Presenter : Jennifer Bratt

SalmonellaSalmonella Stimulate Stimulate Macrophage Macropinocytosis Macrophage Macropinocytosis

and Persist within Spacious and Persist within Spacious PhagosomesPhagosomes

By C.M. Alpuche-Aranda, E.L. Racoosin, J.A. Swanson, S.I. Miller

Things to consider when reading this Things to consider when reading this articlearticle

This article was written in 1994. A This article was written in 1994. A considerable amount of research has been considerable amount of research has been done on this system post publication..done on this system post publication..

The serovar used in this experiment was The serovar used in this experiment was Salmonella enterica Salmonella enterica SerovarSerovar typhimurium typhimurium and the animal model used in this and the animal model used in this experiment is the murine model. experiment is the murine model.

Salmonella enterica Salmonella enterica SerovarSerovar Typhimurium Typhimurium

Gram negative, facultative intracellular rodGram negative, facultative intracellular rod

The pathogenicity of the The pathogenicity of the typhimuriumtyphimurium Serovar is due to its Serovar is due to its ability to invade…ability to invade…

Peyer’s Patch Peyer’s Patch Luminal epithelial cells Luminal epithelial cells macrophages.macrophages.

The cell’s ability to invade macrophages is The cell’s ability to invade macrophages is essential for systemic infection.essential for systemic infection.

How does the cell invade the How does the cell invade the macrophage?macrophage?

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

PhoQ/PhoP, PhoQ/PhoP, pagpags, and s, and prgprgss

What are all these Acronyms?What are all these Acronyms?

PhoQ - Sensor kinase located on the cell PhoQ - Sensor kinase located on the cell surface. Putative function, vacuolar environment surface. Putative function, vacuolar environment detection.detection.

PhoP - Signaling molecule. Phosphorylated PhoP - Signaling molecule. Phosphorylated signaling activator that regulated the expression signaling activator that regulated the expression of the pags and prgs.of the pags and prgs.

pagpag - PhoP-Activated Gene - PhoP-Activated Gene prgprg - PhoP-Repressed Gene - PhoP-Repressed Gene

PhoQ/PhoP detects the macrophage vacuolar PhoQ/PhoP detects the macrophage vacuolar environment…environment…

How does How does S.S. typhimuriumtyphimurium react to it to promote react to it to promote pathogenicity?pathogenicity?

PhoQ/PhoP control the expression of the pags and prgs.PhoQ/PhoP control the expression of the pags and prgs. The The prgprgs are expressed by Salmonella unless inhibited s are expressed by Salmonella unless inhibited

by PhoP. by PhoP.

As long as the cell does not detect that it is inside a As long as the cell does not detect that it is inside a vacuole, vacuole, prgprgs are expressed. s are expressed.

When the cell sensor, PhoP, detects the vacuolar When the cell sensor, PhoP, detects the vacuolar environment, the expression of environment, the expression of prgprgs is blocked and the s is blocked and the expression of expression of pagspags is initiated. is initiated.

How does the cell survive within the How does the cell survive within the macrophage phagosome?macrophage phagosome?

Salmonella induces the macropinocytosis Salmonella induces the macropinocytosis which results in the formation of Spacious which results in the formation of Spacious

Phagosomes (SP) in macrophages.Phagosomes (SP) in macrophages.

The Experimental DesignThe Experimental Design

1.1. Time Lapse Video - Time Lapse Video - SalmonellaSalmonella vs. vs. YersiniaYersinia

2.2. Fluorescent Microscopy – Opsinization Fluorescent Microscopy – Opsinization EffectsEffects

3.3. Time Lapse Video – SP persistence Time Lapse Video – SP persistence

4.4. PhoPPhoPCC mutation effects on SP formation mutation effects on SP formation

Time Lapse VideoTime Lapse Video Salmonella typhimuriumSalmonella typhimurium vs. vs. Yersinia enterocoliticaYersinia enterocolitica

MethodsMethods

Macrophages exposed to either mouse serum opsinized acrophages exposed to either mouse serum opsinized YersiniaYersinia or or SalmonellaSalmonella and recorded by time-lapse video phase contrast and recorded by time-lapse video phase contrast microscopy.microscopy.

