Infection by the human pathogen Legionella pneumophila relies on the translocation of ~300 virulence proteins, termed effectors, which manipulate host-cell processes. However, almost no information exists regarding effectors in other Legionella pathogens. Here we sequenced, assembled and characterized the genomes of 38 Legionella species, and predicted their effector repertoire using a previously validated machine-learning approach. This analysis revealed a treasure trove of 5,885 predicted effectors. The effector repertoire of different Legionella species was found to be largely non-overlapping, and only seven core-effectors were shared among all species studied. Species-specific effectors had atypically low GC content, suggesting exogenous acquisition, possibly from their natural protozoan hosts. Furthermore, we detected numerous novel conserved effector domains, and discovered new domain combinations, which allowed inferring yet undescribed effector functions. The effector collection and network of domain architectures described here can serve as a roadmap for future studies of effector function and evolution.

Legionella and Coxiella are intracellular pathogens that use the virulence-related Icm/Dot type-IVB secretion system to translocate effector proteins into host cells during infection. These effectors were previously shown to contain a C-terminal secretion signal required for their translocation. In this research, we implemented a hidden semi-Markov model to characterize the amino acid composition of the signal, thus providing a comprehensive computational model for the secretion signal. This model accounts for dependencies among sites and captures spatial variation in amino acid composition along the secretion signal. To validate our model, we predicted and synthetically constructed an optimal secretion signal whose sequence is different from that of any known effector. We show that this signal efficiently translocates into host cells in an Icm/Dot-dependent manner. Additionally, we predicted in silico and experimentally examined the effects of mutations in the secretion signal, which provided innovative insights into its characteristics. Some effectors were found to lack a strong secretion signal according to our model. We demonstrated that these effectors were highly dependent on the IcmS-IcmW chaperons for their translocation, unlike effectors that harbor a strong secretion signal. Furthermore, our model is innovative because it enables searching ORFs for secretion signals on a genomic scale, which led to the identification and experimental validation of 20 effectors from Legionella pneumophila, Legionella longbeachae, and Coxiella burnetii. Our combined computational and experimental methodology is general and can be applied to the identification of a wide spectrum of protein features that lack sequence conservation but have similar amino acid characteristics.pathogenomics | type-IV secretion | translocated substrates | Legionnaire disease | Q-fever

c Coxiella burnetii, the causative agent of Q fever, is a human intracellular pathogen that utilizes the Icm/Dot type IVB secretion system to translocate effector proteins into host cells. To identify novel C. burnetii effectors, we applied a machine-learning approach to predict C. burnetii effectors, and examination of 20 such proteins resulted in the identification of 13 novel effectors. To determine whether these effectors, as well as several previously identified effectors, modulate conserved eukaryotic pathways, they were expressed in Saccharomyces cerevisiae. The effects on yeast growth were examined under regular growth conditions and in the presence of caffeine, a known modulator of the yeast cell wall integrity (CWI) mitogen-activated protein (MAP) kinase pathway. In the presence of caffeine, expression of the effectors CBU0885 and CBU1676 caused an enhanced inhibition of yeast growth, and the growth inhibition of CBU0388 was suppressed. Furthermore, analysis of synthetic lethality effects and examination of the activity of the CWI MAP kinase transcription factor Rlm1 indicated that CBU0388 enhances the activation of this MAP kinase pathway in yeast, while CBU0885 and CBU1676 abolish this activation. Additionally, coexpression of CBU1676 and CBU0388 resulted in mutual suppression of their inhibition of yeast growth. These results strongly indicate that these three effectors modulate the CWI MAP kinase pathway in yeast. Moreover, both CBU1676 and CBU0885 were found to contain a conserved haloacid dehalogenase (HAD) domain, which was found to be required for their activity. Collectively, our results demonstrate that MAP kinase pathways are most likely targeted by C. burnetii Icm/Dot effectors.

