Vaccination with the live vaccine strain (LVS) or natural infection with Francisella tularensis confers long-lasting protection against re-infection, and it is almost exclusively dependent on cell-mediated immunity. The extent, if any, to which vaccine-induced and infection-induced immunity differ in quality and persistence remains unclear. In this study, we compared human immune responses resulting from LVS vaccination and tularemia infection. Peripheral blood mononuclear cells from vaccinees and convalescent patients were recall-stimulated and analyzed for proliferation, characterization of cytokine-producing memory T cells, and cytokine secretion. Both vaccination and infection elicited robust antigen-specific T-cell activation, accompanied by coordinated cytokine release. Intracellular staining identified multifunctional CD4 and CD8 T cells after both vaccination and infection. Characterization of memory subsets revealed that vaccination primarily induced transitional and effector memory populations, while natural infection generated broader and more persistent responses, including activation of terminally differentiated TEMRA (Terminal Effector Memory T cells re-expressing CD45RA) cells and higher intracellular expression of MIP-1β by CD4 and CD8 cells. These findings demonstrate that both LVS vaccination and natural infection induce efficacious immune responses to F. tularensis, as evidenced by the presence of long-lasting, multifunctional T cells, but with partially distinct memory profiles. The broader and more durable responses after natural infection highlight areas of relevance to understand the efficacy of existing and future tularemia vaccines.

Autophagy is a crucial mechanism in the host response to intracellular bacterial pathogens during which microorganisms may undergo direct degradation in autophagolysosomes. As a highly virulent intracellular pathogen, Francisella tularensis has developed survival strategies to escape from the phagosome, replicate in the cytosol of mononuclear cells, and avoid degradation within the double-membrane vacuole during the autophagy-mediated response. The aim of this study was to investigate the role of the ATG5 autophagy protein in the host immune response to Francisella tularensis subsp. holarctica, live vaccine strain (LVS), since ATG5 plays an important role in autophagosome formation during canonical autophagy. In vitro experiments were conducted on immortalized bone marrow macrophages subjected to starvation-induced autophagy. Transgenic mice deficient in ATG5 of cells of the myeloid lineage (monocytes/macrophages and granulocytes) were used to analyze the immunological responses after intradermal infection. Cytokine levels were analyzed using Luminex, RT-qPCR, and ELISA, while inflammatory cell infiltration in the lung was analyzed by immunohistochemistry. Our results demonstrate that induced autophagy decreased bacterial replication in vitro. However, ATG5 deficiency in myeloid cells in vivo significantly diminished levels of pro-inflammatory cytokine IFN-γ in the sera, spleen, liver, and lung during Francisella infection. The attenuated pro-inflammatory response also led to significantly reduced macrophage and T cell infiltration in the lung tissue. Our findings also reveal that neutralization of IL-1β in myeloid ATG5ΔMye mice increased susceptibility to tularemia by increasing bacterial burden in organs.

Gram-negative bacteria utilize type VI secretion systems (T6SS) for microbial competition and host interaction. While most pathogens rely on the canonical T6SSi, Francisella species uniquely possess T6SSii. The highly virulent human pathogen Francisella tularensis harbors a distinct T6SSii variant that includes pdpC, encoding a putative effector protein. Bioinformatic analysis revealed a conserved amino acid triad in PdpC, homologous to motifs found in Make Caterpillars Floppy family toxins. To investigate the functional relevance of this triad, site-directed mutagenesis was performed in the live vaccine strain of F. tularensis, substituting each residue with alanine. Mutants showed impaired phagosomal escape, reduced intracellular replication, and marked attenuation in the mouse infection model. Equivalent mutations introduced into F. novicida, a model for T6SS-mediated secretion, confirmed the triad's importance. Mass spectrometry analysis demonstrated that PdpC is secreted in a T6SS-dependent manner. Importantly, the mutations did not affect secretion, and deletion of pdpC did not alter the overall secretion profile. These findings indicate that the conserved triad is essential for PdpC's effector function but dispensable for its secretion. This study highlights a critical motif required for Francisella virulence and provides new insights into the specialized mechanisms of T6SSii effectors.

