Emergence of Mycobacterium canariasense infections in central Israel.

In 2018, Mycobacterium canariasense bloodstream infection was diagnosed in Israel. Further investigation had identified additional five cases in three medical centers, including isolates from blood (1), cornea (1), and sputum (3). Isolates were susceptible to all the antimicrobial tested. All but one isolate was related by whole-genome phylogeny.

Mycobacterium gordonae infecting redclaw crayfish Cherax quadricarinatus.

The redclaw crayfish Cherax quadricarinatus (von Martens, 1868) is one of the most commonly exploited freshwater crayfish species worldwide. Redclaw crayfish are susceptible to a number of pathogens but none have been linked to widespread epizootics. Mycobacterial infections have been sporadically reported in crayfish. In the case described, histopathology and bacterial identification confirmed an opportunistic infection caused by Mycobacterium gordonae in a hatchery of C. quadricarinatus in Israel. Intranuclear inclusion bodies, recorded in cells of the tubular epithelium of the hepatopancreas by histopathology, indicate a co-infection with a viral agent, referable to C. quadricarinatus bacilliform virus (CqBV). To the best of our knowledge this is the first description of mycobacteriosis in redclaw crayfish.

Origins of context-dependent gene repression by capicua.

Receptor Tyrosine Kinase (RTK) signaling pathways induce multiple biological responses, often by regulating the expression of downstream genes. The HMG-box protein Capicua (Cic) is a transcriptional repressor that is downregulated in response to RTK signaling, thereby enabling RTK-dependent induction of Cic targets. In both Drosophila and mammals, Cic is expressed as two isoforms, long (Cic-L) and short (Cic-S), whose functional significance and mechanism of action are not well understood. Here we show that Drosophila Cic relies on the Groucho (Gro) corepressor during its function in the early embryo, but not during other stages of development. This Gro-dependent mechanism requires a short peptide motif, unique to Cic-S and designated N2, which is distinct from other previously defined Gro-interacting motifs and functions as an autonomous, transferable repressor element. Unexpectedly, our data indicate that the N2 motif is an evolutionary innovation that originated within dipteran insects, as the Cic-S isoform evolved from an ancestral Cic-L-type form. Accordingly, the Cic-L isoform lacking the N2 motif is completely inactive in early Drosophila embryos, indicating that the N2 motif endowed Cic-S with a novel Gro-dependent activity that is obligatory at this stage. We suggest that Cic-S and Gro coregulatory functions have facilitated the evolution of the complex transcriptional network regulated by Torso RTK signaling in modern fly embryos. Notably, our results also imply that mammalian Cic proteins are unlikely to act via Gro and that their Cic-S isoform must have evolved independently of fly Cic-S. Thus, Cic proteins employ distinct repressor mechanisms that are associated with discrete structural changes in the evolutionary history of this protein family.

Phosphorylation of the Drosophila melanogaster RNA-binding protein HOW by MAPK/ERK enhances its dimerization and activity.

Drosophila melanogaster Held Out Wings (HOW) is a conserved RNA-binding protein (RBP) belonging to the STAR family, whose closest mammalian ortholog Quaking (QKI) has been implicated in embryonic development and nervous system myelination. The HOW RBP modulates a variety of developmental processes by controlling mRNA levels and the splicing profile of multiple key regulatory genes; however, mechanisms regulating its activity in tissues have yet to be elucidated. Here, we link receptor tyrosine kinase (RTK) signaling to the regulation of QKI subfamily of STAR proteins, by showing that HOW undergoes phosphorylation by MAPK/ERK. Importantly, we show that this modification facilitates HOW dimerization and potentiates its ability to bind RNA and regulate its levels. Employing an antibody that specifically recognizes phosphorylated HOW, we show that HOW is phosphorylated in embryonic muscles and heart cardioblasts in vivo, thus documenting for the first time Serine/Threonine (Ser/Thr) phosphorylation of a STAR protein in the context of an intact organism. We also identify the sallimus/D-titin (sls) gene as a novel muscle target of HOW-mediated negative regulation and further show that this regulation is phosphorylation-dependent, underscoring the physiological relevance of this modification. Importantly, we demonstrate that HOW Thr phosphorylation is reduced following muscle-specific knock down of Drosophila MAPK rolled and that, correspondingly, Sls is elevated in these muscles, similarly to the HOW RNAi effect. Taken together, our results provide a coherent mechanism of differential HOW activation; MAPK/ERK-dependent phosphorylation of HOW promotes the formation of HOW dimers and thus enhances its activity in controlling mRNA levels of key muscle-specific genes. Hence, our findings bridge between MAPK/ERK signaling and RNA regulation in developing muscles.

Challenging diagnosis of Mycolicibacterium cosmeticum/canariasense infection: A case report and literature review.

