Galina Romanov

Yersinia pestis Can Reside in Autophagosomes and Avoid Xenophagy in Murine Macrophages by Preventing Vacuole Acidification

ABSTRACT Yersinia pestis survives and replicates in phagosomes of murine macrophages. Previous studies demonstrated that Y. pestis-containing vacuoles (YCVs) acquire markers of late endosomes or lysosomes in naïve macrophages and that this bacterium can survive in macrophages activated with the cytokine gamma interferon. An autophagic process known as xenophagy, which destroys pathogens in acidic autophagolysosomes, can occur in naïve macrophages and is upregulated in activated macrophages. Studies were undertaken here to investigate the mechanism of Y. pestis survival in phagosomes of naïve and activated macrophages and to determine if the pathogen avoids or co-opts autophagy. Colocalization of the YCV with markers of autophagosomes or acidic lysosomes and the pH of the YCV were determined by microscopic imaging of infected macrophages. Some YCVs contained double membranes characteristic of autophagosomes, as determined by electron microscopy. Fluorescence microscopy showed that ∼40% of YCVs colocalized with green fluorescent protein (GFP)-LC3, a marker of autophagic membranes, and that YCVs failed to acidify below pH 7 in naïve macrophages. Replication of Y. pestis in naïve macrophages caused accumulation of LC3-II, as determined by immunoblotting. While activation of infected macrophages increased LC3-II accumulation, it decreased the percentage of GFP-LC3-positive YCVs (∼30%). A viable count assay showed that Y. pestis survived equally well in macrophages proficient for autophagy and macrophages rendered deficient for this process by Cre-mediated deletion of ATG5, revealing that this pathogen does not require autophagy for intracellular replication. We conclude that although YCVs can acquire an autophagic membrane and accumulate LC3-II, the pathogen avoids xenophagy by preventing vacuole acidification.

Assembly of hereditary amyloid β‐protein variants in the presence of favorable gangliosides

The mechanisms underlying regional amyloid β‐protein (Aβ) deposition in brain remain unclear. Here we show that assembly of hereditary variant Dutch‐ and Italian‐type Aβs, and Flemish‐type Aβ was accelerated by GM3 ganglioside, and GD3 ganglioside, respectively. Notably, cerebrovascular smooth muscle cells, which compose the cerebral vessel wall at which the Dutch‐ and Italian‐type Aβs deposit, exclusively express GM3 whereas GD3 is upregulated in the co‐culture of endothelial cells and astrocytes, which forms the cerebrovascular basement membrane, the site of Flemish‐type Aβ deposition. Our results suggest that regional Aβ deposition is induced by the local gangliosides in the brain.

Deficient cerebral clearance of vasculotropic mutant Dutch/Iowa Double Aß in human AßPP transgenic mice

Cerebral amyloid angiopathy (CAA) is a prominent pathological feature of Alzheimers disease and related familial CAA disorders. However, the mechanisms that account for the cerebral vascular accumulation of amyloid beta-peptide (A beta) have not been defined. Recently, we reported novel transgenic mice (Tg-SwDI) expressing neuronally derived Swedish/Dutch/Iowa vasculotropic mutant human A beta precursor (A betaPP) that develop early-onset and robust accumulation of fibrillar cerebral microvascular A beta. Deficient clearance of Dutch/Iowa mutant A beta from brain across the capillary blood-brain barrier into the circulation may contribute to its potent cerebral accumulation. To further evaluate this theory, we generated a new transgenic mouse (Tg-Sw) that is nearly identical to Tg-SwDI, except lacking the Dutch/Iowa A beta mutations. Tg-Sw and Tg-SwDI mice expressed comparable levels of human A betaPP in brain and not in peripheral tissues. However, Tg-SwDI mice strongly accumulated Dutch/Iowa mutant A beta in brain, particularly in the cerebral microvasculature, whereas Tg-Sw mice exhibited no accumulations of wild-type A beta. Conversely, Tg-SwDI mice had no detectable Dutch/Iowa mutant A beta in plasma whereas Tg-Sw mice exhibited consistent levels of human wild-type A beta in plasma. Together, these findings suggest that while wild-type A beta is readily transported out of brain into plasma, Dutch/Iowa mutant A beta is deficient in this clearance process, likely contributing to its robust accumulation in the cerebral vasculature.

