Mutations of Francisella novicida that Alter the Mechanism of Its Phagocytosis by Murine Macrophages

Infection with the bacterial pathogen Francisella tularensis tularensis (F. tularensis) causes tularemia, a serious and debilitating disease. Francisella tularensis novicida strain U112 (abbreviated F. novicida), which is closely related to F. tularensis, is pathogenic for mice but not for man, making it an ideal model system for tularemia. Intracellular pathogens like Francisella inhibit the innate immune response, thereby avoiding immune recognition and death of the infected cell. Because activation of inflammatory pathways may lead to cell death, we reasoned that we could identify bacterial genes involved in inhibiting inflammation by isolating mutants that killed infected cells faster than the wild-type parent. We screened a comprehensive transposon library of F. novicida for mutant strains that increased the rate of cell death following infection in J774 macrophage-like cells, as compared to wild-type F. novicida. Mutations in 28 genes were identified as being hypercytotoxic to both J774 and primary macrophages of which 12 were less virulent in a mouse infection model. Surprisingly, we found that F. novicida with mutations in four genes (lpcC, manB, manC and kdtA) were taken up by and killed macrophages at a much higher rate than the parent strain, even upon treatment with cytochalasin D (cytD), a classic inhibitor of macrophage phagocytosis. At least 10-fold more mutant bacteria were internalized by macrophages as compared to the parent strain if the bacteria were first fixed with formaldehyde, suggesting a surface structure is required for the high phagocytosis rate. However, bacteria were required to be viable for macrophage toxicity. The four mutant strains do not make a complete LPS but instead have an exposed lipid A. Interestingly, other mutations that result in an exposed LPS core were not taken up at increased frequency nor did they kill host cells more than the parent. These results suggest an alternative, more efficient macrophage uptake mechanism for Francisella that requires exposure of a specific bacterial surface structure(s) but results in increased cell death following internalization of live bacteria

A Francisella Mutant in Lipid A Carbohydrate Modification Elicits Protective Immunity

Francisella tularensis (Ft) is a highly infectious Gram-negative bacterium and the causative agent of the human disease tularemia. Ft is designated a class A select agent by the Centers for Disease Control and Prevention. Human clinical isolates of Ft produce lipid A of similar structure to Ft subspecies novicida (Fn), a pathogen of mice. We identified three enzymes required for Fn lipid A carbohydrate modifications, specifically the presence of mannose (flmF1), galactosamine (flmF2), or both carbohydrates (flmK). Mutants lacking either galactosamine (flmF2) or galactosamine/mannose (flmK) addition to their lipid A were attenuated in mice by both pulmonary and subcutaneous routes of infection. In addition, aerosolization of the mutants (flmF2 and flmK) provided protection against challenge with wild-type (WT) Fn, whereas subcutaneous administration of only the flmK mutant provided protection from challenge with WT Fn. Furthermore, infection of an alveolar macrophage cell line by the flmK mutant induced higher levels of tumor necrosis factor-α (TNF-α) and macrophage inhibitory protein-2 (MIP-2) when compared to infection with WT Fn. Bone marrow–derived macrophages (BMMø) from Toll-like receptor 4 (TLR4) and TLR2/4 knockout mice infected with the flmK mutant also produced significantly higher amounts of interleukin-6 (IL-6) and MIP-2 than BMMø infected with WT Fn. However, production of IL-6 and MIP-2 was undetectable in BMMø from MyD88−/− mice infected with either strain. MyD88−/− mice were also susceptible to flmK mutant infection. We hypothesize that the ability of the flmK mutant to activate pro-inflammatory cytokine/chemokine production and innate immune responses mediated by the MyD88 signaling pathway may be responsible for its attenuation, leading to the induction of protective immunity by this mutant

Norharmane matrix enhances detection of endotoxin by MALDI-MS for simultaneous profiling of pathogen, host, and vector systems