Licensed vaccines against Francisella tularensis, a public health threat in some parts of the world and a potential bioterrorism agent, are lacking in Western countries. Existing tularemia vaccine candidates have not been promising in protecting against the most serious respiratory form of tularemia infection. Previous studies identified a novel live attenuated vaccine candidate, F. tularensis subsp. tularensis ΔclpB, that protected rodents against aerosol challenge with the most virulent biotype of Francisella. Characterization demonstrated that ΔclpB is amenable to modern manufacturing. Here, we evaluated further ΔclpB's protective capacities in Fischer 344 rats and in cynomolgus macaques. Results demonstrated that rats immunized intradermally with ΔclpB survived aerosol F. tularensis challenge with up to 100 median lethal doses administered one year after vaccination, accompanied by reduced clinical signs of infection as well as reduced histopathology and bacterial burdens in lungs and spleens. Moreover, intradermal ΔclpB vaccination protected macaques against at least 500 MLD of aerosol F. tularensis challenge administered one and three months after vaccination; vaccination ameliorated symptoms, bacterial burdens, and tissue pathology when tested one year after vaccination. Given the low incidence of tularemia in nature, these studies therefore lay the foundation for additional animal-based evaluations of efficacy and future safety evaluation of ΔclpB by clinical studies.

IntroductionFor a majority of tularemia patients, serology is the basis for the diagnosis. The aim of this study was to perform an analysis of the samples analyzed at a Swedish reference laboratory for the presence of Francisella tularensis-specific antibody levels in sera from individuals with suspected tularemia. Annual and monthly variations of the total number of samples and proportions of positive samples were analyzed, as well as the influence of age and gender.MethodsWe performed a retrospective analysis of the presence of F. tularensis-specific antibodies in serological samples from patients with suspected tularemia analyzed during the period 2010 - 2022 at the University Hospital of Umeå in Sweden, a national reference laboratory, by use of various statistical methods. In total, some 15,100 serum samples had been analyzed for the presence of IgG and IgM antibodies by ELISA during the 13-year period.ResultsOverall, there were higher number of samples with IgG positive or borderline titers, 2,522 and 921, respectively, than with IgM positive or borderline titers, 1,802 and 409, respectively. Repeated samples were obtained from some 1,930 individuals and approximately a third of the cases, which were initially seronegative, had seroconverted when resampled. Peak number of monthly samples were recorded in August and September, > 3,000. Annual numbers varied greatly and peak numbers were observed in 2015 and 2019, 1,832 and 2,250, respectively, whereas some other years the numbers were 700 – 800. There was also much variation in the annual and monthly percentages of positive samples and they varied between less than 10% to greater than 20%. The highest percentages of positive samples were recorded in September and October. IgG and IgM titers declined with age and these differences were highly significant for IgG titers, with decreasing average titers for each 20-year interval.DiscussionCollectively, the data demonstrate the marked annual and seasonal variations in tularemia sampling occurring in Sweden. Also, the proportion of positive samples increased during months and years with peak number of samples. Another notable finding was that average antibody titers decreased with increased age.

BACKGROUND:Tularemia is an important reemerging disease with a multimodal transmission pattern. Treatment outcomes of current recommended antibiotic regimens (including ciprofloxacin and doxycycline) remain unclear. In this retrospective cohort study, we report clinical, laboratory, geographical, and treatment outcomes of laboratory-confirmed tularemia cases over an 11-year period in Northern Sweden. METHODS:Data from reported tularemia cases (aged >10 years at time of study) in Norrbotten county between 2011 and 2021 were collected through review of electronic medical records and participant questionnaires; 415 of 784 accepted participation (52.9%). Of these, 327 were laboratory-confirmed cases (serology and/or polymerase chain reaction). A multivariable logistic regression model was used to investigate variables associated with retreatment. RESULTS:Median age of participants was 54 years (interquartile range [IQR], 41.5-65) and 49.2% were female. Although ulceroglandular tularemia was the predominant form (n = 215, 65.7%), there were several cases of pulmonary tularemia (n = 40; 12.2%). Inflammatory markers were largely nonspecific, with monocytosis frequently observed (n = 36/75; 48%). Tularemia was often misdiagnosed on presentation (n = 158, 48.3%), with 65 (19.9%) receiving initial inappropriate antibiotics and 102 (31.2%) retreated. Persistent lymphadenopathy was infrequent (n = 22, 6.7%), with 10 undergoing surgical interventions. In multivariable analysis of variables associated with retreatment, we highlight differences in time until receiving appropriate antibiotics (8 [IQR, 3.25-20.75] vs 7 [IQR, 4-11.25] days; adjusted P = .076), and doxycycline-based treatment regimen (vs ciprofloxacin; adjusted P = .084), although this was not significant after correction for multiple comparisons. CONCLUSIONS:We comprehensively summarize clinical, laboratory, and treatment outcomes of type B tularemia. Targeting tularemia requires clinical awareness, early diagnosis, and timely commencement of treatment for an appropriate duration.

