Early Host Cell Targets of Yersinia pestis during Primary Pneumonic Plague

Inhalation of Yersinia pestis causes primary pneumonic plague, a highly lethal syndrome with mortality rates approaching 100%. Pneumonic plague progression is biphasic, with an initial pre-inflammatory phase facilitating bacterial growth in the absence of host inflammation, followed by a pro-inflammatory phase marked by extensive neutrophil influx, an inflammatory cytokine storm, and severe tissue destruction. Using a FRET-based probe to quantitate injection of effector proteins by the Y. pestis type III secretion system, we show that these bacteria target alveolar macrophages early during infection of mice, followed by a switch in host cell preference to neutrophils. We also demonstrate that neutrophil influx is unable to limit bacterial growth in the lung and is ultimately responsible for the severe inflammation during the lethal pro-inflammatory phase

Yersinia pestis Activates Both IL-1β and IL-1 Receptor Antagonist to Modulate Lung Inflammation during Pneumonic Plague

Pneumonic plague is the most rapid and lethal form of Yersinia pestis infection. Increasing evidence suggests that Y. pestis employs multiple levels of innate immune evasion and/or suppression to produce an early “pre-inflammatory” phase of pulmonary infection, after which the disease is highly inflammatory in the lung and 100% fatal. In this study, we show that IL-1β/IL-18 cytokine activation occurs early after bacteria enter the lung, and this activation eventually contributes to pulmonary inflammation and pathology during the later stages of infection. However, the inflammatory effects of IL-1β/IL-1-receptor ligation are not observed during this first stage of pneumonic plague. We show that Y. pestis also activates the induction of IL-1 receptor antagonist (IL-1RA), and this activation likely contributes to the ability of Y. pestis to establish the initial pre-inflammatory phase of disease

With Friends Like These: The Complex Role of Neutrophils in the Progression of Severe Pneumonia

Pneumonia is a leading cause of death from infection in the United States and across the globe. During pulmonary infection, clear resolution of host inflammatory responses occurs in the absence of appreciable lung damage. Neutrophils are the first wave of leukocytes to arrive in the lung upon infection. After activation, neutrophils traffic from the vasculature via transendothelial migration through the lung interstitium and into the alveolar space. Successful pulmonary immunity requires neutrophil-mediated killing of invading pathogens by phagocytosis and release of a myriad of antimicrobial molecules, followed by resolution of inflammation, neutrophil apoptosis, and clearing of dead or dying neutrophils by macrophages. In addition to their antimicrobial role, it is becoming clear that neutrophils are also important modulators of innate and adaptive immune responses, primarily through the release of cytokines and recruitment of additional waves of neutrophils into the airways. Though typically essential to combating severe pneumonia, neutrophil influx into the airways is a double-edged sword: Overzealous neutrophil activation can cause severe tissue damage as a result of the release of toxic agents including proteases, cationic polypeptides, cytokines, and reactive oxygen species (ROS) aimed at killing invading microbes. In extreme cases, the damage caused by neutrophils and other innate immune mediators become the primary source of morbidity and mortality. Here, we review the complex role of neutrophils during severe pneumonia by highlighting specific molecules and processes that contribute to pulmonary immunity, but can also drive progression of severe disease. Depending on the identity of the infectious agent, enhancing or suppressing neutrophil-mediated responses may be key to effectively treating severe and typically lethal pneumonia

Evaluating bacterial secretion using the FRET-based substrate CCF2/AM.

<p>To monitor bacterial secretion, the gene encoding a β-lactamase is cloned in-frame with a portion of the gene encoding a predicted secreted protein to generate a translational fusion. Eukaryotic cells are infected with the bacteria harboring the fusion protein, followed by incubation of infected cells with the fluorescent substrate CCF2/AM (or its analog CCF4/AM). CCF2/AM consists of 7-hydroxycoumarin and fluorescein connected with a cephalosporin linker. Cells can then be examined by flow cytometry, fluorescence microscopy, or using a plate reader in a multiwell format. Under excitation at 405 nm, the 7-hydroxycoumarin and fluorescein moieties of CCF2/AM exhibit FRET, and emission is detected from the acceptor fluorescein as green fluorescence (520 nm). If the target cell has undergone translocation and harbors the bacterial fusion protein, the cephalosporin linker is cleaved by β-lactamase, which disrupts FRET and results in blue fluorescence from 7-hydroxycoumarin at 447 nm.</p

Working toward the Future: Insights into Francisella tularensis Pathogenesis and Vaccine Development

