An antibiotic efflux gene cluster that confers resistance to chloramphenicol, trimethoprim, and ciprofloxacin has been identified in Burkholderia cenocepacia (genomovar III), an important cystic fibrosis pathogen. Five open reading frames have been identified in the cluster. There is apparently a single transcriptional unit, with llpE encoding a lipase-like protein, ceoA encoding a putative periplasmic linker protein, ceoB encoding a putative cytoplasmic membrane protein, and opcM encoding a previously described outer membrane protein. A putative LysR-type transcriptional regulatory gene, ceoR, is divergently transcribed upstream of the structural gene cluster. Experiments using radiolabeled chloramphenicol and salicylate demonstrated active efflux of both compounds in the presence of the gene cluster. Salicylate is an important siderophore produced by B. cepacia complex isolates, and both extrinsic salicylate and iron starvation appear to upregulate ceoR promoter activity, as does chloramphenicol. These results suggest that salicylate is a natural substrate for the efflux pump in B. cenocepacia and imply that the environment of low iron concentration in the cystic fibrosis lung can induce efflux-mediated resistance, even in the absence of antibiotic selective pressure.
Bindu M. Nair, K-John Cheung Jr., Adam Griffith, Jane L. Burns
Outer surface protein C (OspC) is a differentially expressed major surface lipoprotein of Borrelia burgdorferi. ospC is swiftly upregulated when spirochetes leave the Ixodes scapularis tick gut, migrate to the salivary gland, and exit the arthropod vector. Here we show that OspC strongly binds to the tick salivary gland, suggesting a role for OspC in spirochete adherence to this tissue. In vivo studies using a murine model of Lyme borreliosis showed that while OspC F(ab)2 fragments did not influence either the viability of spirochetes or ospC gene expression, they did interfere with B. burgdorferi invasion of tick salivary glands. We then generated ospC knockout spirochetes in an infectious clone of B. burgdorferi and examined them within the vector. OspC-deficient or wild-type spirochetes persisted equally within the gut of unfed ticks and multiplied during the tick engorgement; however, unlike wild-type B. burgdorferi, the mutants were unable to invade salivary glands. Salivary gland colonization of OspC-deficient spirochetes was completely restored when this mutant was complemented in trans with a plasmid harboring the wild-type ospC gene. These studies conclusively demonstrate the importance of OspC in the invasion of tick salivary glands by B. burgdorferi, a critical step in the transmission of spirochetes from the arthropod vector to the mammalian host.
Utpal Pal, Xiaofeng Yang, Manchuan Chen, Linda K. Bockenstedt, John F. Anderson, Richard A. Flavell, Michael V. Norgard, Erol Fikrig
Kaposi sarcoma–associated (KS-associated) herpesvirus (KSHV) infection is linked to the development of both KS and several lymphoproliferative diseases. In all cases, the resulting tumor cells predominantly display latent viral infection. KS tumorigenesis requires ongoing lytic viral replication as well, however, for reasons that are unclear but have been suggested to involve the production of angiogenic or mitogenic factors by lytically infected cells. Here we demonstrate that proliferating cells infected with KSHV in vitro display a marked propensity to segregate latent viral genomes, with only a variable but small subpopulation being capable of stable episome maintenance. Stable maintenance is not due to the enhanced production of viral or host trans-acting factors, but is associated with cis-acting, epigenetic changes in the viral chromosome. These results indicate that acquisition of stable KSHV latency is a multistep process that proceeds with varying degrees of efficiency in different cell types. They also suggest an additional role for lytic replication in sustaining KS tumorigenesis: namely, the recruitment of new cells to latency to replace those that have segregated the viral episome.
Adam Grundhoff, Don Ganem
Invasive aspergillosis is a severe pneumonia that is usually fatal despite currently available therapy. The disease disproportionately afflicts immunocompromised patients, indicating the critical importance of the immune status of the host in this infection, but the defense mechanisms against this pathogen remain incompletely understood. In the current study, we hypothesized that the chemokine ligand monocyte chemotactic protein-1, also designated CC chemokine ligand-2 (MCP-1/CCL2) is necessary for effective host defense against invasive aspergillosis in immunocompromised hosts. We found a rapid and marked induction of MCP-1/CCL2 in the lungs of neutropenic mice with invasive aspergillosis. Neutralizing MCP-1/CCL2 resulted in twofold greater mortality and greater than threefold increase in pathogen burden in the lungs. Neutralization of MCP-1/CCL2 also resulted in reduced recruitment of NK cells to the lungs at early time points, but did not affect the number of other leukocyte effector cells in the lungs. Ab-mediated depletion of NK cells similarly resulted in impaired defenses against the infection, resulting in a greater than twofold increase in mortality and impaired clearance of the pathogen from the lungs. These data establish MCP-1/CCL2–mediated recruitment of NK cells to the lungs as a critical early host defense mechanism in invasive aspergillosis and demonstrate NK cells to be an important and previously unrecognized effector cell in this infection.