Francisella tularensis is the etiological agent of the potentially severe infection tularemia. An existing F: tularensis vaccine, the live vaccine strain (LVS), has been used to protect at-risk personnel, but it is not licensed in any country and it has limited efficacy. Therefore, there is a need of a new, efficacious vaccine. The aim of the study was to perform a detailed analysis of the characteristics of the human immune response to F. tularensis, since this will generate crucial knowledge required to develop new vaccine candidates. Nine individuals were administered the LVS vaccine and peripheral blood mononuclear cells (PBMC) were collected before and at four time points up to one year after vaccination. The properties of the PBMC were characterized by flow cytometry analysis of surface markers and intracellular cytokine staining. In addition, the cytokine content of supernatants from F. tularensis-infected PBMC cultures was determined and the protective properties of the supernatants investigated by adding them to cultures with infected monocyte-derived macrophages (MDM). Unlike before vaccination, PBMC collected at all four time points after vaccination demonstrated F. tularensis-specific cell proliferation, cytokine secretion and cytokine-expressing memory cells. A majority of 17 cytokines were secreted at higher levels by PBMC collected at all time points after vaccination than before vaccination. A discriminative analysis based on IFN-γ and IL-13 secretion correctly classified samples obtained before and after vaccination. Increased expression of IFN-γ, IL-2, and MIP-1β were observed at all time points after vaccination vs. before vaccination and the most significant changes occurred among the CD4 transient memory, CD8 effector memory, and CD8 transient memory T-cell populations. Growth restriction of the highly virulent F. tularensis strain SCHU S4 in MDM was conferred by supernatants and protection correlated to levels of IFN-γ, IL-2, TNF, and IL-17. The findings demonstrate that F. tularensis vaccination induces long-term T-cell reactivity, including TEM and TTM cell populations. Individual cytokine levels correlated with the degree of protection conferred by the supernatants. Identification of such memory T cells and effector mechanisms provide an improved understanding of the protective mechanisms against F. tularensis. mechanisms against F. tularensis.

Interpretation of serological findings in suspected Lyme borreliosis (LB) is challenging and IgM reactivities may have low predictive value. Therefore, if used indiscriminately, there is a risk for incorrect diagnosis of LB. To evaluate the usefulness of IgM titer determination, we performed a study of the prevalence of Borrelia-specific antibodies in serological samples from patients with suspected LB analyzed during the period 2010 - 2021 at the University Hospital of Umeå in Sweden. In total, 19,335 samples had been analyzed for the presence of IgG and IgM antibodies. Overall, there were higher percentages of IgM positive or borderline titers, 1,847 (9.6%) and 905 (4.7%), respectively, than IgG positive or borderline titers, 959 (5.0%) and 406 (2.1%), respectively. Peak number of samples were recorded 2012 - 2013, exceeding 1,800, whereas there were around 1,200 during 2020 - 2021. The peak number of positive IgG and/or positive IgM samples were observed during the period 2015 - 2017 with close to, or above 400, and concomitantly, the proportion of IgG positive samples increased markedly. For IgG positive samples, the increase followed a positive linear time trend (P< 0.001). Peak monthly numbers were observed during August, September, and October. This seasonal increase was significant for the IgG positive group (P< 0.05), but not for the IgM positive/IgG negative group. Repeated samples were obtained from 3,188 individuals and of the initial samples 2,817 were (88%) IgG negative and 2,315 (72%) were IgM negative and of these, 130 (4%) showed IgG seroconversion and 300 (9%) IgM seroconversion. Collectively, the data demonstrate that IgG and/or IgM positive samples represented a minority of all samples, even when repeated sampling had occurred, and IgM positive samples were much more common than IgG positive samples. Thus, the accuracy of the clinical suspicion was low and this will lead to a low predictive value of the analysis, in particular of IgM. These findings question the use of IgM titer determination as a routine analysis.