A Transposon Site Hybridization Screen Identifies galU and wecBC as Important for Survival of Yersinia pestis in Murine Macrophages

Yersinia pestis is able to survive and replicate within murine macrophages. However, the mechanism by which Y. pestis promotes its intracellular survival is not well understood. To identify genes that are important for Y. pestis survival in macrophages, a library comprised of ∼31,500 Y. pestis KIM6+ transposon insertion mutants (input pool) was subjected to negative selection in primary murine macrophages. Genes underrepresented in the output pool of surviving bacteria were identified by transposon site hybridization to DNA oligonucleotide microarrays. The screen identified several genes known to be important for survival of Y. pestis in macrophages, including phoPQ and members of the PhoPQ regulon (e.g., pmrF). In addition, genes predicated to encode a glucose-1-phosphate uridylyltransferase (galU), a UDP-N-acetylglucosamine 2-epimerase (wecB) and a UDP-N-acetyl-d-mannosamine dehydrogenase (wecC) were identified in the screen. Viable-count assays demonstrated that a KIM6+ galU mutant and a KIM6+ wecBC mutant were defective for survival in murine macrophages. The galU mutant was studied further because of its strong phenotype. The KIM6+ galU mutant exhibited increased susceptibility to the antimicrobial peptides polymyxin B and cathelicidin-related antimicrobial peptide (CRAMP). Polyacrylamide gel electrophoresis demonstrated that the lipooligosaccharide (LOS) of the galU mutant migrated faster than the LOS of the parent KIM6+, suggesting the core was truncated. In addition, the analysis of LOS isolated from the galU mutant by mass spectrometry showed that aminoarabinose modification of lipid A is absent. Therefore, addition of aminoarabinose to lipid A and complete LOS core (galU), as well as enterobacterial common antigen (wecB and wecC), is important for survival of Y. pestis in macrophages.

A Protective Epitope in Type III Effector YopE Is a Major CD8 T Cell Antigen during Primary Infection with Yersinia pseudotuberculosis

ABSTRACT Virulence in human-pathogenic Yersinia species is associated with a plasmid-encoded type III secretion system that translocates a set of Yop effector proteins into host cells. One effector, YopE, functions as a Rho GTPase-activating protein (GAP). In addition to acting as a virulence factor, YopE can function as a protective antigen. C57BL/6 mice infected with attenuated Yersinia pestis generate a dominant H2-Kb-restricted CD8 T cell response to an epitope in the N-terminal domain of YopE (YopE69-77), and intranasal vaccination with the YopE69-77 peptide and the mucosal adjuvant cholera toxin (CT) elicits CD8 T cells that are protective against lethal pulmonary challenge with Y. pestis. Because YopE69-77 is conserved in many Yersinia strains, we sought to determine if YopE is a protective antigen for Yersinia pseudotuberculosis and if primary infection with this enteric pathogen elicits a CD8 T cell response to this epitope. Intranasal immunization with the YopE69-77 peptide and CT elicited a CD8 T cell response that was protective against lethal intragastric Y. pseudotuberculosis challenge. The YopE69-77 epitope was a major antigen (∼30% of splenic CD8 T cells were specific for this peptide at the peak of the response) during primary infection with Y. pseudotuberculosis, as shown by flow cytometry tetramer staining. Results of infections with Y. pseudotuberculosis expressing catalytically inactive YopE demonstrated that GAP activity is dispensable for a CD8 T cell response to YopE69-77. Determining the features of YopE that are important for this response will lead to a better understanding of how protective CD8 T cell immunity is generated against Yersinia and other pathogens with type III secretion systems.

Type III Secretion System-Dependent Translocation of Ectopically Expressed Yop Effectors into Macrophages by Intracellular Yersinia pseudotuberculosis

