Qingming Xiong

Autophagosomes induced by a bacterial Beclin 1 binding protein facilitate obligatory intracellular infection

Autophagy, a cytoplasmic catabolic process, plays a critical role in defense against intracellular infection. In turn, evasion or inhibition of autophagy has emerged as an important virulence factor for intracellular pathogens. However, Anaplasma phagocytophilum, the obligatory intracellular bacterium that causes human granulocytic anaplasmosis, replicates in the membrane-bound compartment resembling early autophagosome. Here, we found that Anaplasma translocated substrate 1 (Ats-1), a type IV secretion effector, binds Beclin 1, a subunit of the class III PI3K and Atg14L, and it nucleates autophagosomes with markers of omegasomes, double FYVE-containing protein 1, Atg14L, and LC3. Ats-1 autophagy induction did not activate the starvation signaling pathway of mammalian target of rapamycin. These autophagy proteins were also localized to the Anaplasma inclusion. Ectopically expressed Ats-1 targeted the Anaplasma inclusions and enhanced infection, whereas host cytoplasmic delivery of anti–Ats-1 or Beclin 1 depletion by siRNA suppressed the infection; beclin 1 heterozygous-deficient mice were resistant to Anaplasma infection. Furthermore, Anaplasma growth arrest by the class III PI3K inhibitor 3-methyladenine was alleviated by essential amino acid supplementation. Thus, Anaplasma actively induces autophagy by secreting Ats-1 that hijacks the Beclin 1-Atg14L autophagy initiation pathway likely to acquire host nutrients for its growth.

Cholesterol-Dependent Anaplasma phagocytophilum Exploits the Low-Density Lipoprotein Uptake Pathway

In eukaryotes, intracellular cholesterol homeostasis and trafficking are tightly regulated. Certain bacteria, such as Anaplasma phagocytophilum, also require cholesterol; it is unknown, however, how this cholesterol-dependent obligatory intracellular bacterium of granulocytes interacts with the host cell cholesterol regulatory pathway to acquire cholesterol. Here, we report that total host cell cholesterol increased >2-fold during A. phagocytophilum infection in a human promyelocytic leukemia cell line. Cellular free cholesterol was enriched in A. phagocytophilum inclusions as detected by filipin staining. We determined that A. phagocytophilum requires cholesterol derived from low-density lipoprotein (LDL), because its replication was significantly inhibited by depleting the growth medium of cholesterol-containing lipoproteins, by blocking LDL uptake with a monoclonal antibody against LDL receptor (LDLR), or by treating the host cells with inhibitors that block LDL-derived cholesterol egress from late endosomes or lysosomes. However, de novo cholesterol biosynthesis is not required, since inhibition of the biosynthesis pathway did not inhibit A. phagocytophilum infection. The uptake of fluorescence-labeled LDL was enhanced in infected cells, and LDLR expression was up-regulated at both the mRNA and protein levels. A. phagocytophilum infection stabilized LDLR mRNA through the 3′ UTR region, but not through activation of the sterol regulatory element binding proteins. Extracellular signal–regulated kinase (ERK) was up-regulated by A. phagocytophilum infection, and inhibition of its upstream kinase, MEK, by a specific inhibitor or siRNA knockdown, reduced A. phagocytophilum infection. Up-regulation of LDLR mRNA by A. phagocytophilum was also inhibited by the MEK inhibitor; however, it was unclear whether ERK activation is required for LDLR mRNA up-regulation by A. phagocytophilum. These data reveal that A. phagocytophilum exploits the host LDL uptake pathway and LDLR mRNA regulatory system to accumulate cholesterol in inclusions to facilitate its replication.

Ehrlichia ewingii Infection Delays Spontaneous Neutrophil Apoptosis through Stabilization of Mitochondria

The uncultivable obligate intracellular bacterium Ehrlichia ewingii, previously known only as a canine pathogen, is the most recently recognized agent of human ehrlichiosis. E. ewingii is the only Ehrlichia species known to infect neutrophils. In the blood or in ex vivo culture, neutrophils generally have a short life span. In the present study, we investigated the effect of E. ewingii infection on spontaneous apoptosis of neutrophils. E. ewingii infection significantly delayed dog neutrophil apoptosis during ex vivo culture. The inhibitory effect on neutrophil apoptosis by E. ewingii was reversible on clearance of the organism. By using the fluorescent mitochondrial dyes Mitotracker Red 580 and JC-1, we found that E. ewingii infection stabilized mitochondrial integrity by maintaining mitochondrial membrane potential in neutrophils. These results suggest that E. ewingii delays spontaneous apoptosis of neutrophils via stabilization of host cell mitochondria.

