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. L egionella pneumophila is widespread in natural and man-made water systems and is best known as the etiologic agent of Legionnaires' disease (1-3). Recently, the incidence of pneumonias caused by Gram-negative L. pneumophila has roughly tripled in the United States and elsewhere (4-6). In water, L. pneumophila grows primarily as an intracellular parasite of amoebae (7-11). Human infection occurs mainly by the inhalation of contaminated water droplets originating from a wide range of aerosolgenerating devices (12,13). Yet the first case of person-to-person spread was reported in 2016 (14). In the lungs, L. pneumophila invades and grows in the resident macrophage population and ultimately triggers severe inflammation (3). In both amoebae and macrophages, the bacterium evades the host's degradative lysosomal pathway and replicates to large numbers within a membrane-bound vacuole, the Legionella-containing vacuole (LCV) (15, 16). The Dot/Icm type IV secretion (T4S) system of L. pneumophila plays a major role in pathogenesis (17-19). Indeed, the Dot/Icm apparatus translocates myriad proteins across the LCV membrane and into the host cell cytoplasm, and then the delivered effectors proceed to modulate a wide variety of host processes. One of the earliest and most notable events that has been ascribed to the Dot/Icm system is the recruitment of host GTPases and endoplasmic reticulu...

The Type II Secretion System of Legionella pneumophila Dampens the MyD88 and Toll-Like Receptor 2 Signaling Pathway in Infected Human Macrophages

“…L. pneumophila lsp mutants that lack T2S have an ϳ10-fold reduction in intracellular growth in both U937 cells, a human macrophage-like cell line, and murine macrophages obtained from A/J mice (16)(17)(18). Data from our laboratory have also shown that this reduction in CFU is not due to an entry defect or increased degradation through the phagosome-lysosome pathway but is instead due to a replication defect in LCVs at 4 to 12 h postentry (19).…”

“…The finding that the higher cytokine levels associated with mutant strains are not evident in all species of macrophages suggested that they are not an unspecific by-product of the lspF mutation. The finding that the lspF mutant does not traffic more readily to the phagolysosome than the WT further suggests that it is not subject to greater degradation (19). Nonetheless, to determine if the lspF mutant undergoes increased lysis upon infection of human macrophages, we infected U937 cells with WT and mutant bacteria expressing cell-associated green fluorescent protein (GFP) and then assayed levels of GFP released from bacteria.…”

“…A mutant lacking both flaA and lspF (NU430) was obtained by introducing pGlspF::Km (18) into NU347 by transformation (91) and then selecting for the acquisition of kanamycin resistance. In order to help monitor the behavior of L. pneumophila in macrophages, we utilized previously reported L. pneumophila WT and T2S mutant strains with a GFP-expressing plasmid introduced (19). Legionellae were routinely cultured at 37°C in buffered yeast extract (BYE) broth or on buffered charcoal yeast extract (BCYE) agar, which, when appropriate, contained gentamicin or kanamycin (92).…”

“…The generation of these cell lines was done as previously described (19). Briefly, silencing was achieved by transduction with a vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped lentivirus that encodes an shRNA molecule targeting the gene of interest.…”

“…Also, when a nonreplicating dotA T4S mutant, which is delivered to the degradative lysosomal pathway, was examined, lower, rather than higher, levels of cytokines were seen (16). Finally, as noted above, the lspF mutant is not trafficked to the phagolysosome in either human or murine macrophages (19). Based on these data, we posited that L. pneumophila T2S dampens host signal transduction and cytokine gene transcription.…”