We present the case of an immunocompromised child with Mycolicibacterium cosmeticum/ canariasense infection. Our case highlights the difficulty in adequate speciation. Most isolates described in the literature were identified using 16 s-rRNA PCR, which if performed on our sample would at best be inconclusive. Misidentifications could have a real impact on the body of evidence collected on these isolates thus far.

Mycobacterium intracellulare subsp. chimaera from Cardio Surgery Heating-Cooling Units and from Clinical Samples in Israel Are Genetically Unrelated.

Non-tuberculous mycobacteria (NTM) are opportunistic pathogens that cause illness primarily in the elderly, in the immunocompromised or in patients with underlying lung disease. Since 2013, a global outbreak of NTM infection related to heater-cooler units (HCU) used in cardio-thoracic surgery has been identified. This outbreak was caused by a single strain of Mycobacterium intracellulare subsp. chimaera. In order to estimate the prevalence of this outbreak strain in Israel, we sampled Mycobacterium intracellulare subsp. chimaera from several HCU machines in Israel, as well as from patients, sequenced their genomes and compared them to the outbreak strain. The presence of mixed mycobacteria species in the samples complicated the analysis of obtained sequences. By applying a metagenomic binning strategy, we were able to obtain, and characterize, genomes of single strains from the mixed samples. Mycobacterium intracellulare subsp. chimaera strains were compared to each other and to previously reported genomes from other countries. The strain causing the outbreak related to the HCU machines was identified in several such machines in Israel but not in any clinical sample.

High-Throughput In Vitro Identification of Direct MAPK/Erk Substrates.

Phosphorylation mediated by cellular protein kinases is an effective mechanism employed by an organism to regulate central processes such as cell-cycle progression, metabolic pathways, cytoskeletal function, cell migration and differentiation. Thus, for example, various signaling pathways utilize sequential phosphorylation events to relay external cues from the cell surface to the nucleus, where eventually gene expression profiles are altered and, consequently, changes in cell fates and function are induced. Accordingly, recognizing the direct targets of key effector kinases is of utmost importance for understanding the cellular responses to pathway activity. Here we describe a high-throughput genome-wide proteomics approach aimed at uncovering novel nuclear targets for the single Drosophila MAPK/Erk. Briefly, pools of cDNA are transcribed and translated in vitro in the presence of [35S]Methionine, generating a library of radiolabeled protein pools which are subsequently subjected to biochemical kinase assays using recombinant, active Erk2. Phosphorylated proteins representing potential MAPK/Erk substrates are then detected due to their shifted mobility on SDS-PAGE gels. This protocol can be easily adjusted and applied toward identifying targets of other kinases for which in vitro phosphorylation assays are available.

MAPK substrate competition integrates patterning signals in the Drosophila embryo.

Terminal regions of the Drosophila embryo are patterned by the localized activation of the mitogen-activated protein kinase (MAPK) pathway. This depends on the MAPK-mediated downregulation of Capicua (Cic), a repressor of the terminal gap genes. We establish that downregulation of Cic is antagonized by the anterior patterning morphogen Bicoid (Bcd). We demonstrate that this effect does not depend on transcriptional activity of Bcd and provide evidence suggesting that Bcd, a direct substrate of MAPK, decreases the availability of MAPK for its other substrates, such as Cic. Based on the quantitative analysis of MAPK signaling in multiple mutants, we propose that MAPK substrate competition coordinates the actions of the anterior and terminal patterning systems. In addition, we identify Hunchback as a novel target of MAPK phosphorylation that can account for the previously described genetic interaction between the posterior and terminal systems. Thus, a common enzyme-substrate competition mechanism can integrate the actions of the anterior, posterior, and terminal patterning signals. Substrate competition can be a general signal integration strategy in networks where enzymes, such as MAPK, interact with their multiple regulators and targets.

A MAPK docking site is critical for downregulation of Capicua by Torso and EGFR RTK signaling.

Early Drosophila development requires two receptor tyrosine kinase (RTK) pathways: the Torso and the Epidermal growth factor receptor (EGFR) pathways, which regulate terminal and dorsal-ventral patterning, respectively. Previous studies have shown that these pathways, either directly or indirectly, lead to post-transcriptional downregulation of the Capicua repressor in the early embryo and in the ovary. Here, we show that both regulatory effects are direct and depend on a MAPK docking site in Capicua that physically interacts with the MAPK Rolled. Capicua derivatives lacking this docking site cause dominant phenotypes similar to those resulting from loss of Torso and EGFR activities. Such phenotypes arise from inappropriate repression of genes normally expressed in response to Torso and EGFR signaling. Our results are consistent with a model whereby Capicua is the main nuclear effector of the Torso pathway, but only one of different effectors responding to EGFR signaling. Finally, we describe differences in the modes of Capicua downregulation by Torso and EGFR signaling, raising the possibility that such differences contribute to the tissue specificity of both signals.