Leishmania donovani Infection Causes Distinct Epigenetic DNA Methylation Changes in Host Macrophages

Infection of macrophages by the intracellular protozoan Leishmania leads to down-regulation of a number of macrophage innate host defense mechanisms, thereby allowing parasite survival and replication. The underlying molecular mechanisms involved remain largely unknown. In this study, we assessed epigenetic changes in macrophage DNA methylation in response to infection with L. donovani as a possible mechanism for Leishmania driven deactivation of host defense. We quantified and detected genome-wide changes of cytosine methylation status in the macrophage genome resulting from L. donovani infection. A high confidence set of 443 CpG sites was identified with changes in methylation that correlated with live L. donovani infection. These epigenetic changes affected genes that play a critical role in host defense such as the JAK/STAT signaling pathway and the MAPK signaling pathway. These results provide strong support for a new paradigm in host-pathogen responses, where upon infection the pathogen induces epigenetic changes in the host cell genome resulting in downregulation of innate immunity thereby enabling pathogen survival and replication. We therefore propose a model whereby Leishmania induced epigenetic changes result in permanent down regulation of host defense mechanisms to protect intracellular replication and survival of parasitic cells.     

Anaplasma phagocytophilum AptA modulates Erk1/2 signalling

Anaplasma phagocytophilum causes human granulocytic anaplasmosis, one of the most common tick‐borne diseases in North America. This unusual obligate intracellular pathogen selectively persists within polymorphonuclear leucocytes. In this study, using the yeast surrogate model we identified an A. phagocytophilum virulence protein, AptA (A. phagocytophilum toxin A), that activates mammalian Erk1/2 mitogen‐activated protein kinase. This activation is important for A. phagocytophilum survival within human neutrophils. AptA interacts with the intermediate filament protein vimentin, which is essential for A. phagocytophilum‐induced Erk1/2 activation and infection. A. phagocytophilum infection reorganizes vimentin around the bacterial inclusion, thereby contributing to intracellular survival. These observations reveal a major role for the bacterial protein, AptA, and the host protein, vimentin, in the activation of Erk1/2 during A. phagocytophilum infection.     

Ats-1

Obligatory intracellular life style and a small number of genes for biosynthesis and metabolism necessitate the Gram-negative bacterium, Anaplasma phagocytophilum, to depend on the host cell for nutrients. A. phagocytophilum resides in a membrane-bound inclusion, and secretes a protein, Ats-1 (Anaplasma translocated substrate-1), into the host cell cytoplasm. Ats-1 binds BECN1, a protein critical for autophagy nucleation, and induces autophagosome formation. The autophagosomes traffic to, and fuse with, A. phagocytophilum inclusions, delivering autophagic cargo into the inclusions, which can serve as nutrients for bacterial growth. This finding demonstrates that A. phagocytophilum subverts host cell autophagic machinery to facilitate infection by secreting a BECN1-binding molecule.     

