Francisella Infections Research Articles

A Francisella tularensis-Like Bacterium in Tropical Bed Bugs from Madagascar.

The genus Francisella includes several highly virulent human pathogens and some tick endosymbionts. Francisella infections are acquired by humans through contact with vertebrate animal reservoirs or contaminated water or dust. The species Francisella tularensis can also be transmitted by arthropods including ticks, mosquitoes, and flies. For the first time, we describe the molecular detection of an F. tularensis-like bacterium in bed bugs from samples collected in rural Madagascar. This finding suggests a potential involvement of bed bugs in the ecology of Francisella. The role of bed bugs as possible hosts, reservoirs, or vectors of Francisella spp. should be further investigated.

Host Immunity and Francisella tularensis: A Review of Tularemia in Immunocompromised Patients.

Tularemia, caused by the bacterium Francisella tularensis, is an infrequent zoonotic infection, well known in immunocompetent (but poorly described in immunocompromised) patients. Although there is no clear literature data about the specific characteristics of this disease in immunocompromised patients, clinical reports seem to describe a different presentation of tularemia in these patients. Moreover, atypical clinical presentations added to the fastidiousness of pathogen identification seem to be responsible for a delayed diagnosis, leading to a” loss of chance” for immunocompromised patients. In this article, we first provide an overview of the host immune responses to Francisella infections and discuss how immunosuppressive therapies or diseases can lead to a higher susceptibility to tularemia. Then, we describe the particular clinical patterns of tularemia in immunocompromised patients from the literature. We also provide hints of an alternative diagnostic strategy regarding these patients. In conclusion, tularemia should be considered in immunocompromised patients presenting pulmonary symptoms or unexplained fever. Molecular techniques on pathological tissues might improve diagnosis with faster results.

Increased Resistance to Intradermal Francisella tularensis LVS Infection by Inactivation of the Sts Phosphatases.

The Suppressor of TCR signaling proteins (Sts-1 and Sts-2) are two homologous phosphatases that negatively regulate signaling pathways in a number of hematopoietic lineages, including T lymphocytes. Mice lacking Sts expression are characterized by enhanced T cell responses. Additionally, a recent study demonstrated that Sts-/- mice are profoundly resistant to systemic infection by Candida albicans, with resistance characterized by enhanced survival, more rapid fungal clearance in key peripheral organs, and an altered inflammatory response. To investigate the role of Sts in the primary host response to infection by a bacterial pathogen, we evaluated the response of Sts-/- mice to infection by a Gram-negative bacterial pathogen. Francisella tularensis is a facultative bacterial pathogen that replicates intracellularly within a variety of cell types and is the causative agent of tularemia. Francisella infections are characterized by a delayed immune response, followed by an intense inflammatory reaction that causes widespread tissue damage and septic shock. Herein, we demonstrate that mice lacking Sts expression are significantly resistant to infection by the live vaccine strain (LVS) of F. tularensis Resistance is characterized by reduced lethality following high-dose intradermal infection, an altered cytokine response in the spleen, and enhanced bacterial clearance in multiple peripheral organs. Sts-/- bone marrow-derived monocytes and neutrophils, infected with F. tularensis LVS ex vivo, display enhanced restriction of intracellular bacteria. These observations suggest the Sts proteins play an important regulatory role in the host response to bacterial infection, and they underscore a role for Sts in regulating functionally relevant immune response pathways.

Treatment of Francisella infections via PLGA- and lipid-based nanoparticle delivery of antibiotics in a zebrafish model.

We tested the efficiency of 2 different antibiotics, rifampicin and oxolinic acid, against an established infection caused by fish pathogen Francisella noatunensis ssp. orientalis (F.n.o.) in zebrafish. The drugs were tested in the free form as well as encapsulated into biodegradable nanoparticles, either polylactic-co-glycolic acid (PLGA) nanoparticles or nanostructured lipid carriers. The most promising therapies were PLGA-rifampicin nanoparticles and free oxolinic acid; the PLGA nanoparticles significantly delayed embryo mortality while free oxolinic acid prevented it. Encapsulation of rifampicin in both PLGA and nanostructured lipid carriers enhanced its efficiency against F.n.o. infection relative to the free drug. We propose that the zebrafish model is a robust, rapid system for initial testing of different treatments of bacterial diseases important for aquaculture.

