Juan P. Olano

Pathogenic mechanisms of diseases caused by Rickettsia.

Abstract: The specter of bioterrorism employing genetically engineered Rickettsia resistant to all antibiotics should reawaken the worlds desire to elucidate the pathogenesis of typhus and spotted fever rickettsioses in a search for mechanisms vulnerable to interdiction. The pathogenetic sequence includes rickettsial entry into the dermis, hematogenous dissemination to vascular endothelial cells (most critically in brain and lungs), increased vascular permeability, edema, and immunity mediated by NK cells, IFN‐γ, TNF‐α, RANTES, antibodies, and cytotoxic T lymphocytes. Silverman has demonstrated the role of reactive oxygen species (ROS) produced by R. rickettsii‐infected endothelial cells in peroxidative damage to cell membranes in vitro, and Heinzen has described actin‐based rickettsial intracellular mobility and intercellular spread. At this point the availability of sequences of rickettsial genomes and excellent animal models of rickettsioses have yielded insufficient progress towards the identification of rickettsial virulence factors and knowledge of the importance of injury mediated by ROS, phospholipase A2, protease(s) or other mechanisms in vivo. Attention to the rickettsiosis‐associated procoagulant state led to determination that hemostatic mechanisms largely prevent major hemorrhage without disseminated intravascular coagulation or thrombosis‐mediated ischemia. Particularly lacking is knowledge of early events in vivo at the portal of entry in skin (or lung), of the effects of the inoculum medium (arthropod saliva or feces), mediators produced by infected endothelium under conditions of flow and of the contributions in vivo of immune effectors to pathology, of the role of apoptosis in rickettsial infection, and of the endothelial cell alterations that account for increased vascular permeability. The host cell receptor for the Rickettsia ligand and the mechanism of rickettsial escape from the phagosome need to be elucidated.

Overproduction of TNF-α by CD8+ Type 1 Cells and Down-Regulation of IFN-γ Production by CD4+ Th1 Cells Contribute to Toxic Shock-Like Syndrome in an Animal Model of Fatal Monocytotropic Ehrlichiosis

Human monocytotropic ehrlichiosis (HME) is an emerging, life-threatening, infectious disease caused by Ehrlichia chaffeensis, an obligate intracellular bacterium that lacks cell wall LPS. We have previously developed an animal model of severe HME using a strain of Ehrlichia isolated from Ixodes ovatus ticks (IOE). To understand the basis of susceptibility to severe monocytotropic ehrlichiosis, we compared low and high doses of the highly virulent IOE strain and the less virulent Ehrlichia muris strain that are closely related to E. chaffeensis in C57BL/6 mice. Lethal infections caused by high or low doses of IOE were accompanied by extensive liver damage, extremely elevated levels of TNF-α in the serum, high frequency of Ehrlichia-specific, TNF-α-producing CD8+ T cells in the spleen, decreased Ehrlicha-specific CD4+ T cell proliferation, low IL-12 levels in the spleen, and a 40-fold decrease in the number of IFN-γ-producing CD4+ Th1 cells. All groups contained negligible numbers of IL-4-producing cells in the spleen. Transfer of Ehrlichia-specific polyclonal Abs and IFN-γ-producing Ehrlichia-specific CD4+ and CD8+ type 1 cells protected naive mice against lethal IOE challenge. Interestingly, infection with high dose E. muris provided protection against rechallenge with a lethal dose of IOE. Cross-protection was associated with substantial expansion of IFN-γ-producing CD4+ and CD8+ cells, but not TNF-α-producing CD8+ T cells, a high titer of IgG2a, and a low serum level of TNF-α. In conclusion, uncontrolled TNF-α production by CD8+ T cells together with a weak CD4+ Th1 cell response are associated with immunopathology and failure to clear IOE in the fatal model of HME.