The Anaplasma phagocytophilum effector AmpA hijacks host cell SUMOylation

SUMOylation, the covalent attachment of a member of the small ubiquitin‐like modifier (SUMO) family of proteins to lysines in target substrates, is an essential post‐translational modification in eukaryotes. Microbial manipulation of SUMOylation recently emerged as a key virulence strategy for viruses and facultative intracellular bacteria, the latter of which have only been shown to deploy effectors that negatively regulate SUMOylation. Here, we demonstrate that the obligate intracellular bacterium, Anaplasma phagocytophilum, utilizes an effector, AmpA (A. phagocytophilum post‐translationally modified protein A) that becomes SUMOylated in host cells and this is important for the pathogen's survival. We previously discovered that AmpA (formerly APH1387) localizes to the A. phagocytophilum‐occupied vacuolar membrane (AVM). Algorithmic prediction analyses denoted AmpA as a candidate for SUMOylation. We verified this phenomenon using a SUMO affinity matrix to precipitate both native AmpA and ectopically expressed green fluorescent protein (GFP)‐tagged AmpA. SUMOylation of AmpA was lysine dependent, as SUMO affinity beads failed to precipitate a GFP‐AmpA protein when its lysine residues were substituted with arginine. Ectopically expressed and endogenous AmpA were poly‐SUMOylated, which was consistent with the observation that AmpA colocalizes with SUMO2/3 at the AVM. Only late during the infection cycle did AmpA colocalize with SUMO1, which terminally caps poly‐SUMO2/3 chains. AmpA was also detected in the cytosol of infected host cells, further supporting its secretion and likely participation in interactions that aid pathogen survival. Indeed, whereas siRNA‐mediated knockdown of Ubc9 – a necessary enzyme for SUMOylation – slightly bolstered A. phagocytophilum infection, pharmacologically inhibiting SUMOylation in infected cells significantly reduced the bacterial load. Ectopically expressed GFP‐AmpA served as a competitive agonist against native AmpA in infected cells, while lysine‐deficient GFP‐AmpA was less effective, implying that modification of AmpA lysines is important for infection. Collectively, these data show that AmpA becomes directly SUMOylated during infection, representing a novel tactic for A. phagocytophilum survival.     

Identification and Characterization of Anaplasma phagocytophilum Proteins Involved in Infection of the Tick Vector,Ixodes scapularis

Anaplasma phagocytophilum is an emerging zoonotic pathogen transmitted by Ixodes scapularis that causes human granulocytic anaplasmosis. Here, a high throughput quantitative proteomics approach was used to characterize A. phagocytophilum proteome during rickettsial multiplication and identify proteins involved in infection of the tick vector, I. scapularis. The first step in this research was focused on tick cells infected with A. phagocytophilum and sampled at two time points containing 10–15% and 65–71% infected cells, respectively to identify key bacterial proteins over-represented in high percentage infected cells. The second step was focused on adult female tick guts and salivary glands infected with A. phagocytophilum to compare in vitro results with those occurring during bacterial infection in vivo. The results showed differences in the proteome of A. phagocytophilum in infected ticks with higher impact on protein synthesis and processing than on bacterial replication in tick salivary glands. These results correlated well with the developmental cycle of A. phagocytophilum, in which cells convert from an intracellular reticulated, replicative form to the nondividing infectious dense-core form. The analysis of A. phagocytophilum differentially represented proteins identified stress response (GroEL, HSP70) and surface (MSP4) proteins that were over-represented in high percentage infected tick cells and salivary glands when compared to low percentage infected cells and guts, respectively. The results demonstrated that MSP4, GroEL and HSP70 interact and bind to tick cells, thus playing a role in rickettsia-tick interactions. The most important finding of these studies is the increase in the level of certain bacterial stress response and surface proteins in A. phagocytophilum-infected tick cells and salivary glands with functional implication in tick-pathogen interactions. These results gave a new dimension to the role of these stress response and surface proteins during A. phagocytophilum infection in ticks. Characterization of Anaplasma proteome contributes information on host-pathogen interactions and provides targets for development of novel control strategies for pathogen infection and transmission.     

Variant -and individual dependent nature of persistent Anaplasma phagocytophilum infection

Background

Anaplasma phagocytophilum is the causative agent of tick-borne fever in ruminants and human granulocytotropic anaplasmosis (HGA). The bacterium is able to survive for several months in immune-competent sheep by modifying important cellular and humoral defence mechanisms. Little is known about how different strains of A. phagocytophilum propagate in their natural hosts during persistent infection.

Methods

Two groups of five lambs were infected with each of two 16S rRNA gene variants of A. phagocytophilum, i.e. 16S variant 1 which is identical to GenBank no M73220 and 16S variant 2 which is identical to GenBank no AF336220, respectively. The lambs were infected intravenously and followed by blood sampling for six months. A. phagocytophilum infection in the peripheral blood was detected by absolute quantitative real-time PCR.