Francisella Infection in Cultured Tilapia in Thailand and the Inflammatory Cytokine Response.

Francisella infections developed in freshwater Nile Tilapia Oreochromis niloticus and red tilapia Oreochromis spp. farms in Thailand during 2012-2014. The diseased fish were lethargic and pale in color and showed numerous white nodules in their enlarged spleens. Histopathological examination and electron microscopy suggested that the white nodules were multifocal granulomas consisting of coccobacilli within vacuolated cells. Isolation of Francisella-like bacteria was achieved from 42 of 100 samples, while polymerase chain reaction confirmed Francisella infections in all samples. Analysis of the 16S rRNA gene from samples obtained from three different geographical culture areas revealed more than 99% similarity with F. noatunensis subsp. orientalis. The influence of Francisella infection on inflammatory cytokines was determined on splenic cells of fish intraperitoneally injected with the bacteria (0.8 × 10(5) colony-forming units per fish). Infected tilapia showed significantly greater expression of the pro-inflammatory genes interleukin-1β (IL-1β) and tumor necrotic factor-α (TNF-α) within 24 h postinjection (hpi) and for up to 96 hpi. However, down-regulation of an anti-inflammatory gene, transforming growth factor-β (TGF-β) was observed as early as 24 hpi. This investigation demonstrates that an imbalance between pro- and anti-inflammatory cytokines in response to the infection may account for the substantial number of granulomas in fish hematopoietic tissues that was found in the later stage of the disease. Received September 9, 2015; accepted December 13, 2015.

Treatment of Francisella Infections for Aquaculture using PLGA- and Lipid-based 2Nanoparticle Delivery of Antibiotics in a Zebrafish Model

Treatment of Francisella Infections for Aquaculture using PLGA- and Lipid-based 2Nanoparticle Delivery of Antibiotics in a Zebrafish Model

Morphological analysis of Francisella novicida epithelial cell infections in the absence of functional FipA.

Francisella novicida is a surrogate pathogen commonly used to study infections by the potential bioterrorism agent, Francisella tularensis. One of the primary sites of Francisella infections is the liver where >90% of infected cells are hepatocytes. It is known that once Francisella enter cells it occupies a membrane-bound compartment, the Francisella-containing vacuole (FCV), from which it rapidly escapes to replicate in the cytosol. Recent work examining the Francisella disulfide bond formation (Dsb) proteins, FipA and FipB, have demonstrated that these proteins are important during the Francisella infection process; however, details as to how the infections are altered in epithelial cells have remained elusive. To identify the stage of the infections where these Dsbs might act during epithelial infections, we exploited a hepatocyte F. novicida infection model that we recently developed. We found that F. novicida ΔfipA-infected hepatocytes contained bacteria clustered within lysosome-associated membrane protein 1-positive FCVs, suggesting that FipA is involved in the escape of F. novicida from its vacuole. Our morphological evidence provides a tangible link as to how Dsb FipA can influence Francisella infections.

Interactions of Francisella tularensis with Alveolar Type II Epithelial Cells and the Murine Respiratory Epithelium.

Francisella tularensis is classified as a Tier 1 select agent by the CDC due to its low infectious dose and the possibility that the organism can be used as a bioweapon. The low dose of infection suggests that Francisella is unusually efficient at evading host defenses. Although ~50 cfu are necessary to cause human respiratory infection, the early interactions of virulent Francisella with the lung environment are not well understood. To provide additional insights into these interactions during early Francisella infection of mice, we performed TEM analysis on mouse lungs infected with F. tularensis strains Schu S4, LVS and the O-antigen mutant Schu S4 waaY::TrgTn. For all three strains, the majority of the bacteria that we could detect were observed within alveolar type II epithelial cells at 16 hours post infection. Although there were no detectable differences in the amount of bacteria within an infected cell between the three strains, there was a significant increase in the amount of cellular debris observed in the air spaces of the lungs in the Schu S4 waaY::TrgTn mutant compared to either the Schu S4 or LVS strain. We also studied the interactions of Francisella strains with human AT-II cells in vitro by characterizing the ability of these three strains to invade and replicate within these cells. Gentamicin assay and confocal microscopy both confirmed that F. tularensis Schu S4 replicated robustly within these cells while F. tularensis LVS displayed significantly lower levels of growth over 24 hours, although the strain was able to enter these cells at about the same level as Schu S4 (1 organism per cell), as determined by confocal imaging. The Schu S4 waaY::TrgTn mutant that we have previously described as attenuated for growth in macrophages and mouse virulence displayed interesting properties as well. This mutant induced significant airway inflammation (cell debris) and had an attenuated growth phenotype in the human AT-II cells. These data extend our understanding of early Francisella infection by demonstrating that Francisella enter significant numbers of AT-II cells within the lung and that the capsule and LPS of wild type Schu S4 helps prevent murine lung damage during infection. Furthermore, our data identified that human AT-II cells allow growth of Schu S4, but these same cells supported poor growth of the attenuated LVS strain in vitro. Collectively, these data further our understanding of the role of AT-II cells in Francisella infections.