Serological analysis is the predominant method used to diagnose tularemia, a zoonotic disease caused by the highly virulent bacterium F. tularensis. We determined F. tularensis-specific IgM and IgG antibody titers by an LPS-based ELISA assay on five occasions one to twelve months after onset of ulceroglandular tularemia in 19 individuals. Peak IgM antibody titers were observed at the one-month time point and peak IgG antibody titers at the two-month time point. Both IgG and IgM antibody levels declined linearly thereafter with rather similar kinetics. Compared to the average one-month antibody titers, average IgG titers were not significantly lower before the 12-month time point and IgM titers before the 4-month time point. All, but one average titer, were significantly increased compared to the cut-off of the assay. Average IgG and IgM titers were significantly lower for the group = 69 years old compared to the group < 69 years. Collectively, the data demonstrate a persistence of F. tularensis-specific IgM and IgG antibody titers for at least 12 months after ulceroglandular tularemia. Thus, low, but significantly elevated F. tularensis-specific antibody titers are of limited diagnostic value since they are not indicative of ongoing tularemia.

The Hsp100 family member ClpB is a protein disaggregase which solubilizes and reactivates stress-induced protein aggregates in cooperation with the DnaK/Hsp70 chaperone system. In the pathogenic bacterium Francisella tularensis, ClpB is involved in type VI secretion system (T6SS) disassembly through depolymerization of the IglA-IglB sheath. This leads to recycling and reassembly of T6SS components and this process is essential for the virulence of the bacterium. Here we report the backbone chemical shift assignments and 15N relaxation-based backbone dynamics of the N-terminal substrate-binding domain of ClpB (1-156).

Francisella tularensis is a highly pathogenic intracellular bacterium that causes the disease tularemia. While its ability to replicate within cells has been studied in much detail, the bacterium also encodes a less characterised type 4 pili (T4P) system. T4Ps are dynamic adhesive organelles identified as major virulence determinants in many human pathogens. In F. tularensis, the T4P is required for adherence to the host cell, as well as for protein secretion. Several components, including pilins, a pili peptidase, a secretin pore and two ATPases, are required to assemble a functional T4P, and these are encoded within distinct clusters on the Francisella chromosome. While some of these components have been functionally characterised, the role of PilO, if any, still is unknown. Here, we examined the role of PilO in the pathogenesis of F. novicida. Our results show that the PilO is essential for pilus assembly on the bacterial surface. In addition, PilO is important for adherence of F. novicida to human monocyte-derived macrophages, secretion of effector proteins and intracellular replication. Importantly, the pilO mutant is attenuated for virulence in BALB/c mice regardless of the route of infection. Following intratracheal and intradermal infection, the mutant caused no histopathology changes, and demonstrated impaired phagosomal escape and replication within lung liver as well as spleen. Thus, PilO is an essential virulence determinant of F. novicida.

Francisella tularensis, the causative agent of tularemia, is classified as Tier 1 Select Agent with bioterrorism potential. The efficacy of the only available vaccine, LVS, is uncertain and it is not licensed in the U.S. Previously, by using an approach generally applicable to intracellular pathogens, we identified working correlates that predict successful vaccination in rodents. Here, we applied these correlates to evaluate a panel of SchuS4-derived live attenuated vaccines, namely SchuS4-ΔclpB, ΔclpB-ΔfupA, ΔclpB-ΔcapB, and ΔclpB-ΔwbtC. We combined in vitro co-cultures to quantify rodent T-cell functions and multivariate regression analyses to predict relative vaccine strength. The predictions were tested by rat vaccination and challenge studies, which demonstrated a clear relationship between the hierarchy of in vitro measurements and in vivo vaccine protection. Thus, these studies demonstrated the potential power a panel of correlates to screen and predict the efficacy of Francisella vaccine candidates, and in vivo studies in Fischer 344 rats confirmed that SchuS4-ΔclpB and ΔclpB-ΔcapB may be better vaccine candidates than LVS.