Results

Both 16S rRNA gene variants of A. phagocytophilum established persistent infection for at least six months and showed cyclic bacteraemias, but variant 1 introduced more frequent periods of bacteraemia and higher number of organisms than 16S rRNA gene variant 2 in the peripheral blood.

Conclusion

Organisms were available from blood more or less constantly during the persistent infection and there were individual differences in cyclic activity of A. phagocytophilum in the infected animals. Two 16S rRNA gene variants of A. phagocytophilum show differences in cyclic activity during persistent infection in lambs.     

The Anaplasma phagocytophilum‐occupied vacuole selectively recruits Rab‐GTPases that are predominantly associated with recycling endosomes

Anaplasma phagocytophilum is an obligate intracellular bacterium that infects neutrophils to reside within a host cell‐derived vacuole. The A. phagocytophilum‐occupied vacuole (ApV) fails to mature along the endocytic pathway and is non‐fusogenic with lysosomes. Rab GTPases regulate membrane traffic. To better understand how the bacterium modulates the ApV's selective fusogencity, we examined the intracellular localization of 20 green fluorescent protein (GFP) or red fluorescent protein (RFP)‐tagged Rab GTPases in A. phagocytophilum‐infected HL‐60 cells. GFP‐Rab4A, GFP‐Rab10, GFP‐Rab11A, GFP‐Rab14, RFP‐Rab22A and GFP‐Rab35, which regulate endocytic recycling, and GFP‐Rab1, which mediates endoplasmic reticulum to Golgi apparatus trafficking, localize to the ApV. Fluorescently tagged Rabs are recruited to the ApV upon its formation and remain associated throughout infection. Endogenous Rab14 localizes to the ApV. Tetracycline treatment concomitantly promotes loss of recycling endosome‐associated GFP‐Rabs and acquisition of GFP‐Rab5, GFP‐Rab7, and the lysosomal marker, LAMP‐1. Wild‐type and GTPase‐ deficient versions, but not GDP‐restricted versions of GFP‐Rab1, GFP‐Rab4A and GFP‐Rab11A, localize to the ApV. Strikingly, GFP‐Rab10 recruitment to the ApV is guanine nucleotide‐independent. These data establish that A. phagocytophilum selectively recruits Rab GTPases that are primarily associated with recycling endosomes to facilitate its intracellular survival and implicate bacterial proteins in regulating Rab10 membrane cycling on the ApV.     

SOCS-1 inhibition of type I interferon restrains Staphylococcus aureus skin host defense

The skin innate immune response to methicillin-resistant Staphylococcus aureus (MRSA) culminates in the formation of an abscess to prevent bacterial spread and tissue damage. Pathogen recognition receptors (PRRs) dictate the balance between microbial control and injury. Therefore, intracellular brakes are of fundamental importance to tune the appropriate host defense while inducing resolution. The intracellular inhibitor suppressor of cytokine signaling 1 (SOCS-1), a known JAK/STAT inhibitor, prevents the expression and actions of PRR adaptors and downstream effectors. Whether SOCS-1 is a molecular component of skin host defense remains to be determined. We hypothesized that SOCS-1 decreases type I interferon production and IFNAR-mediated antimicrobial effector functions, limiting the inflammatory response during skin infection. Our data show that MRSA skin infection enhances SOCS-1 expression, and both SOCS-1 inhibitor peptide-treated and myeloid-specific SOCS-1 deficient mice display decreased lesion size, bacterial loads, and increased abscess thickness when compared to wild-type mice treated with the scrambled peptide control. SOCS-1 deletion/inhibition increases phagocytosis and bacterial killing, dependent on nitric oxide release. SOCS-1 inhibition also increases the levels of type I and type II interferon levels in vivo. IFNAR deletion and antibody blockage abolished the beneficial effects of SOCS-1 inhibition in vivo. Notably, we unveiled that hyperglycemia triggers aberrant SOCS-1 expression that correlates with decreased overall IFN signatures in the infected skin. SOCS-1 inhibition restores skin host defense in the highly susceptible hyperglycemic mice. Overall, these data demonstrate a role for SOCS-1-mediated type I interferon actions in host defense and inflammation during MRSA skin infection.     