IglC and PdpA are important for promoting Francisella invasion and intracellular growth in epithelial cells.

The highly infectious bacteria, Francisella tularensis, colonize a variety of organs and replicate within both phagocytic as well as non-phagocytic cells, to cause the disease tularemia. These microbes contain a conserved cluster of important virulence genes referred to as the Francisella Pathogenicity Island (FPI). Two of the most characterized FPI genes, iglC and pdpA, play a central role in bacterial survival and proliferation within phagocytes, but do not influence bacterial internalization. Yet, their involvement in non-phagocytic epithelial cell infections remains unexplored. To examine the functions of IglC and PdpA on bacterial invasion and replication during epithelial cell infections, we infected liver and lung epithelial cells with F. novicida and F. tularensis ‘Type B’ Live Vaccine Strain (LVS) deletion mutants (ΔiglC and ΔpdpA) as well as their respective gene complements. We found that deletion of either gene significantly reduced their ability to invade and replicate in epithelial cells. Gene complementation of iglC and pdpA partially rescued bacterial invasion and intracellular growth. Additionally, substantial LAMP1-association with both deletion mutants was observed up to 12 h suggesting that the absence of IglC and PdpA caused deficiencies in their ability to dissociate from LAMP1-positive Francisella Containing Vacuoles (FCVs). This work provides the first evidence that IglC and PdpA are important pathogenic factors for invasion and intracellular growth of Francisella in epithelial cells, and further highlights the discrete mechanisms involved in Francisella infections between phagocytic and non-phagocytic cells.

Establishment of Three Francisella Infections in Zebrafish Embryos at Different Temperatures

Francisella spp. are facultative intracellular pathogens identified in increasingly diverse hosts, including mammals. F. noatunensis subsp. orientalis and F. noatunensis subsp. noatunensis infect fish inhabiting warm and cold waters, respectively, while F. tularensis subsp. novicida is highly infectious for mice and has been widely used as a model for the human pathogen F. tularensis. Here, we established zebrafish embryo infection models of fluorescently labeled F. noatunensis subsp. noatunensis, F. noatunensis subsp. orientalis, and F. tularensis subsp. novicida at 22, 28, and 32°C, respectively. All infections led to significant bacterial growth, as shown by reverse transcription-quantitative PCR (RT-qPCR), and to a robust proinflammatory immune response, dominated by increased transcription of tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β). F. noatunensis subsp. orientalis was the most virulent, F. noatunensis subsp. noatunensis caused chronic infection, and F. tularensis subsp. novicida showed moderate virulence and led to formation of relatively small granuloma-like structures. The use of transgenic zebrafish strains with enhanced green fluorescent protein (EGFP)-labeled immune cells revealed their detailed interactions with Francisella species. All three strains entered preferentially into macrophages, which eventually assembled into granuloma-like structures. Entry into neutrophils was also observed, though the efficiency of this event depended on the route of infection. The results demonstrate the usefulness of the zebrafish embryo model for studying infections caused by different Francisella species at a wide range of temperatures and highlight their interactions with immune cells.