Brad E. Morrison, Stacy J. Park, Jill M. Mooney, Borna Mehrad
A protective role for antibodies has not previously been described for host defense against the pathogenic fungus Histoplasma capsulatum (Hc). Mouse mAb’s were generated from mice immunized with Hc yeast that binds the cell surface of Hc. Administration of mAb’s before Hc infection reduced fungal burden, decreased pulmonary inflammation, and prolonged survival in a murine infection model. Protection mediated by mAb’s was associated with enhanced levels of IL-4, IL-6, and IFN-γ in the lungs of infected mice. The mAb’s increased phagocytosis of yeast by J774.16 cells through a CR3-dependent process. Ingestion of mAb-opsonized Hc by J774.16 macrophage-like cells was associated with yeast cell growth inhibition and killing. The mAb’s bound to a 17-kDa antigen expressed on the surface of Hc. The antigen was identified as a histone H2B–like protein. This study establishes that mAb’s to a cell surface protein of Hc alter the intracellular fate of the fungus and mediate protection in a murine model of lethal histoplasmosis, and it suggests a new candidate antigen for vaccine development.
Joshua D. Nosanchuk, Judith N. Steenbergen, Li Shi, George S. Deepe Jr., Arturo Casadevall
Cryptococcus neoformans is a fungal pathogen that, after inhalation, can disseminate to the brain. Host alveolar macrophages (AMs) represent the first defense against the fungus. Once phagocytosed by AMs, fungal cells are killed by a concerted mechanism, involving the host-cellular response. If the cellular response is impaired, phagocytosis of the fungus may be detrimental for the host, since C. neoformans can grow within macrophages. Here, we identified a novel cryptococcal gene encoding antiphagocytic protein 1 (App1). App1 is a cryptococcal cytoplasmic protein that is secreted extracellularly and found in the serum of infected patients. App1 does not affect melanin production, capsule formation, or growth of C. neoformans. Treatment with recombinant App1 inhibited phagocytosis of fungal cells through a complement-mediated mechanism, and Δapp1 mutant is readily phagocytosed by AMs. Interestingly, the Δapp1 mutant strain showed a decreased virulence in mice deficient for complement C5 (A/Jcr), but it was hypervirulent in mice deficient for T and NK cells (Tgε26). This study identifies App1 as a novel regulator of virulence for C. neoformans, and it highlights that internalization of fungal cells by AMs increases the dissemination of C. neoformans when the host cellular response is impaired.
Chiara Luberto, Beatriz Martinez-Mariño, Daniel Taraskiewicz, Benjamin Bolaños, Pasquale Chitano, Dena L. Toffaletti, Gary M. Cox, John R. Perfect, Yusuf A. Hannun, Edward Balish, Maurizio Del Poeta
Meningitis occurs when blood-borne pathogens cross the blood-brain barrier (BBB) in a complex interplay between endothelial cells and microbial gene products. We sought to understand the initial response of the BBB to the human meningeal pathogen group B Streptococcus (GBS) and the organism’s major virulence factors, the exopolysaccharide capsule and the β-hemolysin/cytolysin toxin (β-h/c). Using oligonucleotide microarrays, we found that GBS infection of human brain microvascular endothelial cells (HBMEC) induced a highly specific and coordinate set of genes including IL-8, Groα, Groβ, IL-6, GM-CSF, myeloid cell leukemia sequence-1 (Mcl-1), and ICAM-1, which act to orchestrate neutrophil recruitment, activation, and enhanced survival. Most strikingly, infection with a GBS strain lacking β-h/c resulted in a marked reduction in expression of genes involved in the immune response, while the unencapsulated strain generally induced similar or greater expression levels for the same subset of genes. Cell-free bacterial supernatants containing β-h/c activity induced IL-8 release, identifying this toxin as a principal provocative factor for BBB activation. These findings were further substantiated in vitro and in vivo. Neutrophil migration across polar HBMEC monolayers was stimulated by GBS and its β-h/c through a process involving IL-8 and ICAM-1. In a murine model of hematogenous meningitis, mice infected with β-h/c mutants exhibited lower mortality and decreased brain bacterial counts compared with mice infected with the corresponding WT GBS strains.
