Fig 1.
S. marcescens induces blebs and toxicity in human primary corneal cells and on porcine corneal tissue.
(A) Confocal differential interference contrast (DIC) micrographs of human corneal cell line (HCLE) challenged with LB medium (mock) or with S. marcescens strain K904 (MOI = 200 for 2 h). Yellow arrows indicate a bleb extending from one of the cells. Size bar indicates 50 μm. (B) Confocal micrograph of HCLE cells challenged with S. marcescens strain K904 for 2 h and stained with a fluorescent membrane dye. The image shows a side projection of a z-stack of images. White arrow indicates a surface attached HCLE cell and the yellow arrow indicates a membrane bleb. Size bar is 20 μm. (C) SEM micrograph of blebs (yellow arrows) on a HCLE cell speckled with pseudocolored S. marcescens K904 bacteria (red). Size bar is 10 μm. (D) 2-D area of arbitrarily chosen blebs from 6 independent experiments tracked with video microscopy. (E) Primary corneal cells imaged by confocal microscopy with DIC and fluorescent imaging of the same cells stained with viability dye Calcein AM. Yellow arrows indicate blebs. Bar = 20 μm. Mock n = 48, K904 n = 22. (F) SEM micrograph of porcine corneas that had been exposed to naïve contact lenses (Mock, top) or contact lenses coated with wild-type S. marcescens (K904, bottom) prior to fixation. Blebs are indicated with yellow arrows.
Fig 2.
S. marcescens mutations that abrogate cytotoxicity in HCLE cells were isolated and mapped.
(A) Confocal micrographs of primary human corneal epithelial cells challenged with S. marcescens strain K904 wild type and mutant strains isolated for being unable to induce bleb formation (MOI = 200 for 2 h). Three of five mutants are shown, with the other two having an indistinguishable effect on the corneal cells. Differential interference contrast (DIC) and calcein AM viability stained images are shown. (B) Genetic context of transposon insertion mutations that render S. marcescens unable to induce bleb formation in corneal epithelial cells. Downward facing blue arrows indicate transposon insertion sites. Size bar indicates 1000 base pairs.
Fig 3.
S. marcescens shlBA operon is necessary, and ShlA is sufficient, for bleb and cytotoxicity induction.
Confocal micrographs of HCLE cells imaged with differential interference contrast (DIC) and calcein AM viability stain. Yellow arrows indicate blebs extending from corneal cells. The percent of bleb positive cells induced by the indicated treatment are shown. (A) Confocal micrographs of HCLE cells with S. marcescens strain K904 and mutant strains (MOI = 50, 2 h incubation). Vector = pMQ125 or pMQ131; pshlBA = pMQ541; pshlBA::tn = pMQ591. (B) Microscopic evaluation of HCLE cells exposed to E. coli (Top10) with a vector (pMQ175) or shlBA expressing plasmid (pMQ492) at MOI = 50 for 2 h. Cells were alternatively exposed for 3 h to sterile-filtered supernatants from E. coli with the pMQ175 (SUP Control) or pMQ492 (SUP ShlA) plasmids, or partially purified ShlA-containing supernatant fractions from E. coli with the vector negative control (PUR Control) or with pMQ492 (PUR ShlA). Vector = pMQ125; pshlBA = pMQ492.
Fig 4.
A shlBA-like Type Vb secretion system operon from P. mirabilis induces blebbing and cytotoxicity.
Confocal micrographs of HCLE cells imaged with differential interference contrast (DIC) and calcein AM viability stain. Yellow arrows indicate blebs extending from corneal cells. (A) Confocal micrographs of HCLE cells with E. coli strain (Top10), S. marcescens (S. m.) ΔshlB, and P. mirabilis keratitis isolate K2644 and isogenic hmpA mutant strain (MOI = 50, 2 h incubation, 1 hour with E. coli). (B) As in (A), using P. mirabilis keratitis isolate K2675. phpmBA = pMQ601; vector = pMQ132; phpmA = pMQ602.
Fig 5.
S. marcescens gumB gene is necessary but not sufficient for the bleb and cytotoxicity induction, and the gumB mutant strain defect is complemented by igaA-family genes and shlBA.