Alternative Activation of Macrophages and Induction of Arginase Are Not Components of Pathogenesis Mediated by Francisella Species

Virulent Francisella tularensis ssp tularensis is an intracellular, Gram negative bacterium that causes acute lethal disease following inhalation of fewer than 15 organisms. Pathogenicity of Francisella infections is tied to its unique ability to evade and suppress inflammatory responses in host cells. It has been proposed that induction of alternative activation of infected macrophages is a mechanism by which attenuated Francisella species modulate host responses. In this report we reveal that neither attenuated F. tularensis Live Vaccine Strain (LVS) nor virulent F. tularensis strain SchuS4 induce alternative activation of macrophages in vitro or in vivo. LVS, but not SchuS4, provoked production of arginase1 independent of alternative activation in vitro and in vivo. However, absence of arginase1 did not significantly impact intracellular replication of LVS or SchuS4. Together our data establish that neither induction of alternative activation nor expression of arginase1 are critical features of disease mediated by attenuated or virulent Francisella species.

Francisella noatunensis subsp. noatunensis is the aetiological agent of visceral granulomatosis in wild Atlantic cod Gadus morhua

During the 1980s and 1990s wild-caught cod displaying visceral granulomatosis were sporadically identified from the southern North Sea. Presumptive diagnoses at the time included mycobacterial infection, although mycobacteria were never cultivated or observed histologically from these fish. Farmed cod in Norway displaying gross pathology similar to that identified previously in cod from the southern North Sea were recently discovered to be infected with the bacterium Francisella noatunensis subsp, noatunensis. Archived formalin-fixed paraffin-embedded tissues from the original North Sea cases were investigated for the presence of Mycobacterium spp. and Francisella spp. using real-time polymerase chain reaction, DNA sequencing and immunohistochemistry. Whilst no evidence of mycobacterial infection was found, F. noatunensis subsp. noatunensis was identified in association with pathological changes consistent with Francisella infections described from farmed cod in recent years. This study shows that francisellosis occurred in wild-caught cod in the southern North Sea in the 1980s and 1990s and demonstrates that this disease predates intensive aquaculture of cod.

Francisella infections in fish and shellfish

A series of recent reports have implicated bacteria from the family Francisellaceae as the cause of disease in farmed and wild fish and shellfish species such as Atlantic cod, Gadus morhua L., tilapia, Oreochromis spp., Atlantic salmon, Salmo salar L., three-line grunt, Parapristipoma trilineatum (Thunberg), ornamental cichlid species, hybrid striped bass Morone chrysops x M.saxatilis and, recently, a shellfish species, the giant abalone, Haliotisgigantea Gmelin. The range of taxa affected will very probably rise as it is likely that there has been considerable under-reporting to date of these disease agents. In common with other Francisella species, their isolation and culture require specialized solid and liquid media containing cysteine and a source of iron. This likely restricted earlier efforts to identify them correctly as the cause of disease in aquatic animals. The most information to date relates to disease in cod, caused by F.noatunensis and tilapia, caused by F.noatunensis subsp. orientalis (also termed F.asiatica), both causing granulomatous inflammatory reactions. Mortalities in both species can be high and, as the disease can likely be transferred via live fish movements, they pose a significant threat to tilapia and cod aquaculture operations. Although the fish-pathogenic Francisella species are classified in the same genus as the human pathogens F.tularensis, causative agent of tularemia, and F.philomiragia, the risk to humans from the fish and shellfish pathogenic Francisella species is considered very low.

Francisella infections in farmed and wild aquatic organisms

Over the last 10 years or so, infections caused by bacteria belonging to a particular branch of the genus Francisella have become increasingly recognised in farmed fish and molluscs worldwide. While the increasing incidence of diagnoses may in part be due to the development and widespread availability of molecular detection techniques, the domestication of new organisms has undoubtedly instigated emergence of clinical disease in some species. Francisellosis in fish develops in a similar fashion independent of host species and is commonly characterised by the presence of multi-organ granuloma and high morbidity, with varying associated mortality levels. A number of fish species are affected including Atlantic cod, Gadus morhua; tilapia, Oreochromis sp.; Atlantic salmon, Salmo salar; hybrid striped bass, Morone chrysops × M. saxatilis and three-lined grunt, Parapristipoma trilinineatum. The disease is highly infectious and often prevalent in affected stocks. Most, if not all strains isolated from teleost fish belong to either F. noatunensis subsp. orientalis in warm water fish species or Francisella noatunensis subsp. noatunensis in coldwater fish species. The disease is quite readily diagnosed following histological examination and identification of the aetiological bacterium by culture on cysteine rich media or PCR. The available evidence may indicate a degree of host specificity for the various Francisella strains, although this area requires further study. No effective vaccine is currently available. Investigation of the virulence mechanisms and host response shows similarity to those known from Francisella tularensis infection in mammals. However, no evidence exists for zoonotic potential amongst the fish pathogenic Francisella.

