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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
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.
Salmonella enterica Serovar Typhimurium
Gram negative, facultative intracellular rod
The pathogenicity of the typhimurium Serovar is due to its
ability to invade…
 Peyer’s Patch
 Luminal epithelial cells
 macrophages.
The cell’s ability to invade macrophages is
essential for systemic infection.
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.
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
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.
How does the cell survive within the
macrophage phagosome?
Salmonella induces the macropinocytosis
which results in the formation of Spacious
Phagosomes (SP) in macrophages.
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
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 < 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 > 2 min
Enlargement and fusion of bacteria containing phagosomes with other
phagosomes and/or macropinosomes.
T > 35 min
Persistence of phagosomes containing bacteria. Limited shrinkage of some
phagosomes but many continued to fuse with phagosomes and
macropinosomes.
S. typhimurium Video Time-Lapse
Microscopy
SP formation through phagosomal fusion
T=0
T=5
T=25
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 < 10 min
All SP formed deceased significantly in size
We are SO proud of our time-lapse
video images…
Is this phagosomal fusion event attributable to
Salmonella typhimurium?
Is SP formation independent of
opsinization conditions?
Methods
Treat Salmonella with…
1.
2.
3.
4.
Anti-LPS IgG
Normal Mouse Serum containing Complement Proteins
Human Recombinant Mannose Binding Protein
Unopsinized
Visualize with DAPI (DNA fluorescent label)
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 AntiLPS 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.
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
Persistence of Salmonella induced SPs to
compared to non-Salmonella induced
macropinosomes
Methods
Time-lapse video microscopy
1. Expose macrophages to live and heat-killed
2.
3.
Salmonella.
Add gentamycin to culture media to kill all
noninternalized bacteria
Count the number of SP present at t=10min, t=45 min,
t=3.5 h, t=5.5 h.
Persistence of Salmonella induced SP
Comparing this data to dead bacteria, macrophages infected
with live bacteria had 15x as many SP at t=10 min (Data not
shown).
Compare this data to that of M-CSF stimulated macropinosome
formation. M-CSF macropinosomes persist for less than 1015 min.
It is apparent that many SP do shrink over time, but some are
able to persist in the macrophage for many hours.
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.
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)
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.
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
LPS Not Directly Responsible for Induced
Macropinocytosis
 Heat-killed bacteria did not induce
macropinocytosis or SP formation.
 galE mutant formed SP at wt efficiency
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.
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?
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.
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.
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
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
cell’s survival in the phagosome
SP Persistence?
SP formation may be induced but is the variability in SP persistence
due to random fusion events?