The α-pore-forming toxins (α-PFTs) from pathogenic bacteria damage host cell membranes by pore formation. We demonstrate a remarkable, hitherto unknown mechanism by an α-PFT protein from Vibrio cholerae . As part of the MakA/B/E tripartite toxin, MakA is involved in membrane pore formation similar to other α-PFTs. In contrast, MakA in isolation induces tube-like structures in acidic endosomal compartments of epithelial cells in vitro. The present study unravels the dynamics of tubular growth, which occurs in a pH-, lipid-, and concentration-dependent manner. Within acidified organelle lumens or when incubated with cells in acidic media, MakA forms oligomers and remodels membranes into high-curvature tubes leading to loss of membrane integrity. A 3.7 Å cryo-electron microscopy structure of MakA filaments reveals a unique protein-lipid superstructure. MakA forms a pinecone-like spiral with a central cavity and a thin annular lipid bilayer embedded between the MakA transmembrane helices in its active α-PFT conformation. Our study provides insights into a novel tubulation mechanism of an α-PFT protein and a new mode of action by a secreted bacterial toxin.

Bacterial survival within a mammalian host is contingent upon sensing environmental perturbations and initiating an appropriate counter-response. To achieve this, sophisticated molecular machineries are used, where bacterial chaperone systems play key roles. The chaperones are a prerequisite for bacterial survival during normal physiological conditions as well as under stressful situations, e.g., infection or inflammation. Specific stress factors include, but are not limited to, high temperature, osmolarity, pH, reactive oxidative species, or bactericidal molecules. ClpB, a member of class 1 AAA + proteins, is a key chaperone that via its disaggregase activity plays a crucial role for bacterial survival under various forms of stress, in particular heat shock. Recently, it has been reported that ClpB also regulates secretion of bacterial effector molecules related to type VI secretion systems. In this review, the roles of ClpB in stress responses and the mechanisms by which it promotes survival of pathogenic bacteria are discussed.

The outer membrane protects Gram-negative bacteria from the host environment. Lipopolysaccharide (LPS), a major outer membrane constituent, has distinct components (lipid A, core, O-antigen) generated by specialized pathways. In this study, we describe the surprising convergence of these pathways through FlmX, an uncharacterized protein in the intracellular pathogen Francisella. FlmX is in the flippase family, which includes proteins that traffic lipid-linked envelope components across membranes. flmX deficiency causes defects in lipid A modification, core remodeling, and O-antigen addition. We find that an F. tularensis mutant lacking flmX is >1,000,000-fold attenuated. Furthermore, FlmX is required to resist the innate antimicrobial LL-37 and the antibiotic polymyxin. Given FlmX's central role in LPS modification and its conservation in intracellular pathogens Brucella, Coxiella, and Legionella, FlmX may represent a novel drug target whose inhibition could cripple bacterial virulence and sensitize bacteria to innate antimicrobials and antibiotics.

