Susceptibility of immunodeficient mice to aerosol and systemic infection with virulent strains of Francisella tularensis

Previous studies have shown that IFN-gamma, TNF-alpha and NOS-2, but not B cells, are crucial for host defense against primary systemic infection with the attenuated live vaccine strain (LVS) of Francisella tularensis. In this study, we examined the importance of these and additional immune components in host resistance against infection with virulent strains of F. tularensis initiated by systemic and airborne routes. Wild-type (WT) mice and mice deficient in IFN-gamma, TNFR1R2, NOS-2, or B cells were equally susceptible to low dose ( approximately 10 colony forming units) aerosol or intradermal challenge with virulent type B F. tularensis, and succumbed to the infection between days 6 and 8 post-inoculation. Quantitative bacteriology showed that IFN-gamma-/- and B cell-/- mice consistently harbored up to one log(10) more bacteria in their lungs, spleens and livers than WT mice at day 5 post aerosol exposure. Surprisingly, however, compared to other strains of KO mice and WT control mice, IFN-gamma-/- mice showed only mild liver damage as assessed by histopathology and liver function tests. Additional experiments established that even mice with broad immunodeficiency (SCID, neutropenic, splenectomized or thymectomized mice and mice treated with corticosteroid) were no more susceptible to aerosol-initiated infection with virulent type B or type A F. tularensis than immunosufficient control mice. Combined, our results indicate that, unlike LVS, normal type A and type B F. tularensis strains are so extremely virulent that even immunocompetent mice are virtually defenseless to low dose aerosol and intradermal challenges with them.     

Differential susceptibility of Sprague–Dawley and Fischer 344 rats to infection by Francisella tularensis

The type A and B subspecies of Francisella tularensis cause severe disease, tularemia, in humans. However, only the former can be lethal especially if inhaled. It is likely that non-lethal infection is due at least in part to the ability of innate host defenses to control pathogen growth whilst acquired immunity develops. Most common small laboratory animals rapidly succumb to clinical strains of F. tularensis and are, therefore, poor models with which to study innate immunity. In an attempt to improve upon this situation in the present study, Sprague–Dawley and Fischer 344 rats were examined for their ability to combat challenge with type A and B strains of the pathogen. Sprague–Dawley rats were significantly more resistant than Fischer rats to infection with either subspecies. This correlated with the ability of Sprague–Dawley rats to arrest the growth of the pathogen at both the site of challenge and at sites of disseminated infection. The rapidity with which F. tularensis kills susceptible rats and the early onset of control of infection in resistant rats suggests that differences in innate immunity account for these disparate outcomes. Thus, the rat might be a more useful model for studying innate immunity to virulent F. tularensis than other small mammals.     

Francisella tularensis LVS grown in macrophages has reduced ability to stimulate the secretion of inflammatory cytokines by macrophages in vitro

The virulence of Francisella tularensis LVS is determined in part by its ability to invade and replicate within macrophages and stimulate the production of inflammatory cytokines. The present study determined the effects of growing F. tularensis in macrophages on its ability to stimulate cytokine secretion by macrophages. F. tularensis grown in Mueller-Hinton broth (FtB) stimulated the secretion of large amounts of TNF-alpha, IL-12p40, IL-6 and MCP-1/CCL2 when incubated with macrophages overnight. In contrast, F. tularensis released from infected macrophages (FtMac) stimulated very little secretion of these cytokines by primary cultures of murine peritoneal macrophages, human monocytes or macrophage cell lines. Stimulation of nitric oxide production by FtMac was also less than that elicited by FtB. FtMac killed with gentamicin or paraformaldehyde also stimulated low levels of cytokine secretion. FtMac recovered the ability to stimulate cytokine secretion after overnight culture in broth. Infection of macrophages with FtMac inhibited the cytokine response to subsequent stimulation with LPS from Escherichia coli but did not affect Fcgamma receptor-mediated phagocytosis. FtMac were ingested by macrophages at about half the rate of FtB, however, this did not account for the lower cytokine secretion. FtMac and FtB replicated at similar rates within macrophages. Finally, Mice infected with FtMac had a higher mortality rate than those infected with FtB. These results reveal that growth in macrophages causes a reversible phenotypic change in F. tularensis that is associated with decreased stimulation of cytokine secretion, inhibition of LPS-stimulated secretion of inflammatory cytokines by macrophages and increased lethality in mice.     