Critical Role of Cytotoxic T Lymphocytes in Immune Clearance of Rickettsial Infection

ABSTRACT Cytotoxic T-lymphocyte (CTL) activity developed against the major infected target cells of rickettsial infections, endothelial cells and macrophages. Spleen cells from mice immune to Rickettsia conorii exerted specific major histocompatibility complex (MHC) class I-matched CTL activity against R. conorii-infected SVEC-10 endothelial cells, with peak activity on day 10. Similarly, spleen cells from Rickettsia australis-immune mice exerted specific CTL activity against an R. australis-infected macrophage-like cell line. Gamma interferon (IFN-γ) gene knockout mice were more than 100-fold more susceptible to R. australis infection than wild-type C57BL/6 mice. MHC class I gene knockout mice were the most susceptible, more than 50,000-fold more susceptible to a lethal outcome of R. australis infection than wild-type C57BL/6 mice. These results indicate that CTL activity was more critical to recovery from rickettsial infection than were the effects of IFN-γ. The observation that perforin gene knockout mice were more than 100-fold more susceptible than wild-type C57BL/6 mice indicates that perforin-mediated activity accounts for a large component, but not all, of the CTL-mediated antirickettsial effect. CTL activity was expressed by immune CD8 T lymphocytes. Adoptive transfer of immune CD8 T lymphocytes from IFN-γ gene knockout mice intoR. australis-infected IFN-γ gene knockout mice dramatically reduced the infectious rickettsial content in the organs, confirming that CD8 T lymphocytes provide immunity against rickettsiae besides that provided by the secretion of IFN-γ. CTLs appear to be crucial to recovery from rickettsial infection.

Emerging Pathogens: Challenges and Successes of Molecular Diagnostics

More than 50 emerging and reemerging pathogens have been identified during the last 40 years. Until 1992 when the Institute of Medicine issued a report that defined emerging infectious diseases, medicine had been complacent about such infectious diseases despite the alarm bells of infections with human immunodeficiency virus. Molecular tools have proven useful in discovering and characterizing emerging viruses and bacteria such as Sin Nombre virus (hantaviral pulmonary syndrome), hepatitis C virus, Bartonella henselae (cat scratch disease, bacillary angiomatosis), and Anaplasma phagocytophilum (human granulocytotropic anaplasmosis). The feasibility of applying molecular diagnostics to dangerous, fastidious, and uncultivated agents for which conventional tests do not yield timely diagnoses has achieved proof of concept for many agents, but widespread use of cost-effective, validated commercial assays has yet to occur. This review presents representative emerging viral respiratory infections, hemorrhagic fevers, and hepatitides, as well as bacterial and parasitic zoonotic, gastrointestinal, and pulmonary infections. Agent characteristics, epidemiology, clinical manifestations, and diagnostic methods are tabulated for another 22 emerging viruses and five emerging bacteria. The ongoing challenge to the field of molecular diagnostics is to apply contemporary knowledge to facilitate agent diagnosis as well as to further discoveries of novel pathogens.

Human Monocytotropic Ehrlichiosis, Missouri

To determine the incidence, clinical and laboratory characteristics, and utility of molecular diagnosis of human monocytotropic ehrlichiosis (HME) in the primary care setting, we conducted a prospective study in an outpatient primary care clinic in Cape Girardeau, Missouri. One hundred and two patients with a history of fever for 3 days (>37.7°C), tick bite or exposure, and no other infectious disease diagnosis were enrolled between March 1997 and December 1999. HME was diagnosed in 29 patients by indirect immunofluorescent antibody assay and polymerase chain reaction (PCR). Clinical and laboratory manifestations included fever (100%), headache (72%), myalgia or arthralgia (69%), chills (45%), weakness (38%), nausea (38%), leukopenia (60%), thrombocytopenia (56%), and elevated aspartate aminotransferase level (52%). Hospitalization occurred in 41% of case-patients. PCR sensitivity was 56%; specificity, 100%. HME is a prevalent, potentially severe disease in southeastern Missouri that often requires hospitalization. Because clinical presentation of HME is nonspecific, PCR is useful in the diagnosis of acute HME.

