SummaryLegionella pneumophila utilize the Icm/Dot type-IV secretion system to translocate effector proteins into host cells. Some of these effectors were shown before to be regulated at the transcriptional level by the PmrAB and CpxRA two-component systems. In addition, the stationary phase-related regulators LetA and CsrA, which are both members of the same posttranscriptional regulatory cascade, were shown to be involved in L. pneumophila virulence. In this report, we identified two small non-coding RNAs which are part of the LetA-CsrA regulatory cascade and three effector-encoding genes which are directly controlled by this regulatory system. We found that the small non-coding RNAs RsmY and RsmZ, were upregulated by LetA at stationary phase, and relieve the repression of CsrA from its target genes. The three effector-encoding genes were found to be posttranscriptionally upregulated at stationary phase and to contain CsrA regulatory elements that were found to be essential for their stationary phase activation. In addition, rsmY and rsmZ were found to be regulated by the RpoS sigma-factor and the csrA encoding gene was found to be regulated by PmrA. Our results demonstrate that L. pneumophila effectors are regulated at both the transcriptional and the post-transcriptional levels by a complicated regulatory network.

Legionella pneumophila, the causative agent of Legionnaires' disease, is an intracellular human pathogen that utilizes the Icm/ Dot type IVB secretion system to translocate a large repertoire of effectors into host cells. To find coregulated effectors, we performed a bioinformatic genomic screen with the aim of identifying effector-encoding genes containing putative CsrA regulatory elements. The regulation of these genes by the LetAS-RsmYZ-CsrA regulatory cascade was experimentally validated by examining their levels of expression in deletion mutants of relevant regulators and by site-directed mutagenesis of the putative CsrA sites. These analyses resulted in the identification of 26 effector-encoding genes regulated by the LetAS-RsmYZ-CsrA regulatory cascade, all of which were expressed at higher levels during the stationary phase. To determine if any of these effectors is involved in modulating the secretory pathway, they were overexpressed in wild-type yeast as well as in a yeast sec22 deletion mutant, which encodes an R-SNARE that participates in the endoplasmic reticulum (ER)-Golgi trafficking. This examination identified many novel LetAS-RsmYZ-CsrA regulated effectors which are involved in this process. To further characterize the role of these 26 effectors in vesicular trafficking, they were examined in yeast arf and arl deletion mutants, which encode small GTPases that regulate ER-Golgi trafficking. This analysis revealed that the effectors examined manipulate different processes of the secretory pathway. Collectively, our results demonstrate that several of the L. pneumophila effectors which are coregulated in the bacterial cell are involved in the modulation of the same eukaryotic pathway.

Members of the protein phosphatase 2C (PP2C) superfamily are Mg(2+)/Mn(2+)-dependent serine/threonine phosphatases, which are essential for regulation of cell cycle and stress signaling pathways in cells. In this study, a comprehensive genomic analysis of all available metazoan PP2C sequences was conducted. The phylogeny of PP2C was reconstructed, revealing the existence of 15 vertebrate families which arose following a series of gene duplication events. Relative dating of these duplications showed that they occurred in two active periods: before the divergence of bilaterians and before vertebrate diversification. PP2C families which duplicated during the first period take part in different signaling pathways, whereas PP2C families which diverged in the second period display tissue expression differences yet participate in similar signaling pathways. These differences were found to involve variation of expression in tissues which show higher complexity in vertebrates, such as skeletal muscle and the nervous system. Further analysis was performed with the aim of identifying the functional domains of PP2C. The conservation pattern across the entire PP2C superfamily revealed an extensive domain of more than 50 amino acids which is highly conserved throughout all PP2C members. Several insertion or deletion events were found which may have led to the specialization of each PP2C family.

Middle East respiratory syndrome coronavirus, MERS-CoV, was identified in Saudi Arabia in 2012, and as of January 29, 2018, there were 2,123 laboratory-confirmed MERS-CoV cases reported to WHO (WHO, 2018, https://www.who.int/emergencies/mers-cov/en/). Multiple studies suggest that dromedary camels are a source for human MERS-CoV infection. MERS-CoV-specific antibodies have been detected in the serum of dromedary camels across Northern Africa and across the Arabian Peninsula. Israel's geographic location places Israel at risk for MERS-CoV infection. To date, MERS-CoV-related illness has not been reported and the burden of MERS-CoV infection in the Israeli population is unknown. The seroprevalence of MERS-CoV-specific antibodies in Israeli dromedary camels is unknown. The objective of this study was to determine the prevalence of MERS-CoV seropositivity in dromedary camels in Israel. The prevalence of MERS-CoV antibodies in Israeli camels was examined in 71 camel sera collected from four farms across Israel by MERS-CoV-specific microneutralization (Mnt) assay and confirmed by MERS-CoV-specific immunofluorescence assay (IFA). Although this study cannot rule out potential antibody cross-reactivity by IFA, the presence of bovine coronavirus-specific antibodies do not appear to impact detection of MERS-CoV antibodies by Mnt. MERS-CoV neutralizing antibodies were detectable in 51 (71.8%) camel sera, and no association was observed between the presence of neutralizing antibodies and camel age or gender. These findings extend the known range of MERS-CoV circulation in Middle Eastern camels. The high rate of MERS-CoV-specific antibody seropositivity in dromedary camels in the absence of any reported human MERS cases suggests that there is still much to be learned about the dynamics of camel-to-human transmission of MERS-CoV.

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