Recruited macrophages control dissemination of group A Streptococcus from infected soft tissues.

Group A Streptococcus (GAS) causes diverse infections in humans, ranging from mild to life-threatening invasive diseases, such as necrotizing fasciitis (NF), a rapidly progressing deep tissue infection. Despite prompt treatments, NF remains a significant cause of morbidity and mortality, even in previously healthy individuals. The early recruitment of leukocytes is crucial to the outcome of NF; however, although the role of polymorphonuclear neutrophils (PMNs) in host defense against NF is well established, the role of recruited macrophages remains poorly defined. Using a cutaneous murine model mimicking human NF, we found that mice deficient in TNF-α were highly susceptible to s.c. infections with GAS, and a paucity of macrophages, but not PMNs, was demonstrated. To test whether the effects of TNF-α on the outcome of infection are mediated by macrophages/monocytes, we systemically depleted C57BL/6 mice of monocytes by pharmacological and genetic approaches. Systemic monocyte depletion substantially increased bacterial dissemination from soft tissues without affecting the number of recruited PMNs or altering the bacterial loads in soft tissues. Enhanced GAS dissemination could be reverted by either i.v. injection of monocytes or s.c. administration of peritoneal macrophages. These experiments demonstrated that recruited macrophages play a key role in defense against the extracellular pathogen GAS by limiting its spread from soft tissues.

Antisense oligonucleotide splicing modulation as a novel Cystic Fibrosis therapeutic approach for the W1282X nonsense mutation.

BACKGROUND: Antisense oligonucleotide- based drugs for splicing modulation were recently approved for various genetic diseases with unmet need. Here we aimed to generate skipping over exon 23 of the CFTR transcript, to eliminate the W1282X nonsense mutation and avoid RNA degradation induced by the nonsense mediated mRNA decay mechanism, allowing production of partially active CFTR proteins lacking exon 23. METHODS: ∼80 ASOs were screened in 16HBEge W1282X cells. ASO candidates showing significant exon skipping were assessed for their W1282X allele selectivity and the increase of CFTR protein maturation and function. The effect of a highly potent ASO candidates was further analyzed in well differentiated primary human nasal epithelial cells, derived from a W1282X homozygous patient. RESULTS: ASO screening led to identification of several ASOs that significantly decrease the level of CFTR transcripts including exon 23. These ASOs resulted in significant levels of mature CFTR protein and together with modulators restore the channel function following free uptake into these cells. Importantly, a highly potent lead ASOs, efficiently delivered by free uptake, was able to increase the level of transcripts lacking exon 23 and restore the CFTR function in cells from a W1282X homozygote patient. CONCLUSION: The highly efficient exon 23 skipping induced by free uptake of the lead ASO and the resulting levels of mature CFTR protein exhibiting channel function in the presence of modulators, demonstrate the ASO therapeutic potential benefit for CF patients carrying the W1282X mutation with the objective to advance the lead candidate SPL23-2 to proof-of-concept clinical study.

A CRISPR platform for targeted in vivo screens identifies Toxoplasma gondii virulence factors in mice.

Genome-wide CRISPR screening is a powerful tool to identify genes required under selective conditions. However, the inherent scale of genome-wide libraries can limit their application in experimental settings where cell numbers are restricted, such as in vivo infections or single cell analysis. The use of small scale CRISPR libraries targeting gene subsets circumvents this problem. Here we develop a method for rapid generation of custom guide RNA (gRNA) libraries using arrayed single-stranded oligonucleotides for reproducible pooled cloning of CRISPR/Cas9 libraries. We use this system to generate mutant pools of different sizes in the protozoan parasite Toxoplasma gondi and describe optimised analysis methods for small scale libraries. An in vivo genetic screen in the murine host identifies novel and known virulence factors and we confirm results using cloned knock-out parasites. Our study also reveals a potential trans-rescue of individual knock-out parasites in pools of mutants compared to homogenous knock-out lines of the key virulence factor MYR1.

Intrinsically active MEK variants are differentially regulated by proteinases and phosphatases.

MAPK/ERK kinase (MEK) 1/2 are central signaling proteins that serve as specificity determinants of the MAPK/ERK cascade. More than twenty activating mutations have been reported for MEK1/2, and many of them are known to cause diseases such as cancers, arteriovenous malformation and RASopathies. Changes in their intrinsic activity do not seem to correlate with the severity of the diseases. Here we studied four MEK1/2 mutations using biochemical and molecular dynamic methods. Although the studied mutants elevated the activating phosphorylation of MEK they had no effect on the stimulated ERK1/2 phosphorylation. Studying the regulatory mechanism that may explain this lack of effect, we found that one type of mutation affects MEK stability and two types of mutations demonstrate a reduced sensitivity to PP2A. Together, our results indicate that some MEK mutations exert their function not only by their elevated intrinsic activity, but also by modulation of regulatory elements such as protein stability or dephosphorylation.

Direct binding of MEK1 and MEK2 to AKT induces Foxo1 phosphorylation, cellular migration and metastasis.

Crosstalk between the ERK cascade and other signaling pathways is one of the means by which it acquires its signaling specificity. Here we identified a direct interaction of both MEK1 and MEK2 with AKT. The interaction is mediated by the proline rich domain of MEK1/2 and regulated by phosphorylation of Ser298 in MEK1, or Ser306 in MEK2, which we identified here as a novel regulatory site. We further developed a blocking peptide, which inhibits the interaction between MEK and AKT, and when applied to cells, affects migration and adhesion, but not proliferation. The specific mechanism of action of the MEK-AKT complex involves phosphorylation of the migration-related transcription factor FoxO1. Importantly, prevention of the interaction results in a decreased metastasis formation in a breast cancer mouse model. Thus, the identified interaction both sheds light on how signaling specificity is determined, and represents a possible new therapeutic target for metastatic cancer.