Ceramide-1-phosphate is a regulator of Golgi structure and is co-opted by the obligate intracellular bacterial pathogen Anaplasma phagocytophilum.

Many intracellular pathogens structurally disrupt the Golgi apparatus as an evolutionarily conserved promicrobial strategy. Yet, the host factors and signaling processes involved are often poorly understood, particularly for Anaplasma phagocytophilum, the agent of human granulocytic anaplasmosis. We found that A. phagocytophilum elevated cellular levels of the bioactive sphingolipid, ceramide-1-phosphate (C1P), to promote Golgi fragmentation that enables bacterial proliferation, conversion from its non-infectious to infectious form, and productive infection. A. phagocytophilum poorly infected mice deficient in ceramide kinase, the Golgi-localized enzyme responsible for C1P biosynthesis. C1P regulated Golgi morphology via activation of a PKCα/Cdc42/JNK signaling axis that culminates in phosphorylation of Golgi structural proteins, GRASP55 and GRASP65. siRNA-mediated depletion of Cdc42 blocked A. phagocytophilum from altering Golgi morphology, which impaired anterograde trafficking of trans-Golgi vesicles into and maturation of the pathogen-occupied vacuole. Cells overexpressing phosphorylation-resistant versions of GRASP55 and GRASP65 presented with suppressed C1P- and A. phagocytophilum-induced Golgi fragmentation and poorly supported infection by the bacterium. By studying A. phagocytophilum, we identify C1P as a regulator of Golgi structure and a host factor that is relevant to disease progression associated with Golgi fragmentation.IMPORTANCECeramide-1-phosphate (C1P), a bioactive sphingolipid that regulates diverse processes vital to mammalian physiology, is linked to disease states such as cancer, inflammation, and wound healing. By studying the obligate intracellular bacterium Anaplasma phagocytophilum, we discovered that C1P is a major regulator of Golgi morphology. A. phagocytophilum elevated C1P levels to induce signaling events that promote Golgi fragmentation and increase vesicular traffic into the pathogen-occupied vacuole that the bacterium parasitizes. As several intracellular microbial pathogens destabilize the Golgi to drive their infection cycles and changes in Golgi morphology is also linked to cancer and neurodegenerative disorder progression, this study identifies C1P as a potential broad-spectrum therapeutic target for infectious and non-infectious diseases.

Selenomonas sputigena Interactions with Gingival Epithelial Cells That Promote Inflammation.

Increased prevalence and abundance of Selenomonas sputigena have been associated with periodontitis, a chronic inflammatory disease of tooth-supporting tissues, for more than 50 years. Over the past decade, molecular surveys of periodontal disease using 16S and shotgun metagenomic sequencing approaches have confirmed the disease association of classically recognized periodontal pathogens such as Porphyromonas gingivalis, Treponema denticola, and Tannerella forsythia while highlighting previously underappreciated organisms such as Filifactor alocis and S. sputigena. Despite abundant clinical association between S. sputigena and periodontal disease, we have little to no understanding of its pathogenic potential, and virulence mechanisms have not been studied. In this study, we sought to characterize the response of gingival epithelial cells to infection with S. sputigena. Here, we show that S. sputigena attaches to gingival keratinocytes and induces expression and secretion of cytokines and chemokines associated with inflammation and leukocyte recruitment. We demonstrate that S. sputigena induces signaling through Toll-like receptor 2 (TLR2) and TLR4 but evades activation of TLR5. Cytokines released from S. sputigena-infected keratinocytes induced monocyte and neutrophil chemotaxis. These results show that S. sputigena-host interactions have the potential to contribute to bacterially driven inflammation and tissue destruction, the hallmark of periodontitis. Characterization of previously unstudied pathogens may provide novel approaches to develop therapeutics to treat or prevent periodontal disease.

The Obligate Intracellular Bacterial Pathogen Anaplasma phagocytophilum Exploits Host Cell Multivesicular Body Biogenesis for Proliferation and Dissemination.

