Identification of Type 4B Secretion System Substrates That Are Conserved among Coxiella burnetii Genomes and Promote Intracellular Growth

Larson, Carl L.; Pullman, Willis; Beare, Paul A.; Heinzen, Robert A.

doi:10.1128/spectrum.00696-23

“…Its expression is regulated by a PmrA promoter and it includes a putative C-terminal E-Block secretion signal(17). CBU2007 has been recently validated as a secreted C. burnetii effector protein conserved among C. burnetii genomes(18), is important for CCV biogenesis(18) and essential for C. burnetii survival in primary macrophages(19). Nested PCR followed by sequencing(20) indicated that the transposon insertion occurred 711 bp downstream of the gene starting codon.…”

Section: Resultsmentioning

confidence: 99%

“…Phenotypic screen of a library of C. burnetii mutants carrying transposon insertions in genes encoding predicted and validated effector proteins allowed us to pinpoint a transposon insertion in cbu2007 , which largely inhibited LBPA localization at CCVs. CBU2007 is the highest-scoring predicted effector by the S4TE algorithm(16) and has been recently validated as a C. burnetii effector protein(18). CBU2007 is required for intracellular replication(18) and, more importantly, is essential for survival in primary macrophages and virulence in mice(19).…”

Section: Discussionmentioning

confidence: 99%

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The multifunctionCoxiellaeffector Vice stimulates macropinocytosis and interferes with the ESCRT machinery

Bienvenu,

Burette,

Cantet

et al. 2024

Preprint

0

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Intracellular bacterial pathogens divert multiple cellular pathways to establish their niche and persist inside their host.Coxiella burnetii, the causative agent of Q fever, secretes bacterial effector proteins via its Type 4 secretion system to generate aCoxiella-containing vacuole (CCV). Manipulation of lipid and protein trafficking by these effectors is essential for bacterial replication and virulence. Here, we have characterized the lipid composition of CCVs and discovered that the effector Vice interacts with phosphoinositides and membranes enriched in phosphatidylserine (PS) and lysobisphosphatidic acid (LBPA). Remarkably, eukaryotic cells ectopically expressing Vice present compartments that resemble early CCVs in both morphology and composition. We discovered that the biogenesis of these compartments relies on the double function of Vice. The effector protein initially localizes at the plasma membrane of eukaryotic cells where it triggers the internalization of large vacuoles by macropinocytosis. Then, Vice stabilizes these compartments by perturbing the ESCRT machinery and inhibiting the formation of intraluminal vesicles (ILVs). Collectively, our results reveal that Vice is an essentialC. burnetiieffector protein capable of hijacking two major cellular pathways to shape the bacterial replicative niche.Significance statementCoxiella burnetiiis a unique bacterial pathogen that secretes more than a hundred effector proteins to manipulate cellular processes and establish a replicative niche, theCoxiella-containing vacuole (CCV). Our study identified host cell lipids that are actively recruited by the bacterium to the CCV. Using a library of effector mutants, we identified the protein Vice (for Vacuole-inducingCoxiellaeffector) as the first bacterial effector capable of interacting with lysobisphosphatydic acid-enriched membranes and accumulating this lipid to CCVs. We show that Vice is also capable of stimulating macropinocytosis and inhibiting the ESCRT machinery. Together, our data show how a single bacterial effector can manipulate different cellular processes to favor the biogenesis of a bacterial pathogen’s niche.

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The multifunction Coxiella effector Vice stimulates macropinocytosis and interferes with the ESCRT machinery

Bienvenu,

Burette,

Cantet

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

0

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Intracellular bacterial pathogens divert multiple cellular pathways to establish their niche and persist inside their host. Coxiella burnetii , the causative agent of Q fever, secretes bacterial effector proteins via its Type 4 secretion system to generate a Coxiella -containing vacuole (CCV). Manipulation of lipid and protein trafficking by these effectors is essential for bacterial replication and virulence. Here, we have characterized the lipid composition of CCVs and found that the effector Vice interacts with phosphoinositides and membranes enriched in phosphatidylserine and lysobisphosphatidic acid. Remarkably, eukaryotic cells ectopically expressing Vice present compartments that resemble early CCVs in both morphology and composition. We found that the biogenesis of these compartments relies on the double function of Vice. The effector protein initially localizes at the plasma membrane of eukaryotic cells where it triggers the internalization of large vacuoles by macropinocytosis. Then, Vice stabilizes these compartments by perturbing the ESCRT machinery. Collectively, our results reveal that Vice is an essential C. burnetii effector protein capable of hijacking two major cellular pathways to shape the bacterial replicative niche.

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Coxiella burnetii protein CBU2016 supports CCV expansion

Thomas,

Garnish,

Khoo

et al. 2024

Pathogens and Disease

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Coxiella burnetii is a globally distributed obligate intracellular pathogen. Although often asymptomatic, infections can cause acute Q fever with influenza-like symptoms and/or severe chronic Q fever. C. burnetii develops a unique replicative niche within host cells called the Coxiella-containing vacuole (CCV), facilitated by the Dot/Icm type IV secretion system translocating a cohort of bacterial effector proteins into the host. The role of some effectors has been elucidated; however, the actions of the majority remain enigmatic and the list of true effectors is disputable. This study examined CBU2016, a unique C. burnetii protein previously designated as an effector with a role in infection. We were unable to validate CBU2016 as a translocated effector protein. Employing targeted knock-out and complemented strains, we found that the loss of CBU2016 did not cause a replication defect within Hela, THP-1, J774, or iBMDM cells or in axenic media, nor did it affect the pathogenicity of C. burnetii in the Galleria mellonella infection model. Absence of CBU2016 did, however, result in a consistent decrease in the size of CCVs in HeLa cells. These results suggest that although CBU2016 may not be a Dot/Icm effector, it is still able to influence the host environment during infection.

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Identification of Type 4B Secretion System Substrates That Are Conserved among Coxiella burnetii Genomes and Promote Intracellular Growth

Abstract: Coxiella burnetii secretes effector proteins via a T4BSS that are required for successful infection. Over 150 C. burnetii proteins are reported to be T4BSS substrates and often by default considered putative effectors, but few have assigned functions.

Cited by 4 publications

References 48 publications

The multifunctionCoxiellaeffector Vice stimulates macropinocytosis and interferes with the ESCRT machinery

The multifunctionCoxiellaeffector Vice stimulates macropinocytosis and interferes with the ESCRT machinery

The multifunction Coxiella effector Vice stimulates macropinocytosis and interferes with the ESCRT machinery

Coxiella burnetii protein CBU2016 supports CCV expansion

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