Interaction between mammalian cells and Pasteurella multocida B:2. Adherence, invasion and intracellular survival

A Pasteurella multocida B:2 strain from a case of bovine haemorrhagic septicaemia (HS) and a derivative, JRMT12, that was attenuated by a deletion in the aroA gene, were shown to adhere to, invade and survive within cultured embryonic bovine lung (EBL) cells. By comparison, bovine strains of Mannheimia haemolytica serotype A1 and P. multocida serotype A:3, although able to adhere to EBL cells, were not found intracellularly. The B:2 strains were viable intracellularly over a 7 h period, although a steady decline in viability was noted with time. Entry into the mammalian cells was inhibited by cytochalasin D, indicating that cell uptake was by an actin-dependent process. Viability assessment of EBL cells by trypan blue staining indicated that none of the bacterial strains was toxic for the EBL cells. Transmission electron microscopy (TEM) showed that, after entry into the mammalian cells, the B:2 strain resided in a vacuolar compartment. However, only a low percentage of mammalian cells appeared to contain one or more P. multocida B:2, suggesting that only certain EBL cells in the population were capable of being invaded by, or of taking up, the bacteria. TEM showed that P. multocida A:3 and M. haemolytica A:1 were found loosely adhering to the cell surface of EBL cells and were not detected intracellularly. The cell-invasive capacity of P. multocida B:2 may be a virulence property related to its ability to translocate from the respiratory tract into the blood stream.     

Immunization with heat-killed Francisella tularensis LVS elicits protective antibody-mediated immunity

Francisella tularensis (FT) has been classified by the CDC as a category A pathogen because of its high virulence and the high mortality rate associated with infection via the aerosol route. Because there is no licensed vaccine available for FT, development of prophylactic and therapeutic regimens for the prevention/treatment of infection is a high priority. In this report, heat-killed FT live vaccine strain (HKLVS) was employed as a vaccine immunogen, either alone or in combination with an adjuvant, and was found to elicit protective immunity against high-dose FT live vaccine strain (FTLVS) challenge. FT-specific antibodies produced in response to immunization with HKLVS alone were subsequently found to completely protect naive mice against high-dose FT challenge in both infection-interference and passive immunization experiments. Additional passive immunization trials employing serum collected from mice immunized with a heat-killed preparation of an O-antigen-deficient transposon mutant of FTLVS (HKLVS-OAg(neg)) yielded similar results. These findings demonstrated that FT-specific antibodies alone can confer immunity against high-dose FTLVS challenge, and they reveal that antibody-mediated protection is not dependent upon production of LPS-specific antibodies.     

Humoral and cell-mediated immunity to the intracellular pathogen Francisella tularensis

Summary: Francisella tularensis can cause fatal respiratory tularemia in humans and animals and is increasingly being isolated in the United States and several European countries. The correlates of protective immunity against this intracellular bacterium are not known, and currently there are no licensed vaccines available for human use. Cell-mediated immunity has long been believed to be critical for protection, and the importance of humoral immunity is also now recognized. Furthermore, synergy between antibodies, T cell-derived cytokines, and phagocytes appears to be critical to achieve sterilizing immunity against F. tularensis. Thus, novel vaccine approaches should be designed to induce robust antibody and cell-mediated immune responses to this pathogen.     

