DistinctMycobacterium marinumphosphatases determine pathogen vacuole phosphoinositide pattern, phagosome maturation, and escape to the cytosol

Abstract

The causative agent of tuberculosis, Mycobacterium tuberculosis, and its close relative Mycobacterium marinum manipulate phagocytic host cells, thereby creating a replication-permissive compartment termed the Mycobacterium-containing vacuole (MCV). The phosphoinositide (PI) lipid pattern is a crucial determinant of MCV formation and is targeted by mycobacterial PI phosphatases. In this study, we establish an efficient phage transduction protocol to construct defined M. marinum deletion mutants lacking one or three phosphatases, PtpA, PtpB, and/or SapM. These strains were defective for intracellular replication in macrophages and amoebae, and the growth defect was complemented by the corresponding plasmid-borne genes. Fluorescence microscopy of M. marinum-infected Dictyostelium discoideum revealed that MCVs harbouring mycobacteria lacking PtpA, SapM, or all three phosphatases accumulate significantly more phosphatidylinositol-3-phosphate (PtdIns3P) compared with MCVs containing the parental strain. Moreover, PtpA reduced MCV acidification by blocking the recruitment of the V-ATPase, and all three phosphatases promoted bacterial escape from the pathogen vacuole to the cytoplasm. In summary, the secreted M. marinum phosphatases PtpA, PtpB, and SapM determine the MCV PI pattern, compartment acidification, and phagosomal escape.

Mycobacterium marinum produces distinct mycobactin and carboxymycobactin siderophores to promote growth in broth and phagocytes

“…The sources from which intracellular mycobacteria acquire iron are not well understood. Pathogenic mycobacteria, such as M. marinum or M. tuberculosis , replicate in host cells in a distinct, membrane‐bound pathogen vacuole called MCV, followed by rupture of the vacuole membrane and exit of the bacteria to the cytoplasm (Cardenal‐Munoz et al, ; Koliwer‐Brandl et al, ; Russell, ). The MCV represents a metal ion‐poor environment (Chao et al, ), which is depleted of iron due to the activity of NRAMP‐1, a proton‐driven transporter of ferric iron (Bozzaro, Buracco, & Peracino, ; Buracco et al, ; Buracco, Peracino, Andreini, Bracco, & Bozzaro, ; Peracino, Buracco, & Bozzaro, ).…”

“…M. marinum strains lacking mbtB or mbtB and irtAB were impaired for intracellular growth already early (24 hpi) during the infection of RAW 264.7 macrophages, A. castellanii or D. discoideum (Figure ). At these initial stages of infection, the pathogen still resides in an MCV (Koliwer‐Brandl et al, ), and therefore, mycobactins likely play a crucial role for scavenging iron in this membrane‐bound compartment. Exogenously added mycobactins did not rescue the Δ mbtB mutant strain in macrophages or amoebae (Figure ), presumably reflecting efficient killing in bacteriocidal phagolysosomes of mutant bacteria defective for mycobactin biosynthesis.…”

“…M. marinum deletion mutants were generated as previously described in detail (Koliwer‐Brandl et al, ). The allelic exchange substrates needed for specialised phage transduction were generated to replace mbtB and the two neighbouring genes irtA and irtB in M. marinum with a γδres ‐ sacB ‐ hyg ‐ γδres cassette comprising sacB and a Hyg resistance gene flanked by the res sites of the γδ‐resolvase.…”

“…Colours indicate the expression levels of mbt genes (in logFC) of M. marinum during infection at the indicated time points compared with the in vitro control: blue (down-regulated during infection), white (no expression difference), or dark red (up-regulated) 2.3 | Chromosomal deletion of M. marinum mbtB and irtAB by phage transduction To assess the role of mycobactin biosynthesis and transport for M. marinum growth in broth and host cells, we constructed defined deletion mutant strains lacking the gene encoding the putative phenyloxazoline synthase MtbB (mbtB, MMAR_3689), required for an initial step in siderophore synthesis, or lacking mbtB and in addition genes encoding the iron-regulated ABC transporter IrtAB (irtA, MMAR_4037; irtB, MMAR_4036). Using phage transduction (Koliwer-Brandl et al, 2019), we obtained a M. marinum strain, wherein the mbtB gene was replaced by a hygromycin resistance cassette and the sacB gene. In a second transduction step, the unmarked single deletion mutant strain ΔmbtB was generated by removing the res-siteflanked resistance cassette with the resolvase-producing phage phAE7.1 and subsequent counter selection for the absence of sacB.…”

“…marinum is widely used as a versatile M. tuberculosis infection model (Arafah et al, 2013;Bouz & Al Hasawi, 2018;Cardenal-Munoz, Barisch, Lefrancois, Lopez-Jimenez, & Soldati, 2017;Hagedorn, Rohde, Russell, & Soldati, 2009;Hagedorn & Soldati, 2007;Lienard & Carlsson, 2017;Prouty, Correa, Barker, Jagadeeswaran, & Klose, 2003;Shiloh & Champion, 2010;Tükenmez et al, 2019). M. marinum can be handled at biosafety Level 2 and is appreciated for its relatively rapid growth (doubling time of 4-10 hr vs. >20 hr for M. tuberculosis; Clark & Shepard, 1963), the M. tuberculosis-like behaviour during intracellular infection, such as replication within a distinct Mycobacterium-containing vacuole (MCV; Barker, George, Falkow, & Small, 1997;Pozos & Ramakrishnan, 2004;Smith et al, 2008;Tobin & Ramakrishnan, 2008;López-Jiménez et al, 2018;Koliwer-Brandl et al, 2019), as well as its wide spectrum of host cells including macrophages (Barker et al, 1997;Bouley, Ghori, Mercer, Falkow, & Ramakrishnan, 2001;Koliwer-Brandl et al, 2019;Ramakrishnan, Federspiel, & Falkow, 2000) and the amoeba Dictyostelium discoideum (Cardenal-Munoz et al, 2017;Hagedorn et al, 2009;Koliwer-Brandl et al, 2019;Solomon, Leung, & Isberg, 2003) or Acanthamoeba castellanii (Harrison et al, 2013;Kicka et al, 2014).…”