The protein MakA was discovered as a motility-associated secreted toxin from Vibrio cholerae. Here, we show that MakA is part of a gene cluster encoding four additional proteins: MakB, MakC, MakD, and MakE. MakA, MakB, and MakE were readily detected in culture supernatants of wild-type V. cholerae, whereas secretion was very much reduced from a flagellum-deficient mutant. Crystal structures of MakA, MakB, and MakE revealed a structural relationship to a superfamily of bacterial pore-forming toxins. Expression of MakA/B/E in Escherichia coli resulted in toxicity toward Caenorhabditis elegans used as a predatory model organism. None of these Mak proteins alone or in pairwise combinations were cytolytic, but an equimolar mixture of MakA, MakB, and MakE acted as a tripartite cytolytic toxin in vitro, causing lysis of erythrocytes and cytotoxicity on cultured human colon carcinoma cells. Formation of oligomeric complexes on liposomes was observed by electron microscopy. Oligomer interaction with membranes was initiated by MakA membrane binding followed by MakB and MakE joining the assembly of a pore structure. A predicted membrane insertion domain of MakA was shown by site-directed mutagenesis to be essential for toxicity toward C. elegans. Bioinformatic analyses revealed that the makCDBAE gene cluster is present as a genomic island in the vast majority of sequenced genomes of V. cholerae and the fish pathogen Vibrio anguillarum. We suggest that the hitherto-unrecognized cytolytic MakA/B/E toxin can contribute to Vibrionaceae fitness and virulence potential in different host environments and organisms.

Francisella tularensis is a facultative intracellular bacterium and the etiological agent of tularemia, a zoonotic disease. Infection of monocytic cells by F. tularensis can be controlled after activation with IFN-γ; however, the molecular mechanisms whereby the control is executed are incompletely understood. Recently, a key role has been attributed to the Guanylate-binding proteins (GBPs), interferon-inducible proteins involved in the cell-specific immunity against various intracellular pathogens. Here, we assessed the responses of bone marrow-derived murine macrophages (BMDM) and GBP-deficient BMDM to F. tularensis strains of variable virulence; the highly virulent SCHU S4 strain, the human live vaccine strain (LVS), or the widely used surrogate for F. tularensis, the low virulent F. novicida. Each of the strains multiplied rapidly in BMDM, but after addition of IFN-γ, significant GBP-dependent control of infection was observed for the LVS and F. novicida strains, whereas there was no control of the SCHU S4 infection. However, no differences in GBP transcription or translation were observed in the infected cell cultures. During co-infection with F. novicida and SCHU S4, significant control of both strains was observed. Patterns of 18 cytokines were very distinct between infected cell cultures and high levels were observed for almost all cytokines in F. novicida-infected cultures and very low levels in SCHU S4-infected cultures, whereas levels in co-infected cultures for a majority of cytokines showed intermediate levels, or levels similar to those of F. novicida-infected cultures. We conclude that the control of BMDM infection with F. tularensis LVS or F. novicida is GBP-dependent, whereas SCHU S4 was only controlled during co-infection. Since expression of GBP was similar regardless of infecting agent, the findings imply that SCHU S4 has an inherent ability to evade the GBP-dependent anti-bacterial mechanisms.

Inhalation of small numbers of Francisella tularensis subspecies tularensis (Ftt) in the form of small particle aerosols causes severe morbidity and mortality in people and many animal species. For this reason, Ftt was developed into a bona fide biological weapon by the USA, by the former USSR, and their respective allies during the previous century. Although such weapons were never deployed, the 9/11 attack quickly followed by the Amerithrax attack led the U.S. government to seek novel countermeasures against a select group of pathogens, including Ftt. Between 2005–2009, we pursued a novel live vaccine against Ftt by deleting putative virulence genes from a fully virulent strain of the pathogen, SCHU S4. These mutants were screened in a mouse model, in which the vaccine candidates were first administered intradermally (ID) to determine their degree of attenuation. Subsequently, mice that survived a high dose ID inoculation were challenged by aerosol or intranasally (IN) with virulent strains of Ftt. We used the current unlicensed live vaccine strain (LVS), first discovered over 70 years ago, as a comparator in the same model. After screening 60 mutants, we found only one, SCHU S4 ΔclpB, that outperformed LVS in the mouse ID vaccination-respiratory-challenge model. Currently, SCHU S4 ΔclpB has been manufactured under current good manufacturing practice conditions, and tested for safety and efficacy in mice, rats, and macaques. The steps necessary for advancing SCHU S4 ΔclpB to this late stage of development are detailed herein. These include developing a body of data supporting the attenuation of SCHU S4 ΔclpB to a degree sufficient for removal from the U.S. Select Agent list and for human use; optimizing SCHU S4 ΔclpB vaccine production, scale up, and long-term storage; and developing appropriate quality control testing approaches.