Phenotypic and genotypic discrimination of Francisella tularensis ssp. holarctica clades

Francisella tularensis is the causative agent of tularemia, a zoonotic disease with a wide host range. F. tularensis ssp. holarctica (Fth) is of clinical relevance for European countries, including Germany. Whole genome sequencing methods, including canonical Single Nucleotide Polymorphism (canSNP) typing and whole genome SNP typing, have revealed that European Fth strains belong to a few monophyletic populations. The majority of German Fth isolates belong to two basal phylogenetic clades B.6 (biovar I) and B.12 (biovar II). Strains of B.6 and B.12 seem to differ in their pathogenicity, and it has been shown that strains of biovar II are resistant against erythromycin. In this study, we present data corroborating our previous data demonstrating that basal clade B.12 can be divided into clades B.71 and B.72. By applying phylogenetic whole genome analysis as well as proteome analysis, we could verify that strains of these two clades are distinct from one another. This was confirmed by measuring the intensity of backscatter light on bacteria grown in liquid media. Strains belonging to clades B.6, B.71 or B.72 showed clade-specific backscatter growth curves. Furthermore, we present the whole genome sequence of strain A-1341, as a reference genome of clade B.71, and whole proteomes comparison of Fth strains belonging to clades B.6, B.71 and B.72. Further research is necessary to investigate phenotypes and putative differences in pathogenicity of the investigated different clades of Fth to better understand the relationship between observed phenotypes, pathogenicity and distribution of Fth strains.     

Cytotoxic CD4+ T cells specific for Francisella tularensis.

The specific cell-mediated cytotoxicity of tularaemia-immune human T lymphocytes were studied in vitro. Peripheral blood mononuclear cells (PBMC) of six tularaemia-vaccinated healthy subjects were stimulated with F. tularensis LVS whole cell antigen for 6 days and used as effector cells in a conventional 4-h 51Cr release cytotoxicity assay. The target cells were phagocyting autologous monocytes, which were pulsed with F. tularensis or PPD antigen. The specific lysis of the F. tularensis-pulsed cells (42.6% +/- 11.7) was significantly higher (P less than 0.05) than that of the PPD-pulsed ones (22.2% +/- 8.3) or unpulsed control cells (15.9% +/- 5.2). The cytotoxicity was associated with CD4+ F. tularensis-specific T cell clones (TLC), which killed 36.3% +/- 12.3 of the F. tularensis-pulsed targets but only 6.9% +/- 6.5 of the unpulsed control targets. Their lysing was inhibited by monoclonal anti-HLA-DR and anti-HLA-DQ antibodies, but not by CD15 (monocyte/macrophage) antibody. The functional role of CD4+ lymphocytes in tularaemia immunity is discussed.     

Immunoproteomic analysis of the human antibody response to natural tularemia infection with Type A or Type B strains or LVS vaccination

Francisella tularensis is pathogenic for many mammalian species including humans, causing a spectrum of diseases called tularemia. The highly virulent Type A strains have associated mortality rates of up to 60% if inhaled. An attenuated live vaccine strain (LVS) is the only vaccine to show efficacy in humans, but suffers several barriers to licensure, including the absence of a correlate of protection. An immunoproteomics approach was used to survey the repertoire of antibodies in sera from individuals who had contracted tularemia during two outbreaks and individuals from two geographical areas who had been vaccinated with NDBR Lot 11 or Lot 17 LVS. These data showed a large overlap in the antibodies generated in response to tularemia infection or LVS vaccination. A total of seven proteins were observed to be reactive with 60% or more sera from vaccinees and convalescents. A further four proteins were recognised by 30–60% of the sera screened. These proteins have the potential to serve as markers of vaccination or candidates for subunit vaccines.     

Modulation of virulence factors in Francisella tularensis determines human macrophage responses