Defect in efferocytosis leads to alternative activation of macrophages in Francisella infections

The macrophage is a versatile cell type that can sense and respond to a particular need based on the conditions of the microenvironment. Some studies have recently suggested that pathogens can directly influence the polarization of macrophages. As Francisella infections are characterized by intense necrotic infiltrates in the lung as well as in distal sites of infection, we sought to investigate whether pulmonary Francisella infections could cause the polarization of alternatively activated macrophages (M2/aaMs). Our results indicate that Francisella infections can cause the polarization of M2/aaM in vivo and that macrophages can be polarized toward an M2/aaM phenotype more potently if dead cell debris is used for stimulation in the presence and absence of Francisella infections. Finally, we also demonstrate that efferocytosis is inhibited in macrophages infected with Francisella, thus providing a potential explanation for the lack of clearance and eventual accumulation of dead cell debris associated with this disease.

Azithromycin effectiveness against intracellular infections of Francisella

BackgroundMacrolide antibiotics are commonly administered for bacterial respiratory illnesses. Azithromycin (Az) is especially noted for extremely high intracellular concentrations achieved within macrophages which is far greater than the serum concentration. Clinical strains of Type B Francisella (F.) tularensis have been reported to be resistant to Az, however our laboratory Francisella strains were found to be sensitive. We hypothesized that different strains/species of Francisella (including Type A) may have different susceptibilities to Az, a widely used and well-tolerated antibiotic.ResultsIn vitro susceptibility testing of Az confirmed that F. tularensis subsp. holarctica Live Vaccine Strain (LVS) (Type B) was not sensitive while F. philomiragia, F. novicida, and Type A F. tularensis (NIH B38 and Schu S4 strain) were susceptible. In J774A.1 mouse macrophage cells infected with F. philomiragia, F. novicida, and F. tularensis LVS, 5 μg/ml Az applied extracellularly eliminated intracellular Francisella infections. A concentration of 25 μg/ml Az was required for Francisella-infected A549 human lung epithelial cells, suggesting that macrophages are more effective at concentrating Az than epithelial cells. Mutants of RND efflux components (tolC and ftlC) in F. novicida demonstrated less sensitivity to Az by MIC than the parental strain, but the tolC disc-inhibition assay demonstrated increased sensitivity, indicating a complex role for the outer-membrane transporter. Mutants of acrA and acrB mutants were less sensitive to Az than the parental strain, suggesting that AcrAB is not critical for the efflux of Az in F. novicida. In contrast, F. tularensis Schu S4 mutants ΔacrB and ΔacrA were more sensitive than the parental strain, indicating that the AcrAB may be important for Az efflux in F. tularensis Schu S4. F. novicida LPS O-antigen mutants (wbtN, wbtE, wbtQ and wbtA) were found to be less sensitive in vitro to Az compared to the wild-type. Az treatment prolonged the survival of Galleria (G.) mellonella infected with Francisella.ConclusionThese studies demonstrate that Type A Francisella strains, as well as F. novicida and F. philomiragia, are sensitive to Az in vitro. Francisella LPS and the RND efflux pump may play a role in Az sensitivity. Az also has antimicrobial activity against intracellular Francisella, suggesting that the intracellular concentration of Az is high enough to be effective against multiple strains/species of Francisella, especially in macrophages. Az treatment prolonged survival an in vivo model of Francisella-infection.

Lethal pulmonary infection with Francisella novicida is associated with severe sepsis.