ABSTRACT Yersinia pseudotuberculosis is a Gram-negative bacterial pathogen. Virulence in Y. pseudotuberculosis requires the plasmid-encoded Ysc type III secretion system (T3SS), which functions to translocate a set of effectors called Yops into infected host cells. The effectors function to antagonize phagocytosis (e.g., YopH) or to induce apoptosis (YopJ) in macrophages infected with Y. pseudotuberculosis. Additionally, when antiphagocytosis is incomplete and Y. pseudotuberculosis is internalized by macrophages, the bacterium can survive in phagosomes. Previous studies have shown that delivery of effectors into host cells occurs efficiently when Yersinia is extracellular. However, it is not clear whether the T3SS can be utilized by intracellular Y. pseudotuberculosis to translocate Yops. This possibility was investigated here using Y. pseudotuberculosis strains that express YopJ or YopH under the control of an inducible promoter. Bone marrow-derived murine macrophages were infected with these strains under conditions that prevented the survival of extracellular bacteria. Effector translocation was detected by measuring apoptosis or the activities of Yop-β-lactamase fusion proteins. Results showed that macrophages underwent apoptosis when YopJ expression was induced prior to phagocytosis, confirming that delivery of this effector prior to or during uptake is sufficient to cause cell death. However, macrophages also underwent apoptosis when YopJ was ectopically expressed after phagocytosis; furthermore, expression of the translocator YopB from intracellular bacteria also resulted in increased cell death. Analysis by microscopy showed that translocation of ectopically expressed YopH- or YopJ-β-lactamase fusions could be correlated with the presence of viable Y. pseudotuberculosis in macrophages. Collectively, our results suggest that the Ysc T3SS of Y. pseudotuberculosis can function within macrophage phagosomes to translocate Yops into the host cytosol.

Effector CD8+ T Cells Are Generated in Response to an Immunodominant Epitope in Type III Effector YopE during Primary Yersinia pseudotuberculosis Infection

ABSTRACT YopE is a virulence factor that is secreted into host cells infected by Yersinia species. The YopE C-terminal domain has GTPase-activating protein (GAP) activity. The YopE N-terminal domain contains an epitope that is an immunodominant CD8+ T cell antigen during primary infection of C57BL/6 mice with Yersinia pseudotuberculosis. The characteristics of the CD8+ T cells generated in response to the epitope, which comprises YopE amino acid residues 69 to 77 (YopE69–77), and the features of YopE that are important for antigenicity during primary infection, are unknown. Following intravenous infection of naïve C57BL/6 mice with a yopE GAP mutant (the R144A mutant), flow cytometry analysis of splenocytes by tetramer and intracellular cytokine staining over a time course showed that YopE69–77-specific CD8+ T cells producing gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) were generated by day 7, with a peak at day 14. In addition, ∼80% of YopE69–77-specific CD8+ T cells were positive for KLRG1, a memory phenotype marker, at day 21. To determine if residues that regulate YopE activity by ubiquitination or membrane localization affect the antigenicity of YopE69–77, mice were infected with a yopE ubiquitination or membrane localization mutant (the R62K or L55N I59N L63N mutant, respectively). These mutants elicited YopE69–77-specific CD8+ T cells producing IFN-γ and TNF-α with kinetics and magnitudes similar to those of the parental R144A strain, indicating that primary infection primes effector CD8+ T cells independently of the ubiquitination or membrane localization of YopE. Additionally, at day 7, there was an unexpected positive correlation between the numbers of YopE69–77-specific CD8+ T cells and CD11b+ cells, but not between the numbers of YopE69–77-specific CD8+ T cells and bacterial cells, in spleens, suggesting that the innate immune response contributes to the immunodominance of YopE69–77.

P1-037: Protease nexin-2/amyloid ß-protein precursor regulates the extent of cerebral thrombosis

ods: APPDutch mice (overexpressing hAPP E693Q) were crossed with transgenic PS45 (overexpressing hPS1 G384A), BACE54 (overexpressing hBACE1), or APP23 mice (overexpressing hAPP K670N/M671L). All mice were analyzed at 22-24 months of age by immunohistochemistry and Western blotting. Results: APPDutch/PS45 mice develop severe A 42-driven parenchymal amyloidosis with very little vascular amyloid. APPDutch/BACE54 mice reveal more vascular amyloid deposits compared to APPDutch mice and furthermore exhibit parenchymal amyloid. APPDutch/APP23 mice show more vascular amyloid and, interestingly, less parenchymal amyloid compared to APP23 mice. Amyloid deposits in these double-transgenic mice mainly contain A 40 that in turn consists of both mutated and wild-type A , respectively. Conclusions: A 42 and A 40 drive amyloid pathology in different cerebral compartments, i.e. parenchyma versus vasculature. Abundant A 42 provokes the formation of parenchymal amyloid, while a specific increase in A 40 favors the development of vascular amyloid and appears to reduce parenchymal amyloid formation.