Francisella tularensis, the causative agent of tularemia and Category A biodefense agent, is known to replicate within host macrophages, though the pathogenesis of this organism is incompletely understood. We have isolated a variant of F. tularensis live vaccine strain (LVS) based on colony morphology and its effect on macrophages. Human monocyte-derived macrophages produced more tumor necrosis factor alpha (TNFalpha), interleukin (IL)-1beta, IL-6, and IL-12 p40 following exposure to the variant, designated the activating variant (ACV). The immunoreactivity of the lipopolysaccharide (LPS) from both LVS and ACV was comparable to the previously described blue variant and was distinct from the gray variant of LVS. We found, however, the soluble protein fractions of LVS and ACV differed. Further investigation using two-dimensional gel electrophoresis demonstrated higher levels of several proteins in the parental LVS isolate. The differentially expressed proteins featured several associated with virulence in F. tularensis and other pathogens, including intracellular growth locus C (IglC), a sigma(54)-modulation protein family member (YhbH), and aconitase. ACV reverted to the LVS phenotype, indicated by low cytokine induction and high IglC expression, after growth in a chemically defined medium. These data provide evidence that the levels of virulence factors in F. tularensis are modulated based on culture conditions and that this modulation impacts host responses. This work provides a basis for investigation of Francisella virulence factor regulation and the identification of additional factors, co-regulated with IglC, that affect macrophage responses.     

Molecular immunology of experimental primary tularemia in mice infected by respiratory or intradermal routes with type A Francisella tularensis

The type A subspecies of Francisella tularensis is a highly virulent facultative intracellular bacterial pathogen, and a potential biological weapon. Recently, there has been renewed interest in developing new vaccines and therapeutics against this bacterium. Natural cases of disease, tularemia, caused by the type A subspecies are very rare. Therefore, the United States Food and Drug Administration will rely on the so-called Animal Rule for efficacy testing of anti-Francisella medicines. This requires the human disease to be modeled in one or more animal species in which the pathogenicity of the agent is reasonably well understood. Mice are natural hosts for F. tularensis, and might be able to satisfy this requirement. Tularemia pathogenesis appears to be primarily due to the host inflammatory response which is poorly understood at the molecular level. Additionally, the extent to which this response varies depending on host and pathogen genetic background, or by pathogen challenge route or dose is unknown. Therefore, the present study examined sera and infected tissues from C57BL/6 and BALB/c mice challenged by natural intradermal (ID) and respiratory routes with one of two distinct type A strains of the pathogen for cytokine and chemokine responses that might help to explain the morbidity associated with tularemia. The results show that the molecular immune response was mostly similar regardless of the variables examined. For instance, mRNA for the proinflammatory cytokine IL-6, and chemokines KC, and IP-10 was consistently upregulated at all sites of infection. Upregulation of mRNA for several other cytokines and chemokines occurred in a more tissue restricted manner. For instance, IFN-γ was highly upregulated in the skin of BALB/c, but not C57BL/6 mice after ID inoculation of the pathogen, whilst IL-10 mRNA upregulation was only consistently seen in the skin and lungs.     

Delayed presence of alternatively activated macrophages during a Francisella tularensis infection

Francisella tularensis is an intracellular bacterium that has the ability to multiply within the macrophage. The phenotype of a macrophage can determine whether the infection is cleared or the host succumbs to disease. Previously published data has suggested that F. tularensis LVS actively induces the alternative phenotype as a survival mechanism. In these studies we demonstrate that this is not the case for the more virulent strain of F. tularensis SCHU-S4. During an intranasal mouse model of infection, immuno-histochemistry identified that iNOS positive (“classical”) macrophages are present at 72 h post-infection and remain high (supported by CCL-5 release) in numbers. In contrast, arginase/FIZZ-1 positive (“alternative”) cells appear later and in low numbers during the development of the disease tularemia.     