The bacterial or host determinants of lethality associated with respiratory Francisella infections are currently unknown. No exo- or endotoxins that contribute to the severity of this disease have been identified. However, a deregulated host immune response upon infection is characterized by an initial 36- to 48-h delay followed by a rapid and excessive inflammatory response prior to death at 72-120 h. Here, we extend these findings by comparing host immune responses between sublethal and lethal respiratory infections of mice with an attenuated transposon mutant (Mut) of F. novicida (F.n.) strain U112 (sublethal) versus the wild-type (WT) strain (lethal). Infection with WT bacteria, but not the Mut, was characterized by sustained bacteremia and systemic dissemination of the pathogen with temporal increases in bacterial burdens in liver and spleen. Severe pathology with large foci of infiltrates associated with extensive tissue damage was evident in WT-infected lungs, and Mut-infected mice displayed much reduced pathology with intact lung architecture. Similar to other experimental models of severe sepsis, WT- but not the Mut-infected mice exhibited a robust increase in numbers of Gr1+ and CD11b+ cells, while displaying a significant depletion of alphabeta T cells. Further, a dramatic up-regulation of multiple cytokines and chemokines was observed only in lethal WT infection. In addition, an earlier and larger increased expression of S100A9, a known mediator of sepsis, was observed in WT-infected mice. Taken together, these results show that a hyperinflammatory host immune response, culminating in severe sepsis, is responsible for the lethal outcome of respiratory tularemia.

Role of IFNG in Granuloma Formation in a Zebafish Model of Francisella Infection (134.94)

Abstract Members of the bacterial genus Francisella are highly virulent, gram-negative pathogens. New models to study Francisella pathogenesis in evolutionarily distinct species provide comparative insight, as the mechanisms of host resistance and pathogen virulence are not well understood. Using a fish-specific species of Francisella, we recently demonstrated that acutely infected zebrafish undergo a disease process and mount an innate immune response that shares many features with tularemia. In heavily colonized zebrafish immune tissues, bacterial antigens begin to localize to focal regions by five days post-infection as assessed by immunohistochemistry. Currently, we are exploring how Francisella establishes chronic infections in zebrafish. The early focal regions appear to correspond to areas of initial granuloma formation. We hypothesize that the establishment of Francisella-mediated granulomas in zebrafish tissues may represent a host-protective mechanism, confining bacteria and thereby allowing for a strong innate immune response to acute infection to abate. In particular, we are interested in the role that IFNG produced by helper T-cells plays in the formation and maintenance of granulomas during chronic Francisella infections. To do so, we are assessing the formation of granulomas over time in wild type and RAG1 knockout strains of zebrafish via histopathology and immunohistochemistry. In addition, the induction of granuloma formation will also be correlated with the transcriptional response of IFNG and other immune response genes by qRT-PCR in wild type and RAG1 knockout fish. This study will illustrate specific aspects of granuloma formation and host immune responses that are attributable to adaptive immunity.

Lethal pulmonary infection with Francisella novicida causes depletion of αβ T cells from lungs

Respiratory Francisella infections induce a delayed innate immune response followed by a severe sepsis like condition. In this study, mice infected intranasally with Francisella novicida showed a depletion of αβ T cells in lungs while exhibiting large accumulations of other leukocytes correlating with disease severity. The depleted T cells were predominantly CD4 +. The αβ T cells in infected mice showed significantly higher levels of Annexin V binding than those in mock control mice suggesting increased apoptosis of T cells. These results suggest that lack of transition from an innate to adaptive host response is associated with lethality of respiratory tularemia.

Direct repeat‐mediated deletion of a type IV pilin gene results in major virulence attenuation of Francisella tularensis

Francisella tularensis, the causative agent of tularaemia, is a highly infectious and virulent intracellular pathogen. There are two main human pathogenic subspecies, Francisella tularensis ssp. tularensis (type A), and Francisella tularensis ssp. holarctica (type B). So far, knowledge regarding key virulence determinants is limited but it is clear that intracellular survival and multiplication is one major virulence strategy of Francisella. In addition, genome sequencing has revealed the presence of genes encoding type IV pili (Tfp). One genomic region encoding three proteins with signatures typical for type IV pilins contained two 120 bp direct repeats. Here we establish that repeat-mediated loss of one of the putative pilin genes in a type B strain results in severe virulence attenuation in mice infected by subcutaneous route. Complementation of the mutant by introduction of the pilin gene in cis resulted in complete restoration of virulence. The level of attenuation was similar to that of the live vaccine strain and this strain was also found to lack the pilin gene as result of a similar deletion event mediated by the direct repeats. Presence of the pilin had no major effect on the ability to interact, survive and multiply inside macrophage-like cell lines. Importantly, the pilin-negative strain was impaired in its ability to spread from the initial site of infection to the spleen. Our findings indicate that this putative pilin is critical for Francisella infections that occur via peripheral routes.