Anaplasma phagocytophilum is the etiologic agent of the emerging infection, granulocytic anaplasmosis. This obligate intracellular bacterium lives in a host cell-derived vacuole that receives membrane traffic from multiple organelles to fuel its proliferation and from which it must ultimately exit to disseminate infection. Understanding of these essential pathogenic mechanisms has remained poor. Multivesicular bodies (MVBs) are late endosomal compartments that receive biomolecules from other organelles and encapsulate them into intralumenal vesicles (ILVs) using endosomal sorting complexes required for transport (ESCRT) machinery and ESCRT-independent machinery. Association of the ESCRT-independent protein, ALIX, directs MVBs to the plasma membrane where they release ILVs as exosomes. We report that the A. phagocytophilum vacuole (ApV) is acidified and enriched in lysobisphosphatidic acid, a lipid that is abundant in MVBs. ESCRT-0 and ESCRT-III components along with ALIX localize to the ApV membrane. siRNA-mediated inactivation of ESCRT-0 and ALIX together impairs A. phagocytophilum proliferation and infectious progeny production. RNA silencing of ESCRT-III, which regulates ILV scission, pronouncedly reduces ILV formation in ApVs and halts infection by arresting bacterial growth. Rab27a and its effector Munc13-4, which drive MVB trafficking to the plasma membrane and subsequent exosome release, localize to the ApV. Treatment with Nexinhib20, a small molecule inhibitor that specifically targets Rab27a to block MVB exocytosis, abrogates A. phagocytophilum infectious progeny release. Thus, A. phagocytophilum exploits MVB biogenesis and exosome release to benefit each major stage of its intracellular infection cycle: intravacuolar growth, conversion to the infectious form, and exit from the host cell. IMPORTANCE Anaplasma phagocytophilum causes granulocytic anaplasmosis, a globally emerging zoonosis that can be severe, even fatal, and for which antibiotic treatment options are limited. A. phagocytophilum lives in an endosomal-like compartment that interfaces with multiple organelles and from which it must ultimately exit to spread within the host. How the bacterium accomplishes these tasks is poorly understood. Multivesicular bodies (MVBs) are intermediates in the endolysosomal pathway that package biomolecular cargo from other organelles as intralumenal vesicles for release at the plasma membrane as exosomes. We discovered that A. phagocytophilum exploits MVB biogenesis and trafficking to benefit all aspects of its intracellular infection cycle: proliferation, conversion to its infectious form, and release of infectious progeny. The ability of a small molecule inhibitor of MVB exocytosis to impede A. phagocytophilum dissemination indicates the potential of this pathway as a novel host-directed therapeutic target for granulocytic anaplasmosis.

Orientia tsutsugamushi OtDUB Is Expressed and Interacts with Adaptor Protein Complexes during Infection.

Orientia tsutsugamushi is an etiologic agent of scrub typhus, a globally emerging rickettsiosis that can be fatal. The bacterium's obligate intracellular lifestyle requires its interaction with host eukaryotic cellular pathways. The proteins it employs to do so and their functions during infection are understudied. Recombinant versions of the recently characterized O. tsutsugamushi deubiquitylase (OtDUB) exhibit high-affinity ubiquitin binding, mediate guanine nucleotide exchange to activate Rho GTPases, bind clathrin adaptor protein complexes 1 and 2, and bind the phospholipid phosphatidylserine. Whether OtDUB is expressed and its function during O. tsutsugamushi infection have yet to be explored. Here, OtDUB expression, location, and interactome during infection were examined. O. tsutsugamushi transcriptionally and translationally expresses OtDUB throughout infection of epithelial, monocytic, and endothelial cells. Results from structured illumination microscopy, surface trypsinization of intact bacteria, and acetic acid extraction of non-integral membrane proteins indicate that OtDUB peripherally associates with the O. tsutsugamushi cell wall and is at least partially present on the bacterial surface. Analyses of the proteins with which OtDUB associates during infection revealed several known O. tsutsugamushi cell wall proteins and others. It also forms an interactome with adapter protein complex 2 and other endosomal membrane traffic regulators. This study documents the first interactors of OtDUB during O. tsutsugamushi infection and establishes a strong link between OtDUB and the host endocytic pathway.

Functional Characterization of Non-Ankyrin Repeat Domains of Orientia tsutsugamushi Ank Effectors Reveals Their Importance for Molecular Pathogenesis.

