The Legionella genus core effectors display functional conservation among orthologs by themselves or combined with an accessory protein

Wexler, Michael; Zusman, Tal; Linsky, Marika; Lifshitz, Ziv; Segal, G. M.

doi:10.1016/j.crmicr.2022.100105

Cited by 11 publications

(17 citation statements)

References 61 publications

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“…These 44 effectors are likely incorporated either into the membrane of the LCV or that of surrounding organelles to assist in nutrient acquisition, detoxification, and membrane fusion (Figure S1, Table S2). Despite high conservation of these transmembrane effector-encoding (TME) genes across all Legionella species 27, 37 , only lpg2815 , which encodes the metal ion transporter MavN 38, 39 , has been shown to be virulence critical on its own. Hence, as proof-of-concept, we decided to probe this large group of genes with the randomized CRISPRi approach for synthetic lethality.…”

Section: Resultsmentioning

confidence: 99%

“…The copyright holder for this preprint this version posted February 3, 2023. ; https://doi.org/10.1101/2023.02.03.527066 doi: bioRxiv preprint (TME) genes across all Legionella species 27,37 , only lpg2815, which encodes the metal ion transporter MavN 38,39 , has been shown to be virulence critical on its own. Hence, as proof-ofconcept, we decided to probe this large group of genes with the randomized CRISPRi approach for synthetic lethality.…”

Section: Pneumophilamentioning

confidence: 99%

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

Ellis

Myers

Tung

et al. 2023

Preprint

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Pneumophilamentioning

confidence: 99%

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

Ellis

Myers

Tung

et al. 2023

Preprint

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“…Most species bear highly unique effector repertoires, and only nine core effectors with universal conservation are present in Legionella spp. (Gomez-Valero et al, 2019b;Wexler et al, 2022). Effectors often bear eukaryotic domains, likely acquired horizontally from hosts, that are used to interact with or act on their cognate host protein targets.…”

Section: The Dot/icm T4bss Effector Repertoirementioning

confidence: 99%

Molecular regulation of virulence in Legionella pneumophila

Graham,

MacMartin,

de Kievit

et al. 2023

Molecular Microbiology

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Legionella pneumophila is a gram‐negative bacteria found in natural and anthropogenic aquatic environments such as evaporative cooling towers, where it reproduces as an intracellular parasite of cohabiting protozoa. If L. pneumophila is aerosolized and inhaled by a susceptible person, bacteria may colonize their alveolar macrophages causing the opportunistic pneumonia Legionnaires' disease. L. pneumophila utilizes an elaborate regulatory network to control virulence processes such as the Dot/Icm Type IV secretion system and effector repertoire, responding to changing nutritional cues as their host becomes depleted. The bacteria subsequently differentiate to a transmissive state that can survive in the environment until a replacement host is encountered and colonized. In this review, we discuss the lifecycle of L. pneumophila and the molecular regulatory network that senses nutritional depletion via the stringent response, a link to stationary phase‐like metabolic changes via alternative sigma factors, and two‐component systems that are homologous to stress sensors in other pathogens, to regulate differentiation between the intracellular replicative phase and more transmissible states. Together, we highlight how this prototypic intracellular pathogen offers enormous potential in understanding how molecular mechanisms enable intracellular parasitism and pathogenicity.

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“…Only nine effectors [Lpg0086 (LceA), Lpg0103 (VipF), Lpg0107 (RavC), Lpg0140 (CetLp1), Lpg1356 (LceB), Lpg2300 (LegA3/AnkH/AnkW), Lpg2815 (IroT/MavN), Lpg2832 and Lpg3000] are conserved in all analyzed Legionella species [3, 39, 91]. Interestingly, LegA3/AnkH has a companion protein LegA3C, which is not translocated, and only cognate LegA3–LegA3C orthologous pairs from different Legionella species function optimally together [91]. Overall, this low degree of conservation of the effector arsenals suggests that different networks of effectors, which cooperatively ensure intracellular survival, LCV formation and replication, exist.…”

Section: Dot/icm T4bss Effectorsmentioning

confidence: 99%

“…About 65 % of effectors are conserved between L. pneumophila isolates, revealing substantial plasticity, due to deletion, replacement or acquisition of effectors, even between the most frequently used laboratory strains L. pneumophila Philadelphia, Paris, Lens, Corby, 130b and Alcoy [3,90]. Only nine effectors [Lpg0086 (LceA), Lpg0103 (VipF), Lpg0107 (RavC), Lpg0140 (CetLp1), Lpg1356 (LceB), Lpg2300 (LegA3/AnkH/ AnkW), Lpg2815 (IroT/MavN), Lpg2832 and Lpg3000] are conserved in all analyzed Legionella species [3,39,91]. Interestingly, LegA3/AnkH has a companion protein LegA3C, which is not translocated, and only cognate LegA3-LegA3C orthologous pairs from different Legionella species function optimally together [91].…”

Section: General Concepts and Functional Featuresmentioning

confidence: 99%

The Legionella pneumophila Dot/Icm type IV secretion system and its effectors

et al. 2022

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To prevail in the interaction with eukaryotic hosts, many bacterial pathogens use protein secretion systems to release virulence factors at the host–pathogen interface and/or deliver them directly into host cells. An outstanding example of the complexity and sophistication of secretion systems and the diversity of their protein substrates, effectors, is the Defective in organelle trafficking/Intracellular multiplication (Dot/Icm) Type IVB secretion system (T4BSS) of Legionella pneumophila and related species. Legionella species are facultative intracellular pathogens of environmental protozoa and opportunistic human respiratory pathogens. The Dot/Icm T4BSS translocates an exceptionally large number of effectors, more than 300 per L. pneumophila strain, and is essential for evasion of phagolysosomal degradation and exploitation of protozoa and human macrophages as replicative niches. Recent technological advancements in the imaging of large protein complexes have provided new insight into the architecture of the T4BSS and allowed us to propose models for the transport mechanism. At the same time, significant progress has been made in assigning functions to about a third of L. pneumophila effectors, discovering unprecedented new enzymatic activities and concepts of host subversion. In this review, we describe the current knowledge of the workings of the Dot/Icm T4BSS machinery and provide an overview of the activities and functions of the to-date characterized effectors in the interaction of L. pneumophila with host cells.

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The Legionella genus core effectors display functional conservation among orthologs by themselves or combined with an accessory protein

Cited by 11 publications

References 61 publications

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

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

Molecular regulation of virulence in Legionella pneumophila

The Legionella pneumophila Dot/Icm type IV secretion system and its effectors

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