SalmonellaSalmonella

T < 2 minT < 2 min Ruffling and macropinocytosis w/ nonspecific uptake, nascent phagosomes Ruffling and macropinocytosis w/ nonspecific uptake, nascent phagosomes

containing bacteria and macropinosomes considered morphologically containing bacteria and macropinosomes considered morphologically indistinguishable. Both being 2-6 um in diameter, large enough for he bacteria indistinguishable. Both being 2-6 um in diameter, large enough for he bacteria to swim freely inside.to swim freely inside.

T > 2 minT > 2 min Enlargement and fusion of bacteria containing phagosomes with other Enlargement and fusion of bacteria containing phagosomes with other

phagosomes and/or macropinosomes.phagosomes and/or macropinosomes.

T > 35 T > 35 min Persistence of phagosomes containing bacteria. Limited shrinkage of some Persistence of phagosomes containing bacteria. Limited shrinkage of some

phagosomes but many continued to fuse with phagosomes and phagosomes but many continued to fuse with phagosomes and macropinosomes.macropinosomes.

S. typhimuriumS. typhimurium Video Time-Lapse Video Time-Lapse MicroscopyMicroscopy

T=0 T=5 T=25

SP formation through phagosomal fusion

Time Lapse VideoTime Lapse Video SalmonellaSalmonella vs. vs. Yersinia (continued)Yersinia (continued)

Opsinized Opsinized YersiniaYersinia

T = 2 minT = 2 min Small ruffles form adjacent to bacteria Small ruffles form adjacent to bacteria tightly adhered to the macrophage surfaces. Very tightly adhered to the macrophage surfaces. Very limited SP formation through phagosomal fusion.limited SP formation through phagosomal fusion.

T < 10 minT < 10 min All SP formed deceased significantly in sizeAll SP formed deceased significantly in size

We are SO proud of our time-lapse We are SO proud of our time-lapse video images…video images…

Is this phagosomal fusion event attributable to Is this phagosomal fusion event attributable to Salmonella typhimuriumSalmonella typhimurium? ?

Is SP formation independent of Is SP formation independent of opsinization conditions?opsinization conditions?

MethodsMethods

Treat Salmonella with…Treat Salmonella with… 1.1. Anti-LPS IgG Anti-LPS IgG

2.2. Normal Mouse Serum containing Complement ProteinsNormal Mouse Serum containing Complement Proteins

3.3. Human Recombinant Mannose Binding ProteinHuman Recombinant Mannose Binding Protein

4.4. UnopsinizedUnopsinized

Visualize with DAPI (DNA fluorescent label)Visualize with DAPI (DNA fluorescent label)

Results – Is SP formation independent of Results – Is SP formation independent of opsinization conditions?opsinization conditions?

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

• Cells opsinized by Anti LPS IgG entered via ruffling of the Cells opsinized by Anti LPS IgG entered via ruffling of the membrane but were enclosed within smaller phagosomes.membrane but were enclosed within smaller phagosomes.

• The smaller phagosomes containing The smaller phagosomes containing SalmonellaSalmonella opsinized by Anti- opsinized by Anti- LPS IgG began to fuse with macropinosomes enlarging in size until LPS IgG began to fuse with macropinosomes enlarging in size until they resembled visually and numerically the SPs formed by thethey resembled visually and numerically the SPs formed by the complement and MBL opsinized bacteria. complement and MBL opsinized bacteria.

Comparison of Effects of Opsinization of Comparison of Effects of Opsinization of YersiniaYersinia vs. vs. Salmonella on SP FormationSalmonella on SP Formation

Opsinized Yersinia internalization Opsinized Salmonella internalization

Images separated by 2 minutes Images separated by 3.6 minutes

Persistence of Persistence of Salmonella Salmonella induced SPs to induced SPs to compared to non-compared to non-SalmonellaSalmonella induced induced

macropinosomesmacropinosomes

MethodsMethodsTime-lapse video microscopyTime-lapse video microscopy

1.1. Expose macrophages to live and heat-killed Expose macrophages to live and heat-killed SalmonellaSalmonella..

2.2. Add gentamycin to culture media to kill all Add gentamycin to culture media to kill all noninternalized bacterianoninternalized bacteria

3.3. Count the number of SP present at t=10min, t=45 min, Count the number of SP present at t=10min, t=45 min,

t=3.5 h, t=5.5 h.t=3.5 h, t=5.5 h.