Animal Model of Fatal Human Monocytotropic Ehrlichiosis

Human monocytotropic ehrlichiosis caused by Ehrlichia chaffeensis is a life-threatening, tick-borne, emerging infectious disease for which no satisfactory animal model has been developed. Strain HF565, an ehrlichial organism closely related to E. chaffeensis isolated from Ixodes ovatus ticks in Japan, causes fatal infection of mice. C57BL/6 mice became ill on day 7 after inoculation and died on day 9. The liver revealed confluent necrosis, ballooning cell injury, apoptosis, poorly formed granulomas, Kupffer cell hyperplasia, erythrophagocytosis, and microvesicular fatty metamorphosis. The other significant histological findings consisted of marked expansion of the marginal zone and infiltration of the red pulp of the spleen by macrophages, interstitial pneumonitis, and increased numbers of immature myeloid cells and areas of necrosis in the bone marrow. Ehrlichiae were detected by immunohistology and electron microscopy in the liver, lungs, and spleen. The main target cells were macrophages, including Kupffer cells, hepatocytes, and endothelial cells. Apoptosis was detected in Kupffer cells, hepatocytes, and macrophages in the lungs and spleen. This tropism for macrophages and the pathological lesions closely resemble those of human monocytotropic ehrlichiosis for which it is a promising model for investigation of immunity and pathogenesis.

Molecular Characterization of a Glucose-Inhibited Division Gene, gidA, That Regulates Cytotoxic Enterotoxin of Aeromonas hydrophila

ABSTRACT By using a mini-transposon, we obtained two mutated strains of a diarrheal isolate, SSU, of Aeromonas hydrophila that exhibited a 50 to 53% reduction in the hemolytic activity and 83 to 87% less cytotoxic activity associated with the cytotoxic enterotoxin (Act). Act is a potent virulence factor of A. hydrophila and has been shown to contribute significantly to the development of both diarrhea and septicemia in animal models. Subsequent cloning and DNA sequence analysis revealed that transposon insertion occurred at different locations in these two mutants within the same 1,890-bp open reading frame for the glucose-inhibited division gene (gidA). A similar reduction in hemolytic (46%) and cytotoxic (81%) activity of Act was noted in the gidA isogenic mutant of A. hydrophila that was generated by marker exchange mutagenesis. Northern blot analysis revealed that the transcription of the cytotoxic enterotoxin gene (act) was not altered in the gidA transposon and isogenic mutants. However, by generating a chromosomal act::alkaline phosphatase gene (phoA) reporter construct, we demonstrated significantly reduced phosphatase activity in these mutants, indicating the effect of glucose-inhibited division (GidA) protein in modulating act gene expression at the translational level. The biological effects of Act in the gidA mutants were restored by complementation. The virulence of the gidA mutants in mice was dramatically reduced compared to the those of the wild-type (WT) and complemented strains of A. hydrophila. The histopathological examination of lungs, in particular, indicated severe congestion, alveolar hemorrhage, and acute inflammatory infiltrate in the interstitial compartment and the alveolar spaces when mice were infected with the WT and complemented strains. Minimal-to-mild changes were noted in the lungs with the gidA mutants. Taken together, our data indicate for the first time that GidA regulates the most-potent virulence factor of A. hydrophila, Act.

Fc-Dependent Polyclonal Antibodies and Antibodies to Outer Membrane Proteins A and B, but Not to Lipopolysaccharide, Protect SCID Mice against Fatal Rickettsia conorii Infection

ABSTRACT An emphasis on cellular immunity against Rickettsia has led to neglect of analysis of the role of antibody. The availability of an excellent mouse model of spotted fever rickettsiosis enabled investigation of a potential role of antibody in immunity to Rickettsia conorii. C3H severe combined immunodeficiency (SCID) mice were passively transfused with monoclonal antibodies against rickettsial outer membrane protein A (OmpA), OmpB, or lipopolysaccharide (LPS), polyclonal anti-R. conorii serum, Fab fragments of polyclonal antiserum, or no antibodies and then challenged 48 h later with 10 50% lethal doses (LD50) of R. conorii. All mice that received monoclonal antibodies against OmpA and two of four mice that received monoclonal antibodies against OmpB or polyclonal antisera were completely protected, but the recipients of anti-LPS antibodies or the Fab fragments were not protected. Polyclonal antibody treatment of C3H SCID mice that had been infected with 10 LD50 of R. conorii 4 or 5 days earlier prolonged the life of the infected mice from 10.4 to 22.5 days and resulted in decreased levels of infectious rickettsiae in the spleen and liver 24 and 48 h later. Treatment with protective antibodies resulted in the development of large aggregates of R. conorii antigens in splenic macrophages and intraphagolysosomal rickettsial death and digestion. The kinetics of development of antibodies to R. conorii determined by immunoblotting revealed antibodies to LPS on day 6 and antibodies to OmpA and OmpB on day 12, when recovery from the infection had already occurred. Antibodies to particular epitopes of OmpA and OmpB may protect against reinfection, but they may not play a key role in immunity against primary infection. Antibodies might be useful for treating infections with antibiotic-resistant organisms, and some B-cell epitopes should be included in a subunit vaccine.