High-Cholesterol Diet Facilitates Anaplasma phagocytophilum Infection and Up-Regulates Macrophage Inflammatory Protein—2 and CXCR2 Expression in Apolipoprotein E-Deficient Mice

BACKGROUND Anaplasma phagocytophilum is an obligatory intracellular bacterium that infects granulocytes and causes human granulocytic anaplasmosis (HGA). This bacterium requires cholesterol for host cell infection in vitro and incorporates exogenous cholesterol into its membrane. METHODS To understand the role of host cholesterol in A. phagocytophilum infection in vivo, we analyzed the effects of a high-cholesterol diet and reduced apolipoprotein E (apoE) activity on A. phagocytophilum infection in mice. RESULTS A high-cholesterol diet significantly facilitated A. phagocytophilum infection in the spleen, liver, and blood of apoE-deficient (apoE(-/-)) mice, compared with the level of infection in apoE(-/-) mice fed a normal-cholesterol diet or wild-type (WT) mice fed a high- or normal-cholesterol diet. A. phagocytophilum infection induced a significant elevation in the mRNA expression of macrophage inflammatory protein (MIP)-2 and an MIP-2 receptor, CXCR2, in the spleen in apoE(-/-) mice fed a high-cholesterol diet, compared with the other 3 groups. CONCLUSION Our results suggest that high blood cholesterol levels resulting from an interaction between dietary and genetic factors facilitate A. phagocytophilum infection and up-regulate a proinflammatory chemokine and its receptor, which may contribute to HGA pathogenesis.

Identification of 19 Polymorphic Major Outer Membrane Protein Genes and Their Immunogenic Peptides in Ehrlichia ewingii for Use in a Serodiagnostic Assay

ABSTRACT Ehrlichia ewingii, a tick-transmitted rickettsia previously known only as a canine pathogen, was recently recognized as a human pathogen. E. ewingii has yet to be cultivated, and there is no serologic test available to diagnose E. ewingii infection. Previously, a fragment (505 bp) of a single E. ewingii gene homologous to 1 of 22 genes encoding Ehrlichia chaffeensis immunodominant major outer membrane proteins 1 (OMP-1s)/P28s was identified. The purposes of the present study were to (i) determine the E. ewingii omp-1 gene family, (ii) determine each OMP-1-specific peptide, and (iii) analyze all OMP-1 synthesized peptides for antigenicity. Using nested touchdown PCR and a primer walking strategy, we found 19 omp-1 paralogs in E. ewingii. These genes are arranged in tandem downstream of tr1 and upstream of secA in a 24-kb genomic region. Predicted molecular masses of the 19 mature E. ewingii OMP-1s range from 25.1 to 31.3 kDa, with isoelectric points of 5.03 to 9.80. Based on comparative sequence analyses among OMP-1s from E. ewingii and three other Ehrlichia spp., each E. ewingii OMP-1 oligopeptide that was predicted to be antigenic, bacterial surface exposed, unique in comparison to the other E. ewingii OMP-1s, and distinct from those of other Ehrlichia spp. was synthesized for use in an enzyme-linked immunosorbent assay. Plasmas from experimentally E. ewingii-infected dogs reacted significantly with most of the OMP-1-specific peptides, indicating that multiple OMP-1s were expressed and immunogenic in infected dogs. The results support the utility of the tailored OMP-1 peptides as E. ewingii serologic test antigens.

Subversion of NPC1 pathway of cholesterol transport by Anaplasma phagocytophilum.

Intracellular cholesterol amounts, distribution and traffic are tightly regulated to maintain the healthy eukaryotic cell function. However, how intracellular pathogens that require cholesterol, interact with the host cholesterol homeostasis and traffic is not well understood. Anaplasma phagocytophilum is an obligatory intracellular and cholesterol‐robbing bacterium, which causes human granulocytic anaplasmosis. Here we found that a subset of cholesterol‐binding membrane protein, Niemann‐Pick type C1 (NPC1)‐bearing vesicles devoid of lysosomal markers were upregulated in HL‐60 cells infected with A. phagocytophilum, and trafficked to live bacterial inclusions. The NPC1 localization to A. phagocytophilum inclusions was abolished by low‐density lipoprotein (LDL)‐derived cholesterol traffic inhibitor U18666A. Studies using NPC1 siRNA and the cell line with cholesterol traffic defect demonstrated that the NPC1 function is required for bacterial cholesterol acquisition and infection. Furthermore, trans‐Golgi network‐specific soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors, vesicle‐associated membrane protein (VAMP4) and syntaxin 16, which are associated with NPC1 and LDL‐derived cholesterol vesicular transport were recruited to A.  phagocytophilum inclusions, and VAMP4 was required for bacteria infection. Taken together, A. phagocytophilum is the first example of a pathogen that subverts the NPC1 pathway of intracellular cholesterol transport and homeostasis for bacterial inclusion membrane biogenesis and cholesterol capture.