(A,B) Confocal micrographs of primary human corneal and HCLE cells imaged with differential interference contrast (DIC) and calcein AM viability stain after exposure to bacteria for 2 h at MOI = 50, except where noted. Yellow arrows indicate blebs extending from corneal cells. (A) Confocal micrographs of HCLE cells with S. marcescens strain K904, mutant strains, or E. coli strain EC100D pir-116. Vector = pMQ132; pshlBA = pMQ541; pgumB = pMQ480. The percent of bleb positive cells induced by select bacteria are shown. (B) HCLE cells exposed to the gumB mutant strain (MOI = 50) with plasmid-borne igaA-family genes from S. marcescens (pgumB = pMQ480), S. enterica (pigaA = pMQ530), K. pneumoniae (pkumO = pMQ529), E. coli (pyrfF = pMQ531), or P. mirabilis (pumoB = pMQ600). Vector = pMQ132. (C) Relative gene expression using the ΔΔCT method depicts shlA transcript levels in the wild type (K904) and ΔgumB mutant strains at OD600 = 3; *p = 0.0286, Mann-Whitney test.
Fig 6.
GumB regulation of bleb formation and cytotoxicity requires the Rcs signaling system.
(A) Model for the regulatory circuit through which GumB functions to regulate epithelial cell bleb formation, based on this study and DiVenanzio, et al [58]. GumB inhibits (red stop bar) the Rcs-phosphorelay system through which the response regulator RcsB inhibits shlBA expression. The ShlA cytolysin is secreted through the outer membrane by ShlB and is maintained on the bacterial outer membrane or released into the environment where it can form pores in mammalian cell membranes and stimulate bleb formation and cellular death. (B) Confocal micrographs of HCLE cells imaged with differential interference contrast (DIC) and calcein AM viability stain after exposure to bacteria for 2 h at MOI = 50. Yellow arrows indicate blebs extending from corneal cells. Multicopy expression of the rcsC histidine sensor kinase gene confers gumB-like phenotypes to the wild type (prcsC = pMQ514). The ΔgumB mutant bleb-phenotype is suppressed by mutation of the rcsB response regulator gene, and this effect can be complemented by the wild-type rcsB gene on a plasmid (prcsB = pMQ614).
Fig 7.
ShlA induces bleb formation and cytotoxicity via pore formation and subsequent necroptosis.
Confocal micrographs HCLE cells imaged with differential interference contrast (DIC) and calcein AM viability stain after exposure to bacteria for 2 h at MOI = 50. Yellow arrows indicate blebs extending from corneal cells. HCLE cells were exposed to S. marcescens K904 (MOI = 50 for 2 h) with either (A) dextran sulfate 8000 (30 mM, occludes pores caused by pore-forming toxins), (B) necrostatin 5 (100 μM, an inhibitor of necroptosis), or coenzyme Q10 (CoQ10, 0.1 μM, an antioxidant,) or an equal volume of vehicle DMSO incubated with cells for 1 h prior to challenge.
Fig 8.
GumB is necessary for virulence in a Galleria mellonella model of infection via regulation of shlBA.
Shown are survival curves of G. mellonella larvae challenged with S. marcescens. (A) G. mellonella survival over time following injection with 200 CFU is shown (n = 12 for K904, n = 13 for ΔgumB); p<0.001 Log-rank test. (B) Similar survival curves for G. mellonella over time were observed after treatment with either K904 or ΔgumB despite large differences in CFUs injected (n = 14). (C) Survival of larvae injected with the ΔgumB mutant and ΔshlB plasmid with various plasmids as indicated (n = ≥12). Vector = pMQ125; pshlBA = pMQ541.
Fig 9.
GumB is required for survival and proliferation in G. mellonella and in murine phagocytic cells.
(A) Enumeration of S. marcescens K904 wild type and ΔgumB mutant strains 24 h after injection of 103 CFU into G. mellonella. Median and range are shown, n≥3. * indicates significant difference between medians, Mann-Whitney test (p = 0.0286). (B) Growth of K904 and the ΔgumB mutant in G. mellonella homogenates (n = 10). Error bars indicate standard deviation. (C) Enumeration of S. marcescens K904 wild type and ΔgumB mutant strains 24 h growth in heat-treated G. mellonella homogenates, n = 7. Median and interquartile range is shown. (D) Representative experiment describing uptake and proliferation of S. marcescens K904 wild type and ΔgumB mutant strains in RAW macrophage cells (n = 3), mean and standard deviations are shown. *** indicates significant difference by 2-way ANOVA with Tukey’s post-test (p<0.001).
Table 1.
P. mirabilis and S. marcescens strains used in this study.