Salmonella Stimulate Macrophage Macropinocytosis and Persist within Spacious Phagosomes

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Salmonella Stimulate Macrophage Macropinocytosis
and Persist within Spacious Phagosomes
By C.M. Alpuche-Aranda, E.L. Racoosin, J.A.
Swanson, S.I. Miller
Presenter Jennifer Bratt
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Things to consider when reading this article

• This article was written in 1994. A considerable
  amount of research has been done on this system
  post publication..
• The serovar used in this experiment was
  Salmonella enterica Serovar typhimurium and the
  animal model used in this experiment is the
  murine model.

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• Salmonella enterica Serovar Typhimurium
• Gram negative, facultative intracellular rod
• The pathogenicity of the typhimurium Serovar is
  due to its ability to invade
• Peyers Patch
• Luminal epithelial cells
• macrophages.
• The cells ability to invade macrophages is
  essential for systemic infection.

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How does the cell invade the macrophage?

• Prior studies have indicated that the PhoQ / PhoP
  signaling system detects the vacuolar environment
  resulting in transcriptional control of pag and
  prg genes.

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PhoQ/PhoP, pags, and prgs

• What are all these Acronyms?
• PhoQ - Sensor kinase located on the cell surface.
  Putative function, vacuolar environment
  detection.
• PhoP - Signaling molecule. Phosphorylated
  signaling activator that regulated the expression
  of the pags and prgs.
• pag - PhoP-Activated Gene
• prg - PhoP-Repressed Gene

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• PhoQ/PhoP detects the macrophage vacuolar
  environment
• How does S. typhimurium react to it to promote
  pathogenicity?
• PhoQ/PhoP control the expression of the pags and
  prgs.
• The prgs are expressed by Salmonella unless
  inhibited by PhoP.
• As long as the cell does not detect that it is
  inside a vacuole, prgs are expressed.
• When the cell sensor, PhoP, detects the vacuolar
  environment, the expression of prgs is blocked
  and the expression of pags is initiated.

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• How does the cell survive within the macrophage
  phagosome?
• Salmonella induces the macropinocytosis which
  results in the formation of Spacious Phagosomes
  (SP) in macrophages.

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The Experimental Design

1. Time Lapse Video - Salmonella vs. Yersinia
2. Fluorescent Microscopy Opsinization Effects
3. Time Lapse Video SP persistence
4. PhoPC mutation effects on SP formation

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Time Lapse Video Salmonella typhimurium vs.
Yersinia enterocolitica

• Methods
• Macrophages exposed to either mouse serum
  opsinized Yersinia or Salmonella and recorded by
  time-lapse video phase contrast microscopy.
• Salmonella
• T lt 2 min
• Ruffling and macropinocytosis w/
  nonspecific uptake, nascent phagosomes containing
  bacteria and macropinosomes considered
  morphologically indistinguishable. Both being 2-6
  um in diameter, large enough for he bacteria to
  swim freely inside.
• T gt 2 min
• Enlargement and fusion of bacteria
  containing phagosomes with other phagosomes
  and/or macropinosomes.
• T gt 35 min
• Persistence of phagosomes containing
  bacteria. Limited shrinkage of some phagosomes
  but many continued to fuse with phagosomes and
  macropinosomes.

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S. typhimurium Video Time-Lapse Microscopy
SP formation through phagosomal fusion
T0
T5
T25
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Time Lapse Video Salmonella vs. Yersinia
(continued)

• Opsinized Yersinia
• T 2 min
• Small ruffles form adjacent to bacteria
• tightly adhered to the macrophage surfaces.
  Very
• limited SP formation through phagosomal
  fusion.
• T lt 10 min
• All SP formed deceased significantly in
  size

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We are SO proud of our time-lapse video images

• Is this phagosomal fusion event attributable to
  Salmonella typhimurium?

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Is SP formation independent of opsinization
conditions?

• Methods
• Treat Salmonella with
• Anti-LPS IgG
• Normal Mouse Serum containing Complement Proteins
• Human Recombinant Mannose Binding Protein
• Unopsinized
• Visualize with DAPI (DNA fluorescent label)

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Results Is SP formation independent of
opsinization conditions?

• Entry appeared to be identical between cells
  opsinized with complement and unopsinized cells

• Cells opsinized by Anti LPS IgG entered via
  ruffling of the
• membrane but were enclosed within smaller
  phagosomes.
• The smaller phagosomes containing Salmonella
  opsinized by Anti-
• LPS IgG began to fuse with macropinosomes
  enlarging in size until
• they resembled visually and numerically the
  SPs formed by the
• complement and MBL opsinized bacteria.

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Comparison of Effects of Opsinization of Yersinia
vs. Salmonella on SP Formation
Opsinized Yersinia internalization
Opsinized Salmonella internalization
Images separated by 2 minutes
Images separated by 3.6 minutes
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Persistence of Salmonella induced SPs to compared
to non-Salmonella induced macropinosomes

• Methods
• Time-lapse video microscopy
• Expose macrophages to live and heat-killed
  Salmonella.
• Add gentamycin to culture media to kill all
  noninternalized bacteria
• Count the number of SP present at t10min, t45
  min, t3.5 h, t5.5 h.