Orientia tsutsugamushi is a genetically intractable obligate intracellular bacterium, causes scrub typhus, and has one of the largest known armamentariums of ankyrin repeat-containing effectors (Anks). Most have a C-terminal F-box presumed to interact with the SCF ubiquitin ligase complex primarily based on their ability to bind overexpressed Skp1. Whether all F-box-containing Anks bind endogenous SCF components and the F-box residues essential for such interactions has gone unexplored. Many O. tsutsugamushi Ank F-boxes occur as part of a PRANC (pox protein repeats of ankyrin-C-terminal) domain. Roles of the non-F-box portion of the PRANC and intervening sequence region (ISR) that links the ankyrin repeat and F-box/PRANC domains are unknown. The functional relevance of these effectors' non-ankyrin repeat domains was investigated. The F-box was necessary for Flag-tagged versions of most F-box-containing Anks to precipitate endogenous Skp1, Cul1, and/or Rbx1, while the ISR and PRANC were dispensable. Ank toxicity in yeast was predominantly F-box dependent. Interrogations of Ank1, Ank5, and Ank6 established that L1, P2, E4, I9, and D17 of the F-box consensus are key for binding native SCF components and for Ank1 and Ank6 to inhibit NF-κB. The ISR is also essential for Ank1 and Ank6 to impair NF-κB. Ectopically expressed Ank1 and Ank6 lacking the ISR or having a mutagenized F-box incapable of binding SCF components performed as dominant-negative inhibitors to block O. tsutsugamushi NF-κB modulation. This study advances knowledge of O. tsutsugamushi Ank functional domains and offers an approach for validating their roles in infection.

Analysis of Orientia tsutsugamushi promoter activity.

Orientia tsutsugamushi is an obligate intracellular bacterium that causes scrub typhus, a potentially fatal rickettsiosis, and for which no genetic tools exist. Critical to addressing this technical gap is to identify promoters for driving expression of antibiotic resistance and fluorescence reporter genes in O. tsutsugamushi. Such promoters would need to be highly conserved among strains, expressed throughout infection, and exhibit strong activity. We examined the untranslated regions upstream of O. tsutsugamushi genes encoding outer membrane protein A (ompA), 22-kDa type-specific antigen (tsa22) and tsa56. The bacterium transcribed all three during infection of monocytic, endothelial and epithelial cells. Examination of the upstream noncoding regions revealed putative ribosome binding sites, one set of predicted -10 and -35 sequences for ompA and two sets of -10 and -35 sequences for tsa22 and tsa56. Comparison of these regions among geographically diverse O. tsutsugamushi patient isolates revealed nucleotide identities ranging from 84.8 to 100.0%. Upon examination of the candidates for the ability to drive green fluorescence protein expression in Escherichia coli, varying activities were observed with one of the tsa22 promoters being the strongest. Identification and validation of O. tsutsugamushi promoters is an initial key step toward genetically manipulating this important pathogen.

Orientia tsutsugamushi Nucleomodulin Ank13 Exploits the RaDAR Nuclear Import Pathway To Modulate Host Cell Transcription.

Orientia tsutsugamushi is the etiologic agent of scrub typhus, the deadliest of all diseases caused by obligate intracellular bacteria. Nucleomodulins, bacterial effectors that dysregulate eukaryotic transcription, are being increasingly recognized as key virulence factors. How they translocate into the nucleus and their functionally essential domains are poorly defined. We demonstrate that Ank13, an O. tsutsugamushi effector conserved among clinical isolates and expressed during infection, localizes to the nucleus in an importin ß1-independent manner. Rather, Ank13 nucleotropism requires an isoleucine at the thirteenth position of its fourth ankyrin repeat, consistent with utilization of eukaryotic RaDAR (RanGDP-ankyrin repeats) nuclear import. RNA-seq analyses of cells expressing green fluorescent protein (GFP)-tagged Ank13, nucleotropism-deficient Ank13I127R, or Ank13ΔF-box, which lacks the F-box domain essential for interacting with SCF ubiquitin ligase, revealed Ank13 to be a nucleomodulin that predominantly downregulates transcription of more than 2,000 genes. Its ability to do so involves its nucleotropism and F-box in synergistic and mutually exclusive manners. Ank13 also acts in the cytoplasm to dysregulate smaller cohorts of genes. The effector's toxicity in yeast heavily depends on its F-box and less so on its nucleotropism. Genes negatively regulated by Ank13 include those involved in the inflammatory response, transcriptional control, and epigenetics. Importantly, the majority of genes that GFP-Ank13 most strongly downregulates are quiescent or repressed in O. tsutsugamushi-infected cells when Ank13 expression is strongest. Ank13 is the first nucleomodulin identified to coopt RaDAR and a multifaceted effector that functions in the nucleus and cytoplasm via F-box-dependent and -independent mechanisms to globally reprogram host cell transcription. IMPORTANCE Nucleomodulins are recently defined effectors used by diverse intracellular bacteria to manipulate eukaryotic gene expression and convert host cells into hospitable niches. How nucleomodulins enter the nucleus, their functional domains, and the genes that they modulate are incompletely characterized. Orientia tsutsugamushi is an intracellular bacterial pathogen that causes scrub typhus, which can be fatal. O. tsutsugamushi Ank13 is the first example of a microbial protein that coopts eukaryotic RaDAR (RanGDP-ankyrin repeats) nuclear import. It dysregulates expression of a multitude of host genes with those involved in transcriptional control and the inflammatory response being among the most prominent. Ank13 does so via mechanisms that are dependent and independent of both its nucleotropism and eukaryotic-like F-box domain that interfaces with ubiquitin ligase machinery. Nearly all the genes most strongly downregulated by ectopically expressed Ank13 are repressed in O. tsutsugamushi-infected cells, implicating its importance for intracellular colonization and scrub typhus molecular pathogenesis.

