The Salmonella effector SteA contributes to the control of membrane dynamics of Salmonella-containing vacuoles

Abstract

Salmonella enterica serovar Typhimurium is a bacterial pathogen causing gastroenteritis in humans and a typhoid-like systemic disease in mice. S. Typhimurium virulence is related to its capacity to multiply intracellularly within a membrane-bound compartment, the Salmonella-containing vacuole (SCV), and depends on type III secretion systems that deliver bacterial effector proteins into host cells. Here, we analyzed the cellular function of the Salmonella effector SteA. We show that, compared to cells infected by wild-type S. Typhimurium, cells infected by ΔsteA mutant bacteria displayed fewer Salmonella-induced filaments (SIFs), an increased clustering of SCVs, and morphologically abnormal vacuoles containing more than one bacterium. The increased clustering of SCVs and the appearance of vacuoles containing more than one bacterium were suppressed by inhibition of the activity of the microtubule motor dynein or kinesin-1. Clustering and positioning of SCVs are controlled by the effectors SseF and SseG, possibly by helping to maintain a balanced activity of microtubule motors on the bacterial vacuoles. Deletion of steA in S. Typhimurium ΔsseF or ΔsseG mutants revealed that SteA contributes to the characteristic scattered distribution of ΔsseF or ΔsseG mutant SCVs in infected cells. Overall, this shows that SteA participates in the control of SCV membrane dynamics. Moreover, it indicates that SteA is functionally linked to SseF and SseG and suggests that it might contribute directly or indirectly to the regulation of microtubule motors on the bacterial vacuoles.

FIG 1

SteA contributes to the formation of SIFs. HeLa cells were infected for 8 or 14 h with the indicated GFP-expressing S. Typhimurium strains, fixed, and immunostained for LAMP1. (A) Infected cells showing SIFs were counted by immunofluorescence microscopy. Values are the means ± standards errors of the means (n = 3). At least 50 infected cells were analyzed in each experiment. P values were calculated by a two-tailed unpaired Student's t test relative to wt-infected cells for each time postinvasion. (B) Infected HeLa cells were imaged by confocal microscopy for Salmonella expressing GFP (green) and LAMP1 (red). nd, not determined. Bars, 5 μm.

FIG 2

SteA participates in the control of SCV membrane dynamics. HeLa cells were infected for 8 or 14 h with the indicated GFP-expressing strains and fixed. (A and B) Infected HeLa cells were imaged by confocal microscopy for Salmonella expressing GFP (A) or were immunostained for LAMP1 and imaged by confocal microscopy for Salmonella expressing GFP (green) and LAMP1 (red) (B). (C) Infected cells with compact microcolonies were counted by IF microscopy. (D) Infected cells showing apparent abnormal LAMP1 accumulation within the bacterial microcolony were counted by IF microscopy. All values are the means ± standard errors of the means (n = 3). At least 50 infected cells were analyzed in each experiment. P values were calculated by a two-tailed unpaired Student's t test relative to wt-infected cells at each time postinvasion. Bars, 5 μm.

FIG 3

SteA contributes to the normal partitioning of the bacterial vacuole in infected cells. (A) HeLa cells were infected for 14 h with the indicated DsRed-expressing strains, transfected with a plasmid encoding LAMP1-GFP, and fixed. Infected HeLa cells were imaged by confocal microscopy for LAMP1-GFP (green) and Salmonella expressing DsRed (red). (B) HeLa or RAW 264.7 infected cells with abnormal vacuoles, which apparently contained more than a single bacterium, were enumerated by immunofluorescence microscopy. All values are the means ± standard errors of the means (n = 3). At least 50 infected cells were analyzed in each experiment. P values were calculated by a two-tailed unpaired Student's t test relative to wt-infected HeLa or RAW 264.7 cells, except where indicated (comparison of ΔsteA mutant- and ΔsteA ΔinvG mutant-infected cells). (C) Still images from Movie S1 (wt) and Movie S2 (ΔsteA mutant) in the supplemental material. HeLa cells were transfected with pLAMP1-GFP and infected for 8 to 12 h with wt or ΔsteA mutant S. Typhimurium. The images show an infected cell with SCVs containing a single bacterium (wt) and an infected cell showing a large vacuole containing several bacteria (ΔsteA mutant). (D) RAW 264.7 macrophages were infected for 16 h with the indicated GFP-expressing strains, fixed, and immunostained for LAMP1. Infected RAW 264.7 macrophages were imaged by confocal microscopy for Salmonella expressing GFP (green) and LAMP1 (red). In the merged images, the arrows indicate normal vacuoles containing a single bacterium and the arrowheads indicate abnormal vacuoles containing more than one bacterium. Bars, 5 μm.

FIG 4

Early translocation of SteA is not required for the control of SCV membrane dynamics in HeLa cells. HeLa cells were infected for 14 h with S. Typhimurium wt or a ΔsteA mutant strain expressing SteA in trans under the control of the Escherichia coli arabinose-inducible P_BAD promoter (pBAD-SteA-2HA), fixed, and immunostained for Salmonella and LAMP1. The experiment was done in the continuous presence of 0.2% (vol/vol) l-arabinose (+Ara), in its absence (−Ara), or when the sugar was added to the medium at the indicated time points (1 or 4 h p.i.). (A) Infected cells showing SIFs and compact microcolonies were counted by immunofluorescence microscopy. Values are the means ± standard errors of the means (n = 3). At least 50 infected cells were analyzed in each experiment. P values were calculated by a two-tailed unpaired Student's t test relative to the condition with l-arabinose (+ Ara). (B) HeLa cells were imaged by confocal microscopy for Salmonella (green) and LAMP1 (red). Bars, 5 μm.

FIG 5

Inhibition of dynein or kinesin-1 activity suppresses defects in ΔsteA mutant SCVs in HeLa cells. HeLa cells were infected for 14 h with wt and ΔsteA mutant S. Typhimurium; transfected with pLAMP1-GFP plasmid alone, pLAMP1-GFP and pHA-p50 plasmids, or pLAMP1-GFP and pHA-TPR plasmids; fixed; and immunostained for Salmonella and HA. (A) Infected cells with compact microcolonies and abnormal vacuoles were counted by immunofluorescence microscopy. Values are the means ± standard errors of the means (n = 3). At least 50 infected cells were analyzed in each experiment. P values were calculated by a two-tailed unpaired Student's t test. (B) HeLa cells were imaged by confocal microscopy for HA (red), Salmonella (blue), and LAMP1-GFP (green). Bars, 5 μm.

FIG 6

SteA is functionally linked to SPI-2 effectors SseF and SseG. (A to C) HeLa cells were infected for 14 h with the indicated GFP-expressing strains, fixed, and immunostained for the Golgi region with giantin. The infected cells showing microcolonies associated with the Golgi region (A) or compact microcolonies (B) were counted by IF microscopy. (C) HeLa cells were imaged by confocal microscopy for Salmonella expressing GFP (green) and giantin (red). (D and E) HeLa cells were infected with the indicated DsRed-expressing strains for 14 h, transfected with pLAMP1-GFP, and fixed. (D) Infected cells with abnormal vacuoles were counted by IF microscopy. (E) HeLa cells were imaged by confocal microscopy for LAMP1-GFP (green) and Salmonella expressing DsRed (red). All values are the means ± standard errors of the means (n = 3). At least 50 infected cells were analyzed in each experiment. P values were calculated by the Student t test relative to wild-type-infected cells. Bars, 5 μm.