[Abstract unavailable

Francisella tularensis is a gram-negative pathogen that causes life-threatening infections in humans and has potential for use as a biological weapon. The genetic basis of the F. tularensis virulence is poorly understood. This study screened a total of 3,936 transposon mutants of the live vaccine strain for infection in a mouse model of respiratory tularemia by signature-tagged mutagenesis. We identified 341 mutants attenuated for infection in the lungs. The transposon disruptions were mapped to 95 different genes, virtually all of which are also present in the genomes of other F. tularensis strains, including human pathogenic F. tularensis strain Schu S4. A small subset of these attenuated mutants carried insertions in the genes encoding previously known virulence factors, but the majority of the identified genes have not been previously linked to F. tularensis virulence. Among these are genes encoding putative membrane proteins, proteins associated with stress responses, metabolic proteins, transporter proteins, and proteins with unknown functions. Several attenuated mutants contained disruptions in a putative capsule locus which partially resembles the poly-␥-glutamate capsule biosynthesis locus of Bacillus anthracis, the anthrax agent. Deletional mutation analysis confirmed that this locus is essential for F. tularensis virulence.

Francisella tularensis causes systemic disease in humans and other mammals, with high morbidity and mortality associated with inhalation-acquired infection. F. tularensis is a facultative intracellular pathogen, but the scope and significance of cell types infected during disease is unknown. Using flow cytometry, we identified and quantified infected-cell types and assessed the impact of infection on cell populations following inhalation of F. tularensis strains U112, LVS, and Schu S4. Initially, alveolar macrophages comprised over 70% of Schu S4-and LVS-infected cells, whereas approximately 51% and 27% of U112-infected cells were alveolar macrophages and neutrophils, respectively. After 3 days, roughly half of Schu S4-and LVS-and nearly 80% of U112-infected cells were neutrophils. All strains infected CD11b high macrophages, dendritic cells, monocytes, and alveolar type II cells throughout infection. Macrophage, monocyte, and dendritic-cell populations were reduced during U112 infection but not Schu S4 or LVS infection. These results demonstrate directly that F. tularensis is a promiscuous intracellular pathogen in the lung that invades and replicates within cell types ranging from migratory immune cells to structural tissue cells. However, the proportions of cell types infected and the cellular immune response evoked by the human pathogenic strain Schu S4 differ from those of the human avirulent U112.Since 2001, there has been increased interest in understanding pathogens with virulence characteristics that make them dangerous for purposeful release. One such organism is the gram-negative bacterium Francisella tularensis, the etiological agent of tularemia. Virulent strains of Francisella can cause incapacitating or lethal disease in humans, mice, and other mammals (24). Tularemia can be acquired via insect bites (13), by handling infected animal carcasses (30), from contaminated water (11), or by inhalation (15). Inhalation exposure results in the most-acute, rapidly progressing manifestation of disease (6). When inhaled, as few as 10 organisms can cause a debilitating, and potentially fatal, infection in humans (22).Given the seriousness of inhalation-acquired tularemia, surprisingly little is known about Francisella biology in the host lung. Within 1 hour after inhalation, Francisella is found in airway macrophages and dendritic cells (DCs) (3, 4). However, the infection of these cells does not trigger production of tumor necrosis factor alpha or interleukin-6 (3, 4). Instead, Francisella infection induces immunosuppressive mediators, such as transforming growth factor ␤ (TGF-␤) (3) and prostaglandin E2 (31), through yet-unknown mechanisms. F. tularensis also infects other cell types important for host defense against lung infections, such as monocytes (23), neutrophils (16), and alveolar type II (ATII) epithelial cells (10).Growth within host cells is recognized as an important aspect of Francisella pathogenesis; however, the range and scope of cells infected throughout disease in an animal host have not b...

The second-generation antipsychotic olanzapine is effective in reducing psychotic symptoms but can cause extreme weight gain in human patients. We investigated the role of the gut microbiota in this adverse drug effect using a mouse model. First, we used germ-free C57BL/6J mice to demonstrate that gut bacteria are necessary and sufficient for weight gain caused by oral delivery of olanzapine. Second, we surveyed fecal microbiota before, during, and after treatment and found that olanzapine potentiated a shift towards an “obesogenic” bacterial profile. Finally, we demonstrated that olanzapine has antimicrobial activity in vitro against resident enteric bacterial strains. These results collectively provide strong evidence for a mechanism underlying olanzapine-induced weight gain in mouse and a hypothesis for clinical translation in human patients.

Francisella tularensis is a highly virulent intracellular pathogen that invades and replicates within numerous host cell types including macrophages, hepatocytes and pneumocytes. By 24 hours post invasion, F. tularensis replicates up to 1000-fold in the cytoplasm of infected cells. To achieve such rapid intracellular proliferation, F. tularensis must scavenge large quantities of essential carbon and energy sources from the host cell while evading anti-microbial immune responses. We found that macroautophagy, a eukaryotic cell process that primarily degrades host cell proteins and organelles as well as intracellular pathogens, was induced in F. tularensis infected cells. F. tularensis not only survived macroautophagy, but optimal intracellular bacterial growth was found to require macroautophagy. Intracellular growth upon macroautophagy inhibition was rescued by supplying excess nonessential amino acids or pyruvate, demonstrating that autophagy derived nutrients provide carbon and energy sources that support F. tularensis proliferation. Furthermore, F. tularensis did not require canonical, ATG5-dependent autophagy pathway induction but instead induced an ATG5-independent autophagy pathway. ATG5-independent autophagy induction caused the degradation of cellular constituents resulting in the release of nutrients that the bacteria harvested to support bacterial replication. Canonical macroautophagy limits the growth of several different bacterial species. However, our data demonstrate that ATG5-independent macroautophagy may be beneficial to some cytoplasmic bacteria by supplying nutrients to support bacterial growth.