Kelly S. Doran, George Y. Liu, Victor Nizet
Bacillus anthracis lethal toxin (LT) is the major virulence factor of anthrax and reproduces most of the laboratory manifestations of the disease in animals. We studied LT toxicity in BALB/cJ and C57BL/6J mice. BALB/cJ mice became terminally ill earlier and with higher frequency than C57BL/6J mice. Timed histopathological analysis identified bone marrow, spleen, and liver as major affected organs in both mouse strains. LT induced extensive hypoxia. Crisis was due to extensive liver necrosis accompanied by pleural edema. There was no evidence of disseminated intravascular coagulation or renal dysfunction. Instead, analyses revealed hepatic dysfunction, hypoalbuminemia, and vascular/oxygenation insufficiency. Of 50 cytokines analyzed, BALB/cJ mice showed rapid but transitory increases in specific factors including KC, MCP-1/JE, IL-6, MIP-2, G-CSF, GM-CSF, eotaxin, FasL, and IL-1β. No changes in TNF-α occurred. The C57BL/6J mice did not mount a similar cytokine response. These factors were not induced in vitro by LT treatment of toxin-sensitive macrophages. The evidence presented shows that LT kills mice through a TNF-α–independent, FasL-independent, noninflammatory mechanism that involves hypoxic tissue injury but does not require macrophage sensitivity to toxin.
Mahtab Moayeri, Diana Haines, Howard A. Young, Stephen H. Leppla
Serum anti–T cell receptor (TCR) Ab’s are involved in immune regulation directed against pathogenic T cells in experimental models of autoimmune diseases. Our identification of a dominant T cell population expressing the Vβ5.1 TCR gene (TCRBV5-1), which is responsible for the production of pathogenic anti-acetylcholine receptor (AChR) autoantibodies in HLA-DR3 patients with early-onset myasthenia gravis (EOMG), prompted us to explore the occurrence, reactivity, and regulatory role of anti-TCR Ab’s in EOMG patients and disease controls with clearly defined other autoantibodies. In the absence of prior vaccination against the TCR, EOMG patients had elevated anti-Vβ5.1 Ab’s of the IgG class. This increase was restricted largely to EOMG cases with HLA-DR3 and with less severe disease, and it predicted clinical improvement in follow-up studies. EOMG patient sera containing anti-TCR Ab’s bound specifically the native TCR on intact Vβ5.1-expressing cells and specifically inhibited the proliferation and IFN-γ production of purified Vβ5.1-expressing cells to alloantigens in mixed lymphocyte reaction and the proliferation of a Vβ5.1-expressing T cell clone to an AChR peptide, indicating a regulatory function for these Ab’s. This evidence of spontaneously active anti-Vβ5.1 Ab’s in EOMG patients suggests dynamic protective immune regulation directed against the excess of pathogenic Vβ5.1-expressing T cells. Though not sufficient to prevent a chronic, exacerbated autoimmune process, it might be boosted using a TCR peptide as vaccine.
Florence Jambou, Wei Zhang, Monique Menestrier, Isabelle Klingel-Schmitt, Olivier Michel, Sophie Caillat-Zucman, Abderrahim Aissaoui, Ludovic Landemarre, Sonia Berrih-Aknin, Sylvia Cohen-Kaminsky
The worldwide increase in the prevalence of multi-antibiotic–resistant bacteria has threatened the physician’s ability to provide appropriate therapy for infections. The relationship between antimicrobial drug concentration and infecting pathogen population reduction is of primary interest. Using data derived from mice infected with the bacterium Pseudomonas aeruginosa and treated with a fluoroquinolone antibiotic, a mathematical model was developed that described relationships between antimicrobial drug exposures and changes in drug-susceptible and -resistant bacterial subpopulations at an infection site. Dosing regimens and consequent drug exposures that amplify or suppress the emergence of resistant bacterial subpopulations were identified and prospectively validated. Resistant clones selected in vivo by suboptimal regimens were characterized. No mutations were identified in the quinolone resistance–determining regions of gyrA/B or parC/E. However, all resistant clones demonstrated efflux pump overexpression. At base line, MexAB-OprM, MexCD-OprJ, and MexEF-OprN were represented in the drug-resistant population. After 28 hours of therapy, MexCD-OprJ became the predominant pump expressed in the resistant clones. The likelihood of achieving resistance-suppression exposure in humans with a clinically prescribed antibiotic dose was determined. The methods developed in this study provide insight regarding how mathematical models can be used to identify rational dosing regimens that suppress the amplification of the resistant mutant population.
Nelson Jumbe, Arnold Louie, Robert Leary, Weiguo Liu, Mark R. Deziel, Vincent H. Tam, Reetu Bachhawat, Christopher Freeman, James B. Kahn, Karen Bush, Michael N. Dudley, Michael H. Miller, George L. Drusano
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