Persistence of Persistence of SalmonellaSalmonella induced SP induced SP

Comparing this data to dead bacteria, macrophages infected with Comparing this data to dead bacteria, macrophages infected with live bacteria had 15x as many SP at t=10 min (Data not shown).live bacteria had 15x as many SP at t=10 min (Data not shown).

Compare this data to that of M-CSF stimulated macropinosome Compare this data to that of M-CSF stimulated macropinosome formation. M-CSF macropinosomes persist for less than 10-15 formation. M-CSF macropinosomes persist for less than 10-15 min. min.

It is apparent that many SP do shrink over time, but some are able It is apparent that many SP do shrink over time, but some are able to persist in the macrophage for many hours.to persist in the macrophage for many hours.

prgprgs implicated in SP formation by s implicated in SP formation by constitutive constitutive pagpag expression mutant? expression mutant?

MethodsMethods

Count number of SP formed per 80 macrophages of the Count number of SP formed per 80 macrophages of the constitutive pag expression mutant constitutive pag expression mutant PhoPPhoPcc and and wt wt

Salmonella.Salmonella. Use this number to determine % bacteria present in Use this number to determine % bacteria present in

SP.SP.

Compare Compare PhoPPhoPcc mutant to other Salmonella mutants, mutant to other Salmonella mutants, including a including a PhoP nullPhoP null mutant. mutant.

Constitutive pag expressionConstitutive pag expression

Compared to the Compared to the wtwt bacteria, only bacteria, only PhoPPhoPcc mutant had significantly mutant had significantly lower numbers of SP lower numbers of SP formation, comparable to formation, comparable to heat killed bacteria.heat killed bacteria.

PhoPPhoPcc mutants also had mutants also had decreased intracellular decreased intracellular viability. (data not shown)viability. (data not shown)

PhoPPhoPcc revertants formed revertants formed SP at the same efiiciency SP at the same efiiciency as as wt. wt. (data not shown)(data not shown)

PhoPPhoPcc mutant macrophage mutant macrophage phagocytosis phenotypephagocytosis phenotype

Similar phagosome Similar phagosome morphology as morphology as YersiniaYersinia

Fewer ruffle formation, Fewer ruffle formation, smaller phagosomes and smaller phagosomes and less generalized less generalized macropinocytosis.macropinocytosis.

Possible dose effects due Possible dose effects due to less active swimming to less active swimming compensated for with compensated for with higher cell concentration, higher cell concentration, produced no change in produced no change in SP formation.SP formation.

Salmonella LPS Induce SP FormationSalmonella LPS Induce SP Formation

MethodMethod LPS can induce macropinocytosis in BALB/c but not LPS can induce macropinocytosis in BALB/c but not

C3H/HeJ mouse macrophages.C3H/HeJ mouse macrophages.

C3H/HeJ macrophages are LPS resistantC3H/HeJ macrophages are LPS resistant

SP formed equally well in both strains with live SP formed equally well in both strains with live

SalmonellaSalmonella

Heat-killed Heat-killed SalmonellaSalmonella and and galEgalE mutants (LPS deficient) mutants (LPS deficient) compared to compared to wtwt Salmonella in their ability to form SP Salmonella in their ability to form SP

LPS Not Directly Responsible for Induced LPS Not Directly Responsible for Induced

MacropinocytosisMacropinocytosis

Heat-killed bacteria did not induce Heat-killed bacteria did not induce macropinocytosis or SP formation.macropinocytosis or SP formation.

galEgalE mutant formed SP at wt efficiency mutant formed SP at wt efficiency

DiscussionDiscussion

Macrophage uptake of Macrophage uptake of SalmonellaSalmonella Induced ruffling over a large surface results in nonspecific Induced ruffling over a large surface results in nonspecific

uptake of bacteria.uptake of bacteria. Opsinization increased the numbers of bacteria internalized by Opsinization increased the numbers of bacteria internalized by

the macrophage during ruffling.the macrophage during ruffling. Normal mechanisms of uptake were not affected (eg. LPS-IgG Normal mechanisms of uptake were not affected (eg. LPS-IgG

opsinized) with bacteria initially enclosed within small opsinized) with bacteria initially enclosed within small phagosomes, but the phagosomes fused with macropinosomes phagosomes, but the phagosomes fused with macropinosomes and SP. and SP.