Impact of Francisella tularensis pilin homologs on pilus formation and virulence

Francisella tularensis is a facultative intracellular bacterium and the causative agent of tularemia. Virulence factors for this bacterium, particularly those that facilitate host cell interaction, remain largely uncharacterized. However, genes homologous to those involved in type IV pilus structure and assembly, including six genes encoding putative major pilin subunit proteins, are present in the genome of the highly virulent Schu S4 strain. To analyze the roles of three putative pilin genes in pili structure and function we constructed individual pilE4, pilE5, and pilE6 deletion mutants in both the F. tularensis tularensis strain Schu S4 and the Live Vaccine Strain (LVS), an attenuated derivative strain of F. tularensis holarctica. Transmission electron microscopy (TEM) of Schu S4 and LVS wild-type and deletion strains confirmed that pilE4 was essential for the expression of type IV pilus-like fibers by both subspecies. By the same method, pilE5 and pilE6 were dispensable for pilus production. In vitro adherence assays with J774A.1 cells revealed that LVS pilE4, pilE5, and pilE6 deletion mutants displayed increased attachment compared to wild-type LVS. However, in the Schu S4 background, similar deletion mutants displayed adherence levels similar to wild-type. In vivo, LVS pilE5 and pilE6 deletion mutants were significantly attenuated compared to wild-type LVS by intradermal and subcutaneous murine infection, while no Schu S4 deletion mutant was significantly attenuated compared to wild-type Schu S4. While pilE4 was essential for fiber expression on both Schu S4 and LVS, neither its protein product nor the assembled fibers contributed significantly to virulence in mice. Absent a role in pilus formation, we speculate PilE5 and PilE6 are pseudopilin homologs that comprise, or are associated with, a novel type II-related secretion system in Schu S4 and LVS.     

Persistence of cell-mediated immunity three decades after vaccination with the live vaccine strain of Francisella tularensis

The efficacy of many vaccines against intracellular bacteria depends on the generation of cell‐mediated immunity, but studies to determine the duration of immunity are usually confounded by re‐exposure. The causative agent of tularemia, Francisella tularensis, is rare in most areas and, therefore, tularemia vaccination is an interesting model for studies of the longevity of vaccine‐induced cell‐mediated immunity. Here, lymphocyte proliferation and cytokine production in response to F. tularensis were assayed in two groups of 16 individuals, vaccinated 1–3 or 27–34 years previously. As compared to naïve individuals, vaccinees of both groups showed higher proliferative responses and, out of 17 cytokines assayed, higher levels of MIP‐1β, IFN‐γ, IL‐10, and IL‐5 in response to recall stimulation. The responses were very similar in the two groups of vaccinees. A statistical model was developed to predict the immune status of the individuals and by use of two parameters, proliferative responses and levels of IFN‐γ, 91.1% of the individuals were correctly classified. Using flow cytometry analysis, we demonstrated that during recall stimulation, expression of IFN‐γ by CD4⁺CCR7⁺, CD4⁺CD62L⁺, CD8⁺CCR7⁺, and CD8⁺CD62L⁺ cells significantly increased in samples from vaccinated donors. In conclusion, cell‐mediated immunity was found to persist three decades after tularemia vaccination without evidence of decline.     

Salmonella infects B cells by macropinocytosis and formation of spacious phagosomes but does not induce pyroptosis in favor of its survival

We have previously reported that Salmonella infects B cells and survives within endosomal-lysosomal compartments. However, the mechanisms used by Salmonella to enter B cells remain unknown. In this study, we have shown that Salmonella induces its own entry by the induction of localized ruffling, macropinocytosis, and spacious phagosome formation. These events were associated with the rearrangement of actin and microtubule networks. The Salmonella pathogenesis island 1 (SPI-1) was necessary to invade B cells. In contrast to macrophages, B cells were highly resistant to cell death induced by Salmonella. These data demonstrate the ability of Salmonella to infect these non-professional phagocytic cells, where the bacterium can find an ideal intracellular niche to support persistence and the possible dissemination of infection.     

Analyzing time-dependent microarray data using independent component analysis derived expression modes from human macrophages infected with F. tularensis holartica

The analysis of large-scale gene expression profiles is still a demanding and extensive task. Modern machine learning and data mining techniques developed in linear algebra, like Independent Component Analysis (ICA), become increasingly popular as appropriate tools for analyzing microarray data. We applied ICA to analyze kinetic gene expression profiles of human monocyte derived macrophages (MDM) from three different donors infected with Francisella tularensis holartica and compared them to more classical methods like hierarchical clustering. Results were compared using a pathway analysis tool, based on the Gene Ontology and the MeSH database. We could show that both methods lead to time-dependent gene regulatory patterns which fit well to known TNFα induced immune responses. In comparison, the nonexclusive attribute of ICA results in a more detailed view and a higher resolution in time dependent behavior of the immune response genes. Additionally, we identified NFκB as one of the main regulatory genes during response to F. tularensis infection.     