The discovery of novel pathogenic mechanisms engaged during bacterial infections requires the evolution of advanced techniques. Here, we evaluate the dual polarity matrix norharmane (NRM) to improve detection of bacterial lipid A (endotoxin), from host and vector tissues infected with Francisella novicida (Fn). We evaluated NRM for improved detection and characterization of a wide range of lipids in both positive and negative polarities, including lipid A and phospholipids across a range of matrix assisted laser desorption-ionization (MALDI)-coupled applications. NRM matrix improved the limit of detection (LOD) for monophosphoryl lipid A (MPLA) down to picogram-level representing a ten-fold improvement of LOD versus 2,5-dihydroxybenzoic acid (DHB) and 100-fold improvement of LOD versus 9-aminoacridine (9-AA). Improved LOD for lipid A subsequently facilitated detection of the Fn lipid A major ion (m/z 1665) from extracts of infected mouse spleen and the temperature-modified Fn lipid A at m/z 1637 from infected D. variabilis ticks. Finally, we simultaneously mapped bacterial phospholipid signatures within an Fn infected spleen along with exclusively host-derived inositol-based phospholipid (m/z 933) demonstrating co-profiling for the host-pathogen interaction. Expanded use of NRM matrix in other infection models and endotoxin-targeting imaging experiments will improve our understanding of the lipid interactions at the host-pathogen interface

The Fischer 344 Rat Reflects Human Susceptibility to Francisella Pulmonary Challenge and Provides a New Platform for Virulence and Protection Studies

Background: The pathogenesis of Francisella tularensis, the causative agent of tularemia, has been primarily characterized in mice. However, the high degree of sensitivity of mice to bacterial challenge, especially with the human virulent strains of F. tularensis, limits this animal model for screening of defined attenuated vaccine candidates for protection studies. Methods and Findings: We analyzed the susceptibility of the Fischer 344 rat to pulmonary (intratracheal) challenge with three different subspecies (subsp) of F. tularensis that reflect different levels of virulence in humans, and characterized the bacterial replication profile in rat bone marrow-derived macrophages (BMDM). In contrast to the mouse, Fischer 344 rats exhibit a broader range of sensitivity to pulmonary challenge with the human virulent subsp. tularensis and holarctica. Unlike mice, Fischer rats exhibited a high degree of resistance to pulmonary challenge with LVS (an attenuated derivative o

Macrophage Replication Screen Identifies a Novel Francisella Hydroperoxide Resistance Protein Involved in Virulence

Francisella tularensis is a Gram-negative facultative intracellular pathogen and the causative agent of tularemia. Recently, genome-wide screens have identified Francisella genes required for virulence in mice. However, the mechanisms by which most of the corresponding proteins contribute to pathogenesis are still largely unknown. To further elucidate the roles of these virulence determinants in Francisella pathogenesis, we tested whether each gene was required for replication of the model pathogen F. novicida within macrophages, an important virulence trait. Fifty-three of the 224 genes tested were involved in intracellular replication, including many of those within the Francisella pathogenicity island (FPI), validating our results. Interestingly, over one third of the genes identified are annotated as hypothetical, indicating that F. novicida likely utilizes novel virulence factors for intracellular replication. To further characterize these virulence determinants, we selected two hypothetical genes to study in more detail. As predicted by our screen, deletion mutants of FTN_0096 and FTN_1133 were attenuated for replication in macrophages. The mutants displayed differing levels of attenuation in vivo, with the FTN_1133 mutant being the most attenuated. FTN_1133 has sequence similarity to the organic hydroperoxide resistance protein Ohr, an enzyme involved in the bacterial response to oxidative stress. We show that FTN_1133 is required for F. novicida resistance to, and degradation of, organic hydroperoxides as well as resistance to the action of the NADPH oxidase both in macrophages and mice. Furthermore, we demonstrate that F. holarctica LVS, a strain derived from a highly virulent human pathogenic species of Francisella, also requires this protein for organic hydroperoxide resistance as well as replication in macrophages and mice. This study expands our knowledge of Francisella's largely uncharacterized intracellular lifecycle and demonstrates that FTN_1133 is an important novel mediator of oxidative stress resistance