SP formation occurs without LPS stimulationSP formation occurs without LPS stimulation SPs containing SPs containing SalmonellaSalmonella persist for considerably longer than persist for considerably longer than

those without those without SalmonellaSalmonella. . prgprg expression is important for intracellular survival of expression is important for intracellular survival of

SalmonellaSalmonella and is implicated in SP formation. and is implicated in SP formation.

Discussion (Continued)Discussion (Continued)

Epithelial vs. Macrophage Mechanism of InvasionEpithelial vs. Macrophage Mechanism of Invasion Both Epithelial and Macrophage invasion involves membrane Both Epithelial and Macrophage invasion involves membrane

rufflingruffling

Epithelial invasion appears to require cell-to-cell contact and Epithelial invasion appears to require cell-to-cell contact and ruffling occurs in a localized region.ruffling occurs in a localized region.

Macrophage invasion occurs within minutes of exposure with Macrophage invasion occurs within minutes of exposure with generalized ruffling and does not require bacterial adherence.generalized ruffling and does not require bacterial adherence.

Soluble factors responsible for macrophage uptake?Soluble factors responsible for macrophage uptake?Separate mechanisms responsible for Separate mechanisms responsible for SalmonellaSalmonella uptake by uptake by

epithelial cells and macrophages?epithelial cells and macrophages?

Discussion (Continued)Discussion (Continued)Normal and “induced” phagocytosis results in SP Normal and “induced” phagocytosis results in SP

formationformation

Possible theories?Possible theories?

Releases molecules that inhibit phagosome shrinkage.Releases molecules that inhibit phagosome shrinkage. Alters macrophage transport proteins.Alters macrophage transport proteins. Releases an osmotically active solute to increase fluid volume.Releases an osmotically active solute to increase fluid volume. Alters the interaction of the phagosome with other endocytic Alters the interaction of the phagosome with other endocytic

organelles.organelles.

Discussion (Continued)Discussion (Continued)

What we know now.What we know now.

Lysosomal enzyme, cathepsin L, delivery delayed by 30 Lysosomal enzyme, cathepsin L, delivery delayed by 30 minutes.minutes.

IgpA lysosomal membrane protein is present in the SP IgpA lysosomal membrane protein is present in the SP by 40 minutes post uptake.by 40 minutes post uptake.

We can thus infer that the lysosome does fuse with the We can thus infer that the lysosome does fuse with the phagosome at the correct time, so vesicle trafficking has phagosome at the correct time, so vesicle trafficking has probably not been altered.probably not been altered.

Discussion (Continued) Discussion (Continued)

PhoPPhoPCC and related mutants used for determining and related mutants used for determining invasion mechanism.invasion mechanism.

prgsprgs encode proteins responsible for stimulation of encode proteins responsible for stimulation of macrophage SP formation.macrophage SP formation.

PhoPPhoPCC mutants have decreased viability after mutants have decreased viability after phagocytosis by macrophages.phagocytosis by macrophages.

PhoPPhoPCC mutants have decreased SP formation mutants have decreased SP formation comparable to heat-killed comparable to heat-killed SalmonellaSalmonella..

PhoP nullPhoP null mutants express mutants express prgprgs but not s but not pagpags. The s. The mutation only begins to affect viability after acidification mutation only begins to affect viability after acidification of the phagolysosome, when of the phagolysosome, when pagpag expression would expression would normally beginnormally begin

Discussion (Continued)Discussion (Continued)

How can SP formation protect How can SP formation protect SalmonellaSalmonella from the from the macrophage?macrophage?

The macrophage may require the small phagosome volume to kill The macrophage may require the small phagosome volume to kill the the Salmonella.Salmonella.

May aid in diluting the concentration of critical lysosomal enzymes May aid in diluting the concentration of critical lysosomal enzymes Acid tolerant response may be expressed by pags that aids in the Acid tolerant response may be expressed by pags that aids in the

cell’s survival in the phagosomecell’s survival in the phagosome

SP Persistence?SP Persistence?

SP formation may be induced but is the variability in SP persistence SP formation may be induced but is the variability in SP persistence due to random fusion events?due to random fusion events?