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Multiple Legionella Pneumophila Effector Virulence Phenotypes Multiple Legionella pneumophila effector virulence PNAS PLUS phenotypes revealed through high-throughput analysis of targeted mutant libraries Stephanie R. Shamesa,1, Luying Liua, James C. Haveya, Whitman B. Schofielda,b, Andrew L. Goodmana,b, and Craig R. Roya,2 aDepartment of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06519; and bMicrobial Sciences Institute, Yale University School of Medicine, New Haven, CT 06519 Edited by Ralph R. Isberg, Howard Hughes Medical Institute/Tufts University School of Medicine, Boston, MA, and approved October 20, 2017 (received for review May 23, 2017) Legionella pneumophila is the causative agent of a severe pneu- poorly understood. Initial forward genetic screens aimed at identi- monia called Legionnaires’ disease. A single strain of L. pneumo- fying avirulent mutants of L. pneumophila were successful in identi- phila encodes a repertoire of over 300 different effector proteins fying essential components of the Dot/Icm system, but these screens that are delivered into host cells by the Dot/Icm type IV secretion did not identify effector proteins translocated by the Dot/Icm system system during infection. The large number of L. pneumophila ef- (10, 11). It is appreciated that most effectors are not essential for fectors has been a limiting factor in assessing the importance of intracellular replication (12), which is why the genes encoding ef- individual effectors for virulence. Here, a transposon insertion se- fector proteins that are important for virulence were difficult to quencing technology called INSeq was used to analyze replication identify by standard screening strategies that assess intracellular of a pool of effector mutants in parallel both in a mouse model of replication using binary assays that measure plaque formation or infection and in cultured host cells. Loss-of-function mutations in destruction of host cell monolayers (10, 13). Thus, new approaches genes encoding effector proteins resulted in host-specific or broad are required to systematically assess the contribution of indi- virulence phenotypes. Screen results were validated for several vidual L. pneumophila effector proteins during infection. effector mutants displaying different virulence phenotypes using High-throughput sequencing (HTS)-based phenotypic screen- MICROBIOLOGY genetic complementation studies and infection assays. Specifically, ing of bacterial transposon (Tn) mutants has become a powerful loss-of-function mutations in the gene encoding LegC4 resulted technique to assess the contribution of individual genes to bacte- in enhanced L. pneumophila in the lungs of infected mice but rial fitness during host colonization (14). Techniques such as in- not within cultured host cells, which indicates LegC4 augments sertion sequencing (INSeq) (15) and Tn sequencing (TNSeq) (16) bacterial clearance by the host immune system. The effector pro- are massively parallel HTS techniques that enable determination teins RavY and Lpg2505 were important for efficient replication of relative fitness of individual Tn mutants within a mixed pop- within both mammalian and protozoan hosts. Further analysis of ulation. These techniques have been used to generate whole- Lpg2505 revealed that this protein functions as a metaeffector genome mutant populations to identify genes that contribute to that counteracts host cytotoxicity displayed by the effector pro- virulence of several clinically important bacterial pathogens such tein SidI. Thus, this study identified a large cohort of effectors that as Campylobacter jejuni, Haemophilus influenza, Acinetobacter contribute to L. pneumophila virulence positively or negatively baumannii,andPseudomonas aeruginosa (15, 17–20). However, and has demonstrated regulation of effector protein activities by traditional whole-genome screening approaches are susceptible to cognate metaeffectors as being critical for host pathogenesis. Significance type IV secretion | transposon insertion sequencing | bacterial effectors The contribution of individual effectors to Legionella pneumo- acteria of the genus Legionella are inhabitants of fresh water phila virulence has not been systematically examined. This study Band soil environments where they have evolved the capacity employed a parallel high-throughput transposon insertion se- to replicate in a diverse number of protozoan hosts. Although quencing technique called INSeq to probe the L. pneumophila there are over 40 species of Legionella, human infections that effector repertoire and identified multiple effectors that con- progress to a severe pneumonia called Legionnaires’ disease are ’ tribute to virulence in several host organisms, including an ani- most often caused by