Salmonella SPI1 Effector SipA Persists after Entry and Cooperates with a SPI2 Effector to Regulate Phagosome Maturation and Intracellular Replication

Summary Salmonellae employ two type III secretion systems (T3SSs), SPI1 and SPI2, to deliver virulence effectors into mammalian cells. SPI1 effectors, including actin-binding SipA, trigger initial bacterial uptake, whereas SPI2 effectors promote subsequent replication within customized Salmonella-containing vacuoles (SCVs). SCVs sequester actin filaments and subvert microtubule-dependent motors to migrate to the perinuclear region. We demonstrate that SipA delivery continues after Salmonella internalization, with dosage being restricted by host-mediated degradation. SipA is exposed on the cytoplasmic face of the SCV, from where it stimulates bacterial replication in both nonphagocytic cells and macrophages. Although SipA is sufficient to target and redistribute late endosomes, during infection it cooperates with the SPI2 effector SifA to modulate SCV morphology and ensure perinuclear positioning. Our findings define an unexpected additional function for SipA postentry and reveal precise intracellular communication between effectors deployed by distinct T3SSs underlying SCV biogenesis.


Replication and Invasion Assays in Nonphagocytic Cells
The ability of Salmonella strains to invade and replicate within cultured cells was assessed by gentamicin protection. S.typhimurium stationary phase cultures were diluted 1:500 in TY medium and incubated to maximize invasion efficiency (6 h, 37°C, 225 rpm). Washed bacteria were added at a multiplicity of infection (MOI) 100 to serumstarved cells in DMEM supplemented with L-glutamine. After incubation (37°C, 5% CO 2 , 60 min), cells were repeatedly washed with phosphate-buffered saline (PBS) and extracellular bacteria killed by the addition of DMEM supplemented with 100µgml -1 gentamicin, L-glutamine and 10% FCS (v/v) (37°C, 5% CO 2 , 60 min). Cells were washed again with PBS and incubated further (DMEM containing 10% FCS, L-glutamine and 7µgml -1 gentamicin), or lysed in 10mM Tris-Cl, pH 7.4, 0.5% (v/v) Triton X-100. Serial cell lysate dilutions were plated onto LB agar.

Replication Assays in Macrophages
Macrophages were seeded into 24-well tissue culture plates at a density of 4 x 10 5 cells per well 24 hours before use. Salmonella were cultured at 37°C, 225 rpm until they reached an OD 600 of 2.0, diluted 1:2 and opsonized in DMEM/RPMI 1640 containing FCS and 10% normal mouse serum at 37°C for 20 min. Bacteria were added to the macrophages at a MOI of ~100, centrifuged at 500rpm for 5 min at room temperature and incubated for a further 25 min at 37°C in 5% CO 2 . The macrophages were washed once with DMEM/RPMI 1640 containing FCS and 100µgml -1 gentamicin and incubated in this medium for 1 h. The medium was replaced with DMEM/RPMI 1640 containing FCS and 16µgml -1 gentamicin and incubated in this medium for the remainder of the experiment. Cells were lysed and plated as previously described.

Infection of Transfected Cells
GFP-InvB and GFP p50/dynamitin transfectants were infected 24 h after transfection. Transfectants expressing SipA, SipA-N and SipA-C were seeded onto coverslips 24 h post-transfection and selected using 500µgml -1 geneticin in growth medium. Cells were grown to 70-80% confluency prior to S.typhimurium infection. Expression of these constructs did not adversely affect cell number or viability.
Cm, BFA, bafilomycin A1, ATA, CD and LB were purchased from Sigma and MG132 from Calbiochem. ATA and MG132 do not affect S.typhimurium viability or invasion rate, and unless otherwise stated, agents were retained throughout subsequent incubation and washing steps.

Figure S6. Primary Sequence Similarity between SipA and Cellular Proteins Involved in Intracellular Trafficking
Upper, Sequence alignment comparing SipA residues 121-180 and eukaryotic proteins derived from BLAST database searches using the BLOSUM62 matrix (existence 11; extension 1). Numbers in brackets denote multiple hits between the index and target proteins. Invariant and similar residues are highlighted in red and orange, respectively. Primary sequence identity is >35%. Lower, left, Region of sequence homology mapped onto the SipA 48-264 crystal structure (Lilic et al., 2006). Residues 121-180 are shown in blue, with amino acids identical to H.sapiens restin (CLIP-170) in red with side-chains. Right, 'Rear view' image, in which the left image is rotated 180º about the vertical axis. Consistent with our finding that GFP-InvB binding failed to impede SipA function, residues 121-180 are remote from the chaperone-binding domain.