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.

bThe novel biochemical test, the Rapidec Carba NP (RCNP), was evaluated using carbapenemase-and non-carbapenemase-producing Enterobacteriaceae isolates. The RCNP test was compared with the Carba NP test (CNP) and the modified Hodge test. Compared to the CNP test, the RCNP test had identical sensitivity (96%) and lower specificity (93% versus 100%). The medium used to culture the isolates significantly affected test sensitivity and specificity. The RCNP test was quicker and easier to perform than the other tests. Increasing resistance to carbapenems, which are most often the last line of therapy, is now emerging at an alarming rate in Enterobacteriaceae (1). Detection of carbapenemase-producing Enterobacteriaceae (CPE) in the clinical laboratory is of major importance for implementing infection control measures in a timely manner (2) and possibly also for therapeutic considerations (3). The molecular-based techniques used to identify CPE are time consuming and require specialized laboratory equipment and skills, whereas many of the phenotypic methods (e.g., the modified Hodge test [MHT]) have low sensitivity and specificity (as reviewed in reference 4). Thus, there is a need for a more accurate, efficient, and easy-to-use method.The goals of this study were to assess a new, commercial assay, the Rapidec Carba NP test (RCNP) (bioMérieux, France) as a method for the detection of carbapenemases in Enterobacteriaceae in Israel. We compared the sensitivity and specificity of the RCNP test and the time and skill level of the clinical staff required to perform it with two tests: the Carba NP (CNP) (5, 6) and the MHT (7,8), the method that is most commonly used for detection of carbapenemase in Israel.We examined a collection of 98 strains that were isolated from surveillance and clinical cultures from Israeli patients from 2012 to 2014. The collection included diverse strains of the Enterobacteriaceae family; the species were identified using the Vitek 2 system (bioMérieux, France) ( Table 1). Antimicrobial susceptibility testing for ertapenem, imipenem, and meropenem was performed by agar dilution with all study strains, and results were interpreted using the Clinical and Laboratory Standards Institute guidelines (Table 1) (9). Of the 98 strains examined, 69 were carbapenemaseproducing Enterobacteriaceae (CPE) that tested positive by PCR for one of the following genes: bla KPC (10), bla NDM (11), bla , bla VIM (13), or bla IMI (14). The results of the other tests were compared to the PCR results whenever a carbapenemase gene was detected. The remaining 29 strains were non-carbapenemaseproducing, carbapenem-resistant Enterobacteriaceae (NP-CRE), defined by a negative PCR (for the genes above and for the bla IMP [15] gene) and a negative CNP test (routinely used with the CHROMagar KPC media [CHROMagar, Paris, France] in our lab, as described below).The RCNP test was conducted according to the manufacturer's instructions, and change in color was visualized after 30 min and 2 h of incubation. CNP was performed with modific...

CG 258 clade 2 has retained its predominance despite increased diversification. Although interchanging of CG 258 strains occurred between most PACHs, local spread is the leading cause of its dissemination.

Highlights The Legionella genus contains nine core effectors. Three Legionella pneumophila core effectors are required for intracellular growth. The Legionella genus core effectors display functional conservation among orthologs. One Legionella core effector requires an accessory protein to perform its function.

The goal of this study was to compare the molecular features of bovine- and human community-acquired extended-spectrum β-lactamase (ESBL)-producing Escherichia coli in Israel. Bovine ESBL-producing E. coli were isolated during a point-prevalence study from the main farming locations throughout Israel. Human ESBL-producing E. coli isolates were collected from community-acquired urinary tract infection cases. Molecular typing was done initially by repetitive extragenic palindromic-PCR. Representative isolates were subjected to next-generation sequencing (NGS) and analyzed for multilocus sequence typing (MLST), core genome MLST (cgMLST), bla gene allele, and mobile genetic elements (MGEs) surrounding it. Out of the 287 bovine- and 104 community-derived ESBL-producing E. coli isolates, 44 and 26 isolates were subjected to NGS, respectively. Both populations exhibited a diverse but distinct clonal structure with predominance of several sequence types (STs); two clones, ST-10/167 (n = 13) and ST-38 (n = 8), were present. cgMLST analysis of these clones revealed that the majority of isolates exhibited phylogenetic distance (PD) of >178 gene difference from their closest isolate, with the exception of five isolates that exhibited PD of <24 gene difference, including two bovine- to three community-derived isolates. Hence, clonal transmission of ESBL-producing E. coli between cattle and the community, although uncommon, is likely to have occurred. The bla gene was identified in 52/70 (74%) isolates from both cattle and the community and was surrounded by MGEs that were composed mostly of either the Tn3 or IS1380 families. Thus, MGEs are likely to play an important role in the exchange of resistance genes.