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Persistence of Salmonella induced SP

• Comparing this data to dead bacteria, macrophages
  infected with live bacteria had 15x as many SP at
  t10 min (Data not shown).
• Compare this data to that of M-CSF stimulated
  macropinosome formation. M-CSF macropinosomes
  persist for less than 10-15 min.
• It is apparent that many SP do shrink over time,
  but some are able to persist in the macrophage
  for many hours.

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prgs implicated in SP formation by constitutive
pag expression mutant?

• Methods
• Count number of SP formed per 80 macrophages of
  the constitutive pag expression mutant PhoPc and
  wt Salmonella.
• Use this number to determine bacteria present
  in SP.
• Compare PhoPc mutant to other Salmonella mutants,
  including a PhoP null mutant.

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Constitutive pag expression

• Compared to the wt bacteria, only PhoPc mutant
  had significantly lower numbers of SP formation,
  comparable to heat killed bacteria.
• PhoPc mutants also had decreased intracellular
  viability. (data not shown)
• PhoPc revertants formed SP at the same efiiciency
  as wt. (data not shown)

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PhoPc mutant macrophage phagocytosis phenotype

• Similar phagosome morphology as Yersinia
• Fewer ruffle formation, smaller phagosomes and
  less generalized macropinocytosis.
• Possible dose effects due to less active swimming
  compensated for with higher cell concentration,
  produced no change in SP formation.

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Salmonella LPS Induce SP Formation

• Method
• LPS can induce macropinocytosis in BALB/c but not
  C3H/HeJ mouse macrophages.
• C3H/HeJ macrophages are LPS resistant
• SP formed equally well in both strains with live
  Salmonella
• Heat-killed Salmonella and galE mutants (LPS
  deficient) compared to wt Salmonella in their
  ability to form SP

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LPS Not Directly Responsible for Induced
Macropinocytosis

• Heat-killed bacteria did not induce
  macropinocytosis or SP formation.
• galE mutant formed SP at wt efficiency

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Discussion

• Macrophage uptake of Salmonella
• Induced ruffling over a large surface results in
  nonspecific uptake of bacteria.
• Opsinization increased the numbers of bacteria
  internalized by the macrophage during ruffling.
• Normal mechanisms of uptake were not affected
  (eg. LPS-IgG opsinized) with bacteria initially
  enclosed within small phagosomes, but the
  phagosomes fused with macropinosomes and SP.
• SP formation occurs without LPS stimulation
• SPs containing Salmonella persist for
  considerably longer than those without
  Salmonella.
• prg expression is important for intracellular
  survival of Salmonella and is implicated in SP
  formation.

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Discussion (Continued)

• Epithelial vs. Macrophage Mechanism of Invasion
• Both Epithelial and Macrophage invasion involves
  membrane ruffling
• Epithelial invasion appears to require
  cell-to-cell contact and ruffling occurs in a
  localized region.
• Macrophage invasion occurs within minutes of
  exposure with generalized ruffling and does not
  require bacterial adherence.
• Soluble factors responsible for macrophage
  uptake?
• Separate mechanisms responsible for Salmonella
  uptake by epithelial cells and macrophages?

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Discussion (Continued)

• Normal and induced phagocytosis results in SP
  formation
• Possible theories?
• Releases molecules that inhibit phagosome
  shrinkage.
• Alters macrophage transport proteins.
• Releases an osmotically active solute to increase
  fluid volume.
• Alters the interaction of the phagosome with
  other endocytic organelles.

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Discussion (Continued)

• What we know now.
• Lysosomal enzyme, cathepsin L, delivery delayed
  by 30 minutes.
• IgpA lysosomal membrane protein is present in the
  SP by 40 minutes post uptake.
• We can thus infer that the lysosome does fuse
  with the phagosome at the correct time, so
  vesicle trafficking has probably not been
  altered.

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Discussion (Continued)

• PhoPC and related mutants used for determining
  invasion mechanism.
• prgs encode proteins responsible for stimulation
  of macrophage SP formation.
• PhoPC mutants have decreased viability after
  phagocytosis by macrophages.
• PhoPC mutants have decreased SP formation
  comparable to heat-killed Salmonella.
• PhoP null mutants express prgs but not pags. The
  mutation only begins to affect viability after
  acidification of the phagolysosome, when pag
  expression would normally begin

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Discussion (Continued)

• How can SP formation protect Salmonella from the
  macrophage?
• The macrophage may require the small
  phagosome volume to kill the Salmonella.
• May aid in diluting the concentration of critical
  lysosomal enzymes
• Acid tolerant response may be expressed by pags
  that aids in the cells survival in the phagosome
• SP Persistence?
• SP formation may be induced but is the
  variability in SP persistence due to random
  fusion events?