Presenter : Jennifer Bratt Salmonella Stimulate Macrophage Macropinocytosis and Persist within Spacious Phagosomes By C.M. Alpuche-Aranda, E.L. Racoosin, J.A. Swanson, S.I. Miller

Things to consider when reading this article  This article was written in 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. 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. PhoP C mutation effects on SP formation

Time Lapse Video Salmonella typhimurium vs. Yersinia enterocolitica Methods acrophages exposed to either mouse serum opsinized Yersinia or Salmonella and recorded by time-lapse video phase contrast microscopy. 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. 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. Enlargement and fusion of bacteria containing phagosomes with other phagosomes and/or macropinosomes. T > 35 T > 35 min Persistence of phagosomes containing bacteria. Limited shrinkage of some phagosomes but many continued to fuse with phagosomes and macropinosomes. 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 T=0 T=5 T=25 SP formation through phagosomal fusion

Time Lapse Video Salmonella vs. Yersinia (continued) Opsinized Yersinia T = 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 min All SP formed deceased significantly in size 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. Anti-LPS IgG 2. Normal Mouse Serum containing Complement Proteins 3. Human Recombinant Mannose Binding Protein 4. 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 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 Salmonella opsinized by Anti- The smaller phagosomes containing Salmonella 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 the they 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 Yersinia vs. Salmonella on SP Formation Opsinized Yersinia internalizationOpsinized 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 Salmonella. 2. Add gentamycin to culture media to kill all noninternalized bacteria 3. 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 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? 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 PhoP c and wt Salmonella. Use this number to determine % bacteria present in SP. Compare PhoP c mutant to other Salmonella mutants, including a PhoP null mutant.

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

PhoP c 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) PhoP C and related mutants used for determining invasion mechanism.  prgs encode proteins responsible for stimulation of macrophage SP formation.  PhoP C mutants have decreased viability after phagocytosis by macrophages.  PhoP C 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. 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? SP formation may be induced but is the variability in SP persistence due to random fusion events?