Anaplasma phagocytophilum Rab10‐dependent parasitism of the trans‐Golgi network is critical for completion of the infection cycle

Anaplasma phagocytophilum is an emerging human pathogen and obligate intracellular bacterium. It inhabits a host cell‐derived vacuole and cycles between replicative reticulate cell (RC) and infectious dense‐cored (DC) morphotypes. Host–pathogen interactions that are critical for RC‐to‐DC conversion are undefined. We previously reported that A. phagocytophilum recruits green fluorescent protein (GFP)‐tagged Rab10, a GTPase that directs exocytic traffic from the sphingolipid‐rich trans‐Golgi network (TGN) to its vacuole in a guanine nucleotide‐independent manner. Here, we demonstrate that endogenous Rab10‐positive TGN vesicles are not only routed to but also delivered into the A. phagocytophilum‐occupied vacuole (ApV). Consistent with this finding, A. phagocytophilum incorporates sphingolipids while intracellular and retains them when naturally released from host cells. TGN vesicle delivery into the ApV is Rab10 dependent, up‐regulates expression of the DC‐specific marker, APH1235, and is critical for the production of infectious progeny. The A. phagocytophilum surface protein, uridine monophosphate kinase, was identified as a guanine nucleotide‐independent, Rab10‐specific ligand. These data delineate why Rab10 is important for the A. phagocytophilum infection cycle and expand the understanding of the benefits that exploiting host cell membrane traffic affords intracellular bacterial pathogens.     

Essential Domains of Anaplasma phagocytophilum Invasins Utilized to Infect Mammalian Host Cells

Anaplasma phagocytophilum causes granulocytic anaplasmosis, an emerging disease of humans and domestic animals. The obligate intracellular bacterium uses its invasins OmpA, Asp14, and AipA to infect myeloid and non-phagocytic cells. Identifying the domains of these proteins that mediate binding and entry, and determining the molecular basis of their interactions with host cell receptors would significantly advance understanding of A. phagocytophilum infection. Here, we identified the OmpA binding domain as residues 59 to 74. Polyclonal antibody generated against a peptide spanning OmpA residues 59 to 74 inhibited A. phagocytophilum infection of host cells and binding to its receptor, sialyl Lewis x (sLe^x-capped P-selectin glycoprotein ligand 1. Molecular docking analyses predicted that OmpA residues G61 and K64 interact with the two sLe^x sugars that are important for infection, α2,3-sialic acid and α1,3-fucose. Amino acid substitution analyses demonstrated that K64 was necessary, and G61 was contributory, for recombinant OmpA to bind to host cells and competitively inhibit A. phagocytophilum infection. Adherence of OmpA to RF/6A endothelial cells, which express little to no sLe^x but express the structurally similar glycan, 6-sulfo-sLe^x, required α2,3-sialic acid and α1,3-fucose and was antagonized by 6-sulfo-sLe^x antibody. Binding and uptake of OmpA-coated latex beads by myeloid cells was sensitive to sialidase, fucosidase, and sLe^x antibody. The Asp14 binding domain was also defined, as antibody specific for residues 113 to 124 inhibited infection. Because OmpA, Asp14, and AipA each contribute to the infection process, it was rationalized that the most effective blocking approach would target all three. An antibody cocktail targeting the OmpA, Asp14, and AipA binding domains neutralized A. phagocytophilum binding and infection of host cells. This study dissects OmpA-receptor interactions and demonstrates the effectiveness of binding domain-specific antibodies for blocking A. phagocytophilum infection.