L. pneumophila (1). Legionnaires disease mal model of Legionnaires’ disease. Importantly, this study results from inhalation of Legionella-contaminated aerosols and demonstrates that effector proteins contribute to host virulence subsequent bacterial replication within alveolar macrophages. both positively and negatively by controlling intracellular repli- Bacterial replication occurs in a specialized Legionella-contain- cation and influencing host immune responses, which demon- ing vacuole (LCV) that evades fusion with lysosomes and asso- strates that the subtle alterations in the effector repertoire ciates intimately with the host endoplasmic reticulum (ER) (2, of a single L. pneumophila strain can greatly impact host 3). Formation of the LCV and intracellular bacterial replication pathogenicity. is dependent on the Dot/Icm type IV secretion system (T4SS) (4, 5), which translocates bacterial effector proteins into the host cell Author contributions: S.R.S., A.L.G., and C.R.R. designed research; S.R.S., L.L., and J.C.H. where they subvert normal host processes to promote pathogen performed research; W.B.S. and A.L.G. contributed new reagents/analytic tools; S.R.S., replication (6). The Philadelphia-1 strain of L. pneumophila was L.L., A.L.G., and C.R.R. analyzed data; and S.R.S. and C.R.R. wrote the paper. isolated from the eponymous Legionnaires’ disease outbreak that The authors declare no conflict of interest. occurred in 1976 (7), and this strain has been shown to encode This article is a PNAS Direct Submission. over 300 different effector proteins (8). Published under the PNAS license. Genome sequencing studies have demonstrated a high degree 1Present address: Division of Biology, Kansas State University, Manhattan, KS 66506. of plasticity in the effector repertoires encoded by between dif- 2To whom correspondence should be addressed. Email: [email protected]. ferent strains of L. pneumophila and different Legionella species This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (9). How the effector repertoire influences host virulence remains 1073/pnas.1708553114/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1708553114 PNAS Early Edition | 1of9 Downloaded by guest on September 25, 2021 population bottlenecks, which result in stochastic changes in mu- INSeq Analysis of the EMP Identifies Factors That Control L. pneumophila tant abundance that are unrelated to fitness (21). In most animal Virulence. A mouse model of Legionnaires’ disease was used to models of Legionnaires’ disease, these bottlenecks would likely determine whether individual mutants with known virulence occur using populations containing more than 1,000 different phenotypes could be identified after INSeq analysis of the mutants, which complicates using whole-genome INSeq ap- EMP. Specifically, the EMP was screened after intranasal in- − − proaches for assessing the contribution of effector proteins in oculation of C57BL/6 (WT) mice and NLRC4 / mice. INSeq host pathogenicity. was used to profile the output EMP population after 48 h of To circumvent the challenges associated with whole-genome infection, which is when replication of L. pneumophila peaks in mutant screening, INSeq technology was used to sequence an the lungs of mice (27). The distribution of mutants in the output arrayed L. pneumophila Tn mutant library and determine where populations was compared with the input populations (Fig. 1B individual Tn insertion mutants were located in the arrayed li- and Fig. S2). brary. From these data, mutants deficient in individual effector There was a significant increase in the proportion of flaA::Tn genes were clonally isolated to generate an effector mutant pool mutants in the lungs of C57BL/6 mice in the output population (EMP) that was used to assess the fitness of individual effector at 48 h, which indicated that the flagellin-deficient mutant had a mutants using both a mouse model of Legionnaires’ disease and competitive advantage over other mutants in the EMP (Fig. S2 cultured host cells. This systematic analysis revealed distinct and Dataset S3). By contrast, flaA::Tn mutants did not display a virulence phenotypes for individual effector mutants and a competitive advantage in NLRC4-deficient mice (Dataset S3). complex relationship between the L. pneumophila effector gene Thus, INSeq analysis of the EMP successfully determined the repertoire and host virulence. competitive advantage flaA::Tn mutants have in escaping flagellin- mediated activation of the NAIP5/NLRC4 inflammasome. Using Results C57BL/6 mice, there was a significant decrease in the output Generation of the L. pneumophila EMP. To produce a pool of population at 48 h for mutants with Tn insertions in genes en- L. pneumophila mutants where specific effector genes were
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