Surface-Expressed Enolase Contributes to the Pathogenesis of Clinical Isolate SSU of Aeromonas hydrophila

In this study, we demonstrated that the surface-expressed enolase from diarrheal isolate SSU of Aeromonas hydrophila bound to human plasminogen and facilitated the latters tissue-type plasminogen activator-mediated activation to plasmin. The bacterial surface-bound plasmin was more resistant to the action of its specific physiological inhibitor, the antiprotease alpha(2)-antiplasmin. We found that immunization of mice with purified recombinant enolase significantly protected the animals against a lethal challenge dose of wild-type (WT) A. hydrophila. Minimal histological changes were noted in organs from mice immunized with enolase and then challenged with WT bacteria compared to severe pathological changes found in the infected and nonimmunized group of animals. This correlated with the smaller bacterial load of WT bacteria in the livers and spleens of enolase-immunized mice than that found in the nonimmunized controls. We also showed that the enolase gene could potentially be important for the viability of A. hydrophila SSU as we could delete the chromosomal copy of the enolase gene only when another copy of the targeted gene was supplied in trans. By site-directed mutagenesis, we altered five lysine residues located at positions 343, 394, 420, 427, and 430 of enolase in A. hydrophila SSU; the mutated forms of enolase were hyperexpressed in Escherichia coli, and the proteins were purified. Our results indicated that lysine residues at positions 420 and 427 of enolase were crucial in plasminogen-binding activity. We also identified a stretch of amino acid residues ((252)FYDAEKKEY(260)) in the A. hydrophila SSU enolase involved in plasminogen binding. To our knowledge, this is the first report of the direct involvement of surface-expressed enolase in the pathogenesis of A. hydrophila SSU infections and of any gram-negative bacteria in general.

Braun Lipoprotein (Lpp) Contributes to Virulence of Yersiniae: Potential Role of Lpp in Inducing Bubonic and Pneumonic Plague

ABSTRACT Yersinia pestis evolved from Y. pseudotuberculosis to become the causative agent of bubonic and pneumonic plague. We identified a homolog of the Salmonella enterica serovar Typhimurium lipoprotein (lpp) gene in Yersinia species and prepared lpp gene deletion mutants of Y. pseudotuberculosis YPIII, Y. pestis KIM/D27 (pigmentation locus minus), and Y. pestis CO92 with reduced virulence. Mice injected via the intraperitoneal route with 5 × 107 CFU of the Δlpp KIM/D27 mutant survived a month, even though this would have constituted a lethal dose for the parental KIM/D27 strain. Subsequently, these Δlpp KIM/D27-injected mice were solidly protected against an intranasally administered, highly virulent Y. pestis CO92 strain when it was given as five 50% lethal doses (LD50). In a parallel study with the pneumonic plague mouse model, after 72 h postinfection, the lungs of animals infected with wild-type (WT) Y. pestis CO92 and given a subinhibitory dose of levofloxacin had acute inflammation, edema, and masses of bacteria, while the lung tissue appeared essentially normal in mice inoculated with the Δlpp mutant of CO92 and given the same dose of levofloxacin. Importantly, while WT Y. pestis CO92 could be detected in the bloodstreams and spleens of infected mice at 72 h postinfection, the Δlpp mutant of CO92 could not be detected in those organs. Furthermore, the levels of cytokines/chemokines detected in the sera were significantly lower in animals infected with the Δlpp mutant than in those infected with WT CO92. Additionally, the Δlpp mutant was more rapidly killed by macrophages than was the WT CO92 strain. These data provided evidence that the Δlpp mutants of yersiniae were significantly attenuated and could be useful tools in the development of new vaccines.