRISPRi recapitulated the growth defect of deletion strains. By altering the position of crRNA-encoding spacers within the CRISPR array, our platform achieved the gradual depletion of targets that was mirrored by the severity in phenotypes. Multiplex CRISPRi thus holds great promise for probing large sets of genes in bulk in order to decipher virulence strategies of L. pneumophila and other bacterial pathogens.

Effects of Exercise With and Without Energy Replacement on Substrate Utilization in the Fasting State.

Objective: The present study aimed to examine the interactive effect of exercise and energy balance on energy expenditure and substrate utilization.Method: Seven men and 7 women underwent three 2-day experimental protocols in a random order. Each protocol consisted of no exercise (NE), exercise only (EO), or exercise with a matched energy replacement (ER) on day 1 followed by metabolic testing that occurred after a 12-hour overnight fasting on day 2. Both EO and ER involved treadmill running at 60% maximal oxygen uptake (VO2max) that induced an energy expenditure of ∼ 500 kcal. The replacement meal used in ER contained ∼ 500 kcal made up of 45% carbohydrate, 30% fat, and 25% protein. During metabolic testing, oxygen uptake (VO2), heart rate (HR), respiratory exchange ratio (RER), and rates of carbohydrate (COX) and fat oxidation (FOX) were determined in three successive 15-minute periods including rest and exercise at 50% and 70% VO2max.Results: No differences in VO2 and HR were found at rest among NE, EO, and ER. However, RER was lower in EO than NE (0.840 ± 0.014 vs 0.889 ± 0.012, p < 0.05), COX (g·min-1) was lower in ER than NE (0.144 ± 0.016 vs 0.197 ± 0.019, p < 0.05), and FOX (g·min-1) was higher in EO or ER than NE (0.054 ± 0.010 or 0.057 ± 0.009 vs 0.034 ± 0.007, p < 0.05). No treatment effects were observed for all variables at either intensity.Conclusions: This study demonstrates that an exercise of moderate intensity can increase resting fat oxidation even when the exercise-induced energy expenditure is balanced by energy intake. This finding suggests that muscle action is vital in augmenting fat utilization.