Establishment of lethal inhalational infection with Francisella tularensis (tularaemia) in the common marmoset (Callithrix jacchus)

Susceptibility and lethality studies of inhalational tularaemia were undertaken using the common marmoset ( Callithrix jacchus ) to determine its suitability as a non-human primate model. Pairs of marmosets were exposed to varying challenge doses of Francisella tularensis by the airborne route and monitored for up to 14 days postchallenge (p.c.). Lethal infection was achieved following a retained dose of less than 10 bacterial colony-forming units (CFU). However, precise LD₅₀ determination was not possible. The model was characterized using a target challenge dose of approximately 100 CFU. Increased core body temperature was the first indicator of disease, at approximately 2.5 days p.c. Overt clinical signs were first observed 12–18 h after the temperature increase. Significantly decreased activity was observed after approximately 3 days. All animals succumbed to infection between 4.5 and 7 days p.c. At postmortem examination, gross pathology was evident in the liver, spleen and lungs of all animals and high bacterial numbers were detected in all the organs assessed. Bacteraemia was demonstrated in all animals postmortem. Histopathological observations included severe suppurative bronchopneumonia, severe multifocal pyogranulomatous hepatitis, splenitis and lymphadenitis. Tularaemia disease progression in the common marmoset therefore appears to be consistent with the disease seen in humans and other animal models. The common marmoset may therefore be considered a suitable model for further studies of inhalational tularaemia.     

In vitro models of acute and long-term continuous infection of human respiratory epithelial cells with Chlamydophila pneumoniae have opposing effects on host cell apoptosis

Persistent infection with the obligate intracellular pathogen Chlamydophila pneumoniae has been implicated in the pathogenesis of many chronic diseases, but its mechanism remains unclear. Many pathogens have been found to modulate cellular apoptosis in order to survive and multiply. Chlamydial species were shown to both induce and inhibit host cell apoptosis depending on the experimental conditions. We utilized in vitro models of acute and long-term continuous (LTC) infection with the same cell line (HEp-2) and chlamydial isolate (TW-183) used in both models. Host cell apoptosis in infected and uninfected cells was assessed by fluorescence microscopy and flow cytometry. While acute infection induced apoptosis 72 h post-infection, LTC-infected cells had low rates of apoptosis and showed resistance to different exogenous inducers of apoptosis (sorbitol, serum withdrawal, hydrogen peroxide) when compared to uninfected cells. Chronicity of infection appears to be a critical factor in the modulation of host cell apoptosis by C. pneumoniae. Induction of apoptosis may help to propagate the infection, while inhibition of apoptosis could help protect the organism in chronic infection.     

应用短串联重复序列分析技术对我国土拉弗菌的分型研究

目的 对我国土拉弗菌进行基因分型研究。方法 应用短串联重复序列(SSTR)分析技术对我国不同地区分离出的5株土拉弗菌菌株进行分型。SSTR9和SSTRl6两个区域扩增后测序，并将结果与国外其它分离株进行比较。结果 5株菌在SSTR9区重复序列的个数分别为21、9、12、9、15，片段长度分别为396bp、2S8bp、315bp、2886p和342bp。在SSTR16区域扩增序列完全相同，拷贝数为2，片段长度为361bp。其中4株菌重复序列多态性与瑞典、芬兰等欧洲株接近，而分离自新疆的114菌株在SSTR9区存在独特的重复序列多态性，可能是我国特有的基因型。结论 5个分离株均属于B型土拉弗菌，与型特异性引物分型结果一致。PCR-SSTR分析可作为国内土拉弗菌分型及进行个体菌株区分的可靠方法。     

B cell precursors are targets for Salmonella infection

We previously reported that, in mice, B cells are a reservoir for bacteria during Salmonella infection. Here, we show that, within the bone marrow, B cells and their precursors are targeted for infection by Salmonella enterica serovar typhimurium. Our data suggest that B cells within the bone marrow may be a bacterial niche during chronic Salmonella infection.