Orientia tsutsugamushi modulates cellular levels of NF-κB inhibitor p105.

BACKGROUND: Scrub typhus is a neglected tropical disease that threatens more than one billion people. If antibiotic therapy is delayed, often due to mis- or late diagnosis, the case fatality rate can increase considerably. Scrub typhus is caused by the obligate intracellular bacterium, Orientia tsutsugamushi, which invades phagocytes and endothelial cells in vivo and diverse tissue culture cell types in vitro. The ability of O. tsutsugamushi to replicate in the cytoplasm indicates that it has evolved to counter eukaryotic host cell immune defense mechanisms. The transcription factor, NF-κB, is a tightly regulated initiator of proinflammatory and antimicrobial responses. Typically, the inhibitory proteins p105 and IκBα sequester the NF-κB p50:p65 heterodimer in the cytoplasm. Canonical activation of NF-κB via TNFα involves IKKß-mediated serine phosphorylation of IκBα and p105, which leads to their degradation and enables NF-κB nuclear translocation. A portion of p105 is also processed into p50. O. tsutsugamushi impairs NF-κB translocation into the nucleus, but how it does so is incompletely defined. PRINCIPAL FINDINGS: Western blot, densitometry, and quantitative RT-PCR analyses of O. tsutsugamushi infected host cells were used to determine if the pathogen's ability to inhibit NF-κB is linked to modulation of p105. Results demonstrate that p105 levels are elevated several-fold in O. tsutsugamushi infected HeLa and RF/6A cells with only a nominal increase in p50. The O. tsutsugamushi-stimulated increase in p105 is bacterial dose- and protein synthesis-dependent, but does not occur at the level of host cell transcription. While TNFα-induced phosphorylation of p105 serine 932 proceeds unhindered in infected cells, p105 levels remain elevated and NF-κB p65 is retained in the cytoplasm. CONCLUSIONS: O. tsutsugamushi specifically stabilizes p105 to inhibit the canonical NF-κB pathway, which advances understanding of how it counters host immunity to establish infection.

Functional inhibition or genetic deletion of acid sphingomyelinase bacteriostatically inhibits Anaplasma phagocytophilum infection in vivo.

Anaplasma phagocytophilum infects neutrophils to cause granulocytic anaplasmosis. It poorly infects mice deficient in acid sphingomyelinase (ASM), a lysosomal enzyme critical for cholesterol efflux, and wild-type mice treated with desipramine that functionally inhibits ASM. Whether inhibition or genetic deletion of ASM is bacteriostatic or bactericidal for A. phagocytophilum and desipramine's ability to lower pathogen burden requires a competent immune system were unknown. Anaplasma phagocytophilum-infected severe combined immunodeficiency disorder (SCID) mice were administered desipramine or PBS, followed by the transfer of blood to naïve wild-type mice. Next, infected wild-type mice were given desipramine or PBS followed by transfer of blood to naïve SCID mice. Finally, wild-type or ASM-deficient mice were infected and blood transferred to naïve SCID mice. The percentage of infected neutrophils was significantly reduced in all desipramine-treated or ASM-deficient mice and in all recipients of blood from these mice. Infection was markedly lower in ASM-deficient and desipramine-treated wild-type mice versus desipramine-treated SCID mice. Yet, infection was never ablated. Thus, ASM activity contributes to optimal A. phagocytophilum infection in vivo, pharmacologic inhibition or genetic deletion of ASM impairs infection in a bacteriostatic and reversible manner and A. phagocytophilum is capable of co-opting ASM-independent lipid sources.