Ehrlichia secretes Etf-1 to induce autophagy and capture nutrients for its growth through RAB5 and class III phosphatidylinositol 3-kinase.

Ehrlichia chaffeensis is an obligatory intracellular bacterium that causes a potentially fatal emerging zoonosis, human monocytic ehrlichiosis. E. chaffeensis has a limited capacity for biosynthesis and metabolism and thus depends mostly on host-synthesized nutrients for growth. Although the host cell cytoplasm is rich with these nutrients, as E. chaffeensis is confined within the early endosome-like membrane-bound compartment, only host nutrients that enter the compartment can be used by this bacterium. How this occurs is unknown. We found that ehrlichial replication depended on autophagy induction involving class III phosphatidylinositol 3-kinase (PtdIns3K) activity, BECN1 (Beclin 1), and ATG5 (autophagy-related 5). Ehrlichia acquired host cell preincorporated amino acids in a class III PtdIns3K-dependent manner and ehrlichial growth was enhanced by treatment with rapamycin, an autophagy inducer. Moreover, ATG5 and RAB5A/B/C were routed to ehrlichial inclusions. RAB5A/B/C siRNA knockdown, or overexpression of a RAB5-specific GTPase-activating protein or dominant-negative RAB5A inhibited ehrlichial infection, indicating the critical role of GTP-bound RAB5 during infection. Both native and ectopically expressed ehrlichial type IV secretion effector protein, Etf-1, bound RAB5 and the autophagy-initiating class III PtdIns3K complex, PIK3C3/VPS34, and BECN1, and homed to ehrlichial inclusions. Ectopically expressed Etf-1 activated class III PtdIns3K as in E. chaffeensis infection and induced autophagosome formation, cleared an aggregation-prone mutant huntingtin protein in a class III PtdIns3K-dependent manner, and enhanced ehrlichial proliferation. These data support the notion that E. chaffeensis secretes Etf-1 to induce autophagy to repurpose the host cytoplasm and capture nutrients for its growth through RAB5 and class III PtdIns3K, while avoiding autolysosomal killing.

Evaluation of peptide- and recombinant protein-based assays for detection of anti-Ehrlichia ewingii antibodies in experimentally and naturally infected dogs.

OBJECTIVE To evaluate microtiter-plate format ELISAs constructed by use of different diagnostic targets derived from the Ehrlichia ewingii p28 outer membrane protein for detection of E ewingii antibodies in experimentally and naturally infected dogs. SAMPLE POPULATION Serum samples from 87 kenneled dogs, 9 dogs experimentally infected with anti-E ewingii, and 180 potentially naturally exposed dogs from Missouri. PROCEDURES The capacities of the synthetic peptide and truncated recombinant protein to function as detection reagents in ELISAs were compared by use of PCR assay, western blot analysis, and a full-length recombinant protein ELISA. Diagnostic targets included an E ewingii synthetic peptide (EESP) and 2 recombinant proteins: a full-length E ewingii outer membrane protein (EEp28) and a truncated E ewingii outer membrane protein (EETp28) RESULTS A subset of Ehrlichia canis-positive samples cross-reacted in the EEp28 ELISA; none were reactive in the EESP and EETp28 ELISAs. The EESP- and EETp28-based ELISAs detected E ewingii seroconversion at approximately the same time after infection as the EEp28 ELISAs. In afield population, each of the ELISAs identified the same 35 samples as reactive and 27 samples as nonreactive. Anaplasma and E can is peptides used in a commercially available ELISA platform did not detect anti-E ewingii antibodies in experimentally infected dogs. CONCLUSIONS AND CLINICAL RELEVANCE The EESP and EETp28 ELISAs were suitable for specifically detecting anti-E ewingii antibodies in experimentally and naturally infected dogs.

The prenylation inhibitor manumycin A reduces the viability of Anaplasma phagocytophilum.