Summary: Francisella tularensis is a facultative intracellular gram-negative pathogen and the etiological agent of the zoonotic disease tularemia. Recent advances in the field of Francisella genetics have led to a rapid increase in both the generation and subsequent characterization of mutant strains exhibiting altered growth and/or virulence characteristics within various model systems of infection. In this review, we summarize the major properties of several Francisella species, including F. tularensis and F. novicida, and provide an up-to-date synopsis of the genes necessary for pathogenesis by these organisms and the determinants that are currently being targeted for vaccine development

In Vivo Himar1-Based Transposon Mutagenesis of Francisella tularensis

Francisella tularensis is the intracellular pathogen that causes human tularemia. It is recognized as a potential agent of bioterrorism due to its low infectious dose and multiple routes of entry. We report the development of a Himar1-based random mutagenesis system for F. tularensis (HimarFT). In vivo mutagenesis of F. tularensis live vaccine strain (LVS) with HimarFT occurs at high efficiency. Approximately 12 to 15% of cells transformed with the delivery plasmid result in transposon insertion into the genome. Results from Southern blot analysis of 33 random isolates suggest that single insertions occurred, accompanied by the loss of the plasmid vehicle in most cases. Nucleotide sequence analysis of rescued genomic DNA with HimarFT indicates that the orientation of integration was unbiased and that insertions occurred in open reading frames and intergenic and repetitive regions of the chromosome. To determine the utility of the system, transposon mutagenesis was performed, followed by a screen for growth on Chamberlain's chemically defined medium (CDM) to isolate auxotrophic mutants. Several mutants were isolated that grew on complex but not on the CDM. We genetically complemented two of the mutants for growth on CDM with a newly constructed plasmid containing a nourseothricin resistance marker. In addition, uracil or aromatic amino acid supplementation of CDM supported growth of isolates with insertions in pyrD, carA, or aroE1 supporting the functional assignment of genes within each biosynthetic pathway. A mutant containing an insertion in aroE1 demonstrated delayed replication in macrophages and was restored to the parental growth phenotype when provided with the appropriate plasmid in trans. Our results suggest that a comprehensive library of mutants can be generated in F. tularensis LVS, providing an additional genetic tool to identify virulence determinants required for survival within the host

YopE-TEM translocation in the lung during pneumonic plague.

<p>(A) Groups of mice were inoculated intranasally with 10⁶ CFU of <i>Y. pestis</i> expressing YopE-TEM or PBS alone (mock), followed by staining with CCF2-AM. Lungs were harvested at 12 hpi, and live cells were gated to evaluate blue/green fluorescence by flow cytometry. Cells showing blue fluorescence have been injected with YopE-TEM. Histograms show representative data from a single mouse; (B) Total injection events (cells exhibiting blue fluorescence) in the lungs of mice inoculated with 10⁶ CFU <i>Y. pestis</i> YopE-TEM; (C) The percentage of total cells in the lung injected with YopE-TEM was evaluated for samples shown in (B) at 6, 12, and 24 hpi; (D) Lung bacterial burden was evaluated in mice inoculated with 10⁶ CFU <i>Y. pestis</i> YopE-TEM. (E) Live injected cells in lungs of mice inoculated with 10⁶ CFU <i>Y. pestis</i> YopE-TEM were gated for identification of alveolar macrophages (F480⁺CD11b^low/midCD11c^high), interstitial macrophages (F480⁺CD11b^highCD11c^mid/low), F480⁺CD11b^highCD11c^high macrophages, monocytes (F480⁻CD11b^highLy-6G⁻), neutrophils (F480⁻CD11b^highLy-6G⁺), and CD11b high and low dendritic cells (F480⁻ CD11c⁺); All data are representative of at least three independent experiments with three to five mice at each time point. Error bars represent SEM.</p

Pre-treatment of mice with clodrosome or neutrophil-depleting antibody α-Ly-6G.

<p>(A) Total YopE-TEM injection events 6 hpi in mice inoculated with 10⁶ CFU <i>Y. pestis</i> YopE-TEM with and without prior alveolar macrophage depletion with clodrosome. (B) Total YopE-TEM injection events 24 hpi in <i>Y. pestis</i>-infected mice with and without pre-treatment with neutrophil-depleting antibody. (C) Identity of injected cells 6 hpi in mice with and without prior clodrosome treatment. (D) Identity of injected cells 24 hpi with and without pre-treatment with neutrophil-depleting antibody. Data are representative of at least three independent experiments with three to five mice at each time point. Error bars represent SEM for all graphs.</p