Ehrlichia chaffeensis EplA Interaction With Host Cell Protein Disulfide Isomerase Promotes Infection.

Ehrlichia chaffeensis is an obligate intracellular bacterium that invades monocytes to cause the emerging and potentially severe disease, monocytic ehrlichiosis. Ehrlichial invasion of host cells, a process that is essential for the bacterium's survival and pathogenesis, is incompletely understood. In this study, we identified ECH_0377, henceforth designated as EplA (E. chaffeensis PDI ligand A) as an E. chaffeensis adhesin that interacts with host cell protein disulfide isomerase (PDI) to mediate bacterial entry into host cells. EplA is an outer membrane protein that E. chaffeensis expresses during growth in THP-1 monocytic cells. Canine sera confirmed to be positive for exposure to Ehrlichia spp. recognized recombinant EplA, indicating that it is expressed during infection in vivo. EplA antiserum inhibited the bacterium's ability to infect monocytic cells. The EplA-PDI interaction was confirmed via co-immunoprecipitation. Treating host cell surfaces with antibodies that inhibit PDI and/or thioredoxin-1 thiol reductase activity impaired E. chaffeensis infection. Chemical reduction of host cell surfaces, but not bacterial surfaces with tris(2-carboxyethyl)phosphine (TCEP) restored ehrlichial infectivity in the presence of the PDI-neutralizing antibody. Antisera specific for EplA C-terminal residues 95-104 (EplA95-104) or outer membrane protein A amino acids 53-68 (OmpA53-68) reduced E. chaffeensis infection of THP-1 cells. Notably, TCEP rescued ehrlichial infectivity of bacteria that had been treated with anti-EplA95-104, but not anti-EcOmpA53-68. These results demonstrate that EplA contributes to E. chaffeensis infection of monocytic cells by engaging PDI and exploiting the enzyme's reduction of host cell surface disulfide bonds in an EplA C-terminus-dependent manner and identify EplA95-104 and EcOmpA53-68 as novel ehrlichial receptor binding domains.

Serologic Evidence for the Exposure of Eastern Coyotes (Canis latrans) in Pennsylvania to the Tick-Borne Pathogens Borreliella burgdorferi and Anaplasma phagocytophilum.

Lyme disease and anaplasmosis are tick-borne bacterial diseases caused by Borreliella and Anaplasma species, respectively. A comprehensive analysis of the exposure of eastern coyotes (Canis latrans) in the northeastern United States to tick-borne pathogens has not been conducted. In this report, we assess the serological status of 128 eastern coyotes harvested in Pennsylvania in 2015 and 2017 for antibodies to Borreliella burgdorferi and Anaplasma phagocytophilum Immunoblot and dot blot approaches were employed to test each plasma sample by using cell lysates and recombinant proteins as detection antigens. The results demonstrate high seropositivity incidences of 64.8% and 72.7% for B. burgdorferi and A. phagocytophilum, respectively. Antibodies to both pathogens were detected in 51.5% of the plasma samples, indicating high potential for coinfection. Antibodies to the B. burgdorferi proteins DbpB, VlsE, DbpA, BBA36, and OspF (BBO39) were detected in 67.2, 63.3, 56.2, 51.6, and 48.4% of the plasma samples, respectively. Antibodies to the A. phagocytophilum P44 and P130 proteins were detected in 72.7 and 60.9% of the plasma samples, respectively.IMPORTANCE The incidence of Lyme disease (Borreliella burgdorferi) and anaplasmosis (Anaplasma phagocytophilum) are increasing in North America and Europe. The causative agents of these debilitating tick-transmitted infections are maintained in nature in an enzootic cycle involving Ixodes ticks and diverse mammals and birds. It has been postulated that predators directly or indirectly influence the dynamics of the enzootic cycle and disease incidence. Here, we demonstrate high seropositivity of eastern coyotes for B. burgdorferi and A. phagocytophilum As coyotes become established in urban and suburban environments, interactions with humans, companion animals, and urban/suburban wildlife will increase. Knowledge of the pathogens that these highly adaptable predators are exposed to or carry, and their potential to influence or participate in enzootic cycles, is central to efforts to reduce the risk of tick-borne diseases in humans and companion animals.

