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Type II Secretion Is Necessary for Optimal Association of the Legionella-Containing Vacuole with Macrophage Rab1B but Enhances Intracellular Replication Mainly by Rab1B-Independent Mechanisms.

ABSTRACT: Previously, we documented that type II secretion (T2S) promotes intracellular infection of macrophages by Legionella pneumophila In the present study, we identified infection events that are modulated by T2S by comparing the behaviors of wild-type and T2S mutant bacteria in murine bone marrow-derived macrophages and human U937 cells. Although the two strains behaved similarly for entry into the host cells and evasion of lysosomal fusion, the mutant was impaired in the ability to initiate replication between 4 and 8 h postentry and to grow to large numbers in the Legionella-containing vacuole (LCV), as evident at 12 h. At 4 h postinoculation, mutant LCVs had a significantly reduced association with Rab1B, a host GTPase that facilitates the tethering of endoplasmic reticulum (ER)-derived vesicles to LCVs. The mutant did not lose expression or translocation of six type IV secretion effectors (e.g., SidM) that are well known for mediating Rab1B association with the LCV, indicating that T2S promotes the interaction between the LCV and Rab1B via a novel mechanism. Interestingly, the mutant's growth defect was exacerbated in macrophages that had been depleted of Rab1B by short hairpin RNA (shRNA) treatment, indicating that T2S also potentiates events beyond Rab1B association. In support of this, a sidM lspF double mutant had an intracellular growth defect that was more dramatic than that of the lspF mutant (and a sidM mutant) and showed a growth difference of as much as a 400-fold compared to the wild type. Together, these data reveal a new role for T2S in intracellular infection that involves both Rab1B-dependent and Rab1B-independent processes.

SUBMITTER: White RC

PROVIDER: S-EPMC5116710 | biostudies-literature | 2016 Dec

REPOSITORIES: biostudies-literature

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Type II Secretion Is Necessary for Optimal Association of the Legionella-Containing Vacuole with Macrophage Rab1B but Enhances Intracellular Replication Mainly by Rab1B-Independent Mechanisms.

White Richard C RC Cianciotto Nicholas P NP

Infection and immunity 20161118 12

Previously, we documented that type II secretion (T2S) promotes intracellular infection of macrophages by Legionella pneumophila In the present study, we identified infection events that are modulated by T2S by comparing the behaviors of wild-type and T2S mutant bacteria in murine bone marrow-derived macrophages and human U937 cells. Although the two strains behaved similarly for entry into the host cells and evasion of lysosomal fusion, the mutant was impaired in the ability to initiate replica ...[more]

PMID: 27600508

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Project description:Coxiella burnetii is an obligate intracellular gammaproteobacterium and zoonotic agent of Q fever. We previously identified 15 small noncoding RNAs (sRNAs) of C. burnetii One of them, CbsR12 (Coxiella burnetiismall RNA 12), is highly transcribed during axenic growth and becomes more prominent during infection of cultured mammalian cells. Secondary structure predictions of CbsR12 revealed four putative CsrA-binding sites in stem loops with consensus AGGA/ANGGA motifs. We subsequently determined that CbsR12 binds to recombinant C. burnetii CsrA-2, but not CsrA-1, proteins in vitro Moreover, through a combination of in vitro and cell culture assays, we identified several in trans mRNA targets of CbsR12. Of these, we determined that CbsR12 binds and upregulates translation of carA transcripts coding for carbamoyl phosphate synthetase A, an enzyme that catalyzes the first step of pyrimidine biosynthesis. In addition, CbsR12 binds and downregulates translation of metK transcripts coding for S-adenosylmethionine synthetase, a component of the methionine cycle. Furthermore, we found that CbsR12 binds to and downregulates the quantity of cvpD transcripts, coding for a type IVB effector protein, in mammalian cell culture. Finally, we found that CbsR12 is necessary for expansion of Coxiella-containing vacuoles and affects growth rates in a dose-dependent manner in the early phase of infecting THP-1 cells. This is the first characterization of a trans-acting sRNA of C. burnetii and the first example of a bacterial sRNA that regulates both CarA and MetK synthesis. CbsR12 is one of only a few identified trans-acting sRNAs that interacts with CsrA.IMPORTANCE Regulation of metabolism and virulence in C. burnetii is not well understood. Here, we show that C. burnetii small RNA 12 (CbsR12) is highly transcribed in the metabolically active large-cell variant compared to the nonreplicative small-cell variant. We show that CbsR12 directly regulates several genes involved in metabolism, along with a type IV effector gene, in trans In addition, we demonstrate that CbsR12 binds to CsrA-2 in vitro and induces autoaggregation and biofilm formation when transcribed ectopically in Escherichia coli, consistent with other CsrA-sequestering sRNAs. These results implicate CbsR12 in the indirect regulation of a number of genes via CsrA-mediated regulatory activities. The results also support CbsR12 as a crucial regulatory component early on in a mammalian cell infection.

Dataset Information

Type II Secretion Is Necessary for Optimal Association of the Legionella-Containing Vacuole with Macrophage Rab1B but Enhances Intracellular Replication Mainly by Rab1B-Independent Mechanisms.

Publications

Type II Secretion Is Necessary for Optimal Association of the Legionella-Containing Vacuole with Macrophage Rab1B but Enhances Intracellular Replication Mainly by Rab1B-Independent Mechanisms.

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