Cathepsin B modulates lysosomal biogenesis and host defense against Francisella novicida infection

Lysosomal cathepsins regulate an exquisite range of biological functions, and their deregulation is associated with inflammatory, metabolic, and degenerative diseases in humans. In this study, we identified a key cell-intrinsic role for cathepsin B as a negative feedback regulator of lysosomal biogenesis and autophagy. Mice and macrophages lacking cathepsin B activity had increased resistance to the cytosolic bacterial pathogen Francisella novicida. Genetic deletion or pharmacological inhibition of cathepsin B down-regulated mechanistic target of rapamycin activity and prevented cleavage of the lysosomal calcium channel TRP ML1. These events drove transcription of lysosomal and autophagy genes via transcription factor EB, which increased lysosomal biogenesis and activation of autophagy initiation kinase ULK1 for clearance of the bacteria. Our results identified a fundamental biological function of cathepsin B in providing a checkpoint for homeostatic maintenance of lysosome populations and basic recycling functions in the cell

Epidemiology and Antimicrobial Resistance of Salmonella sp. Isolated from Dogs and Cats in Northeastern Thailand

During the period December 2009-November 2010, 600 fecal samples were collected from 250 diarrheal and 250 non-diarrheal dogs, 50 diarrheal and 50 non-diarrhea cats. It was found that 11.6 and 13.2% of diarrheal and non-diarrheal dogs and 8.0 and 10.0% of diarrheal and non-diarrheal cats were infected with Salmonella, respectively. The five most common serovars in dogs were S. stanley, S. rissen, S. enterica ser 4, 5, 12 :i:-, S. weltevreden and S. tryphimurium (14.5, 12.9, 11.3, 11.3 and 9.7%, respectively). The five most common serovars in cats were S. weltevreden, S. eastbourne, S. typhimurium, S. virchow and S. hvittingfoss (44.4, 22.2, 11.1, 11.1 and 11.1%, respectively). Isolates from dogs were resistant to amoxicillin (43.5%), gentamicin (8.1%), nalidixic acid (9.7%), sulphamethoxazole/trimethoprim (12.9%) and tetracycline (43.5%). The isolates from cats were resistant to amoxicillin (25%) and tetracycline (25%). Detection of Salmonella sp. in dogs and cats without clinical signs indicated that the animals were in carrier stage and potentially able to pass the disease to their owners

Severe anaphylaxis after pelvic examination: a case report of dual latex and chlorhexidine allergies

Abstract Background Natural rubber latex and chlorhexidine have previously been identified as causative substances in perioperative anaphylaxis. A pelvic examinations is generally considered noninvasive, however, this procedure is rarely associated with severe allergic reactions. We reported a rare case of dual latex and chlorhexidine allergies which caused anaphylaxis after pelvic examination in a woman with a history of latex-related fruits allergy. Case presentation A 54-year-old woman had severe anaphylaxis after a pelvic examination due to dual latex and chlorhexidine (CHX) allergies. The gynecologist used CHX for the vaginal preparation and wore latex-containing gloves with lubricating gel during the examination. In vivo and in vitro tests revealed CHX sensitization by a positive skin prick test to chlorhexidine at a very low concentration (0.002 mg/mL), and a positive basophil activation test to CHX. Latex allergy was confirmed by a positive specific IgE to latex and a positive glove-use test at 20 min. An analysis of specific IgE to latex component revealed positive results for Hev b 1, 5, 6.02, and 11. As she also had a past history of fruit allergy, prick-to-prick testing with latex-related fruits was performed. The results were positive for avocado, banana, jackfruit, kiwi, and longan. Conclusions Concomitant mucosal exposure of both natural rubber latex and CHX in highly sensitized patients during pelvic examinations can lead to severe anaphylaxis. Pre-procedural screening for an allergy to latex or CHX, or to any other allergen, should be performed in patients where there is suspicion of a specific allergy due to a previous allergic reaction. Increased awareness of these two allergens in all healthcare settings may improve patient safety