Anaplasma phagocytophilum is an obligately intracellular bacterium and is the causative agent of human granulocytic anaplasmosis (HGA), an emerging and major tick-borne disease in the USA and other parts of the world. This study showed that the prenylation inhibitor manumycin A effectively blocked A. phagocytophilum infection in host cells (HL-60 or RF/6A cells). A. phagocytophilum infection activated extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase in host cells, and manumycin A treatment reduced ERK activation in A. phagocytophilum-infected host cells. As ERK activation is required for A. phagocytophilum infection, we examined whether manumycin A inhibited the bacteria directly or through host ERK signalling. Treatment of A. phagocytophilum alone with manumycin A significantly reduced the bacterial infectivity of host cells and bacterial viability in the absence of host cells, whereas pre-treatment of host cells did not inhibit bacterial infection in host cells. The inhibitory effect of manumycin A on A. phagocytophilum infection in host cells was achieved even at a concentration 100 times lower than that required for effective inhibition of mammalian cell signalling. These results suggested that manumycin A directly inactivates the bacterium, resulting in reduced infection and ERK1/2 activation. Thus, the manumycin group of drugs may have a therapeutic potential for HGA.

EtpE Binding to DNase X Induces Ehrlichial Entry via CD147 and hnRNP-K Recruitment, Followed by Mobilization of N-WASP and Actin

ABSTRACT Obligate intracellular bacteria, such as Ehrlichia chaffeensis, perish unless they can enter eukaryotic cells. E. chaffeensis is the etiological agent of human monocytic ehrlichiosis, an emerging infectious disease. To infect cells, Ehrlichia uses the C terminus of the outer membrane invasin entry-triggering protein (EtpE) of Ehrlichia (EtpE-C), which directly binds the mammalian cell surface glycosylphosphatidyl inositol-anchored protein, DNase X. How this binding drives Ehrlichia entry is unknown. Here, using affinity pulldown of host cell lysates with recombinant EtpE-C (rEtpE-C), we identified two new human proteins that interact with EtpE-C: CD147 and heterogeneous nuclear ribonucleoprotein K (hnRNP-K). The interaction of CD147 with rEtpE-C was validated by far-Western blotting and coimmunoprecipitation of native EtpE with endogenous CD147. CD147 was ubiquitous on the cell surface and also present around foci of rEtpE-C-coated-bead entry. Functional neutralization of surface-exposed CD147 with a specific antibody inhibited Ehrlichia internalization and infection but not binding. Downregulation of CD147 by short hairpin RNA (shRNA) impaired E. chaffeensis infection. Functional ablation of cytoplasmic hnRNP-K by a nanoscale intracellular antibody markedly attenuated bacterial entry and infection but not binding. EtpE-C also interacted with neuronal Wiskott-Aldrich syndrome protein (N-WASP), which is activated by hnRNP-K. Wiskostatin, which inhibits N-WASP activation, and cytochalasin D, which inhibits actin polymerization, inhibited Ehrlichia entry. Upon incubation with host cell lysate, EtpE-C but not an EtpE N-terminal fragment stimulated in vitro actin polymerization in an N-WASP- and DNase X-dependent manner. Time-lapse video images revealed N-WASP recruitment at EtpE-C-coated bead entry foci. Thus, EtpE-C binding to DNase X drives Ehrlichia entry by engaging CD147 and hnRNP-K and activating N-WASP-dependent actin polymerization. IMPORTANCE Ehrlichia chaffeensis, an obligate intracellular bacterium, causes a blood-borne disease called human monocytic ehrlichiosis, one of the most prevalent life-threatening emerging tick-transmitted infectious diseases in the United States. The survival of Ehrlichia bacteria, and hence, their ability to cause disease, depends on their specific mode of entry into eukaryotic host cells. Understanding the mechanism by which E. chaffeensis enters cells will create new opportunities for developing effective therapies to prevent bacterial entry and disease in humans. Our findings reveal a novel cellular signaling pathway triggered by an ehrlichial surface protein called EtpE to induce its infectious entry. The results are also important from the viewpoint of human cell physiology because three EtpE-interacting human proteins, DNase X, CD147, and hnRNP-K, are hitherto unknown partners that drive the uptake of small particles, including bacteria, into human cells. Ehrlichia chaffeensis, an obligate intracellular bacterium, causes a blood-borne disease called human monocytic ehrlichiosis, one of the most prevalent life-threatening emerging tick-transmitted infectious diseases in the United States. The survival of Ehrlichia bacteria, and hence, their ability to cause disease, depends on their specific mode of entry into eukaryotic host cells. Understanding the mechanism by which E. chaffeensis enters cells will create new opportunities for developing effective therapies to prevent bacterial entry and disease in humans. Our findings reveal a novel cellular signaling pathway triggered by an ehrlichial surface protein called EtpE to induce its infectious entry. The results are also important from the viewpoint of human cell physiology because three EtpE-interacting human proteins, DNase X, CD147, and hnRNP-K, are hitherto unknown partners that drive the uptake of small particles, including bacteria, into human cells.