Immunization against Anaplasma phagocytophilum Adhesin Binding Domains Confers Protection against Infection in the Mouse Model.

Anaplasma phagocytophilum causes granulocytic anaplasmosis, a debilitating infection that can be fatal in the immunocompromised. It also afflicts animals, including dogs, horses, and sheep. No granulocytic anaplasmosis vaccine exists. Because A. phagocytophilum is an obligate intracellular bacterium, inhibiting microbe-host cell interactions that facilitate invasion can disrupt infection. The binding domains of A. phagocytophilum adhesins A. phagocytophilum invasion protein A (AipA), A. phagocytophilum surface protein (Asp14), and outer membrane protein A (OmpA) are essential for optimal bacterial entry into host cells, but their relevance to infection in vivo is undefined. In this study, C57BL/6 mice were immunized with a cocktail of keyhole limpet hemocyanin-conjugated peptides corresponding to the AipA, Asp14, and OmpA binding domains in alum followed by challenge with A. phagocytophilum The bacterial peripheral blood burden was pronouncedly reduced in immunized mice compared to controls. Examination of pre- and postchallenge sera from these mice revealed that immunization elicited antibodies against AipA and Asp14 peptides but not OmpA peptide. Nonetheless, pooled sera from pre- and postchallenge groups, but not from control groups, inhibited A. phagocytophilum infection of HL-60 cells. Adhesin domain immunization also elicited interferon gamma (IFN-γ)-producing CD8-positive (CD8+) T cells. A follow-up study confirmed that immunization against only the AipA or Asp14 binding domain was sufficient to reduce the bacterial peripheral blood load in mice following challenge and elicit antibodies that inhibit A. phagocytophilum cellular infection in vitro These data demonstrate that AipA and Asp14 are critical for A. phagocytophilum to productively infect mice, and immunization against their binding domains elicits a protective immune response.

Binding of Host Cell Surface Protein Disulfide Isomerase by Anaplasma phagocytophilum Asp14 Enables Pathogen Infection.

Diverse intracellular pathogens rely on eukaryotic cell surface disulfide reductases to invade host cells. Pharmacologic inhibition of these enzymes is cytotoxic, making it impractical for treatment. Identifying and mechanistically dissecting microbial proteins that co-opt surface reductases could reveal novel targets for disrupting this common infection strategy. Anaplasma phagocytophilum invades neutrophils by an incompletely defined mechanism to cause the potentially fatal disease granulocytic anaplasmosis. The bacterium's adhesin, Asp14, contributes to invasion by virtue of its C terminus engaging an unknown receptor. Yeast-two hybrid analysis identified protein disulfide isomerase (PDI) as an Asp14 binding partner. Coimmunoprecipitation confirmed the interaction and validated it to be Asp14 C terminus dependent. PDI knockdown and antibody-mediated inhibition of PDI reductase activity impaired A. phagocytophilum infection of but not binding to host cells. Infection during PDI inhibition was rescued when the bacterial but not host cell surface disulfide bonds were chemically reduced with tris(2-carboxyethyl)phosphine-HCl (TCEP). TCEP also restored bacterial infectivity in the presence of an Asp14 C terminus blocking antibody that otherwise inhibits infection. A. phagocytophilum failed to productively infect myeloid-specific-PDI conditional-knockout mice, marking the first demonstration of in vivo microbial dependency on PDI for infection. Mutational analyses identified the Asp14 C-terminal residues that are critical for binding PDI. Thus, Asp14 binds and brings PDI proximal to A. phagocytophilum surface disulfide bonds that it reduces, which enables cellular and in vivo infection.IMPORTANCEAnaplasma phagocytophilum infects neutrophils to cause granulocytic anaplasmosis, an emerging potentially fatal disease and the second-most common tick-borne illness in the United States. Treatment options are limited, and no vaccine exists. Due to the bacterium's obligatory intracellular lifestyle, A. phagocytophilum survival and pathogenesis are predicated on its ability to enter host cells. Understanding its invasion mechanism will yield new targets for preventing bacterial entry and, hence, disease. We report a novel entry pathway in which the A. phagocytophilum outer membrane protein Asp14 binds host cell surface protein disulfide isomerase via specific C-terminal residues to promote reduction of bacterial surface disulfide bonds, which is critical for cellular invasion and productive infection in vivo Targeting the Asp14 C terminus could be used to prevent/treat granulocytic anaplasmosis. Our findings have broad implications, as a thematically similar approach could be applied to block infection by other intracellular microbes that exploit cell surface reductases.

Functional inhibition of acid sphingomyelinase disrupts infection by intracellular bacterial pathogens.

Intracellular bacteria that live in host cell-derived vacuoles are significant causes of human disease. Parasitism of low-density lipoprotein (LDL) cholesterol is essential for many vacuole-adapted bacteria. Acid sphingomyelinase (ASM) influences LDL cholesterol egress from the lysosome. Using functional inhibitors of ASM (FIASMAs), we show that ASM activity is key for infection cycles of vacuole-adapted bacteria that target cholesterol trafficking-Anaplasma phagocytophilum, Coxiella burnetii, Chlamydia trachomatis, and Chlamydia pneumoniae. Vacuole maturation, replication, and infectious progeny generation by A. phagocytophilum, which exclusively hijacks LDL cholesterol, are halted and C. burnetii, for which lysosomal cholesterol accumulation is bactericidal, is killed by FIASMAs. Infection cycles of Chlamydiae, which hijack LDL cholesterol and other lipid sources, are suppressed but less so than A. phagocytophilum or C. burnetii A. phagocytophilum fails to productively infect ASM-/- or FIASMA-treated mice. These findings establish the importance of ASM for infection by intracellular bacteria and identify FIASMAs as potential host-directed therapies for diseases caused by pathogens that manipulate LDL cholesterol.

Correction: Naimi, W.A., et al. Differential Susceptibility of Male versus Female Laboratory Mice to Anaplasma phagocytophilum Infection. Trop. Med. Infect. Dis. 2018, 3, 78.

The authors wish to make the following corrections to this paper [...].

The Obligate Intracellular Bacterium Orientia tsutsugamushi Targets NLRC5 To Modulate the Major Histocompatibility Complex Class I Pathway.

Orientia tsutsugamushi is an obligate intracellular bacterium that infects mononuclear and endothelial cells to cause the emerging global health threat scrub typhus. The ability of O. tsutsugamushi to survive in monocytes facilitates bacterial dissemination to endothelial cells, which can subsequently lead to several potentially fatal sequelae. As a strict intracellular pathogen that lives in the cytoplasm of host cells, O. tsutsugamushi has evolved to counter adaptive immunity. How the pathogen does so and the outcome of this strategy in monocytes versus endothelial cells are poorly understood. This report demonstrates that O. tsutsugamushi reduces cellular levels of NOD-, LRR-, and CARD-containing 5 (NLRC5), a recently identified specific transactivator of major histocompatibility complex class I (MHC-I) component gene expression, to inhibit MHC-I biosynthesis. Importantly, the efficacy of this approach varies with the host cell type infected. In nonprofessional antigen-presenting HeLa and primary human aortic endothelial cells, the O. tsutsugamushi-mediated reduction of NLRC5 results in lowered MHC-I component transcription and, consequently, lower total and/or surface MHC-I levels throughout 72 h of infection. However, in infected THP-1 monocytes, which are professional antigen-presenting cells, the reductions in NLRC5 and MHC-I observed during the first 24 h reverse thereafter. O. tsutsugamushi is the first example of a microbe that targets NLRC5 to modulate the MHC-I pathway. The differential ability of O. tsutsugamushi to modulate this pathway in nonprofessional versus professional antigen-presenting cells could influence morbidity and mortality from scrub typhus.

Outer Membrane Protein A Conservation among Orientia tsutsugamushi Isolates Suggests Its Potential as a Protective Antigen and Diagnostic Target.

Scrub typhus threatens one billion people in the Asia-Pacific area and cases have emerged outside this region. It is caused by infection with any of the multitude of strains of the bacterium Orientia tsutsugamushi. A vaccine that affords heterologous protection and a commercially-available molecular diagnostic assay are lacking. Herein, we determined that the nucleotide and translated amino acid sequences of outer membrane protein A (OmpA) are highly conserved among 51 O. tsutsugamushi isolates. Molecular modeling revealed the predicted tertiary structure of O. tsutsugamushi OmpA to be very similar to that of the phylogenetically-related pathogen, Anaplasma phagocytophilum, including the location of a helix that contains residues functionally essential for A. phagocytophilum infection. PCR primers were developed that amplified ompA DNA from all O. tsutsugamushi strains, but not from negative control bacteria. Using these primers in quantitative PCR enabled sensitive detection and quantitation of O. tsutsugamushi ompA DNA from organs and blood of mice that had been experimentally infected with the Karp or Gilliam strains. The high degree of OmpA conservation among O. tsutsugamushi strains evidences its potential to serve as a molecular diagnostic target and justifies its consideration as a candidate for developing a broadly-protective scrub typhus vaccine.

Differential Susceptibility of Male Versus Female Laboratory Mice to Anaplasma phagocytophilum Infection.

Human granulocytic anaplasmosis (HGA) is a debilitating, non-specific febrile illness caused by the granulocytotropic obligate intracellular bacterium called Anaplasma phagocytophilum. Surveillance studies indicate a higher prevalence of HGA in male versus female patients. Whether this discrepancy correlates with differential susceptibility of males and females to A. phagocytophilum infection is unknown. Laboratory mice have long been used to study granulocytic anaplasmosis. Yet, sex as a biological variable (SABV) in this model has not been evaluated. In this paper, groups of male and female C57Bl/6 mice that had been infected with A. phagocytophilum were assessed for the bacterial DNA load in the peripheral blood, the percentage of neutrophils harboring bacterial inclusions called morulae, and splenomegaly. Infected male mice exhibited as much as a 1.85-fold increase in the number of infected neutrophils, which is up to a 1.88-fold increase in the A. phagocytophilum DNA load, and a significant increase in spleen size when compared to infected female mice. The propensity of male mice to develop a higher level of A. phagocytophilum infection is relevant for studies utilizing the mouse model. This stresses the importance of including SABV and aligns with the observed higher incidence of infection in male versus female patients.

Orientia tsutsugamushi uses two Ank effectors to modulate NF-κB p65 nuclear transport and inhibit NF-κB transcriptional activation.

Orientia tsutsugamushi causes scrub typhus, a potentially fatal infection that threatens over one billion people. Nuclear translocation of the transcription factor, NF-κB, is the central initiating cellular event in the antimicrobial response. Here, we report that NF-κB p65 nuclear accumulation and NF-κB-dependent transcription are inhibited in O. tsutsugamushi infected HeLa cells and/or primary macrophages, even in the presence of TNFα. The bacterium modulates p65 subcellular localization by neither degrading it nor inhibiting IκBα degradation. Rather, it exploits host exportin 1 to mediate p65 nuclear export, as this phenomenon is leptomycin B-sensitive. O. tsutsugamushi antagonizes NF-κB-activated transcription even when exportin 1 is inhibited and NF-κB consequently remains in the nucleus. Two ankyrin repeat-containing effectors (Anks), Ank1 and Ank6, each of which possess a C-terminal F-box and exhibit 58.5% amino acid identity, are linked to the pathogen's ability to modulate NF-κB. When ectopically expressed, both translocate to the nucleus, abrogate NF-κB-activated transcription in an exportin 1-independent manner, and pronouncedly reduce TNFα-induced p65 nuclear levels by exportin 1-dependent means. Flag-tagged Ank 1 and Ank6 co-immunoprecipitate p65 and exportin 1. Both also bind importin ß1, a host protein that is essential for the classical nuclear import pathway. Importazole, which blocks importin ß1 activity, abrogates Ank1 and Ank6 nuclear translocation. The Ank1 and Ank6 regions that bind importin ß1 also mediate their transport into the nucleus. Yet, these regions are distinct from those that bind p65/exportin 1. The Ank1 and Ank6 F-box and the region that lies between it and the ankyrin repeat domain are essential for blocking p65 nuclear accumulation. These data reveal a novel mechanism by which O. tsutsugamushi modulates the activity and nuclear transport of NF-κB p65 and identify the first microbial proteins that co-opt both importin ß1 and exportin 1 to antagonize a critical arm of the antimicrobial response.